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https://openalex.org/W2127083139
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https://ajod.org/index.php/ajod/article/download/40/59
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English
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Inclusion of vulnerable groups in health policies: Regional policies on health priorities in Africa
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African journal of disability
| 2,013
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cc-by
| 9,099
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Inclusion of vulnerable groups in health policies:
Regional policies on health priorities in Africa Authors:
Margie Schneider1,2
Arne Henning Eide 3
Mutamad Amin 4
Malcom MacLachlan5
Hasheem Mannan6
Affiliations:
1Psychology Department,
Stellenbosch University,
South Africa
2Centre for Social
Development in Africa,
University of Johannesburg,
South Africa
3SINTEF Technology and
Society, Norway
4Ahfad University for
Women, Omdurman, Sudan
5Centre for Rehabilitation
Studies, Stellenbosch
University, South Africa
6Centre for Global Health and
School of Psychology, Dublin,
Ireland
Correspondence to:
Margie Schneider
Email:
margie_who@mweb.co.za
Postal address:
Flat 13 Derby Court, 1
Gordon Rd, Kenilworth 7708,
South Africa
Dates:
Received: 07 July 2012
Accepted: 07 Oct. 2012
Published: 22 Jan. 2013
How to cite this article:
Schneider, M., Eide, A.H.,
Amin, M., MacLachlan,
M. & Mannan, H., 2013,
‘Inclusion of vulnerable
groups in health policies:
Regional policies on health
priorities in Africa’, African
Journal of Disability 2(1), Art. #40, 9 pages. http://dx.doi. org/10.4102/ajod.v2i1.40 Authors:
Margie Schneider1,2
Arne Henning Eide 3
Mutamad Amin 4
Malcom MacLachlan5
Hasheem Mannan6
Affiliations:
1Psychology Department,
Stellenbosch University,
South Africa
2Centre for Social
Development in Africa,
University of Johannesburg,
South Africa
3SINTEF Technology and
Society, Norway
4Ahfad University for
Women, Omdurman, Sudan
5Centre for Rehabilitation
Studies, Stellenbosch
University, South Africa
6Centre for Global Health and
School of Psychology, Dublin,
Ireland
Correspondence to:
Margie Schneider
Email:
margie_who@mweb.co.za
Postal address:
Flat 13 Derby Court, 1
Gordon Rd, Kenilworth 7708,
South Africa
Dates:
Received: 07 July 2012
Accepted: 07 Oct. 2012
Published: 22 Jan. 2013
How to cite this article:
Schneider, M., Eide, A.H.,
Amin, M., MacLachlan,
M. & Mannan, H., 2013,
‘Inclusion of vulnerable
groups in health policies:
Regional policies on health
priorities in Africa’, African
Journal of Disability 2(1), Art. #40, 9 pages. http://dx.doi. org/10.4102/ajod.v2i1.40
Scan this QR
code
ith o r
Read online: Background: If access to equitable health care is to be achieved for all, policy documents must
mention and address in some detail different needs of groups vulnerable to not accessing such
health care. If these needs are not addressed in the policy documents, there is little chance that
they will be addressed at the stage of implementation. Objectives: This paper reports on an analysis of 11 African Union (AU) policy documents
to ascertain the frequency and the extent of mention of 13 core concepts in relation to 12
vulnerable groups, with a specific focus on people with disabilities. Method: The paper applied the EquiFrame analytical framework to the 11 AU policy
documents. Original Research
Page 1 of 9 Original Research
Page 1 of 9 Original Research Page 1 of 9 Inclusion of vulnerable groups in health policies:
Regional policies on health priorities in Africa The 11 documents were analysed in terms of how many times a core concept
was mentioned and the extent of information on how the core concept should be addressed
at the implementation level. Each core concept mention was further analysed in terms of the
vulnerable group in referred to. Results: The analysis of regional AU policies highlighted the broad nature of the reference
made to vulnerable groups, with a lack of detailed specifications of different needs of different
groups. This is confirmed in the highest vulnerable group mention being for ‘universal’. The
reading of the documents suggests that vulnerable groups are homogeneous in their needs,
which is not the case. There is a lack of recognition of different needs of different vulnerable
groups in accessing health care. Conclusion: The need for more information and knowledge on the needs of all vulnerable
groups is evident. The current lack of mention and of any detail on how to address needs of
vulnerable groups will significantly impair the access to equitable health care for all. 1.Disability as a holistic experience is a barrier to accessing health care but disability is created by a combination of a person’s health
condition and a range of barriers and facilitators in the person’s life context. Introduction Correspondence to:
Margie Schneider
Email:
margie_who@mweb.co.za
Postal address:
Flat 13 Derby Court, 1
Gordon Rd, Kenilworth 7708,
South Africa
Dates:
Received: 07 July 2012
Accepted: 07 Oct. 2012
Published: 22 Jan. 2013 If health policies are to be instruments for realising equity in health, they must specifically
document and address the needs of groups vulnerable, or at least potentially vulnerable, in
their access to equitable health care services. This paper analyses a series of regional African
policy documents to understand the extent to which these documents mention and detail these
vulnerability needs, and address the barriers to access to health care. Acknowledging barriers to people accessing health care has important implications for providing
equitable access to health care and ensuring the best possible outcomes in relation to wellbeing
and social justice. Disability is one such barrier.1 Disability is a common human experience and,
in interaction with crucial environmental barriers, can and does limit equitable access to health
care. Not accessing health care further exacerbates existing health problems, and puts people
with activity limitations at risk for developing new health problems that could easily have been
prevented. For example, if a person in a wheelchair is unable to access the health service they
will not visit the facility when having bronchitis, leading to further complications if not treated
or resolved. Disability is not the only reason why people are limited in their access to equitable
health care, but is the focus of this paper. Other groups that are often limited in their access to
equitable health care include people living in poverty, women-headed households, orphans and
other children with special needs, ethnic minorities, youth, displaced people and people with
chronic illnesses (Dixon Woods et al. 2005; Goudge et al. 2009; Makwiza et al. 2009; Panter-Brick
2002; Perry et al. 2007; Reichard, Sacco & Turnbull 2004; Ridde 2008). How to cite this article:
Schneider, M., Eide, A.H.,
Amin, M., MacLachlan,
M. & Mannan, H., 2013,
‘Inclusion of vulnerable
groups in health policies:
Regional policies on health
priorities in Africa’, African
Journal of Disability 2(1), Art. #40, 9 pages. http://dx.doi. org/10.4102/ajod.v2i1.40 A number of health care priorities have been identified for the African continent by the African
Union (AU), including tuberculosis, malaria, HIV and/or AIDS, other infectious diseases, and
sexual and reproductive health. Original Research This yielded 18 core
concepts, to which were added three identified by the United
Nations Committee on Economic, Social and Cultural Rights
(2000) as being important components of determinants of
equitable access to health care. These additional three core
concepts were accountability, quality and access, including
equitable access to health care as a basic human right (Gilson
et al. 2008; Russel & Gilson 2006). While all 21 core concepts
were considered in the analysis (Table 1), this paper focuses
on 13 core concepts (marked with an asterisk*) that were
identified in the regional policies beyond an occasional
mention in one or two policy documents.3 Equitable access to health care is more likely when issues
from both health-focused and vulnerability-focused policy
documents are integrated into single policy documents with
integrated implementation plans. This ensures that issues of
vulnerability are mainstreamed and included in all policies
rather than being treated as a special case and as a separate
and often ‘afterthought’ issue. We argue that mainstreaming
at the level of policy documents is a step in the direction of
ensuring equitable access to health care truly for all. Buse et al. (2007:1) describe how ‘[p]olicy analysis can
contribute to meeting health objectives and untangling the
complex forces of power and process that underpin change’. Thus, to meet the health objectives of equitable access to
health care by vulnerable groups, we need to understand the
extent to which their needs are integrated into health related
policies, as a step towards understanding the ‘complex
forces’ that influence this integration and lead (or not) to
equitable access to health care. If vulnerable groups and their
specific needs are not visible or are seen as a separate issue
not for inclusion in a ‘mainstream’ policy, their needs are not
likely to be addressed and integrated into such ‘mainstream’
policies, and even less at the implementation and budget
allocation stages. Thus, it is of interest to determine the extent
to which such groups are included in policy documents in
order to advocate for such inclusion if required. The EquitAble project2 has as one of its aims to determine the
impact of a range of vulnerability factors in equitable access
to health care with a specific focus on the impact of disability. Original Research Original Research TABLE 1: EquiFrame: Key questions and key language of core concepts. Number
Core concept
Definition/Key question
1. Non-discrimination
Does the policy support the rights of vulnerable
groups with equal opportunity in receiving
health care? 2. Individualised
services
Does the policy support the rights of vulnerable
groups with individually tailored services to
meet their needs and choices? 3. Entitlement
Does the policy indicate how vulnerable groups
may qualify for specific benefits relevant to
them? TABLE 1: EquiFrame: Key questions and key language of core concepts. Number
Core concept
Definition/Key question
1. Non-discrimination
Does the policy support the rights of vulnerable
groups with equal opportunity in receiving
health care? 2. Individualised
services
Does the policy support the rights of vulnerable
groups with individually tailored services to
meet their needs and choices? 3. Entitlement
Does the policy indicate how vulnerable groups
may qualify for specific benefits relevant to
them? strategies and plans of action to guide AU member states
in managing these health priorities at a national level. In
addition to these health-focused documents, there are series
of policy documents for sectors of the population seen as
being vulnerable and at risk for not accessing the necessary
health care services. These include documents related to
women, youth, elderly people, children and people with
disabilities, as shown in the list of documents reviewed and
provided below. This reflects recognition of the potential
vulnerability of certain groups within populations. Cultural Rights (2000:1) describes health as ‘a fundamental
human right indispensable for the exercise of other human
rights’ and sets out the four intersecting elements ensuring
equitable access to health care services: accessibility,
availability, acceptability and quality. This is the definition of
equitable health care access adopted by the EquitAble project
and used in the development of the policy analysis framework
called EquiFrame (Mannan et al. 2011; MacLachlan et al. 2011). EquiFrame (Mannan et al. 2011) is a policy analysis framework
that uses a set of core concepts that, if mentioned in a policy,
‘informs the analyst concerning what the policy is [and] what
it is intended to accomplish’ (Stowe & Turnbull 2001:206). The
core concepts were extracted from work done specifically on
disability policy analysis within a human rights framework
(Reichard et al. 2004; Stowe & Turnbull 2001; Turnbull, Beegle
& Stowe 2001; Turnbull & Stowe 2001). Original Research The first stage of the EquitAble project comprised an analysis
of policy documents to determine whether the identified
vulnerability factors are integrated into existing policies on
health care provision. The national policies of four African
countries (Sudan, Namibia, Malawi and South Africa),
regional policies developed by the AU for its member states,
and a range of international policies were analysed. The
policies considered are those related to health care service
provision and managing specific health care priorities in
the African region. This paper reports on the analysis of the
regional policies developed by the AU for its member states. The analysis of individual country policies are presented in a
separate paper (Mannan et al. 2012). Vulnerable groups are ‘social groups who experience
limited resources and consequent high relative risk for
morbidity and premature mortality’ (Flaskerud & Winslow
1998:69). The focus of the EquiFrame (Mannan et al. 2011) is
vulnerability, specifically in relation to accessing health care. Vulnerable groups included in the EquitAble analysis were
women, children, elderly people, ethnic minorities, displaced
people, people suffering from some illnesses and people with
disabilities. Other groups, such as sexual minorities, can
also be considered vulnerable but were not included in the
analysis. The focus of the analysis was on developing and
using a policy analysis tool rather than being fully exhaustive
of all vulnerable groups. Vulnerability is a complex process whereby individual
characteristics of a person or group of people put that person
at risk of not accessing, for example, health care. The risk
factor in itself does not cause limited access, but rather the
interaction of the factor with a range of other factors external
to the person results in an outcome of lack of access. Thus, 3.The remaining 7 core concepts were mentioned very rarely or not at all. Introduction Accordingly, the AU has developed a number of policies, Scan this QR
code with your
smart phone or
mobile device
to read online. Read online: Read online: Scan this QR
code with your
smart phone or
mobile device
to read online. Copyright: © 2013. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License. doi:10.4102/ajod.v2i1.40 http://www.ajod.org Page 2 of 9 4.When using the term ‘policy/ies’ the reference intended includes strategy documents,
plans of action, and similar documents that provide guidance and set out principles
for users of the documents. Equitable access to health care and EquiFrame The United Nations Committee on Economic, Social and 2.The full title of the EquitAble project is ‘Enabling universal and equitable access
to healthcare for vulnerable people in resource poor settings in Africa’ and it is
funded by the European Union Funding Programme 7 (FP7). Further information is
obtainable at http://www.sintef.no/Projectweb/Equitable/. 3.The remaining 7 core concepts were mentioned very rarely or not at all. http://www.ajod.org doi:10.4102/ajod.v2i1.40 Original Research
Page 3 of 9 Page 3 of 9 TABLE 2: List and definition of vulnerable groups. Number
Vulnerable group
Attributes or definitions
1. Limited resources
Referring to poor people or people living in poverty. 2. Increased relative risk for morbidity
Referring to people with one of the top 10 illnesses, identified by WHO, as occurring within the relevant country. 3. Mother child mortality
Referring to factors affecting maternal and child health (Children 0−5 years). 4. Women-headed household
Referring to households headed by a woman. 5. Children (with special needs)
Referring to children marginalised by special contexts, such as orphans or children on the street. 6. Aged
Referring to older age. 7. Youth
Referring to younger age without identifying gender. 8. Ethnic minorities
Referring to non-majority groups in terms of culture, race or ethnic identity. 9. Displaced populations
Referring to people who, because of civil unrest or unsustainable livelihoods, have been displaced from their previous
residence. 10. Living away from services
Referring to people living far from health services, either in time or distance. 11. Suffering from chronic
illness
Referring to people who have an illness which requires continuing need for care. 12. People with disability
Referring to persons with disabilities, including physical, sensory, intellectual or mental health conditions, and including
synonyms of disability. 13
Universal
Making general reference to vulnerable groups without being specific enough to be categorised under any one group. WHO, World Health Organization. TABLE 2: List and definition of vulnerable groups. Making general reference to vulnerable groups without being specific enough to be categorised under any one group. having an impairment does not in itself determine access
to health care, but the interaction of having an impairment,
such as loss of lower limb mobility, long distances to the
health facility, poor or expensive transport, and inaccessible
buildings all contribute to an outcome of inequitable access
to health care for that person. Equitable access to health care and EquiFrame The role of policies is to ensure
that these external factors are set out and elucidated in order
to ensure effective implementation. This needs to be done for
the different needs of different vulnerable groups. and extent of mention of the identified vulnerable groups
and core concepts. Methodology The research design is exploratory in nature and consists of
reviews and analysis of key health-focused policies using
the EquiFrame matrix of 13 of the 21 core concepts and 13
vulnerable groups, including the ‘universal’ category. The EquiFrame matrix provides a detailed analysis of which
core concept is mentioned for which vulnerable groups and
the nature of the mention, as set out in Appendix 1, as an
example. The nature of the mention was rated according
to a scale, as set out below. The higher the rating the more
comprehensive and detailed the nature of the mention. Ratings of 3 and 4 would be required in order to address the
different needs of different vulnerable groups: Mannan et al. (2011) identified the 12 key vulnerable groups
from a review of the literature. An additional category
‘universal’, was included to capture information clearly
referring to vulnerable groups but not sufficiently specific in
its mention to allocate to one of the other 12 groups (Table 2). Two of the groups were omitted from this regional analysis –
‘increased relative risk for morbidity’ and ‘mother and child
mortality’ – as these are more complex and require access to
health care to be given a diagnosis of a health condition for
the first, or a range of factors outside of a narrow scope of
health care for the second. 0 = Concept not mentioned at all 0 = Concept not mentioned at all
1 = Concept only mentioned but not developed (incidental)
2 = Concept mentioned and explained (notable)
3 = Specific policy actions identified to address concept (centra
4 = Intention to monitor concept expressed (fundamental)
N/A = Not relevant 1 = Concept only mentioned but not developed (incident 2 = Concept mentioned and explained (notable) 3 = Specific policy actions identified to address concept (central) 4 = Intention to monitor concept expressed (fundamental) Original Research Original Research accordingly. For the purpose of this report, the documents
reviewed will be referred to as policy documents. accordingly. For the purpose of this report, the documents
reviewed will be referred to as policy documents. the actual term. For example, ‘limited resources’ and ‘poor’
were noted as being about the same vulnerable group. Once the policy was analysed and the ratings set out on the
matrix, the 11 policies were summarised with respect to the
number of core concepts mentioned and for which vulnerable
group these were mentioned, as set out in Appendix 2. Criteria for selection of regional policies There were a number of criteria applied to the selection of
the policy documents. The exploratory nature of the research
allowed us to select key policies to use in the exploration
rather than being exhaustive. In addition the scope of the
policy had to be health or health care services, be a current
document (even if dated from 10 to 15 years ago), and be
regionally focused in their scope. The documents were
obtained from a review of the health and health related
sections of the AU website. If a policy was a general one, those sections that made
reference to health and health care and the preamble and
guiding principles were identified within the policy and
analysed in detail. For example, if a document dealt with
issues ranging from health, education and employment,
then only the preamble and the specific health clauses were
considered for analysis. The language used in the policy was
the basis for the analysis. The following 11 documents were selected and analysed. All
are available from the AU website (AU n.d.): 1. African charter on human and people’s rights – Banjul,
Organisation for African Unity (OAU) document, 1981. 1. African charter on human and people’s rights – Banjul,
Organisation for African Unity (OAU) document, 1981. If there was more than one occurrence of a core concept for
a particular vulnerable group, all instances were recorded
under the ‘number column of the matrix’ but only the highest
rating was included under the relevant vulnerable group. The analysis presented below focuses on the number of
times a core concept was mentioned, followed by a separate
description of the rating. y
2. African charter on the rights and welfare of the child
– 1990, entered into force 1999 – OAU. 2. African charter on the rights and welfare of the child
– 1990, entered into force 1999 – OAU. 3. Protocol to the African charter on human and people’s
rights on the rights of women in Africa. – 1995 adopted
in 2003. 3. Protocol to the African charter on human and people’s
rights on the rights of women in Africa. – 1995 adopted
in 2003. 4. Africa summit on roll back Malaria – 25 April 2000, Abuja
– OAU document. 4. Africa summit on roll back Malaria – 25 April 2000, Abuja
– OAU document. The vulnerable groups 11. Africa health strategy: 2007–2015 – AU document,
April 2007. The 11 documents together mention all 13 vulnerable groups
(VGs) (Table 3). However, the most commonly mentioned
VGs across all policies are ‘universal’ (in 10 policies) and
‘youth’ (7 policies). The other VGs were only mentioned 1−3
times across the 11 policies. The least mentioned VGs (only Ethical considerations 5. Abuja declaration on HIV and/or AIDS, TB and other
related infections diseases – OAU, April 2001. As this was a document review no ethical clearance was
required. 6. Pan-African forum for children – OAU document –
May 2001. 6. Pan-African forum for children – OAU document –
May 2001. Results and discussion 7. Maputo declaration on HIV/AIDS, tuberculosis, Malaria
and other related infectious diseases – July 2003 – AU
document. The analysis confirmed that the regional documents provide, in
general, broad guidelines for individual countries to develop
their own national level policies and guidelines. Very
few implementation plans or monitoring and evaluation
suggestions were made. While guidelines for these latter
components should be provided in regional policies, the
detail would be the domain of the individual country policies. 8. Draft continental policy framework for the promotion
of sexual and reproductive health and rights in Africa. –
AU, October 2005. 9. Plan of action on sexual and reproductive health and
rights (Maputo Plan of Action) 2007–2010 – AU
document, September 2006. 10. AU youth charter – AU, July 2006. Aim and objectives N/A = Not relevant Regional policies refer to policies developed within Africa
and by the main African regional body, the AU. The AU is
a body that brings together African countries and develops
policies on issues related to specific African continent needs
and problems. The aim of this paper is to review the extent to which
policies4 addressing regional health priorities document
the needs of vulnerable groups, as reflected in the core
concepts. The underlying assumption is that their inclusion
within policies will increase the likelihood of effective and
integrated management on the ground. This assumption
is not tested in this paper but is addressed in later papers
from the EquitAble project that look at the realised access to
health care services in four African countries. The focus is on
a review of the actual policy documents only and not of the
policy development process or its implementation. The documents analysed for this report are recommendations
from the AU Assembly to member countries, with the
aim of providing guidelines for member states to develop
their own national level policies. The documents reviewed
include charters, protocols, declarations, policy frameworks,
strategies and a plan of action. These documents deal with
continent-wide issues of importance in relation to health,
such as HIV and/or AIDS, Malaria, TB, and sexual and
reproductive health services. The objectives of the paper are to: 1. Identify relevant health policies for the region of Africa
developed by the African Union. 2. Review these policy documents in relation to the frequency 2. Review these policy documents in relation to the frequency These documents are usually prepared by experts who
present them to the council of ministers from member
states (in this case Ministers of Health) to review and adopt doi:10.4102/ajod.v2i1.40 http://www.ajod.org Page 4 of 9 Analytical process The policies were analysed by two researchers independently,
followed by a comparison of the ratings used. Differences
were discussed and a consensus reached on what rating
to use. The core concepts were rated under the vulnerable
group referred to in the policy. If no mention was made of
any particular vulnerable group, but the reference to these
groups was clear (e.g. ‘all vulnerable groups’), the rating was
placed under ‘universal’. Thus each policy has a resulting
completed matrix. (See Appendix 3 for an example of such an
analysis). The mentioning of a core concept was rated from 1
to 4 depending on the nature of the mention as set out above. The analysis consisted of scanning the written text for
mentions of core concepts and vulnerable groups. Terms that
were contained in the definition of the concepts of groups
were noted as mentions, and, in addition, different terms but
that had the same meaning. The focus was on the meaning of
the core concept or vulnerable groups rather than strict use of TABLE 3: Number of mentions for each vulnerable group in 11 regional policies. Vulnerable groups
Number of times
mentioned
Number of policies in
which VG is mentioned
Limited resources
0
0
Women-headed households
0
0
Ethnic minorities
0
0
Elderly
4
3
Displaced populations
5
2
Living far from services
5
3
With disabilities (including youth
with disabilities)
7
4
Children with special needs
9
4
With chronic illness
10
3
Youth (without disabilities)
40
7
Universal
148
10
VG, vulnerable groups. TABLE 3: Number of mentions for each vulnerable group in 11 regional policies. Vulnerable groups
Number of times
mentioned
Number of policies in
which VG is mentioned
Limited resources
0
0
Women-headed households
0
0
Ethnic minorities
0
0
Elderly
4
3
Displaced populations
5
2
Living far from services
5
3
With disabilities (including youth
with disabilities)
7
4
Children with special needs
9
4
With chronic illness
10
3
Youth (without disabilities)
40
7
Universal
148
10
VG, vulnerable groups. TABLE 3: Number of mentions for each vulnerable group in 11 regional policies. Vulnerable groups
Number of times
Number of policies in http://www.ajod.org doi:10.4102/ajod.v2i1.40 Page 5 of 9 Original Research TABLE 4: Number of core concepts mentioned in 11 regional policies (ranked by
total number of CC mentioned). Analytical process Core concept
Number of policies
mentioning core concept
(out of 11 policies)
Total number of core
concept mentions
1. Protection from harm
6
17
2. Prevention
9
23
3. Autonomy
0
0
4. Privacy
0
0
5. Non-discrimination
6
22
6. Cultural responsiveness
3
8
7. Coordination of services
6
32
8. Capacity building
7
42
9. Individualised service
6
22
10. Accountability
3
12
11. Quality
6
14
12. Access
6
28
13. Efficiency
2
15
CC, core concept. TABLE 4: Number of core concepts mentioned in 11 regional policies (ranked by
total number of CC mentioned). mentioned in 1 policy) were ‘limited resources’, ‘women-
headed households’ and ‘displaced populations’. The high rate of mention for youth refers to youth generally
and does not include youth with disabilities, who were
counted under ‘with disabilities’. This number is explained
in part by the inclusion of the AU Youth Charter. When
looking at youth specifically, the analysis shows that 25
mentions of this vulnerable group were spread across 6
policies, excluding the Youth Charter, and 14 mentions in
the Youth Charter in relation to health care. This suggests
that issues of youth access to health care are reasonably well
addressed in most policies and that the Youth Charter does
address health care access as one of its concerns. The Youth
Charter includes three mentions of youth with disabilities
(counted under ‘with disabilities’), and one of ‘living far from
services’. This finding is congruent with the fact that policies
that address specific groups, such as youth, will address
needs of that group and will not address needs of some of
the other vulnerable groups. mention of ‘non-discrimination’ and ‘individualised services’. Both are mentioned in six policies. Mentions of these two
core concepts remain general (e.g. ‘no discrimination for
anyone’ and ‘services relevant for all’) rather than specifying
different needs of different vulnerable groups, especially
disabled people. The large number of ‘universal’ mentions (relative to the
other 12 specific vulnerable groups) highlights the nature
of the policy content, which remains broad and lacking in
specificity in relation to those sectors of the population who
may struggle to access health care. For example, a policy
would mention ‘all vulnerable groups’ but not specify which
these would include. Core concepts The frequency of occurrence of each of the 13 core concepts in
the 11 documents is presented below (Table 4). The numbers
in the first column are those allocated to the core concepts as
set out in Table 1 above. The last column gives the number
of times each core concept was mentioned in total across all
the policies. Across the 11 policies, ‘capacity building’ was the
most frequently mentioned core concept (42) followed
by ‘coordination of services’. Apart from ‘autonomy’
and ‘privacy’, which were never mentioned, ‘cultural
responsiveness’ was mentioned least. These results reflect
the scope of the policies as pushing for capacity building of
health care providers and ensuring that services are provided
in a coordinated manner. ‘Access’ is mentioned 28 times
across 6 policies. However, as shown in the more detailed
analysis on ‘access’ below, the majority of these mentions are
about a broad notion of ‘equitable access to health care’ in
some form or another. Making services physically accessible
and providing information in an accessible format are not
reflected as being important components of health care
provision, as they are not mentioned specifically. This trend
is counteracted to some extent by the relatively frequent Core concepts that were coded as having a specific policy
action were ‘protection from harm’, ‘prevention’, ‘non-
discrimination’, ‘capacity building’, ‘individualised services’,
‘accountability’, ‘quality’ and ‘access’. Rating of mentioned core concepts The majority of core concepts were only mentioned
(rating 1) or mentioned and explained (rating 2). Very
few were mentioned together with a specific policy action
identified to address the core concept (rating 3). None of
the mentioned core concepts included any discussion on
intentions to monitor the core concept addressed (rating 4). Only four policies had ratings of ‘3’ identifying any policy
action. These were the Pan African Forum for Children (May
2001), the draft continental policy framework for promotion
of sexual and reproductive health rights in Africa (October
2005), AU Youth Charter (July 2006) and the African health
strategy (2007). Analytical process Whilst it is acknowledged that this is
a step in the right direction, there remains a gap as to how
the needs of such groups are to be met within the scope of
a policy. It sounds more like a token mention of vulnerable
groups than a serious consideration of the aspects to be
addressed in order to meet their needs. Furthermore, the lack
of specificity obscures the different needs of different groups. These results all reflect the broad nature of these regional
documents and their emphasis on building strong health care
services that provide equitable access for all but with little
unpacking of what equitable access entails for individual
vulnerable groups or what ‘for all’ really means. Conclusions and recommendations This lack, furthermore, may reflect poor
or no knowledge of the required specifications and/or the
human rights implications of not addressing specific needs The analysis of regional AU policies highlighted the broad
nature of the reference made to vulnerable groups, with a
lack of detailed specifications of different needs of different
groups. This is confirmed in the highest vulnerable group TABLE 5: Analysis of access mentions in eleven regional documents. TABLE 5: Analysis of access mentions in eleven regional documents. d i 10 4102/ j d 2i1 40
htt //
j d
TABLE 5: Analysis of access mentions in eleven regional documents. Regional Policy Document
Number of mentions
Content of mentions
1. African charter on human and people’s rights – Banjul, OAU document 1981
0
–
2. Protocol to the African charter on human and people’s rights on the rights of
women in Africa – 1995, adopted in 2003
1
• Access to health care services 1
3. Maputo declaration on HIV/AIDS, Tuberculosis, Malaria and other related
infections diseases – July 2003 – AU document
1
• Financial (affordable drugs) 1
4. African charter on the rights and welfare of the child – 1990, entered into
force 1999 – OAU
2
• Physical (buildings) 1
• Information (training) 1
5. Abuja declaration on HIV/AIDS, TB and other related infections diseases –
OAU – April 2001
3
• Access to health care services 1
• Financial (Affordable drugs and vaccines) 2
6. Africa summit on roll back malaria – 25 April 2000, Abuja – OAU document
3
• Access to health care services 2
• Financial access (resources required) 1
7. Pan-African forum for children – OAU document – May 2001
3
• Information 1
• Access to health care services 1
• Financial (Affordable drugs) 1
8. AU Youth Charter – AU July 2006
6
• Access to health care services (equitable access;
programmes to address health pandemics; timely access to
health care) 4
• Information (youth friendly services) 1
• Physical (infrastructure and mobility for access) 1
9. Africa health strategy: 2007 – 2015 – AU document, April 2007
8
• Services (essential health care) 4
• Financial (essential drugs; cost; exempt from taxes) 3
• Physical (distance and time to reach services) 1
10. Conclusions and recommendations The first objective of this paper was to identify relevant AU
policies for the African region. We identified 11 policies that
are current in their application, ranging from ones focused on
specific groups (e.g. Youth Charter) through to one focused
on specific issues (e.g. Draft continental policy framework for
the promotion of sexual and reproductive health and rights
in Africa). These policies are not exhaustive by any means,
but provide a way to start the process of policy analysis
proposed in this paper. Given the usefulness of this analysis
in highlighting gaps in these 11 policies, the recommendation
is to continue this type of analysis on a wider range of health
and other policies. In total there were 28 mentions of ‘access’, ranging from zero
to nine mentions per document (Table 5). The more general
category of ‘equitable access to services’ is the largest of
the mentioned components, confirming the broad-stroke
nature of the regional policies. While this category does not
specifically refer to any of the three components – physical,
financial and information access – they are implicit in the
phrase ‘access to universal and equitable health care services’
or variations on that phrase. However, given the discussion
above, it may be insufficient for these to be mentioned
implicitly, and it would be preferable for these policies to
make more explicit what is required in order to ensure access
to universal and equitable health. The second objective of reviewing these policies in relation to
core concepts and vulnerable groups showed that there are
important gaps in the way the core concepts and vulnerable
groups are addressed in these policies. The very general
nature of reference to vulnerable groups and the limited
mention of core concepts suggests that there is limited scope
for guiding countries in developing their national policies. The issue of affordable drugs or access to drugs have been
coded under ‘financial’ because of its overt reference to
financial aspects of this reference – affordable. The lack of further specifications in the ‘access to universal
and equitable health care’ may lead to lack of implementation
in a context where there are many implementation issues
to be considered. Page 6 of 9 11 policies) relative to the other three. The relevant core
concept is ‘access’. 11 policies) relative to the other three. The relevant core
concept is ‘access’. of different vulnerable groups. Given the argument that
policy documents must make clear reference to the needs
of vulnerable groups and ways to meet these through
implementation as one of the components of ensuring
universal and equitable access to health, it is worrying that
the regional policy documents fail to provide more detailed
specifications. There are different components to accessibility – physical,
financial and information. The references to ‘access’ in
the 11 documents were reviewed in relation to these three
components. An additional type of mention is a more
general point about ‘access to services’ which is common in
the regional documents. The documents frequently make
reference to the need for access to health care services but
without any further specifications of what this entails. Original Research Original Research Page 6 of 9 Analysis of access references The notion of accessibility is one of the four components that
ensure equitable access to health care, as discussed at the
start of this paper, with the other three being acceptability,
availability and quality. Accessibility is crucial in facilitating
equalisation of opportunities for people with disabilities,
making it important to look at this aspect in more detail. The
further analysis of access only is driven largely by the high
number of mentions of this core concept (28 mentions across doi:10.4102/ajod.v2i1.40
http://www.ajod.org http://www.ajod.org doi:10.4102/ajod.v2i1.40 References African Union (AU), n.d., viewed 18 October 2009, from http://www.africa-union.org/
root/au/AboutAu/au_in_a_nutshell_en.htm The regional nature of the policies may explain some of the
lack of detail concerning relevant policy actions and intention
to monitor, as this would be more suitable in national level
policies. However, one would like to see regional policies
provide guidelines on these at least. In particular, the number
of ratings of 3 and 4 should be increased. Increased numbers
of 3 ratings would ensure that specific policy actions are
identified to address the concept, and of 4 ratings that these
policy actions are taken seriously through an intention to
monitor the concept. Buse, K., Dickinson, C., Gilson, L. & Murray, S., 2007, ‘How can the analysis of
power and process in policy-making improve health outcomes?’ Briefing paper
(October), Overseas Development Institute, London. Flaskerud, J.H. & Winslow, B.J., 1998, ‘Conceptualizing vulnerable populations
health-related research’, Nursing Research 47(2), 69−78. http://dx.doi. org/10.1097/00006199-199803000-00005 Goudge, J., Gilson, L., Russell, S., Gumede, T. & Mills, A., 2009, ‘Affordability, availability
and acceptability barriers to health care for the chronically ill: Longitudinal case
studies from South Africa’, BMC Health Services Research 9(75), n.p. http://www. biomedcentral.com/1472-6963/9/75 MacLachlan, M., Mannan, H., El Tayeb, S., El Khatim, A., Swartz, L., Munthali, A., van
Rooy, G., McVeigh, J., Eide, A.H. & Schneider, M., 2011, ‘EquiFrame: A framework
for analysis of the inclusion of human rights and vulnerable groups in health
policies’, Health & Human Rights 13(2), 1−20. Makwiza, I., Nyirenda, L., Bongololo, G., Banda, T., Chimzizi, R. & Theobald, S., 2009,
‘Who has access to counselling and testing and anti-retroviral therapy in Malawi
– An equity analysis’, International Journal for Equity in Health 8(13), n.p. http://
www.equityhealthj.com/content/8/1/13 The recent focus on the importance of mainstreaming
disability in the discussion on the millennium development
goals (MDGs) reflects, firstly, the lack of consideration of
disability within the MDGs generally, and secondly, should
be an impetus for ensuring that the needs of disabled people
and other vulnerable groups are made more explicit in
policies. Mannan, H., Amin, M., MacLaclahn, M. with El Tayeb, S., El Khatim, A., Bedri, N.,
McVeigh, J., Swartz, L., Munthali, A., Van Rooy, G., Eide, A. & Schneider M.,
2011, The EquiFrame manual, The Global Health Press, Dublin. http://dx.doi. org/10.1177/1044207312439103 Mannan, H., McVeigh, J., MacLaclan M., Amin M., Swartz, L., Munthali, A. Competing interests Turnbull, H.R. & Stowe, M.J., 2001, ‘A taxonomy for organizing the core concepts
according to their underlying principles’, Journal of Disability Policy Studies 12(3),
177–97. http://dx.doi.org/10.1177/104420730101200304 The authors declare that they have no financial or personal
relationships which may have inappropriately influenced
them in writing this paper. United Nations Economic and Social Council, 2000, Substantive Issues Arising in
the Implementation of the International Covenant on Economic, Social and
Cultural Rights, General Comment No. 14, The Right to the Highest Attainable
Standard of Health (Article 12 of the International Covenant on Economic, Social
and Cultural Rights), viewed 2 July 2012, from http://www.unhchr.ch/tbs/doc. nsf/%28symbol%29/E.C.12.2000.4.En. Acknowledgments Ridde, V., 2008, ‘The problem of the worst-off is dealt with after all other issues’:
The equity and health policy implementation gap in Burkina Faso’, Social Science
& Medicine 66(6), 1368–78. http://dx.doi.org/10.1016/j.socscimed.2007.10.026 This research was funded by the European Commission
Framework Programme 7, Project Title: Enabling Universal
and Equitable Access to Healthcare for Vulnerable People
in Resource Poor Settings in Africa, Grant Agreement
No.: 223501. Russell, S. & Gilson, L., 2006, ‘Are health services protecting the livelihoods of the urban
poor in Sri Lanka? Findings from two low-income areas of Colombo’, Social Science
& Medicine 63(7), 1732–44. http://dx.doi.org/10.1016/j.socscimed.2006.04.017 Stowe, M.J. & Turnbull, H.R., 2001, ‘Tools for analyzing policy on the books and policy
on the streets’, Journal of Disability Policy Studies 12(3), 206–214. http://dx.doi. org/10.1177/104420730101200306 Turnbull, H.R., Beegle, G. & Stowe, M.J., 2001,‘The core concepts of disability policy
affecting families who have children with disabilities’, Journal of Disability Policy
Studies 12(3), 133–43. http://dx.doi.org/10.1177/104420730101200302 References & van Rooy,
G., 2012, ‘Core concepts of human rights and inclusion of vulnerable groups in the
disability and rehabilitation policies of Malawi, Namibia, Sudan and South Africa’,
Journal of Disability Policy Studies 23(2), 67−81. Panter-Brick, C., 2002, ’Street children, human rights, and public health: A critique
and future directions’, Annual Review of Anthropology 31, 147–71. http://dx.doi. org/10.1146/annurev.anthro.31.040402.085359 One of the aims of the AU is to ‘mainstream gender in all
programmes and activities of the union.’ (AU n.d.). Alongside
the mainstreaming of gender, there needs to be mainstreaming
of disability needs and those of other vulnerable groups in all
the policies and related documentation. The focus should be
on diversity management to address all needs of all people. Perry, H.B., King-Schultz, L.W., Aftab, A.S. & Bryant, J.H., 2007, ‘Health equity issues at
the local level: Socio-geography, access, and health outcomes in the service area
of the Hôpital Albert Schweitzer-Haiti’, International Journal for Equity in Health
6(7), n.p. http://www.equityhealthj.com/content/6/1/7 Reichard, A., Sacco, T. M. & Turnbull, R., 2004, ‘Access to health care for individuals
with developmental disabilities from minority backgrounds’, Mental Retardation
42(6), 459–70. http://dx.doi.org/10.1352/0047-6765(2004)42<459:ATHCFI>2.0. CO;2 Original Research Original Research M.S. (Stellenbosch University and University of Johannesburg)
and A.H.E. (SINTEF) did the analysis of the regional policies. M.A. (Trinity College Dublin), H.M. (Trinity College Dublin)
and M.M. (Afhad University for Women) were team leaders
for the development of EquiFrame. M.S. drafted the paper
and all authors contributed extensive comments. mention being for ‘universal’. The reading of the documents
suggests that vulnerable groups are homogeneous in their
needs, which is not the case. The needs of a people living
far from a health facility are not the same as those for a
deaf person attending a clinic. The first requires adequate
transport while the second requires an accessible form of
communication, such as sign language. This type of nuance
is lost in general mentions of vulnerable groups. Conclusions and recommendations Draft continental policy framework for the promotion of sexual and
reproductive health and rights in Africa – AU, October 2005
9
• Access to health care services (universal access) 6
• Financial (affordable supply and use of ARVs; increasing
resources for services) 2 (information for youth) 1
11. Plan of action on sexual and reproductive health and rights (Maputo Plan of
Action) 2007–2010 – AU document, September 2006
9
• Access to health care services (universal access) 7
• Financial (basic medicines available; quality and affordable
services) 2
AU, African Union; OAU, Organisation for African Unity; ARV, Antiretroviral drugs. doi:10.4102/ajod.v2i1.40 Page 7 of 9 Appendix 1 Appendix 1 p
p
y
Policy Title: Africa health strategy: 2007−2015. AU document, April 2007. Concepts
Number of
mentions
Limited
resources
Increase RR of
morbidity
MCM
WHH
Children (with
special needs)
Aged
Youth
Ethnic
minorities
Displaced
populations
Living away from
services
Suffering from
chronic illness
Disabled
Universal
Protection from harm
1
0
0
2
0
0
0
0
0
0
0
0
0
1
Prevention
5
0
0
1
0
0
0
1
0
1
0
0
0
1
Autonomy
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Privacy
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Participation
5
0
0
0
0
0
0
0
0
0
0
0
0
3
Liberty
-
0
0
0
0
0
0
0
0
0
0
0
0
Non-discrimi-nation
8
0
0
2
0
0
0
0
0
0
0
0
0
2
Cultural res-ponsiveness
5
0
0
0
0
0
0
0
0
0
0
0
0
1
Family resources
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Family Support
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Integration
1
0
0
0
0
0
0
-
0
1
0
0
0
1
Contribution
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Coordination of services
18
0
0
0
0
0
0
0
0
0
0
0
0
18
Capacity building
20
0
0
2
0
0
0
0
0
0
0
0
0
3
Capability based services
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Individualised services
4
0
0
0
0
0
0
0
0
1
0
0
0
1
Account-ability
9
0
0
0
0
0
0
0
0
0
0
0
0
3
Quality
7
0
0
0
0
0
0
0
0
0
0
0
0
3
Access
8
0
0
0
0
0
0
0
0
0
0
0
0
3
Efficiency
8
0
0
0
0
0
0
0
0
2
0
0
0
2
Number of times VG
mentioned
-
0
0
6
0
0
1
1
0
4
0
0
1
78
WHH, women headed households; MCM, mother/child mortality; RR, relative risk. Authors’ contributions Woods, D., Kirk, M., Agarwal, D., Annandale, S., Arthur, E., Harvey, T., Hsu, J., Katbamna,
R., Olsen, S., Smith, R., Riley, L., & Sutton, A., 2005, Vulnerable groups and access
to healthcare: A critical interpretive review, National Co-ordinating Centre for NHS
Service Delivery and Organization R & D, London. Appendix starts on next page → Appendix starts on next page → doi:10.4102/ajod.v2i1.40
http://www.ajod.org http://www.ajod.org doi:10.4102/ajod.v2i1.40 Original Research
Page 8 of 9 Appendix 2 Appendix 1 Maputo declaration on
HIV/AIDS, Tuberculosis,
Malaria and other related
infectious diseases – July
2003
-
-
-
-
-
-
-
-
Service
coordination
and
collaboration
– 2
Prof and
system
capacity
building – 2
Access – 1
-
Prevention and amelioration
– 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 1
8. Draft Continental policy
framework
for the promotion of
sexual and reproductive
health rights – AU October
2005
-
-
-
Individualised
appropriate
services – 1
Prevention and
amelioration – 1
Cultural
responsiveness – 1
Service
coordination and
collaboration – 1
Individualised
appropriate
services – 1
-
-
-
Prevention
and
amelioration
– 1
Service
coordination
and
collaboration
– 1
Individualised
appropriate
services – 1
Individualised
appropriate services
– 1
Protection from harm – 3
Prevention and amelioration – 1
Anti-discrimination – 2
Cultural responsiveness – 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 2
Individualised appropriate
services – 1
Accountability – 1
Quality – 3
Access – 1
9. Plan of action on sexual
and reproductive
health and rights (Maputo
Plan of Action) 2007–2010
-
-
-
-
Individualised
appropriate
services – 1
-
-
-
Service
coordination
and
collaboration
– 1
Access – 1
-
Prevention and amelioration – 1
Anti-discrimination – 2
Cultural responsiveness – 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 3
Individualised appropriate
services – 1
Accountability – 2
Quality – 1
Access – 2
10. AU Youth Charter – AU
July 2006
-
-
-
-
Protection from
harm – 5
prevention and
amelioration – 4
Anti-
discrimination-4
Access – 1
-
-
Access–1
-
Anti-
discrimination – 2
Access – 1
- appropriate
services – 2
Quality – 1
7. Maputo declaration on
HIV/AIDS, Tuberculosis,
Malaria and other related
infectious diseases – July
2003
-
-
-
-
-
-
-
-
Service
coordination
and
collaboration
– 2
Prof and
system
capacity
building – 2
Access – 1
-
Prevention and amelioration
– 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 1
8. Appendix 1 Maputo declaration on
HIV/AIDS, Tuberculosis,
Malaria and other related
nfectious diseases – July
2003
-
-
-
-
-
-
-
-
Service
coordination
and
collaboration
– 2
Prof and
system
capacity
building – 2
Access – 1
-
Prevention and amelioration
– 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 1
8. Draft Continental policy
ramework
or the promotion of
sexual and reproductive
health rights – AU October
2005
-
-
-
Individualised
appropriate
services – 1
Prevention and
amelioration – 1
Cultural
responsiveness – 1
Service
coordination and
collaboration – 1
Individualised
appropriate
services – 1
-
-
-
Prevention
and
amelioration
– 1
Service
coordination
and
collaboration
– 1
Individualised
appropriate
services – 1
Individualised
appropriate services
– 1
Protection from harm – 3
Prevention and amelioration – 1
Anti-discrimination – 2
Cultural responsiveness – 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 2
Individualised appropriate
services – 1
Accountability – 1
Quality – 3
Access – 1
9. Plan of action on sexual
and reproductive
health and rights (Maputo
Plan of Action) 2007–2010
-
-
-
-
Individualised
appropriate
services – 1
-
-
-
Service
coordination
and
collaboration
– 1
Access – 1
-
Prevention and amelioration – 1
Anti-discrimination – 2
Cultural responsiveness – 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 3
Individualised appropriate
services – 1
Accountability
2 APPENDIX 2 (Continues...): Summary analysis of selected vulnerable groups and core concepts in 11 regional policies. Policy
LR
WHH
Children with
special needs
Elderly
Youth
Ethnic
mino-rities
Displaced
pop
Living far
from services
With chronic
illness
With disabilities
Universal
6. Pan-African forum for
children –
OAU document – May
2001
Prof and systems
capacity building
– 2
Individualised
appropriate
services – 1
Protection from
harm – 2
Prevention and
amelioration – 3
Anti-discrimination
– 3
Prof and systems
capacity building
– 2
Individualised
appropriate
services – 2
Quality – 1
Prof and
systems
capacity
building – 2
-
-
-
Access – 2
7. Appendix 1 APPENDIX 2: Summary analysis of selected vulnerable groups and core concepts in 11 regional policies. li
hild
i h
ld
l
h
h i
i
l
d
i i
f
i h h
i
i h di
biliti
i
l /
APPENDIX 2: Summary analysis of selected vulnerable groups and core concepts in 11 regional policies. Policy
LR
WHH
Children with
special needs
Elderly
Youth
Ethnic
mino-rities
Displaced
pop
Living far
from services
With chronic
illness
With disabilities
Universal
1. Abuja declaration on
HIV/AIDS
-
-
Prevention and
amelioration - 1§
-
Prevention and
amelioration −1
Prevention and
amelioration – 2
-
-
-
-
-
Anti-discrimination – 1
Service coordination and
collaboration – 3
Prevention and amelioration
– 1
Prof and system capacity
building – 2
Access – 3
Quality – 1
2. African charter on the
rights
and welfare of the child
– 1990
-
-
Anti-
discrimination-1
services – 2
Access – 1
-
-
-
-
-
-
-
Anti-discrimination – 2
Individualised appropriate
services – 2
Access – 1
3. Protocol to the African
charter on human and
people’s rights on the
rights of women in Africa –
1995, adopted in 2003
-
-
-
Individualised
appropriate
services – 1
-
-
-
Individualised
appropriate
services – 1
-
Individualised
appropriate
services – 1
Prevention and amelioration
– 1
Individualised appropriate
services – 1
Access – 1
4. African charter on
human and
people’s rights – 1981
-
-
-
Protection from
harm – 1
Prevention &
amelioration- 1
-
-
-
-
-
Protection
from harm – 1
Prevention
and amelioration
– 1
Protection from harm – 1
Prevention and amelioration
– 1
5. Africa summit on roll back
Malaria – 25 April 2000
-
-
Protect from
harm – 1
-
Protection from
harm – 1
Prof and systems
capacity
building – 2
Efficiency – 1
-
-
Efficiency – 3
-
-
Protection from harm – 1
Anti-discrimination – 1
Service coordination and
collaboration – 3
Prof and systems and capacity
building – 2
Quality – 1
Access – 3
Efficiency – 3
OAU, Organisation for African Unity; AU, African Union; LR, limited resources; WHH, women headed households. Appendix 2 continues on next page → doi:10.4102/ajod.v2i1.40 doi:10.4102/ajod.v2i1.40 Original Research
Page 9 of 9 Original Research
Page 9 of 9 Page 9 of 9 pp
p
services – 2
Quality – 1
7. Appendix 1 Draft Continental policy
framework
for the promotion of
sexual and reproductive
health rights – AU October
2005
-
-
-
Individualised
appropriate
services – 1
Prevention and
amelioration – 1
Cultural
responsiveness – 1
Service
coordination and
collaboration – 1
Individualised
appropriate
services – 1
-
-
-
Prevention
and
amelioration
– 1
Service
coordination
and
collaboration
– 1
Individualised
appropriate
services – 1
Individualised
appropriate services
– 1
Protection from harm – 3
Prevention and amelioration – 1
Anti-discrimination – 2
Cultural responsiveness – 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 2
Individualised appropriate
services – 1
Accountability – 1
Quality – 3
Access – 1
9. Plan of action on sexual
and reproductive
health and rights (Maputo
Plan of Action) 2007–2010
-
-
-
-
Individualised
appropriate
services – 1
-
-
-
Service
coordination
and
collaboration
– 1
Access – 1
-
Prevention and amelioration – 1
Anti-discrimination – 2
Cultural responsiveness – 1
Service coordination and
collaboration – 1
Prof and system capacity
building – 3
Individualised appropriate
services – 1
Accountability – 2
Quality – 1
Access – 2
10. AU Youth Charter – AU
July 2006
-
-
-
-
Protection from
harm – 5
prevention and
amelioration – 4
Anti-
discrimination-4
Access – 1
-
-
Access–1
-
Anti-
discrimination – 2
Access – 1
-
11. Africa health strategy:
2007–2015
-
-
-
-
Prevention and
amelioration – 1
-
Prevention and
amelioration
– 1
Individualised
appropriate
services -2
-
-
-
Protection from harm – 1
Prevention and amelioration – 2
Anti-discrimination – 4
Cultural responsiveness – 5
Service coordination and
collaboration – 18
Prof and system capacity building
– 20
Individualised appropriate
i
2 doi:10.4102/ajod.v2i1.40 doi:10.4102/ajod.v2i1.40
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https://openalex.org/W2955129910
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https://www2.ia-engineers.org/conference/index.php/iciae/iciae2019/paper/download/2082/1344
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English
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Design of Routing Algorithm for Wireless Sensor Networks based on Clustering Structure
| null | 2,019
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Abstract Based on the deep research of wireless sensor network
routing algorithm, proposed a MEEMLC-LEACH (More
Energy-Efficient
Multi-Levels
Clustering
LEACH)
optimized routing algorithm. The main contents include:
Adopting
the
three-level
clustering
method
and
incorporating the energy factor into the calculation formula
of the electoral cluster head node, shortening the distance of
data transmission between the member node and the cluster
head, the cluster head node and the BS;When the initial state
is completely the same, concluded the network lifetime of
the MEEMLC-LEACH algorithm is about 1.4 times that of
the LEACH algorithm by calculating the total energy
consumption of each frame; Finally, using MATLAB to
simulate the optimization algorithm by using direct routing
(Direct), LEACH and MEEMLC-LEACH three routing
methods, Visually shows the superiority of the optimization
algorithm in balancing energy consumption and prolonging
network lifetime. ZigBee
technology
is
a
short-range
wireless
communication technology, which has been widely used in
recent years because of its outstanding advantages of small
size and low power consumption. In the practical application
of wireless sensor network technology for agriculture,
transportation, medical care, home monitoring, etc., the
effective transmission of data is a core part, which is directly
related to whether the entire system can be continuously
monitored and operated(2-5). Data transmission relies on
efficient routing algorithms. In recent years, research on
routing algorithms has never been interrupted, and LEACH
algorithm (Low Energy Adaptive Clustering Hierarchy) has
been widely used as a low-power adaptive algorithm(6-10). Keywords: agricultural internet of things; routing algorithm;
wireless sensor network a Yangzhou university, 196 huayang west road,Yangzhou and 225127, China
*Corresponding Author：zhangzh@yzu edu cn *Corresponding Author：zhangzh@yzu.edu.cn *Corresponding Author：zhangzh@yzu.edu.cn research hotspot(1). The agricultural Internet of Things is a
wireless sensor network built by a large number of wireless
sensor
nodes
through
routing
algorithms. The
communication data is used to transmit monitoring data to
each other, and the data is collected on the detection platform. Finally, a complete agricultural information detection system
was constructed. 2. The origin of the LEACH algorithm Among the clustering routing algorithms, the LEACH
algorithm is the most representative. LEACH is essentially a
complete distributed routing protocol. Therefore, it does not
require global information. The algorithm gives a threshold
in each cycle, and each node randomly assigns a value in the
interval [0,1]. By comparing the value with the threshold
(whether it is less than), it is determined whether the node is
in the cluster head node. The successful calculation method
is shown in formula (1): DOI: 10.12792/iciae2019.024 Design of routing algorithm for wireless sensor networks based on
clustering structure Zhenghua Zhanga,*,Zheng Gonga,Jie Xua,Jiafeng Zhanga Proceedings of the 7th IIAE International Conference on Industrial Application Engineering 2019 Proceedings of the 7th IIAE International Conference on Industrial Application Engineering 2019 © 2019 The Institute of Industrial Applications Engineers, Japan. 3.2 Energy consumption model Where p is the percentage of the number of cluster head
nodes in the total number of nodes, which represents the
possibility that any node is elected as the cluster head; r is
the number of rounds currently circulating(11-14). Send the l bit length data to the node with distance d ,
and the energy consumed can satisfy the formula (2): However, LEACH still has some shortcomings. The
algorithm does not consider the residual energy of the node
and the distribution and density of each node when electing
the cluster head. Excessive randomness sometimes shortens
the life of some nodes or selects the cluster head away from
the base station, which will result in uneven energy
consumption. When the number of remaining surviving
nodes cannot complete the routing task, the network lifetime
will also be shortened(15-17). At the same time, LEACH can
not guarantee the position and number of cluster heads per
round. The formation of clusters in the basic LEACH is
random, which leads to uneven distribution of clusters in the
network. 2
( , )
Tx
elec
amp
E
l d
lE
l
d
e
=
+
（2） （2） When it is necessary to send the same length of data to
the same node, the energy consumed by the single-hop
method is generally greater than the total energy consumed
by the intermediate node(19). When the node receives the data, the energy consumption
is independent of the distance d , and the energy consumed
by the node receiving the data from the neighboring node l
bit of distance d satisfies the formula (3): ( )
Rx
elec
E
l
lE
=
（3） （3） For each wireless node, the energy consumption of the
data transceiver part accounts for a large proportion of the
total energy consumption, so other parts of the energy
consumption are ignored here. In this paper, only the
communication energy consumption is considered in the
analysis. 1. Introduction In recent years, China's agricultural informatization
process is accelerating with the introduction of concepts such
as “smart agriculture”, at the same time the precision
agriculture characterized by automation and intelligence is
an inevitable choice for current and future agricultural
development. Among them, the agricultural Internet of
Things based on wireless sensor networks has become a © 2019 The Institute of Industrial Applications Engineers, Japan. 115 DOI: 10.12792/iciae2019.024 Proceedings of the 7th IIAE International Conference on Industrial Application Engineering 2019 monitoring area. monitoring area. monitoring area. 𝑇(𝑛) = {
𝑝
1−𝑝[𝑟𝑚𝑜𝑑(1/𝑝)] 𝑛∈𝐺
0 𝑜𝑡ℎ𝑒𝑟𝑠
（1） 4. Design of MEEMLC-LEACH algorithm Since the focus of this paper is on the design of routing
algorithms, in order to design the algorithm conveniently and
quickly and analyze its superiority, now we make the
following assumptions:(1)The energy of each node is
limited.This chapter aims to design a self-organizing routing
method for balanced energy and to maximize network
lifetime (18). Therefore, first assume that the initial energy of
each node is a fixed value, and it will not increase during the
monitoring process, so that it is convenient to observe the
energy
consumption
of
each
node
through
simulation.(2)Each node has perceptual ability.All sensor
nodes need to obtain their own location and the location of
other neighboring nodes within a short distance through a
corresponding mechanism (such as GPS) to calculate their
own distance to the base station.(3)Each node can accurately
detect its own energy level, and the power of the wireless
antenna is controllable and adjustable (guarantee that it can
directly communicate with the BS after adjusting to a certain
power).(4)The monitoring area is an N×N rectangular area,
and the nodes are relatively evenly distributed in the © 2019 The Institute of Industrial Applications Engineers, Japan. 4.3
MEEMLC-LEACH
algorithm
specific
implementation method The routing algorithm is further described below in
conjunction with the model. Fig.1. Schematic diagram
Layer 3 node
cluster head
Layer 3 node
cluster head
Base station
USB
Wireless transceiver
Wireless transceiver
Farmland monitoring area
Layer 2 node
cluster head
Layer 2 node
cluster head
Layer 1 node
cluster head
Layer 1 node
cluster head
First layer
member node
First layer
member node
First layer
guest node
First layer
guest node Fig.1. Schematic diagram
Layer 3 node
cluster head
Layer 3 node
cluster head
Base station
USB
Wireless transceiver
Wireless transceiver
Farmland monitoring area
Layer 2 node
cluster head
Layer 2 node
cluster head
Layer 1 node
cluster head
Layer 1 node
cluster head
First layer
member node
First layer
member node
First layer
guest node
First layer
guest node Algorithm 1 MEEMLC-LEACH algorithm 4.2 MEEMLC-LEACH algorithm data transmission
process Fig.1. Schematic diagram Fig.1. Schematic diagram FIG. 1 is a schematic structural diagram of a wireless
sensor network, where a wireless ad hoc network includes a
plurality of aggregation nodes, a plurality of cluster head
nodes, a plurality of member nodes, and a plurality of guest
nodes. FIG. 1 is a schematic structural diagram of a wireless
sensor network,where a wireless ad hoc network includes a
plurality of aggregation nodes, a plurality of cluster head
nodes, As shown in Figure 1, all nodes are layered and each
layer is elected as a cluster head node. This shows that in the
practical application of building a wireless network to
construct an agricultural Internet of Things, it is important
that the large number of nodes in the monitoring area are self-
organized in a layered manner. Before the ad hoc network,
all nodes need to initialize and build their own neighbor node
information list. The content of the message packet broadcast
by each node to the neighbor node includes: a node ID, a
node residual energy RE, and a node-to-base station distance
DtoBS. When each node is initialized, the initial content is: the
node level ClusterLevel is 0, the cluster head node is the base
station BS, the campaign cluster head result is failed, and the
node transmission radius is
opt
TR
. The data collection phase mainly includes two
steps:intra-cluster transmission and data transmission
between cluster heads. Algorithm 2 gives the main flow of
data transmission. Algorithm 2: Data Transfer Process
1: Non-cluster head nodes within each cluster aggregate data to their
cluster head nodes;
The transmission order is : Guest node member node cluster
head node
2: Each cluster head node transmits the respective data upwards
according to the layer value from low to high, until it is delivered to the
BS. Finally, all monitoring data is gathered at the base station BS. 4.1 Clustering process of MEEMLC-LEACH algorithm The generation of clusters is the main process of dynamic
ad hoc networks. Algorithm 1 gives the main flow of
MEEMLC-LEACH algorithm. © 2019 The Institute of Industrial Applications Engineers, Japan. 116 Proceedings of the 7th IIAE International Conference on Industrial Application Engineering 2019 Proceedings of the 7th IIAE International Conference on Industrial Appl
Algorithm 1: MEEMLC-LEACH algorithm
1: Initialize each node;
2: Elect the cluster head node of the current layer among the
remaining unclustered nodes; firstly, elect the cluster head node of the
first layer, and the initial transmission radius of the first layer is
opt
TR
. 3: selecting member nodes of each cluster head within the
transmission radius of the layer;
4: selecting a guest node of each member node within a transmission
radius of the layer;
5: Update cluster head information;
The main update is: layer value n plus 1, transmission radius
TR doubled :
=
2n
opt
TR TR
´
. 6: Repeat steps 2-5 until the transmission radius is equal to the radius
of the entire monitoring network, which is cover the entire network. 7: Wait for the end of current round data transmission and restart the
next round. Algorithm 1 MEEMLC-LEACH algorithm Step 2 mainly performs data collection and aggregation
between clusters. Each cluster head node collects data of the
member nodes managed by itself according to a
predetermined time, and aggregates the collected data and its
own data to the cluster head node of the upper layer. Finally,
the cluster head node collects all the data and sends it to the
BS. © 2019 The Institute of Industrial Applications Engineers, Japan. 4.4 Energy consumption analysis of MEEMLC-LEACH
algorithm. The cluster head node of the top layer is the aggregation
node. The transmission radius of the aggregation node covers
the whole network. Each aggregation node uploads the
merged data to the base station through wireless transmission
and reception, and the base station transmits it to the upper
computer for display. From the top cluster head down, each
lower cluster head transfers the data of the nodes in the
cluster to the cluster head in turn. This transmission
mechanism can reduce the hop count during data
transmission, reduce the energy consumption of the network,
and shorten the transmission delay. According to the comparison of the data collection
methods, both LEACH and MEEMLC-LEACH send
monitoring data to the BS repeatedly in a frame manner at
corresponding time slots. Here, we deduct and analyze the
total energy consumption of the two routing methods in one
frame according to the premise hypothesis and the energy
consumption model. It is known that there are n nodes in the
monitoring area evenly distributed in the rectangular area of
l Each layer of nodes is divided into three levels, namely
cluster head nodes, member nodes and guest nodes. Each
node's message list contains its own hierarchical information. For member nodes, when they receive a cluster head
generation message, first, determine whether the level in the
cluster head generation message and its own level satisfies
the relationship: Vj(ClusterLevel)+1 = Vi (ClusterLevel). If
not, the member node discards directly without any response,
otherwise the cluster head generation message of the cluster
head node is saved in its own candidate cluster head list SCH. If the member node finds that there are multiple cluster head
node messages in the candidate cluster head list SCH, which is
there are multiple cluster heads in the neighboring node
within the transmission radius then compared the ratio of the
remaining energy RE of each cluster head in the candidate
cluster head list SCH to their distance DtoBS from the base
station: RE/DtoBS. In the end, the node with the highest ratio is
successful and becomes the cluster head of the node. If there
is only one cluster head node message in the candidate
cluster head list SCH, which is only one cluster head node in
the neighbor node is within the transmission radius, the
member node directly joins the cluster. Algorithm 2 data transmission process Step 1 mainly performs the collection and aggregation
process in the cluster. Each member node collects and
aggregates the data of the guest nodes in the data and the
communication radius according to the predetermined time,
and then sends the data to the cluster head node of the
cluster(20). © 2019 The Institute of Industrial Applications Engineers, Japan. © 2019 The Institute of Industrial Applications Engineers, Japan. 117 Proceedings of the 7th IIAE International Conference on Industrial Application Engineering 2019 4.4 Energy consumption analysis of MEEMLC-LEACH
algorithm. For the node that has
just joined and the node that is moving, if the transmission
radius of the cluster head node is exceeded, these nodes are
called guest nodes and indirectly join the cluster through the
member nodes. after the end of the cluster head election and
before the end of one round of transmission. Therefore, in
addition to receiving the data of the subordinate cluster head
fusion, the cluster head nodes of each layer also need to
receive the data of the member nodes and the guest nodes in
the cluster, after further merged and upload them to the upper
cluster head N×N, and the amount of data sent by each node takes a l bit. For LEACH, a total of k cluster head nodes are evenly
distributed, and the probability formula of each node being
elected as a cluster head node in each round is: k
p
n
=
(4) (4) For a frame of LEACH, each cluster head node receives
data sent by other member nodes in the cluster, and sends the
data directly to the BS. Under the premise of uniform
distribution, the number of member nodes managed by each
cluster head node Satisfy formula (5): non CH
n
k
N
k
-
-
=
（5） （5） Therefore, the energy consumption of each cluster head
node is: (
). 4.4 Energy consumption analysis of MEEMLC-LEACH
algorithm. ( )
( ,
)
CH
Rx
Tx
toBS
n
k
E
E
l
E
l d
k
-
=
+
(6) (6) Substituting
2
( )
( , )
Rx
elec
Tx
elec
amp
E
l
lE
E
l d
lE
l
d
=
=
+
into the above
formula, you get: 2
( )
( , )
Rx
elec
Tx
elec
amp
E
l
lE
E
l d
lE
l
d
=
=
+
into the above Substituting 2
CH
elec
amp
toBS
n
E
lE
l
d
k
e
=
+
（7） （7） Since each node is evenly distributed in the monitoring
area, the distance from the cluster head node to the base
station BS is estimated as follows: 2
2
2
2
0
0
1
(
)
(
)
(
)
N
N
toBS
BS
BS
E d
x x
y y
dxdy
M
=
−
+
−
（8） In （8） In a frame, each non-cluster head node only needs to send data © 2019 The Institute of Industrial Applications Engineers, Japan. © 2019 The Institute of Industrial Applications Engineers, Japan. 4.4 Energy consumption analysis of MEEMLC-LEACH
algorithm. cluster
CH
non CH
n
k
E
E
E
k
-
-
=
+
(11) MEEMLC
LEACH
R
Tx toCH
Tx toBS
E
E
E
E
-
-
-
=
+
+
(19) H
(11) Under the premise that the initial state and the initial energy
are exactly the same, when using the MEEMLC-LEACH
algorithm and the LEACH algorithm, the relationship
between the lifetimes of the networks is estimated as follows: Under the premise that the initial state and the initial energy
are exactly the same, when using the MEEMLC-LEACH
algorithm and the LEACH algorithm, the relationship
between the lifetimes of the networks is estimated as follows: Equation (12) gives the total energy consumption of the
entire monitoring area in one frame: LEACH
cluster
E
kE
=
(12) MEEMLC LEACH
MEEMLC LEACH
LEACH
LEACH
MEEMLC LEACH
LEACH
E
T
E
E
E
T
E
E
-
-
-
=
=
(20)
5.Simulation and Result Analysis of
MEEMLC-LEACH Algorithm MEEMLC LEACH
MEEMLC LEACH
LEACH
LEACH
MEEMLC LEACH
LEACH
E
T
E
E
E
T
E
E
-
-
-
=
=
(20) 2
2
(2
)
(
)
(
)
2
elec
amp
amp
toBS
N
l
n
k E
n
k
k
E d
k
=
−
+
−
+
(20) In MEEMLC-LEACH, each node shortens the distance
of sending data by establishing parent-child relationship with
each other. This multi-hop routing approach increases the
overall energy consumption of the network in receiving data,
but greatly reduces the total energy consumption in
transmitting data. The energy that the entire monitoring
network consumes on the received data in each frame is at
most: 5.Simulation and Result Analysis of
MEEMLC-LEACH Algorithm In order to evaluate the performance of MEEMLC-
LEACH algorithm, this paper chooses MATLAB to simulate
the realization process of the designed routing algorithm. The calculation of
( )
T n takes the formula (3), The value
of the parameter p is 5%, and the parameters used in the
simulation are shown in Table 1. (
1)
( )
(
1)
R
Rx
elec
E
n
E
l
l n
E
=
-
=
-
(13) (13) That is each node participates in the routing and forwarding
process of data. 4.4 Energy consumption analysis of MEEMLC-LEACH
algorithm. 118 Proceedings of the 7th IIAE International Conference on Industrial Application Engineering 2019 to the cluster head node of the cluster in which it is located,
and there is no receiving task, so the energy consumption of
each non-cluster head node is: 2
2
4
(
)
to
parent
N
E d
n
p
-
=
(16) (16) Therefore: Therefore: 2
( ,
)
non CH
Tx
toCH
elec
amp
toCH
E
E
l d
lE
l
d
e
-
=
=
+
(9) 2
4
(
)[
]
Tx toCH
elec
amp
N
E
l n
k E
n
e
p
-
=
-
+
(17) (17) The estimated distance between the non-cluster head
node and the cluster head node is: Similarly, in a frame, the total energy consumed by the
cluster head node in sending data to the cluster head node is: 2
2
[
]
2
toCH
N
E d
k
p
=
(10) 2
4
(
1)
Tx toBS
elec
amp
M
E
l k
E
k
−
=
−
+
(18) (10) In summary, the energy consumption of a cluster in one
frame is: Thus, the total energy consumption consumed by the
monitored area in each frame is approximately: (
). 4.4 Energy consumption analysis of MEEMLC-LEACH
algorithm. But the energy that the non-cluster node
consumes on the transmitted when sending data is: (
)
( ,
)
Tx toCH
Tx
to parent
E
n k E E
l d
−
−
=
−
(14) (14) Cause each sensor node is evenly distributed, the algorithm
ensures that each node can find its own parent node within
one hop communication radius (including the guest node
looking for member nodes and member nodes looking for
cluster head nodes)
. It is assumed here that all non-cluster
nodes successfully find the parent node within the single hop
range, then: 2
2
(
). 4
to
parent
E d
n
N
p
-
=
(15) 2
2
(
). 4
to
parent
E d
n
N
p
-
=
(15)
which is: (15) which is: © 2019 The Institute of Industrial Applications Engineers, Japan. © 2019 The Institute of Industrial Applications Engineers, Japan. 119 Proceedings of the 7th IIAE International Conference on Industrial Application Engineering 2019 Table 1. Parameter values in the simulation
parameter
value
parameter
value
Monitring
area
(0,0)-
(200,200)
Eelec/(nJ/bit)
50
Base station
coordinates
(100,300)
εamp/(pJ/(bit.m
2))
10
Total
number
ofnodes
200
ε'amp/(pJ/(bit.m
4))
0.001
3
Initial
energy
of
the node
2
d0/m
87
Packet sizel
2000
Ebf/(nJ/(bit.sig
nal))
5 Table 1. Parameter values in the simulation
parameter
value
parameter
value
Monitring
area
(0,0)-
(200,200)
Eelec/(nJ/bit)
50
Base station
coordinates
(100,300)
εamp/(pJ/(bit.m
2))
10
Total
number
ofnodes
200
ε'amp/(pJ/(bit.m
4))
0.001
3
Initial
energy
of
the node
2
d0/m
87
Packet sizel
2000
Ebf/(nJ/(bit.sig
nal))
5 for the 200 initial nodes, when the direct transmission
method is adopted, there are basically no nodes in the
network after about 600 frames; When the traditional
LEACH routing mode is adopted, although the nodes within
1000 frames can basically work normally, the number of
nodes surviving thereafter is drastically reduced, and the
network is basically paralyzed after 1500 frames. When the
MEEMLC-LEACH
optimized
routing
algorithm designed in this paper is adopted, the entire
wireless sensor network can operate normally within 3500
frames. 4.4 Energy consumption analysis of MEEMLC-LEACH
algorithm. First of all, this is because changing the long-
distance single-hop route to a short-distance multi-hop route
can reduce part of the energy consumption without changing
the transmission distance; Secondly, the algorithm equalizes
the energy consumption, so that the energy consumption
speed of each node is synchronized, and some nodes are
prevented from dying too quickly due to "hot spots" and the
like, thereby achieving the purpose of prolonging the
network life. According to the value of the parameters in the table, the
estimation result can be given according to the analysis result
of formula (20). Compared with the traditional algorithm, the
network survival time after using MEEMLC-LEACH
algorithm is as shown in formula (21): Simulation 2: Energy Balance Performance Simulation MEEMLC LEACH
LEACH
T
T
-
» 1.45 (21) MEEMLC LEACH
LEACH
T
T
-
» 1.45 (21) In order to test the superiority of the MEEMLC-LEACH
algorithm in equalizing energy consumption, the direct
transmission (Direct), LEACH and MEEMLC-LEACH
routing methods are used to simulate the total energy
consumption of the entire network. The total network energy
consumption under the three routing protocols increases as
the number of frames increases (time) as shown in Figure 3. © 2019 The Institute of Industrial Applications Engineers, Japan. Simulation 1: Energy-saving simulation Firstly, in order to test the performance of the MEEMLC-
LEACH algorithm in energy saving, the direct transit
(Direct), LEACH and MEEMLC-LEACH routing methods
are used to simulate the network lifetime. The number of
nodes surviving in the three routing protocols changes as the
number of frames increases (time) as shown in Figure 2. Fig.3. Comparison of energy consumption of the
network
0
200
400
600
800
1000
1200
1400
1600
0
50
100
150
200
250
300
350
400
a
b
c
Frame/s
整个网络消耗的能量/J
Direct
LEACH
MEEMLC-LEACH 0
200
400
600
800
1000
1200
1400
1600
0
50
100
150
200
250
300
350
400
a
b
c
Frame/s
整个网络消耗的能量/J
Direct
LEACH
MEEMLC-LEACH Fig.2. Comparison of the number of nodes surviving in
the network
It can be seen from the simulation results of FIG. 2 that
0
500
1000
1500
2000
2500
3000
3500
4000
0
20
40
60
80
100
120
140
160
180
200
a
b
c
Frame/s
Node/s
Direct
LEACH
MEEMLC-LEACH Fig.3. Comparison of energy consumption of the
network Fig.2. Comparison of the number of nodes surviving in
the network It can be seen from the simulation results in Fig. 3 that in
the initial stage, the MEEMLC-LEACH algorithm consumes It can be seen from the simulation results of FIG. 2 that © 2019 The Institute of Industrial Applications Engineers, Japan. 120 Proceedings of the 7th IIAE International Conference on Industrial Application Engineering 2019 Kumar Bhoi. Heterogeneous fault diagnosis for wireless
sensor networks[J]. Ad Hoc Networks,2018,69. more energy than the traditional algorithm, because the
algorithm needs to consume a certain amount of energy in
sensing and acquiring its own position and neighbor node
location and region division. However, after 200s, the
improved algorithm has some improvement compared with
the other two methods. This is because the optimization
algorithm adopts the three-level clustering and multi-layer
transmission
method,
which
greatly
shortens
the
communication distance of each node to transmit data. (5) Ahmed Al‐Baz,Ayman El‐Sayed. A new algorithm for
cluster head selection in LEACH protocol for wireless sensor
networks[J]. International Journal of Communication
Systems, 2018,31(1). (6)Alexander Cherepanov,Igor
Tyshchenko,Mariia
Popova,Dmitriy Vakhnin. Building
Energy
Efficient
Wireless Sensor Networks[J]. International Journal of
Electronics and Telecommunications,2017,63(1). It can be seen from the simulation results that the
MEEMLC-LEACH algorithm has certain optimization and
improvement in reducing energy consumption and balancing
energy consumption, effectively extending the life of
wireless sensor networks. (7)Carlos Abreu,Francisco Miranda,P.M. Mendes. Simulation 1: Energy-saving simulation Smart
context-aware QoS-based admission control for biomedical
wireless sensor networks[J]. Journal of Network and
Computer Applications,2017,88. (8)H.LUO,Y.,.ZOU,J.X.LU,Z.H.YU,et.al. Research
on
Multi-parameter Routing Equilibrium Mechanism in
Wireless Sensor Networks[J].Computer Science and
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mechanism makes the replacement of the cluster head nodes
more reasonable, and the energy consumption is maximized
by minimizing the clusters. When campaigning for the
cluster head node, the MEEMLC-LEACH algorithm
improves the threshold calculation by taking the energy
factor into consideration, which makes the election of the
cluster head node more reasonable and further prolongs the
survival time of the whole network. (10) Osama Moh’d Alia. Dynamic relocation of mobile base
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harmony
search
algorithm[J]. Information
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Length of sick leave – Why not ask the sick-listed? Sick-listed individuals predict their length of sick leave more accurately than professionals
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BioMed Central BioMed Central BioMed Central Open Acc
Research article
Length of sick leave – Why not ask the sick-listed? Sick-listed
individuals predict their length of sick leave more accurately than
professionals
Nils Fleten*1, Roar Johnsen2 and Olav Helge Førde1 Open Access p
Nils Fleten*1, Roar Johnsen2 and Olav Helge Førde1 Address: 1Department of Community Medicine, University of Tromsø, Tromsø, N-9037, Norway and 2Department of Community Medicine and
General Practice, Norwegian University of Science and Technology, Trondheim, Norway Email: Nils Fleten* - Nils.Fleten@ism.uit.no; Roar Johnsen - Roar.Johnsen@medisin.ntnu.no; Olav Helge Førde - Olav.Helge.Forde@ism.uit.no
* Corresponding author Received: 24 May 2004
Accepted: 12 October 2004 This article is available from: http://www.biomedcentral.com/1471-2458/4/46 ;
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 respectively. Musculoskeletal disorders were the main rea-
son for sick leaves (83% of the cases). respectively. Musculoskeletal disorders were the main rea-
son for sick leaves (83% of the cases). g
The increasing rate of sick leave experienced in most West-
ern countries challenges insurance companies, employers,
and public authorities to identify measures to reduce bur-
dens at the individual, workplace and societal levels. A total of 495 randomly selected persons received a ques-
tionnaire on the expected length of their ongoing sick
leave period. The answer categories were: less than 4
weeks, 4 to 7 weeks, 8 to 11 weeks, 12 to 15 weeks, 16 to
25 weeks, 26 to 51 weeks, and at least 1 year. Some 152
persons (30.7%), called the responder group, returned the
questionnaire with this question filled in. To reduce the expenses of sick leave and the risk of expul-
sion from work, the Norwegian government introduced
legislation in 1993 that anticipated early and more vigor-
ous interventions of the Norwegian National Insurance
Scheme [1]. The Norwegian Public Report no. 27 [2],
2000, underscored the importance of early intervention
by the National Insurance Offices (NIOs). A major chal-
lenge for the NIOs is to identify newly sick-listed individ-
uals at risk of prolonged sick leave, and who are therefore
potential candidates for rehabilitating interventions. Based on SC1s available after 14 days of sick leave, two
NIO officers without formal medical competence, but
experienced in working with sick-listed persons, and two
experienced physicians working part time as insurance
medical officers (NIO medical consultants), assessed the
expected length in each of the 993 ongoing sick leave
cases. In 496 randomly chosen cases, the NIO assessors
had additional access to information on sick leave periods
during the previous 3 years. Of potentially 1986 assess-
ments in each profession, the officers and medical con-
sultants had 18 and 25 missing assessments, respectively. The selection process is currently based on information in
medical sickness certificates supplied by access to the reg-
ister of previous sickness benefits. A medical sickness cer-
tificate (Sickness Certificate 1; SC1) is required if sick
leave exceeds 3 days, and after 8 weeks an extended med-
ical certificate is mandatory (Sickness Certificate 2; SC2)
[3]. Abstract Background: The knowledge of factors accurately predicting the long lasting sick leaves is sparse,
but information on medical condition is believed to be necessary to identify persons at risk. Based
on the current practice, with identifying sick-listed individuals at risk of long-lasting sick leaves, the
objectives of this study were to inquire the diagnostic accuracy of length of sick leaves predicted in
the Norwegian National Insurance Offices, and to compare their predictions with the self-
predictions of the sick-listed. Methods: Based on medical certificates, two National Insurance medical consultants and two
National Insurance officers predicted, at day 14, the length of sick leave in 993 consecutive cases
of sick leave, resulting from musculoskeletal or mental disorders, in this 1-year follow-up study. Two months later they reassessed 322 cases based on extended medical certificates. Self-
predictions were obtained in 152 sick-listed subjects when their sick leave passed 14 days. Diagnostic accuracy of the predictions was analysed by ROC area, sensitivity, specificity, likelihood
ratio, and positive predictive value was included in the analyses of predictive validity. Results: The sick-listed identified sick leave lasting 12 weeks or longer with an ROC area of 80.9%
(95% CI 73.7–86.8), while the corresponding estimates for medical consultants and officers had
ROC areas of 55.6% (95% CI 45.6–65.6%) and 56.0% (95% CI 46.6–65.4%), respectively. The
predictions of sick-listed males were significantly better than those of female subjects, and older
subjects predicted somewhat better than younger subjects. Neither formal medical competence,
nor additional medical information, noticeably improved the diagnostic accuracy based on medical
certificates. Conclusion: This study demonstrates that the accuracy of a prognosis based on medical
documentation in sickness absence forms, is lower than that of one based on direct communication
with the sick-listed themselves. Page 1 of 11
(page number not for citation purposes) BMC Public Health 2004, 4:46 http://www.biomedcentral.com/1471-2458/4/46 http://www.biomedcentral.com/1471-2458/4/46 Observed length of sick leaves The reference standard lengths of individual sick leaves
within 1 year were collected from the National Sickness
Benefit Register. Sick leaves interrupted by only 1–2 days
without sickness benefits, typically on weekends, were
registered as a single period. The observed length of sick
leave thus comprised the total period of continuous full-
time or part-time absence due to sickness within 1 year. Based on the current practice with identifying sick-listed
individuals at risk of long-lasting sick leaves, the objec-
tives of this study were to inquire diagnostic accuracy of
predictions within the NIOs, and to compare their predic-
tions with the self-predictions of the sick-listed. Background In addition to diagnosis and certified period, the
majority of SC1s contain information on the occupation
and employee, whereas information on chronic disease,
previous sick leave episodes, prognosis and comments are
more scattered. SC2s include updated medical informa-
tion on work ability, planned diagnostics and treatments,
and on the prognosis. The value of this information as a
guideline for selective intervention has, however, never
been established, either as an indicator of potential pro-
longed absence, or as an indicator of the need for occupa-
tional or vocational rehabilitation [4]. SC2s became available in 322 of the 459 cases where sick
leave exceeded 8 weeks, and the NIO assessors reassessed
these cases. Reproducibility of assessments by medical consultants
were analysed in 20 cases reassessed by the two NIO med-
ical consultants, and assessed by another eight of their
colleagues. Page 2 of 11
(page number not for citation purposes) Methods
b The diagnostic accuracy of predicted lengths was com-
pared on the basis of sensitivity, specificity, likelihood
ratio and the area under the receiver operating character-
istics curves (ROC area) [6,7]. The non-parametric stand-
ard error and 95% CI for the ROC area were calculated in
SPSS-11. The ROC curve represents plots of the true-posi-
tive rate (sensitivity) and the false positive rate (1 – specif-
icity) at the average of two consecutive categories of the
assessments (>= 0 weeks, >= 4 weeks, >= 8 weeks etc). The
ROC curves of the mean assessment by NIO officers and
medical consultants include even intermediate points rep-
resenting half categories. In October and November 1997 and March and April
1998, newly sick-listed persons with musculoskeletal or
mental disorders (ICPC, L- and P- diagnoses) [5] were
included consecutively if they were certified sick for longer
than 2 weeks (Figure 1). Five hundred persons were
included in each period. The study took place in the cities
of Tromsø and Harstad in Northern Norway. The total
length of sickness benefits was registered during the fol-
lowing year in the National Sickness Benefit Register. Missing data on the length of sick leave reduced the
number of included subjects to 993. The mean ages of
these 391 men and 602 women were 41.4 and 39.7 years, Page 2 of 11
(page number not for citation purposes) Page 2 of 11
(page number not for citation purposes) BMC Public Health 2004, 4:46 http://www.biomedcentral.com/1471-2458/4/46 Inclusions The National Insurance Offices in the cities of Harstad and Tromsø included consecutively
993 newly sick-listed persons, certified sick beyond 14 days due to musculoskeletal or
mental disorders, during October–November 1997 and February–March 1998. Expected length after 14 days of sick leave (N=993)
Pre-selected answers categories: < 4 weeks, 4-7 weeks, 8-11 weeks, 12-15 weeks, 1
weeks, 26-51 weeks and at least one year Expected length after 14 days of sick leave (N=993) Expected length after 14 days of sick leave (N=993)
Pre-selected answers categories: < 4 weeks, 4-7 weeks, 8-11 weeks, 12-15 weeks, 16-25
weeks, 26-51 weeks and at least one year Pre-selected answers categories: < 4 weeks, 4-7 weeks, 8-11 weeks, 12-15 weeks, 16
weeks, 26-51 weeks and at least one year National Insurance assessments (N=993)
Two National Insurance officers and two National
Insurance medical consultants individually assessed
expected length of the 993 ongoing sick leaves. Randomly chosen the assessments were based on: Self-assessments (N=495)
Randomly chosen 495 of the
included sick-listed persons were
invited to assess their expected
length of sick leave as their sick
leave passed 14 days. x
Only Sickness Certificate(s) 1 available at 14
days of sick leave
(193 male and 304 females). No answer
N=343 x
Sickness Certificate(s) 1 available at 14 days
of sick leave and the record of sickness
benefits the last 3 years. (198 males and 298 females). x
Sickness Certificate(s) 1 available at 14 days
of sick leave and the record of sickness
benefits the last 3 years. (198 males and 298 females). Responders N=152 Half the
answers were received within 7
days, and 80 % within 12 days from
their sick leave passed 14 days Missing extended medical
certificate (Sickness Certificate 2)
at 8 weeks N=137 Missing extended medical
certificate (Sickness Certificate 2)
at 8 weeks N=137 Returned to work within 8 weeks
N=534 Returned to work within 8 weeks
N=534 Expected length after 8 weeks of sick leave (N=322) Pre-selected answers categories: 8-11 weeks, 12-15 weeks, 16-25 weeks, 26-51 weeks and
at least one year. After 8 weeks of sick leave, the two National Insurance officers and two National Insurance
di
l
l
d h
d l
h
f h
322 i k l
h After 8 weeks of sick leave, the two National Insurance officers and two National Insurance
medical consultants reassessed the expected lengths of the 322 sick leaves where an
extended medical certificate (Sickness Certificate 2) was received. Observed length of the included sick leave periods (N=993)
The actual lengths of the sick leaves were collected from the National Sickness Register
after one year. Flow-chart
Figure 1
Flow-chart. Flow-chart of inclusion, and the different assessments of expected length, of the included sick leaves after 2 and 8
weeks of sick leave. low-chart
Figure 1
Flow-chart. Receiver operating characteristics of prediction p
g
p
The sick-listed subjects predicted sick leaves equal to or
longer than 12 weeks more accurately than the NIO med-
ical consultants and officers, as shown by the ROC curve
in Figure 2. The differences in ROC area between respond-
ers and non-responders were most marked among
younger subjects and in females (Table 2). Generally, the
length of sick leave was predicted more accurately in older
subjects than in younger subjects, and better in males than
in females. Access to past history of sick leaves improved
the ROC area of NIO consultants from 60.6% (95% CI
51.3–69.9%) to 75.4% (95% CI 68.2–82.6%) in male
sick-listed, but did not improve the ROC area in assess-
ments of female sick-listed. The mean length of the sick leave in the responder group
was 107.4 days (95% confidence interval, CI, 88.7–126.1
days), compared to 92.4 days in the 343 non-responders. Stratified analysis revealed longer mean sick leaves among
responders 40 years and younger, of 109.3 days (95% CI
81.4–134.5 days), compared to the 79.3 days (95% CI Table 1: Categorical distribution of observed and predicted length of sick leave. Observed and predicted length of sick leaves in seven
categories for all participants (n = 993) compared to the responder group (n= 152). The assessments of National Insurance medical
consultants and officers are grouped according to proportions of persons predicted in each category. Table 1: Categorical distribution of observed and predicted length of sick leave. Observed and predicted length of sick leaves in seven
categories for all participants (n = 993) compared to the responder group (n= 152). The assessments of National Insurance medical
consultants and officers are grouped according to proportions of persons predicted in each category. categories for all participants (n = 993) compared to the responder group (n= 152). The assessments of National Insurance medical
consultants and officers are grouped according to proportions of persons predicted in each category. Expected length after 14 days of sick leave (N=993) Flow-chart of inclusion, and the different assessments of expected length, of the included sick leaves after 2 and 8
weeks of sick leave Page 3 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2458/4/46 http://www.biomedcentral.com/1471-2458/4/46 BMC Public Health 2004, 4:46 The predictive validity is presented as sensitivity, specifi-
city, positive predictive value (PPV) and likelihood ratio
at different thresholds, cut-offs, in predicted length [8]. Reliability of predicted length was analysed with agree-
ment between assessors, the kappa value [9,10]. 65.6–93.1 days) in non-responders. Stratification on gen-
der or musculoskeletal or mental disorders did not reveal
any significant differences in the length of sick leave
between responders and non-responders. All assessors, including the sick-listed themselves, system-
atically overestimated the length of short sick leaves (last-
ing 4–11 weeks) and underestimated the length of long
sick leaves (exceeding 16 weeks; Table 1). The proportions
of sick leaves lasting longer than 8, 12 or 26 weeks did not
differ significantly between the responder group and the
rest. Approval The Regional Ethical Committee approved the protocol,
and the Norwegian Data Inspectorate licensed the neces-
sary register of sick-listed subjects. Results The mean observed continuous sickness absence was
100.8 days (median 48 days). Sick leaves in females lasted
a mean of 105.1 days, compared to 94.6 days in men
(medians 55 and 43 days, respectively). The mean length
among persons with musculoskeletal disorders was 90.2
days in 335 males and 108.6 days in 489 females. The
mean length among persons with mental disorders was
120.6 days in 56 males and 90.0 days in 113 females. Receiver operating characteristics of prediction All participants Proportion according to
Responder group Proportion according to
Length of
sick leave
categories
Observed
length %
Assessed by medical
consultants %
95% CI
Assessed by
officers %
95% CI
Observed
length %
95% CI
Assessed by medical
consultants %
95% CI
Assessed by
officers %
95% CI
Assessed by
sick-listed %
95% CI
< 4 weeks
31.7
27.6
25.7–29.7
18.9
17.2–20.7
29.6
22.5–37.5
32.2
27.0–37.8
20.9
16.4–25.9
25.0
18.3–32.7
4–7 weeks
22.0
41.8
39.6–44.0
36.8
34.7–39.0
25.0
18.3–32.7
40.9
35.3–46.7
33.4
28.1–39.1
36.2
28.6–44.4
8–11 weeks
12.9
20.3
18.6–22.2
25.4
23.5–27.4
7.2
3.7–12.6
18.3
14.1–23.1
26.8
21.9–32.2
15.1
9.8–21.8
12–15 weeks
6.2
7.0
5.9–8.3
13.7
12.2–15.3
3.9
1.5–8.4
6.3
3.8–9.7
13.6
9.9–18.0
10.5
6.1–16.5
16–25 weeks
9.3
1.0
0.6–1.6
1.9
1.3–2.6
13.2
8.2–19.6
0.7
0.1–2.4
2.6
1.2–5.2
5.9
2.7–10.9
26–51 weeks
6.8
0.7
0.4–1.2
0.7
0.4–1.2
9.9
5.6–15.8
0.3
0.0–1.8
0.0
0.0–1.2
1.3
0.2–4.7
>= 52 weeks
11.1
1.5
1.0–2.1
2.5
1.9–3.3
11.2
6.7–17.3
1.3
0.4–3.2
2.6
1.2–5.2
5.9
2.7–10.9 BMC Public Health 2004, 4:46 http://www.biomedcentral.com/1471-2458/4/46 ROC curves of identifying sick leaves lasting at least 12 weeks
Figure 2
ROC curves of identifying sick leaves lasting at least 12 weeks. The ROC curve of ability to identify sick leaves lasti
at least 12 weeks, plotted at the average of two consecutive categories, in length predicted by sick-listed (n = 152), and mea
length predicted by National Insurance officers and medical consultants in the responder group (n = 149, 150) and for all th
data (n= 972, 975). The points representing cut-offs in predicted length >= 4 weeks (red), >= 8 weeks (pink) and >= 12 wee
(blue) are identified. >=4 weeks
>= 8 weeks
>= 12 weeeks
0
0,2
0,4
0,6
0,8
1
0
0,2
0,4
0,6
0,8
1
1-specificity
Sensitivity
Sick-listed
Consultants responders
Consultants total
Officers responders
Officers total ROC curves of identifying sick leaves lasting at least 12 weeks
Figure 2
ROC curves of identifying sick leaves lasting at least 12 weeks. Receiver operating characteristics of prediction The ROC curve of ability to identify sick leaves last
at least 12 weeks, plotted at the average of two consecutive categories, in length predicted by sick-listed (n = 152), and me
>=4 weeks
>= 8 weeks
>= 12 weeeks
0
0,2
0,4
0,6
0,8
1
0
0,2
0,4
0,6
0,8
1
1-specificity
Sensitivity
Sick-listed
Consultants responders
Consultants total
Officers responders
Officers total 0 0,2 ROC curves of identifying sick leaves lasting at least 12 weeks
Figure 2
ROC curves of identifying sick leaves lasting at least 12 weeks. The ROC curve of ability to identify sick leaves lasting
at least 12 weeks, plotted at the average of two consecutive categories, in length predicted by sick-listed (n = 152), and mean
length predicted by National Insurance officers and medical consultants in the responder group (n = 149, 150) and for all the
data (n= 972, 975). The points representing cut-offs in predicted length >= 4 weeks (red), >= 8 weeks (pink) and >= 12 weeks
(blue) are identified. Changing the observed length to be identified from 12
weeks to 8 or 26 weeks did not significantly change the
diagnostic accuracy as assessed by the ROC area. The sick-
listed identified sick leaves lasting 8 weeks or longer with a ROC area of 79.5% (95% CI 72.2–85.6%), and sick
leaves lasting 26 weeks or longer with a ROC area of
75.5% (95% CI 67.9–82.1%). Sick-listed persons with
mental disorders or with neck, or shoulder and arm Page 5 of 11
(page number not for citation purposes) Page 5 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2458/4/46 BMC Public Health 2004, 4:46 Table 2: ROC area of identifying sick leaves lasting at least 12 weeks. The ability to identify sick leaves lasting at least 12 weeks in the
responder group (n = 152) and in all participants (N = 993), presented as ROC area, calculated from length of sick leave predicted by
sick-listed, and mean length predicted by National Insurance medical consultants and officers. The range of the individual National
Insurance ROC areas is presented for all participants. Receiver operating characteristics of prediction Medical consultants
Officers
Self-assessed
Responders n = 152
Responders n
= 149
All participants n = 972
Responders n
= 150
All participants n = 975
Sick-listed
n
ROC area
ROC area
ROC area
Range individual
ROC area
ROC area
Range individual
N
95% CI
95% CI
95% CI
ROC area
95% CI
95% CI
ROC area
All
152
80.9
55.6
64.6
59.6–64.2
56.0
61.4
55.6–65.6
993
73.7–86.8
45.6–65.6
60.8–68.3
46.6–65.4
57.7–65.1
17–40
years of age
78
508
76.4
65.6–87.2
43.0
28.8–57.2
57.2
51.7–62.8
54.5–57.8
48.9
35.4–62.5
57.4
52.0–62.9
51.9–57.8
41–67
years of age
74
485
85.7
77.2–94.2
68.3
54.8–81.8
70.7
65.8–75.6
63.4–70.1
62.5
49.4–75.6
65.1
60.1–70.1
56.1–73.4
Males
56
90.9
63.0
68.7
62.8–68.3
59.6
63.6
56.5–71.8
391
83.4–98.4
47.3–78.7
62.8–74.6
44.3–74.9
57.5–69.8
Females
96
74.7
50.9
62.0
57.7–61.5
54.0
60.3
52.9–61.8
602
64.8–84.7
38.1–63.8
57.2–66.8
42.0–65.9
55.6–64.9 Table 2: ROC area of identifying sick leaves lasting at least 12 weeks. The ability to identify sick leaves lasting at least 12 weeks in the
responder group (n = 152) and in all participants (N = 993), presented as ROC area, calculated from length of sick leave predicted by
sick-listed, and mean length predicted by National Insurance medical consultants and officers. The range of the individual National
Insurance ROC areas is presented for all participants. Table 2: ROC area of identifying sick leaves lasting at least 12 weeks. The ability to identify sick leaves lasting at least 12 weeks in the
responder group (n = 152) and in all participants (N = 993), presented as ROC area, calculated from length of sick leave predicted by
sick-listed, and mean length predicted by National Insurance medical consultants and officers. The range of the individual National
Insurance ROC areas is presented for all participants. The sensitivity of identifying sick leaves lasting at least 26
weeks was generally low when medical consultants and
officers predicted on the basis of SC1s. (Table 4). The sen-
sitivity was improved somewhat by introducing SC2
information, but the effects on likelihood ratio and PPV if
prevalence corrected, were minor. The sensitivity of identifying sick leaves lasting at least 26
weeks was generally low when medical consultants and
officers predicted on the basis of SC1s. (Table 4). The sen-
sitivity was improved somewhat by introducing SC2
information, but the effects on likelihood ratio and PPV if
prevalence corrected, were minor. disorders, were most accurate in their assessment (Figure
3). Page 6 of 11
(page number not for citation purposes) Receiver operating characteristics of prediction This was in contrast to NIO assessors, who demon-
strated the lowest predictive ability in these diagnostic
groups, particularly in responders. The impact on diag-
nostic accuracy of knowing the occupation was small. Sensitivity, specificity, predictive value and likelihood ratio The
ROC area are presented with blue bars of 95% CI in the responder group (n = 152/), and red bars without horizontal lines
between upper and lower individual ROC area of the NIO assessors for all sick leaves (n = /958). 0
10
20
30
40
50
60
70
80
90
100
Mental
disorders
n=20/165
Neck
disorders
n=25/147
Shoulder
and arm
n=20/150
Back
disorders
n=44/229
Injuries
n=12/69
Other L-
diorders
n=31/198
ROC area in %
Sick-listed
NIO assessors of responders
NIO assessors of all sick leaves Sick-listed
NIO assessors of responders
NIO assessors of all sick leaves ROC area in different diagnostic groups
Figure 3
ROC area in different diagnostic groups. ROC area representing ability to identify sick leaves 12 weeks or longer in dif-
ferent diagnostic groups, calculated on length predicted by sick-listed, and mean of lengths predicted by NIO assessors. The
ROC area are presented with blue bars of 95% CI in the responder group (n = 152/), and red bars without horizontal lines
between upper and lower individual ROC area of the NIO assessors for all sick leaves (n = /958). g
g
p
g
ROC area in different diagnostic groups. ROC area representing ability to identify sick leaves 12 weeks or longer in dif-
ferent diagnostic groups, calculated on length predicted by sick-listed, and mean of lengths predicted by NIO assessors. The
ROC area are presented with blue bars of 95% CI in the responder group (n = 152/), and red bars without horizontal lines
between upper and lower individual ROC area of the NIO assessors for all sick leaves (n = /958). Table 3: Predictive validity – identifying sick leaves lasting at least 12 weeks. Predictive validity of identifying sick leaves that lasted at
least 12 weeks, using 8 weeks as the cut-off in length as predicted by the sick-listed, medical consultants and officers. The prediction
based on the Sickness Certificate 2 (SC2) used a cut-off in predicted length of at least 12 weeks. Sensitivity, specificity, PPV, and
likelihood ratio data for NIO assessors are presented as means with 95% CI. Sensitivity, specificity, predictive value and likelihood ratio Sensitivity, specificity, predictive value and likelihood ratio
The sick-listed subjects predicted their sick leaves with
higher sensitivity and PPV than the NIO assessors (Tables
3, 4). Male sick-listed predicted sick leaves lasting at least
12 weeks with a sensitivity of 0.82% (95% CI 0.60–0.95)
and a PPV of 0.78 (95% CI 0.56–0.93) using predicted
length of at least 8 weeks. The corresponding sensitivity
and PPV of female sick-listed were both 0.61 (95% CI
0.44–0.77). According to the results, the effects of the different predic-
tive strategies can be illustrated by considering a program
designed to intervene in all cases where the subject is
expected to be sick-listed for more than 12 weeks at 14
days of sick leave. Out of every 1000 sick-listed persons,
333 will be sick-listed for more than 12 weeks according
to the prevalence in this study. The random selection of
333 persons will include 111 true positives, while 333
persons selected by officers will include 133 of the 333
persons that will be sick-listed at least 12 weeks. The eval-
uation of 1000 sick-listed individuals thus increases the
number of true positives by 22 in a selection of 333 sick-
listed persons. The alternative strategy of asking the sick-
listed themselves will include 210 true positives in a selec-
tion of 333 persons. Duration of at least 8 weeks was the preferable cut-off in
predicted length, to identify sick leaves lasting at least 12
weeks (Table 3). A predicted length of at least 12 weeks
reduced the sensitivity in all the data to 0.17 in medical
consultants and 0.25 in officers. The corresponding
improvement in PPV was modest, reaching 0.54 in medi-
cal consultants and 0.45 in officers. Using a predicted
length of at least 4 weeks would have markedly reduced
the specificity (Figure 2). Reliability and reproducibility of the predicted length
Agreement between medical consultants in their initial
prediction of sick leaves lasting at least 12 weeks, was fair, Page 6 of 11
(page number not for citation purposes) Page 6 of 11
(page number not for citation purposes) BMC Public Health 2004, 4:46 http://www.biomedcentral.com/1471-2458/4/46 ROC area in different diagnostic groups
Figure 3
ROC area in different diagnostic groups. ROC area representing ability to identify sick leaves 12 weeks or longer in dif-
ferent diagnostic groups, calculated on length predicted by sick-listed, and mean of lengths predicted by NIO assessors. Sensitivity, specificity, predictive value and likelihood ratio Predicted length
n assessments
Sensitivity
(95% CI)
Specificity
(95% CI)
Likelihood ratio
(95% CI)
PPV1
(95% CI)
PPV adjusted to prevalence
33.4% (95% CI)
Sick-listed
152
0.69
(0.56–0.84)
0.80
(0.70–0.87)
3.4
(1.9–6.2)
0.68
(0.54–0.79)
0.63
(0.49–0.76)
Medical consultants
Responder group
301
0.35
(0.26–0.44)
0.78
(0.71–0.84)
1.6
(1.0–2.5)
0.49
(0.38–0.61)
0.44
(0.33–0.56)
Medical consultants All
participants
1961
0.42
(0.38–0.45)
0.75
(0.73–0.77)
1.7
(1.4–1.9)
0.45
(0.41–0.49)
Officers Responder group
302
0.53
(0.44–0.62)
0.59
(0.51–0.66)
1.3
(0.9–1.8)
0.44
(0.36–0.53)
0.39
(0.31–0.48)
Officers All participants
1968
0.53
(0.49–0.57)
0.60
(0.58–0.63)
1.3
(1.2–1.5)
0.40
(0.37–0.43)
Medical consultants SC2
637
0.85
(0.82–0.88)
0.44
(0.36–0.52)
1.5
(1.2–1.9)
0.82
(0.79–0.86)
0.43
(0.39–0.48)
Officers SC2
636
0.88
(0.86–0.91)
0.33
(0.26–0.41)
1.3
(1.1–1.7)
0.80
(0.77–0.84)
0.40
(0.35–0.44)
1The prevalence of sick leaves lasting at least 12 weeks was 38.2% in the responder group (n = 152), 33.4% for all participants (n = 993), and 72.1%
in the SC2 group (n= 322). Table 3: Predictive validity – identifying sick leaves lasting at least 12 weeks. Predictive validity of identifying sick leaves that lasted at
least 12 weeks, using 8 weeks as the cut-off in length as predicted by the sick-listed, medical consultants and officers. The prediction
based on the Sickness Certificate 2 (SC2) used a cut-off in predicted length of at least 12 weeks. Sensitivity, specificity, PPV, and
likelihood ratio data for NIO assessors are presented as means with 95% CI. Table 3: Predictive validity – identifying sick leaves lasting at least 12 weeks. Predictive validity of identifying sick leaves that lasted at
least 12 weeks, using 8 weeks as the cut-off in length as predicted by the sick-listed, medical consultants and officers. The prediction
based on the Sickness Certificate 2 (SC2) used a cut-off in predicted length of at least 12 weeks. Sensitivity, specificity, PPV, and
likelihood ratio data for NIO assessors are presented as means with 95% CI. 1The prevalence of sick leaves lasting at least 12 weeks was 38.2% in the responder group (n = 152), 33.4% for all participants (n = 993), and 72.1%
in the SC2 group (n= 322) 1The prevalence of sick leaves lasting at least 12 weeks was 38.2% in the responder group (n = 152), 33.4% for all participants (n = 993), and 72.1%
in the SC2 group (n= 322). Sensitivity, specificity, predictive value and likelihood ratio Page 7 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2458/4/46 BMC Public Health 2004, 4:46 Table 4: Predictive validity – identifying sick leaves lasting at least 26 weeks. Predictive validity of the ability to identify sick leaves lasting
at least 26 weeks, using 8, 12 or 26 weeks, as cut-offs in length as predicted by the sick-listed, medical consultants or officers. Sensitivity,
specificity, PPV and likelihood ratio data for NIO assessors are presented as means for length predicted on Sickness Certificates 1 and
Sickness Certificates 2 (SC2). Predicted length
n assess-ments
Sensitivity
(95% CI)
Specificity
(95% CI)
Likelihood ratio
(95% CI)
PPV1
(95% CI)
PPV adjusted to
prevalence 17.9% (95% CI)
Sick-listed >= 8 weeks
152
0.69
(0.50–0.84)
0.69
(0.60–0.77)
2.2
(1.3–3.9)
0.37
(0.25–0.51)
0.33
(0.21–0.47)
Sick-listed >= 12 weeks
152
0.50
(0.32–0.68)
0.83
(0.75–0.90)
3.0
(1.5–6.1)
0.44
(0.28–0.62)
0.40
(0.24–0.58)
Sick-listed >= 26 weeks
152
0.28
(0.14–0.47)
0.98
(0.94–1.00)
16.9
(3.5–160)
0.82
(0. 48–0.98)
0.78
(0.44–0.95)
Consultants >= 8 weeks
1961
0.44
(0.39–0.49)
0.72
(0.70–0.74)
1.6
(1.3–1.9)
0.25
(0.22–0.29)
Consultants >= 12 weeks
1961
0.20
(0.16–0.24)
0.92
(0.90–0.93)
2.4
(1.8–3.2)
0.35
(0.28–0.41)
Consultants >= 26 weeks
1961
0.07
(0.04–0.10)
0.99
(0.98–0.99)
2.8
(1.5–5.4)
0.54
(0.38–0.69)
Officers >= 8 weeks
1968
0.55
(0.50–0.60)
0.58
(0.56–0.61)
1.3
(1.1–1.5)
0.22
19.3–24.9
Officers >= 12 weeks
1968
0.26
(0.21–0.31)
0.83
(0.81–0.85)
1.6
(1.3–2.0)
0.25
(0.20–0.29)
Officers >= 26 weeks
1968
0.06
(0.04–0.09)
0.98
(0.97–0.98)
1.5
(0.9–2.6)
0.34
(0.23–0.48)
SC2
Consultants >= 12 weeks
637
0.89
(0.86–0.93)
0.29
(0.25–0.34)
1.3
(1.1–1.5)
0.44
(0.40–0.48)
0.22
(0.18–0.25)
Consultants >= 26 weeks
637
0.24
(0.19–0.29)
0.96
(0.93–0.98)
5.9
(3.4–11.0)
0.79
(0.68–0.87)
0.56
(0.41–0.70)
Officers >= 12 weeks
636
0.89
(0.85–0.93)
0.21
(0.17–0.25)
1.1
(0.9–1.3)
0.41
(0.37–0.45)
0.20
(0.16–0.23)
Officers >= 26 weeks
636
0.28
(0.22–0.34)
0.90
(0.87–0.93)
2.8
(1.9–4.3)
0.64
(0.54–0.73)
0.38
(0.28–0.49)
1The prevalence of sick leaves lasting at least 26 weeks was 21.1% in the responder group (n = 152), 17.9% for all the data (n = 993), and 38.5% in
the SC2 group (n = 322). Table 4: Predictive validity – identifying sick leaves lasting at least 26 weeks. Predictive validity of the ability to identify sick leaves lasting
at least 26 weeks, using 8, 12 or 26 weeks, as cut-offs in length as predicted by the sick-listed, medical consultants or officers. Sensitivity, specificity, predictive value and likelihood ratio Sensitivity,
specificity, PPV and likelihood ratio data for NIO assessors are presented as means for length predicted on Sickness Certificates 1 and
Sickness Certificates 2 (SC2). es lasting at least 26 weeks was 21.1% in the responder group (n = 152), 17.9% for all the data (n = 993), and 38.5% in 1The prevalence of sick leaves lasting at least 26 weeks was 21.1% in the responder group (n = 152), 17.9% for all the data (n = 993), and 38.5% in
the SC2 group (n = 322). with a kappa of 0.31 (95% CI 0.20–0.43). The corre-
sponding kappa value between officers was 0.05 (95% CI
-0.05–0.14). tice in Norwegian NIOs. Instead, the sick-listed them-
selves predicted their length of sick leaves far more
accurately, but this information is not routinely sought. In the prediction of sick leaves lasting at least 12 weeks
based on the SC2, agreement was moderate between med-
ical consultants (kappa = 0.42, 95% CI 0.29–0.54) and
fair between officers (kappa = 0.26, 95% CI 0.10–0.42). The corresponding agreements in the prediction of sick
leaves lasting at least 26 weeks were moderate between
medical consultants (kappa = 0.55, 95% CI 0.40–0.70)
and fair between insurance officers (kappa = 0.31, 95% CI
0.17–0.47). Representativeness The officers in the present study were selected from expe-
rienced officers who had shown an interest in the field of
sick leave. This might introduce a bias of overestimating
the officers' general ability to predict the length of sick
leaves. The performances of the two medical consultants
were representative of eight of their colleagues who partic-
ipated in the reproducibility part of the study. We there-
fore consider the diagnostic accuracy of the assessors to be
representative of their professional groups, or at least not
underestimated due to bias. Although the diagnostic accu-
racy varied within each group, the main conclusion of bet-
ter predictive ability among the sick-listed, was challenged
neither by comparing with the mean length predicted by
assessors, nor by comparing with the best-performing
NIO assessor. The differences in diagnostic accuracy, between the two
participating medical consultants and their eight col-
leagues in the reproducibility group, were not significant. Page 8 of 11
(page number not for citation purposes) Why did the sick-listed make better predictions? Why did the sick listed make better predictions? If the lengths of sick leaves were predominantly related to
loss of function caused by sickness, in line with the
legislation, we would expect that the medical consultants'
professional competence would favour them in predic-
tions of the lengths of sick leaves. The differences we
observed between medical consultants and officers in
mean ROC area, were however minor. Furthermore, we
could not demonstrate any significant differences in diag-
nostic accuracy between medical consultants and officers
when aggregate information on disease, treatment, func-
tion related to work, and prognoses were available in the
SC2. The improvement in ROC area with this aggregated
information was minor, with the area just reaching 70%,
which is considered borderline useful for some purposes
[7]. The result is in line with Bjørndal's findings of low
prognostic impact of the SC2 [15], and is supported by
findings of a low predictive power of symptoms and signs
in neck and shoulder disorders [16]. The better prediction
of the length of sick leave by the sick-listed themselves, is
supported by studies that have identified different non-
disease determinants of sick leave, such as job satisfaction
[17], attitudes towards pain [18], irreplaceability [19] and
psychosocial work environment [20-22]. Studies identify-
ing that at least the initial sickness certification is
predominantly patient controlled [23,24] indicate the
competence of the sick-listed. Self-rated health seems to
be an independent predictor of return to work [17], disa-
bility pension [25] and early retirement [26]. Our findings
can be interpreted as indicating that the subjective percep-
tion of sickness and work ability is more predictive of the
length of sick leave, than the apparently more objective
description in medical terms. The differences in predictive
ability were especially significant in persons with mental
and neck disorders, while the NIO assessors performed
equal to the sick-listed in the more clear-cut injuries with
more standardised treatment and prognosis. Mental dis- The marginal predictive ability and modest agreement
between NIO assessors questions the use of resources in
selection based on information from medical certificates. The predictions of medical consultants tend to be better
than those of officers, but not to an extent that makes it
more meaningful to use medical consultants in the selec-
tion process, rather than officers. Discussion The results of the present study question any practical
value of using information in medical sickness certificates
in predicting the length of sick leave, as is the current prac- Page 8 of 11
(page number not for citation purposes) http://www.biomedcentral.com/1471-2458/4/46 http://www.biomedcentral.com/1471-2458/4/46 BMC Public Health 2004, 4:46 The distributions of gender and diagnosis among the 993
persons included in the study were comparable with those
in the National Sickness Benefits Register. The findings of
longer sick leaves in women with musculoskeletal disor-
ders, and longer sick leaves in men with mental disorders,
are consistent with the Register and other studies [11-13]. orders, with high prevalence in the population, and an
increasing cause of absence [27], are of special interest
[13]. This increasing prevalence of sick leaves indicates the
presence of factors separate from the diagnosis criteria. It
seems that the more clear-cut the disease and the recom-
mended treatment, the lesser the gain in predictive ability
achieved by asking the sick-listed, and vice versa. The
modest gain in predictive ability caused by introducing
more medical information by the inclusion of the SC2
supports this interpretation. A more complete description
of symptoms and treatment does not necessarily give bet-
ter prognostic information when this includes little
knowledge of the consequences related to occupation,
and the effects of treatment are undocumented or, at best,
marginal. The low responder rate among the sick-listed introduced a
possible selection bias, although we could not identify
any selection bias in gender, age, diagnosis or occupation
[14]. If there was a selection towards more predictable sick
leaves, this should have been reflected in the assessments
of officers and medical consultants. The general trend of
lower diagnostic accuracy of NIO assessors in the
responder group indicates that if any selection bias con-
tributes to the results, it is an underestimate of the self-
predictive ability. Diagnostic accuracy – practical implication
The Norwegian NIO is obliged by legislation to perform
early intervention on the sick-listed in an effort to reduce
the length of sick leave and the risk of expulsions from
work. Limited resources and the large number of sick-
listed individuals make selection desirable before any
intervention is initiated. An alternative to selection on the
basis of medical certificates is to communicate directly
with the sick-listed themselves. This selection for interven-
tion by NIOs might be seen as screening. The aim is to
reach – at an acceptable cost – as many as possible of
those that might profit from intervention. The potential
individual gain by intervention will be greater when
longer lasting sick leaves can be anticipated, and greater
the sooner individual intervention programs are
established. Competing interests 14. Fleten N, Johnsen R, Ostrem BS: Sykmeldte tror tiltak på
arbeidsplassen kan redusere sykefravær [Sick-listed patients
think job adjustments might reduce sick-leaves]. Tidsskr Nor
Laegeforen 1999, 119:3730-3734. The author N.F. is part-time employed as National Insur-
ance medical consultant. g
15. Bjorndal A: Oppfølging av langtidssykemeldte. En under-
søkelse av en kohort fra Moss kommune. [Follow-up of per-
sons on long-term sick-leave. A cohort study in the city of
Moss]. Tidsskr Nor Laegeforen 1994, 114:2857-2862. Authors' contributions NF was in charge of designing and running the study, and
performed most of the analyses and the writing of this
manuscript. RJ actively supervised all parts of the study,
and OHF contributed to planning and writing. All authors
read and approved the final version of the manuscript. ]
g
16. Viikari-Juntura E, Takala E, Riihimaki H, Martikainen R, Jappinen P:
Predictive validity of symptoms and signs in the neck and
shoulders. J Clin Epidemiol 2000, 53:800-808. J
p
17. van-der-Giezen AM, Bouter LM, Nijhuis FJ: Prediction of return-
to-work of low back pain patients sicklisted for 3-4 months. Pain 2000, 87:285-294. 18. Lofvander M: Attitudes towards pain and return to work in
young immigrants on long- term sick leave. Scand J Prim Health
Care 1999, 17:164-169. Conclusions Sick-listed individuals predicted their length of sick leave
far more accurately than did NIO medical consultants and
officers based on information from sickness certificates
and the history of past sick leaves. The predictions of sick-
listed males were better than those of females, and older
persons predicted better than younger persons. The avail-
ability of more information, as through the SC2, had only
a minor effect on the predictive ability of the medical con-
sultants and officers. Neither reliability nor validity of
their predictions was satisfactory. 6. Hanley JA, McNeil BJ: The meaning and use of the area under a
receiver operating characteristic (ROC) curve. Radiology 1982,
143:29-36. 7. Swets JA: Measuring the accuracy of diagnostic systems. Sci-
ence 1988, 240:1285-1293. 8. Moons KG, Stijnen T, Michel BC, Buller HR, Van Es GA, Grobbee DE,
Habbema JD: Application of treatment thresholds to diagnos-
tic-test evaluation: an alternative to the comparison of areas
under receiver operating characteristic curves. Med Decis
Making 1997, 17:447-454. g
9. Altman GD: Practical statistics for medical research. 1st edi-
tion. Chapman and Hall; 1991:396-409. 10. Fleiss JL: The Measurement of Interrater Agreement. In Statis-
tical Methods for Rates and Proportions Volume 13. 2nd edition. New
York, John Wiley; 1981:212-236. This study demonstrates the need to re-consider the diag-
nostic usefulness of documentation on sickness absences,
and supports a change in strategy from collecting more
medical information to more direct communication with
the sick-listed themselves, for effective and early interven-
tions to prevent long sick leaves and expulsions from
work. J
y
11. Brage S, Nygard JF, Tellnes G: The gender gap in musculoskele-
tal-related long-term sickness absence in Norway. Scand J Soc
Med 1998, 26:34-43. 12. Hensing G, Brage S, Nygard JF, Sandanger I, Tellnes G: Sickness
absence with psychiatric disorders--an increased risk for
marginalisation among men? Soc Psychiatry Psychiatr Epidemiol
2000, 35:335-340. 13. Sandanger I, Nygard JF, Brage S, Tellnes G: Relation between
health problems and sickness absence: gender and age differ-
ences--a comparison of low-back pain, psychiatric disorders,
and injuries. Scand J Public Health 2000, 28:244-252. http://www.biomedcentral.com/1471-2458/4/46 http://www.biomedcentral.com/1471-2458/4/46 BMC Public Health 2004, 4:46 5. such tests should be compared with the results of crude
self-estimated length. Tellnes G, Brage S, Haland EM, Brodholt A: Hvilke symptomer og
plager forer til sykmelding? ICPC-koding av pasientenes
egne vurderinger i allmennpraksis. [What symptoms and
complaints result in sick-listing? ICPC-coding of patients'
own opinion in general practice]. Tidsskr Nor Laegeforen 1992,
112:1985-1988. Why did the sick-listed make better predictions? With limited resources for intervention, it might be more
cost effective to identify those whose sick listing will last
longer than 26 weeks instead of 12 weeks. Based on self-
reporting, eight out of ten would be true positives, and
one fourth of the individuals would be reached. To reach
the same number of true positives at 14 days of sick leave,
the ratio of true positives would be reversed from eight
out of ten, to two or three out of ten, if the selection were
based on medical certificates. In the search for tests predicting long-lasting sick leaves,
such as The Örebro Musculoskeletal Pain Questionnaire
[28], the present study indicates that the results of any Page 9 of 11
(page number not for citation purposes) Page 9 of 11
(page number not for citation purposes) Acknowledgements The authors want to thank the Norwegian Ministry Of Health and Social
Affairs for funding the study from July 1997 to December 1999, canalized
through the National Insurance Administration (project no. 13345). 19. Aronsson G, Gustafsson K, Dallner M: Sick but yet at work. An
empirical study of sickness presenteeism [In Process
Citation]. J Epidemiol Community Health 2000, 54:502-509. ] J p
y
20. Kivimaki M, Elovainio M, Vahtera J: Workplace bullying and sick-
ness absence in hospital staff. Occup Environ Med 2000,
57:656-660. This study could not have been performed without the support and contri-
bution of the county and local National Insurance Offices in Troms. 21. Niedhammer I, Bugel I, Goldberg M, Leclerc A, Gueguen A: Psycho-
social factors at work and sickness absence in the Gazel
cohort: a prospective study. Occup Environ Med 1998, 55:735-741. Page 10 of 11
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2000]. Oslo; 2001. 28. Linton SJ, Boersma K: Early identification of patients at risk of
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ity of the Orebro Musculoskeletal Pain Questionnaire. Clinical
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Norwegian National Insurance Administration; Planning and
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2000]. Oslo; 2001.
28.
Linton SJ, Boersma K: Early identification of patients at risk of
developing a persistent back problem: The predictive valid-
ity of the Orebro Musculoskeletal Pain Questionnaire. Clinical
Journal of Pain 2003, 19:80-86. Pre-publication history The pre-publication history for this paper can be accessed
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High neopterin and IP-10 levels in cerebrospinal fluid are associated with neurotoxic tryptophan metabolites in acute central nervous system infections
|
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Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327
https://doi.org/10.1186/s12974-018-1366-3 Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327
https://doi.org/10.1186/s12974-018-1366-3 RESEARCH
Open Access
High neopterin and IP-10 levels in
cerebrospinal fluid are associated with
neurotoxic tryptophan metabolites in acute
central nervous system infections
Else Quist-Paulsen1,2*
, Pål Aukrust3,4,5,6,7, Anne-Marte Bakken Kran9,2,5, Oona Dunlop10, Vidar Ormaasen1,
Birgitte Stiksrud1,2, Øivind Midttun8, Thor Ueland3,5,6,7, Per Magne Ueland8, Tom Eirik Mollnes2,6,7,11,13,14,15
and Anne Ma Dyrhol-Riise1,2,6,12 RESEARCH
Open Access
High neopterin and IP-10 levels in
cerebrospinal fluid are associated with
neurotoxic tryptophan metabolites in acute
central nervous system infections
Else Quist-Paulsen1,2*
, Pål Aukrust3,4,5,6,7, Anne-Marte Bakken Kran9,2,5, Oona Dunlop10, Vidar Ormaasen1,
Birgitte Stiksrud1,2, Øivind Midttun8, Thor Ueland3,5,6,7, Per Magne Ueland8, Tom Eirik Mollnes2,6,7,11,13,14,15
and Anne Ma Dyrhol-Riise1,2,6,12 High neopterin and IP-10 levels in
cerebrospinal fluid are associated with
neurotoxic tryptophan metabolites in acute
central nervous system infections Else Quist-Paulsen1,2*
, Pål Aukrust3,4,5,6,7, Anne-Marte Bakken Kran9,2,5, Oona Dunlop10, Vidar Ormaasen1,
Birgitte Stiksrud1,2, Øivind Midttun8, Thor Ueland3,5,6,7, Per Magne Ueland8, Tom Eirik Mollnes2,6,7,11,13,14,15
and Anne Ma Dyrhol-Riise1,2,6,12 * Correspondence: e.q.paulsen@studmed.uio.no * Correspondence: e.q.paulsen@studmed.uio.no
1Department of Infectious Diseases, Oslo University Hospital, Ullevaal
Hospital, P. O. Box 4956 Nydalen, N-0450 Oslo, Norway
2Institute of Clinical Medicine, University of Oslo, Oslo, Norway
Full list of author information is available at the end of the article * Correspondence: e.q.paulsen@studmed.uio.no
1Department of Infectious Diseases, Oslo University Hospital, Ullevaal
Hospital, P. O. Box 4956 Nydalen, N-0450 Oslo, Norway
2Institute of Clinical Medicine, University of Oslo, Oslo, Norway
Full list of author information is available at the end of the article Abstract Background: The host response to intruders in the central nervous system (CNS) may be beneficial but could also
be harmful and responsible for neurologic symptoms and sequelae in CNS infections. This immune response
induces the activation of the kynurenine pathway (KP) with the production of neuroactive metabolites. Herein, we
explored cytokine and KP responses in cerebrospinal fluid (CSF) and serum in patients with encephalitis, aseptic,
and bacterial meningitis. Methods: Cytokines were measured in CSF and serum by multiplex assay in adult patients with encephalitis of
infectious, autoimmune or unknown etiology (n = 10), aseptic meningitis (ASM, n = 25), acute bacterial meningitis
(ABM, n = 6), and disease control patients with similar symptoms but without pleocytosis in CSF (n = 42). Liquid
chromatography-tandem mass spectrometry (LC-MS/ MS) was used to measure KP metabolites in CSF and serum. Results: A characteristic pattern of increasing cytokine levels and KP metabolites was found in CSF from encephalitis
to ASM, with the highest levels in ABM. In ASM and ABM, most inflammatory mediators, including IL-6, IL-8, and IFN-
inducible protein-10 (IP-10), showed markedly elevated levels in CSF compared with serum, indicating
production within the CNS. In contrast to most mediators, the highest level of IP-10 was found in the ASM
group, suggesting a potential role for IP-10 in aseptic/viral meningitis. Neopterin and IP-10 were associated
with marked changes in KP metabolites in CSF with increasing kynurenine/tryptophan ratio reflecting
indoleamine 2,3-dioxygenase activity. Neopterin, a marker of IFN-γ activity, was associated with an unfavorable
balance between neuroprotective and neurotoxic tryptophan metabolites. Conclusion: We show that parenchymal and meningeal inflammations in CNS share a characteristic cytokine
profile with a general immune response in the CSF with limited influence from the systemic circulation. IFN-γ
activity, assessed by neopterin and IP-10 levels, may play a role in the activation of the KP pathway in these
patients, potentially mediating neurotoxic effects. Keywords: Encephalitis, Aseptic meningitis, Bacterial meningitis, Cytokines, Chemokines, Kynurenine tryptophan
pathway, Indoleamine 2,3-dioxygenase, Neopterin Background indoleamine 2,3-dioxygenase (IDO) which is upregu-
lated by inflammatory cytokines, mainly by interferon
gamma (IFN-γ) [20], linking T cell activation to the
regulation of the KP. Background
The host inflammatory response to intruders to the
central nerve system (CNS) plays an important role
for neuronal injury in encephalitis and meningitis. The cytokine profiles of aseptic meningitis (ASM) and
acute bacterial meningitis (ABM) have been investi-
gated in several studies, in general showing increased
levels of inflammatory mediators [1–8]. However, for
encephalitis, inflammatory responses have mainly been
evaluated
for
patients
with
herpes
simplex
virus
(HSV) infection [9–13]. Thus, comparison of cytokine
levels in encephalitis, ASM and ABM and control pa-
tients are scarce. Moreover, most studies have focused
on a limited number of inflammatory markers, and
few studies have examined parallel samples of serum
and cerebrospinal fluid (CSF) from the same patients. It is known that the inflammation activates the
kynurenine pathway (KP) resulting in the depletion of
tryptophan (TRP) and formation of metabolites with
potential
neurotoxic
(e.g.,
quinolinic
acid
[QA],
3-hydroxykynurenine
[3-HK])
and
neuroprotective
(e.g., kynurenic acid [KYNA]) effects (Fig. 1) [14, 15]. The activation of the KP seems to also have immune
modulating effects,
resulting in
inhibition
of TH1
cells, activation of regulatory T cells (Tregs) and inhib-
ition of natural killer (NK) cells [16–19]. In the CNS,
the
rate-limiting
enzyme
for
TRP
catabolism
is The host inflammatory response to intruders to the
central nerve system (CNS) plays an important role
for neuronal injury in encephalitis and meningitis. The cytokine profiles of aseptic meningitis (ASM) and
acute bacterial meningitis (ABM) have been investi-
gated in several studies, in general showing increased
levels of inflammatory mediators [1–8]. However, for
encephalitis, inflammatory responses have mainly been
evaluated
for
patients
with
herpes
simplex
virus
(HSV) infection [9–13]. Thus, comparison of cytokine
levels in encephalitis, ASM and ABM and control pa-
tients are scarce. Moreover, most studies have focused
on a limited number of inflammatory markers, and
few studies have examined parallel samples of serum
and cerebrospinal fluid (CSF) from the same patients. © The Author(s). 2018 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. Page 2 of 14 Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Background g
Altered cytokine levels and associated imbalance of
neurotoxic and neuroprotective metabolites in the KP
have been suggested to contribute to the pathogenesis of
several chronic conditions in the CNS, such as schizo-
phrenia [21, 22], bipolar disorders [23, 24], Parkinson’s
disease [25], Alzheimer’s [26] and Huntington’s disease
[27], and AIDS-related dementia [28, 29], as well as in
traumatic brain injury [30]. However, there is limited
data on the role of KP in acute infections like encephal-
itis and meningitis, although increased levels of KYNA
have been shown in patients with HSV encephalitis,
Lyme borreliosis, tick-borne encephalitis, and bacterial
meningitis [4, 31–33]. Moreover, altered tryptophan me-
tabolism has been linked to disease severity in tubercu-
lous meningitis [34]. It is known that the inflammation activates the
kynurenine pathway (KP) resulting in the depletion of
tryptophan (TRP) and formation of metabolites with
potential
neurotoxic
(e.g.,
quinolinic
acid
[QA],
3-hydroxykynurenine
[3-HK])
and
neuroprotective
(e.g., kynurenic acid [KYNA]) effects (Fig. 1) [14, 15]. The activation of the KP seems to also have immune
modulating effects,
resulting in
inhibition
of TH1
cells, activation of regulatory T cells (Tregs) and inhib-
ition of natural killer (NK) cells [16–19]. In the CNS,
the
rate-limiting
enzyme
for
TRP
catabolism
is The aim of the present study was to elucidate the in-
flammatory network and KP metabolites in ASM and
ABM, characterized by meningeal inflammation, and in
encephalitis, characterized by brain parenchymal inflam-
mation. Parallel samples of serum and CSF were exam-
ined in patient groups and control patients, i.e., patients
with similar symptoms but without CSF pleocytosis or
other signs of CNS infection. Fig. 1 Schematic presentation of the KP pathway. IDO is the main enzyme responsible for the TRP catabolism in CNS. KYN is further degraded
into the neuroprotective NMDAr antagonist KYNA by KAT, or by KMO and KYNU into the neurotoxic metabolites of 3-HK and QA. QA is an
agonist of the NMDA receptor. Abbreviations: AA, anthranilic acid; 3-HAA, 3-hydroxyanthranilic acid; HAO, 3-hydroxyanthranilic acid oxidase; 3-HK,
3-hydroxykynurenine; IDO, indoleamine-2,3-dioxygenase; KAT, kynurenine aminotransferase; KMO, kynurenine 3-monooxygenase; KYN, kynurenine;
KYNA, kynurenic acid; KYNU, kynureninase; QA, quinolinic acid; PIC, picolinic acid; TRP, tryptophan; XA, xanthurenic acid. Bold box indicates
neuroprotective metabolite, dashed boxes indicate neurotoxic metabolites in the KP pathway Fig. 1 Schematic presentation of the KP pathway. IDO is the main enzyme responsible for the TRP catabolism in CNS. Study participants and study design Study participants and study design y p
p
y
g
This cross-sectional study was performed at the Oslo Uni-
versity Hospital (OUS), Ullevaal, a regional hospital for 2.7
million people. Patients were eligible for inclusion if they
had (1) onset of symptoms of CNS infection within less
than 7 days and (2) clinical indication for a diagnostic
lumbar puncture (LP). Patients were included between
January 2014 and December 2015. CSF leukocyte counts
≥5 × 106 /L was found in 68 patients. Of these, 23 did not
fulfill the case definition of encephalitis of viral, auto-
immune or unknown cause, aseptic meningitis (ASM), or
bacterial meningitis (ABM) (Additional file 1: Table S1). In four patients, the time from CSF sampling until centri-
fugation was > 10 h, rendering a total of 41 patients with
CNS infection (Additional file 2: Figure S1). The control
group consisted of age- and gender-matched patients with
similar symptoms, but without signs of CNS inflamma-
tion, i.e., no pleocytosis and no microbiological agent
detected in their CSF (n = 42). In the control group, pa-
tients with delirium, chronic or acute psychiatric disease,
Parkinson’s disease, Huntington’s disease, CNS malig-
nancy, dementia, epilepsy or seizures, cerebral vascular
disease, transient global amnesia, and septicemia were not
included. For detailed case definitions, see Additional file 1:
Table S1. Flowchart of the study population and the over-
view of analyses performed for the various groups are
shown in Additional file 2: Figure S1. Sampling of CSF and serum CSF samples were collected in endotoxin-free polypro-
pylene tubes and stored at 4 °C until centrifugation at
2000×g for 10 min. Serum samples were collected in
endotoxin-free tubes without any additives, allowed to
clot at room temperature, and centrifuged at 3000×g for
10 min. Supernatants from CSF and serum were centri-
fuged within 10 h after collection and immediately fro-
zen in triplicates of approximately 700 μL each at −80 °
C. All analyses were performed on previously unthawed
samples. For six patients, no serum was available. Background KYN is further degraded
into the neuroprotective NMDAr antagonist KYNA by KAT, or by KMO and KYNU into the neurotoxic metabolites of 3-HK and QA. QA is an
agonist of the NMDA receptor. Abbreviations: AA, anthranilic acid; 3-HAA, 3-hydroxyanthranilic acid; HAO, 3-hydroxyanthranilic acid oxidase; 3-HK,
3-hydroxykynurenine; IDO, indoleamine-2,3-dioxygenase; KAT, kynurenine aminotransferase; KMO, kynurenine 3-monooxygenase; KYN, kynurenine;
KYNA, kynurenic acid; KYNU, kynureninase; QA, quinolinic acid; PIC, picolinic acid; TRP, tryptophan; XA, xanthurenic acid. Bold box indicates
neuroprotective metabolite, dashed boxes indicate neurotoxic metabolites in the KP pathway Fig. 1 Schematic presentation of the KP pathway. IDO is the main enzyme responsible for the TRP catabolism in CNS. KYN is further degraded
into the neuroprotective NMDAr antagonist KYNA by KAT, or by KMO and KYNU into the neurotoxic metabolites of 3-HK and QA. QA is an
agonist of the NMDA receptor. Abbreviations: AA, anthranilic acid; 3-HAA, 3-hydroxyanthranilic acid; HAO, 3-hydroxyanthranilic acid oxidase; 3-HK,
3-hydroxykynurenine; IDO, indoleamine-2,3-dioxygenase; KAT, kynurenine aminotransferase; KMO, kynurenine 3-monooxygenase; KYN, kynurenine;
KYNA, kynurenic acid; KYNU, kynureninase; QA, quinolinic acid; PIC, picolinic acid; TRP, tryptophan; XA, xanthurenic acid. Bold box indicates
neuroprotective metabolite, dashed boxes indicate neurotoxic metabolites in the KP pathway Page 3 of 14 Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Mass spectrometry analyses of tryptophan metabolites in
CSF and serum All patients, or their next of kin when the patient was
not able to consent, gave written informed consent to
participate in the study. The study was approved by The
Regional Committees for Medical and Health Research
Ethics (REC South East, reference number 2011/2578)
and the ethical council of the hospital. Tryptophan and kynurenine metabolites were measured
only in the patients with encephalitis, ASM with con-
firmed viral etiology (viral meningitis, VM), ABM, and
controls (Additional file 2: Figure S1). Concentrations of
TRP, kynurenine [KYN], anthranilic acid [AA], KYNA,
3-HK, 3-hydroxyanthranilic acid [3-HAA], xanthurenic
acid [XA], QA, picolinic acid [PIC], and neopterin were
analyzed
in
CSF
and
serum
by
liquid
chromatography-tandem mass spectrometry (LC-MS/
MS) by Bevital AS [35, 36]. For TRP, nine patients had
levels in CSF below the lower limit of detection (LOD). As this may represent a finding rather than a limitation
by the analysis, these were set equal to the LOD
(0.4 μM) in the statistical analyses and in the calculation
of the KYN/TRP ratio (KYN (nmol)/TRP (μmol)) as an
index of IDO activity. XA was not detected in CSF for
46 of the 50 patients and was not included in the
analysis. Microbiological diagnostics For all included patients, CSF leukocyte counts (CSF
WBC), CSF protein, and CSF glucose were measured. Bacterial culture of CSF and analyses for identification
of causative agent were performed in all individuals by a
panel of specific PCR for common neurotropic virus and
bacteria. Methods Multiplex analyses of soluble markers in CSF and serum
A multiplex cytokine assay (Bio-Plex Pro Human Cytokine
27-plex Panel; Bio-Rad laboratories Inc. Hercules, CA)
was used to measure the concentrations of 27 different cy-
tokines: tumor necrosis factor (TNF), IFN-γ, interleukin
(IL)-1β, IL-1 receptor antagonist (IL-1Ra), IL-2, IL-4, IL-5,
IL-6, IL-7, IL-8/CXCL8, IL-9, IL-10, IL-12(p70), IL-13,
IL-15,
IL-17A,
monocyte
chemoattractant
protein
(MCP)-1/CCL2,
IFN-inducible
protein-10
(IP-10)/
CXCL10,
eotaxin/CCL11,
macrophage
inflammatory
protein-1α and -1β (MIP-1α/CCL3, MIP-1β/CCL4), regu-
lated on activation, normal T cell expressed and secreted
(RANTES/CCL5), granulocyte-colony stimulating factor
(G-CSF), granulocyte-macrophage CSF (GM-CSF), basic
fibroblast growth factor (FGF), platelet-derived growth
factor (PDGF), and vascular endothelial growth factor
(VEGF). The assay was performed using the instructions
of the manufacturer. CSF samples were tested undiluted. Only cytokines with less than 20% missing values were
included in further analyses. Undetectable levels were
assigned the lowest detectable level (LDL) measured in
the cohort for the respective marker. In the CSF samples,
IFN-γ, IL-5, PDGF, Basic FGF, and RANTES, and for
serum, IL-2, IL-5, IL-7, IL-15, G-CSF, GM-CSF, and FGF
were excluded from further analyses based on the criteria
stated above. Multiplex analyses of soluble markers in CSF and serum Statistical methods Continuous data are presented as median (IQR, inter-
quartile range). Due to lack of normal distribution, ana-
lysis of variance (ANOVA) with the Kruskal-Wallis test
for multiple groups was used. If the Kruskal-Wallis test
revealed significant differences, the Mann-Whitney U Page 4 of 14 Page 4 of 14 Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Quist-Paulsen et al. Journal of Neuroinflammation are presented in Table 1. There were no significant
differences in gender or age between the patient
groups. In the CNS infection group, four patients re-
ported a history of depression, but only two of these
received antidepressant drugs. The etiology of enceph-
alitis was known for four patients (40%), three viral
(adenovirus, HSV1, varicella-zoster virus [VZV]) and
one N-methyl-D-aspartate receptor [NMDAr] enceph-
alitis. Of the 25 patients with ASM, eight were diag-
nosed with enterovirus in CSF, six patients suffered
from HSV2 meningitis, one patient seroconverted and
had positive IgM in CSF for Toscana virus, and for
one patient, intrathecal antibody production of IgG
against Borrelia burgdorferi was detected. For patients
with
ABM,
Streptococcus
pneumoniae
(n
= 2),
Staphylococcus aureus (n = 2), Neisseria meningitidis
(n = 1), and Haemophilus influenzae (n = 1) were de-
tected in CSF by growth and/or PCR, and for all test was used to compare pairs of groups. To limit type
II statistical errors, no correction for multiple compari-
sons was made in this explorative study. P values < 0.05
were considered statistically significant. Categorical vari-
ables are expressed as counts (percentages) and analyzed
by Pearson’s chi-square test. Correlations were analyzed
using Spearman’s rank correlation coefficient. All data
analyses were performed in SPSS version 24 (IBM Corp. Armonk, NY, USA) and graphs generated by GraphPad
Prism 7 (GraphPad, San Diego, USA). test was used to compare pairs of groups. To limit type
II statistical errors, no correction for multiple compari-
sons was made in this explorative study. P values < 0.05
were considered statistically significant. Categorical vari-
ables are expressed as counts (percentages) and analyzed
by Pearson’s chi-square test. Correlations were analyzed
using Spearman’s rank correlation coefficient. All data
analyses were performed in SPSS version 24 (IBM Corp. Armonk, NY, USA) and graphs generated by GraphPad
Prism 7 (GraphPad, San Diego, USA). Study participant characteristics Ten patients had encephalitis of viral, autoimmune,
or unknown etiology according to the case definition
(Additional file 1: Table S1), 25 patients were diag-
nosed with ASM, six patients with ABM, and 42 were
control patients. Characteristics of the study group Table 1 Patient characteristics and clinical presentation
Parameter
Encephalitis (n
10)
A Table 1 Patient characteristics and clinical presentation
Parameter
Encephalitis (n = 10)
ASM (n = 25)
ABM (n = 6)
Controls (n = 42)
p valuea
Gender, males (%)
4 (40)
10 (40)
4 (67)
13 (31)
0.385
Age, years
43.5 (30, 72)
35 (28, 48)
52 (41, 68)
31 (22, 41)
0.054
Hospital stay, days
19 (11, 42)b
3 (1.5, 5.5)c
19 (14, 33)b, d
2.0 (1.0, 4.0)
< 0.001
Comorbidity (%)
Immunodeficiencye
2 (30)b
1 (4)c
1 (17)b
–
0.029
Psychiatric disorder
2 (20)b
2 (8)
–
–
0.046
Etiology known (%)f
4/10 (40)
16/25 (64)
6/6 (100)
–
< 0.001
Headache (%)
7/10 (70)b
24/25 (96)c
3/4 (75)
39/41 (95)
0.037
Neck stiffness (%)g
2/10 (20)
12/25 (48)
3/5 (60)
12/42 (29)
0.175
Objective fever (%)h
7/10 (70)
17/25 (68)
5/6 (83)
20/42 (48)
0.168
Focal neurology (%)
5/9 (56)b
2/19 (10)c
1/6 (17)
1/24 (4)
0.003
GCS ≤14 (%)
10/10 (100)b
2/25 (8)c
5/6 (83)b,d
6/42 (14)
< 0.001
CSF WBC (× 106/L)
25 (9.5, 92)b
179 (26, 271)b, c
212 (91, 1434)b, c
1.0 (1.0, 2.0)
< 0.001
CSF protein (g/L)
0.57 (0.4, 0.9)b
0.59 (0.4, 0.8)b
2.2 (0.8, 5.9)b, c, d
0.26 (0.2, 0.3)
< 0.001
CSF glucose (mmol/L)
3.6 (3.3, 4.4)
3.5 (3.0, 3.7)
3.6 (0.4, 6.3)
3.5 (3.2, 3.7)
0.341
Glucose ratio
0.6 (0.5, 0.7)
0.6 (0.5, 0.6)b
0.4 (0.1, 0.6)b
0.6 (0.6, 0.7)
0.009
Albumin ratio
8.4 (6.5,14)b
9.4 (6.1, 13)b
79 (36, 137)b, c, d
4.0 (2.6, 5.0)
< 0.001
Blood WBC (× 109/L)
8.6 (7.4, 11)
8.2 (6.1, 11)
14 (8.5, 18)
10 (6.7, 12)
0.173
CRP, serum (mg/L)
6 (0.9, 86)
3.5 (1.3, 12)b
169 (96, 446)b, c, d
16 (2.1, 77)
0.001
CSF WBC white blood cell count in CSF, glucose ratio CSF glucose/serum glucose, albumin ratio CSF albumin/serum albumin. Significant p-values are marked
in bold. Cytokine profiles in CSF and serum Cytokines analyzed in parallel in CSF and serum
showed
distinct
patterns
for
the
different
patient
groups. Overall, the highest levels of CSF cytokines
were found in
patients
with
ABM, including the
prototypical inflammatory cytokines TNF, IL-1β and
IL-6, inflammatory chemokines (e.g., IL-8, MCP-1,
MIP-1α and MIP-1β), cytokines with potent effect on
T cell function (e.g., IP-10, IL-7, IL-9 and IL-15), and
growth
factors
(e.g.,
VEGF
and
G-CSF)
(Fig. 2,
Additional file 3: Table S2). The typical cytokine pat-
tern in CSF was an increase from disease controls
without CNS infection to patients with encephalitis
and ASM with the highest levels in those with ABM. In contrast to this pattern, the CXC chemokine IP-10
showed the highest median level in the ASM group
(Fig. 2). Although lower than in patients with ABM
and ASM, patients with encephalitis had higher levels
for most cytokines in CSF compared to the control
group, with no significant difference in the levels
between encephalitis cases with or without known eti-
ology (data not shown). In order to relate the cytokine profile to tryptophan
metabolism (Fig. 1), metabolites of the KP in the CSF
were examined in patients with encephalitis (n = 10),
those in the ASM group with verified viral cause (VM, n
= 12), ABM (n = 6), and controls (n = 22). The median
levels of most KP metabolites were higher in patients
with encephalitis, VM, and ABM compared with the
controls, with the highest median levels observed in the
ABM group (Fig. 4, Additional file 5: Table S4). The only
exception was TRP, which was lowest in the VM group
(Fig. 4). g
The KYN/TRP ratio was higher in all CNS infections
compared to the controls (p = 0.009), indicating an in-
creased conversion of TRP (Fig. 4). Moreover, to evalu-
ate the relationship between putatively neuroprotective
(KYNA) and neurotoxic (3-HK and QA) KP metabolites,
we calculated the KYNA/(3-HK + QA) ratio showing de-
creased levels for patients with encephalitis indicating a
net neurotoxic effect of TRP metabolites in these pa-
tients (Fig. 4). g
Most KP metabolites were present at higher concentra-
tions in serum (Additional file 5: Table S4, Additional file 6:
Figure S2) than in CSF (Fig. 2), but with less significant
differences between the groups. Study participant characteristics Import-
antly, the majority of the control group had fever,
headache, and neck stiffness, similar to most of the
patients with CNS infection. Cytokine profiles in CSF and serum For the total group with
CNS infection (encephalitis, VM and ABM), CSF levels
correlated with serum levels for 3-HK (Rho 0.6, p < 0.001),
QA (0.4, p = 0.04), and PIC (0.7, p < 0.001), for all other
KP metabolites, there was no significant correlation be-
tween CSF and serum levels. Furthermore, all CSF/serum
ratios for KP metabolites, except for TRP, KYNA, and PIC
ratio, were positively correlated with CSF WBC count,
while only the ratio of PIC was correlated with CSF albu-
min/serum ratio (Additional file 7: Table S5). The encephalitis group and the control group gener-
ally showed lower cytokine levels in CSF than in serum,
with exceptions for IL-6 and IL-8 in the encephalitis
group and MCP-1 and IP-10 for both groups (Figs. 2
and 3, Additional file 3: Table S2). In contrast, the
groups with meningeal involvement and in particular the
ABM group displayed markedly higher CSF levels than
serum levels for most cytokines. For some of the cyto-
kines, the CSF levels were more than tenfold higher than
in serum (e.g., IL-6, IL-8, IP-10). In general, serum levels
did not display the striking and significant differences
between the patient groups and controls as seen in CSF
(Fig. 3). In fact, although all patients in the ABM group
had a positive blood culture, only TNF, IL-6, IL-8,
IL-1Ra, and MIP-1α demonstrated higher serum levels
than in the control group. Study participant characteristics D t
h
di
(IQR)
b
/
(%) Data shown are median (IQR) or numbers/n (%) Data shown are median (IQR) or numbers/n (%) ap values for one way analysis of variance (Kruskal-Wallis)
b y
y
< 0.05 for analysis with Mann-Whitney U test (MWU) in comparison with the control group p
y
y
bp < 0.05 for analysis with Mann-Whitney U test (MWU) in comparison with the control group
c der treatment for or treated for cancer within the last year (including hematological malignancies), HIV infection or diabetes mellitus t
munosuppressive or immune modulating drugs pp
g
g
fCausing agents for encephalitis were 3 viral (adenovirus, HSV1, VZV) and 1 NMDAr encephalitis. ABM; Streptococcus pneumonia (n = 2), Staphylococcus aureus (n =
2), Neisseria meningitides (n = 1), and Haemophilus influenzae (n = 1). ASM; HSV2 (n = 6), enterovirus (n = 8), Toscana virus (n = 1), and 1 patient had intrathecal
antibody production of IgG against Borrelia burgdorferi
gNeck stiffness was assessed by a physician before LP fCausing agents for encephalitis were 3 viral (adenovirus, HSV1, VZV) and 1 NMDAr encephalitis. ABM; Streptococcus pneumonia (n = 2), Staphylococcus aureus (n =
2), Neisseria meningitides (n = 1), and Haemophilus influenzae (n = 1). ASM; HSV2 (n = 6), enterovirus (n = 8), Toscana virus (n = 1), and 1 patient had intrathecal
antibody production of IgG against Borrelia burgdorferi
gNeck stiffness was assessed by a physician before LP y
p y
hFever was defined as either ≥38 °C upon admission or within 24 h after admission, or measured to ≥38 °C by the patient prior to admission Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Page 5 of 14 Quist-Paulsen et al. Journal of Neuroinflammation the examined mediators (Additional file 4: Table S3). Finally, except for MCP-1, all cytokines in CSF corre-
lated positively with the CSF leukocyte counts, and
all, except for MCP-1 and IP-10, correlated with CSF/
serum albumin ratio. Collectively, these data under-
score the limited information obtained from a serum
sample in contrast to that obtained from CSF when
examining the
inflammatory
network
in
infectious
CNS diseases. these patients, the causative bacteria was also de-
tected in blood culture. Patients with encephalitis pre-
sented with less headache and more focal neurology
compared to the other groups, and impairment of
consciousness
was
observed
in
significantly
fewer
patients with ASM and in the control group. Markers of IFN-γ activation correlate with activation of
the KP When we analyzed the CNS infections all together
(encephalitis, ASM, and ABM), a significant correl-
ation between serum and CSF levels were found only
for TNF (Rho 0.4, p = 0.03), IL-1Ra (Rho 0.4, p =
0.03), IL-6 (Rho 0.5, p = 0.004), and MCP-1 (Rho 0.3,
p = 0.04) suggesting intrathecal production of most of IFN-γ is known to have a major influence on the KP
[37, 38]. In the present study, IFN-γ levels in CSF
were below detection level in 58% of the cases. How-
ever, neopterin, being a reliable and stable marker of
IFN-γ activity [39], was markedly elevated in CSF in
all
groups
of
patients
with
neuroinflammation Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Page 6 of 14 Quist-Paulsen et al. Journal of Neuroinflammation Fig. 2 (See legend on next page.) Fig. 2 (See legend on next page.) Fig. 2 (See legend on next page.) Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Page 7 of 14 (See figure on previous page.)
Fig. 2 Cytokines in CSF in patients with encephalitis (Enc, n = 10), aseptic meningitis (ASM, n = 25), bacterial meningitis (ABM, n = 6) in comparison with
controls (Ctr, n = 42). Data shown are medians with IQR and all were significant by the Kruskal-Wallis test. Comparisons of two groups were analyzed by
using Mann-Whitney U test. Asterisks above patient groups indicate significant difference vs controls, asterisks above horizontal lines indicate significant
differences between individual groups (Mann-Whitney U test): *p < 0.05, **p < 0.01, and ***p < 0.001. Values below the detection limit were set to the
lowest detectable level for that analyte compared with controls (Fig. 4). Notably, when all
CNS infections were analyzed together, CSF level of
neopterin was strongly positively correlated with IDO
(measured as the KYN/TRP ratio) and inversely cor-
related with the KYNA/(3-HK + QA) ratio (Fig. 5). This
suggests
an
association
between
high
IFN-γ
activity and net neurotoxic effects of KP metabolites. A positive correlation was also seen between IDO
and IP-10, another IFN-
related cytokine, but no
significant correlation was found between IP-10 and
KYNA/(3-HK + QA) ratio (Fig. 5). catabolism of TRP. Interestingly, patients with encephalitis
and viral meningitis had an unfavorable balance between
neuroprotective and neurotoxic TRP metabolites. Markers of IFN-γ activation correlate with activation of
the KP These
changes in KP metabolites were associated with CSF levels
of neopterin, and for the KYN/TRP ratio also with IP-10,
suggesting a link between IFN-γ and altered KP metabo-
lites in these patients. The influx of inflammatory cells and the resulting
dysregulation of cytokine networks may be detrimen-
tal during CNS infection and is thought to contribute
to
neurological
complications
[12,
42–44]. CNS
inflammation is a complex process, depending on
anatomical site (parenchyma vs meninges), cell type
being involved (e.g., infiltrating leukocytes in acute in-
fection vs brain-derived cells), and causing agent (e.g.,
viral vs bacterial). In this study, we describe findings
from patients with acute inflammation in the CNS,
mainly of infectious cause. The levels of cytokines in
CSF were generally higher for all infectious groups
compared to control, and strongly correlated with
each other, demonstrating a general inflammatory re-
sponse in patients with acute CNS infections. The
pattern of increasing levels of inflammatory markers
from encephalitis to aseptic meningitis and finally to
bacterial meningitis indicates increased inflammation
in all subgroup of patients, but with a more modest
CSF inflammation in patients with encephalitis. Not
surprisingly, the highest levels of most of the media-
tors were found in patients with bacterial meningitis
and there was a CSF/serum ratio > 1 of most media-
tors in patients with ABM, despite bacteremia. Fur-
thermore, most cytokines were correlated with CSF
WBC count, but not with corresponding serum levels,
which implies the inflammatory response in the CNS
to be independent of a peripheral immune response
with no or minimal influx or efflux between the two
compartments. These results indicate a predominately
intrathecal production of these mediators in CNS in-
fections. IL-6, IL-8, and TNF in CSF have in several
studies been found to be elevated in meningitis and
have even been proposed as biomarkers in CSF for
bacterial meningitis [6, 45]. We found a general in-
crease for most of the inflammatory mediators in
both ASM and ABM, and our study does not support
the use of one particular cytokine as a diagnostic
marker to distinguish ABM from other CNS condi-
tions, including ASM. Association of cytokines and metabolites in the KP and
clinical variables Abnormal regulation of the KP metabolites has been re-
lated to depression [40, 41]. However, excluding the two
patients in the encephalitis group who reported existing
or recent depression from the correlation analyses did
not influence the findings (data not shown). Although
the Glasgow Coma Scale (GCS) is a crude tool, it reflects
the severity of critically ill patients. Among patients with
aseptic meningitis, all but two had a GCS at 15; thus,
only patients with encephalitis and bacterial meningitis
were included in correlation analyses with GCS. We did
not find any significant correlations between cytokine
levels or KP metabolites in CSF and GCS in these
patients (data not shown). However, the number of pa-
tients was too low to make any firm conclusion. Discussion 3 Cytokines in serum in patients with encephalitis (Enc, n = 10), aseptic meningitis (ASM, n = 20), bacterial meningitis (ABM, n = 6) in comparison with
controls (Ctr, n = 41). Data shown are medians with IQR. Asterisks above patient groups indicate significant difference vs controls, asterisks above horizontal
lines indicate significant differences between individual groups (Mann-Whitney U test): *p < 0.05, **p < 0.01, and ***p < 0.001. Values below the detection
limit were set to the lowest detectable level for that analyte detectable TRP levels. The strong correlation of KYN/
TRP ratio with neopterin and IP-10 indicates that this
IDO activity is driven by IFN-γ. IDO has been found to
exhibit an immunosuppressive effect by upregulation of
Tregs and downregulation of Th17 cells, which could be
relevant in CNS infections, especially in ASM where T
cells are of particular importance. In contrast to the as-
sociation with IDO activity, neopterin, but not IP-10,
was associated with a “neurotoxic” KYNA/(3-HK + QA)
ratio in patients with CNS infection. The lack of correl-
ation of IP-10 to KYNA/(3-HK + QA) could be related
to low statistical power. However, whereas IP-10 is in-
duced by IFN- γ in several cell types including mono-
cytes, stromal cells, and endothelial cells [59], neopterin
is almost selectively produced in monocytes/macro-
phages, and through its relation to tetrahydrobiopterin,
neopterin may be more closely related to tryptophan
metabolism than IP-10 [60, 61]. In contrast to most of the inflammatory markers,
IP-10 levels were comparable in patients with ASM and
ABM. IP-10 is secreted by several cell types in response
to IFN-γ, and experimental studies have shown that
IP-10 is highly induced in the CNS (e.g., West Nile in-
fections [46], HIV encephalitis [47], HSV encephalitis
[48], enteroviral encephalitis [3, 49], Japanese encephal-
itis [50], and tick-borne encephalitis [TBE] [51]). In-
creased levels have also been reported in patients
suffering from TBE [52], neuroborreliosis [53], entero-
virus infection [3], HSV meningitis, and HSV encephal-
itis [9]. IP-10 is a chemoattractant for monocytes and
macrophages and promotes T cell recruitment, especially
of CD8+ T cells [46, 54, 55]. Although IP-10 has been
linked to viral clearance in the CNS [48, 55], IP-10 has
also been described to promote apoptosis of neurons
and excessive production has been associated with more
severe outcome [47, 50, 51]. Discussion In the present study, we
found an association between IP-10 and the KYN/TRP
ratio, indicating increased IDO activity and catabolism
of TRP with potential harmful effects on CNS. Thus,
our present data may further support a potential role for
this chemokine in CNS infections, particularly in ASM. This should be further investigated. Finally, when looking at the CSF/serum ratios of KP
metabolites, we found very narrow ranges in the control
group,
suggesting
strict
control
of
KP
in
healthy
subjects. Studies of the inflammatory profile in human CNS
infections including both meningeal and parenchymal
infections are relatively scarce [62–65], and comparisons
between different studies are hampered by the diversity
of causing agents and divergent inclusion criteria. Never-
theless, knowledge of immunological mechanism is piv-
otal in order to develop better diagnostic and potentially
therapeutic tools for these patients. To our knowledge,
the measurement of a large panel of metabolites in the
KP in both serum and CSF, with parallel analyses of a
wide range of cytokines and related mediators, have not
been reported for patients with these conditions, espe-
cially not for encephalitis. Moreover, the correlation in
present study of IP-10 and neopterin with the KP metab-
olites and the decreased KYNA/(3-HK + QA) ratio in
encephalitis are interesting findings that, as far as we are
aware of, have not been reported in these clinical rele-
vant CNS infections. However, the present study also
has some limitations. The subgroups of patients, and in
particular patients with ABM and encephalitis, were
small and too small for sub-analyses on causing agents. Moreover, the etiology was unknown for 60% of patients
with encephalitis with few patients with confirmed viral
cause. Besides, due to lack of reliable measures of sever-
ity and outcome, together with the relatively low number
of patients with CNS infection, we cannot make any Neurologic dysfunction is common in patients with
encephalitis and bacterial meningitis, and dysregulation
of the KP has been shown in syndromes and disorders
with certain overlap in symptoms [21, 30, 32, 56, 57]. Herein, we compared these metabolites in patients with
a stringent definition of etiology; encephalitis, confirmed
viral meningitis, and bacterial meningitis. Recent litera-
ture has shown that in HSV encephalitis, a low level of
the neuroprotective metabolite KYNA was associated
with more severe outcome [31]. In TB meningitis, low
TRP was associated with a better survival rate [34]. Discussion In the present study, we demonstrate a gradual increase in
levels of a wide range of cytokines, including chemokines,
interleukins, interferons, and growth factors from enceph-
alitis to ASM and finally to ABM as compared with con-
trols, reflecting the level of CSF inflammation seen in
patients with these CNS infections. Moreover, in ASM
and ABM, the levels were much higher in CSF than in
serum for most of the mediators, even though the patients
with ABM had positive blood cultures for the actual
pathogen. Notably, in contrast to most of the mediators,
IP-10 levels in CSF had the highest median value in the
ASM group, indicating a potential role for this chemokine
in aseptic meningitis. Finally, these changes in inflamma-
tory mediators were associated with marked changes in
KP metabolites in CSF. In particular, all groups of CNS in-
fections had increased IDO activity assessed by KYN/TRP
ratio compared with controls, indicating an increased Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327
Page 8 of 14 Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Page 8 of 14 Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Page 9 of 14 (See figure on previous page.)
Fig. 3 Cytokines in serum in patients with encephalitis (Enc, n = 10), aseptic meningitis (ASM, n = 20), bacterial meningitis (ABM, n = 6) in comparison with
controls (Ctr, n = 41). Data shown are medians with IQR. Asterisks above patient groups indicate significant difference vs controls, asterisks above horizontal
lines indicate significant differences between individual groups (Mann-Whitney U test): *p < 0.05, **p < 0.01, and ***p < 0.001. Values below the detection
limit were set to the lowest detectable level for that analyte (See figure on previous page.)
Fig. 3 Cytokines in serum in patients with encephalitis (Enc, n = 10), aseptic meningitis (ASM, n = 20), bacterial meningitis (ABM, n = 6) in comparison with
controls (Ctr, n = 41). Data shown are medians with IQR. Asterisks above patient groups indicate significant difference vs controls, asterisks above horizontal
lines indicate significant differences between individual groups (Mann-Whitney U test): *p < 0.05, **p < 0.01, and ***p < 0.001. Values below the detection
limit were set to the lowest detectable level for that analyte (See figure on previous page.)
Fig. Discussion In
our study, the ratio of the neuroprotective metabolite
KYNA to the sum of neurotoxic metabolites 3-HK and
QA was lower for patients with encephalitis compared
to the other groups, which could indicate a stronger ac-
tivation of the KMO branch in encephalitis. In fact,
studies are ongoing regarding the inhibition of several
steps in the KP, including studies on centrally available
KMO inhibitors [58]. In the present study, we observed very low levels of
TRP for several patients with CNS infection, especially
in patients with ASM. We hypothesize that this results
from increased IDO activity, as these patients had a sig-
nificantly higher level of KYN compared to patients with Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327
Page 10 of 14 Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Page 10 of 14 conclusion regarding the use of these cytokines and me-
and no pleocytosis, but many of these patients suffered
Fig. 4 Neopterin, kynurenine metabolites, and ratios in patients with encephalitis (Enc, n = 10), viral meningitis (VM, n = 12), and bacterial
meningitis (ABM, n = 6) in comparison with controls (Ctr, n = 22). Data shown are median with IQR, and all were significant in the analysis of
variance with the Kruskal-Wallis test. Comparisons of two groups were analyzed by Mann-Whitney U test. Asterisks above patient groups indicate
significant difference vs controls, asterisks above horizontal lines indicate significant differences between individual groups (Mann-Whitney U test):
*p < 0.05, **p < 0.01, and ***p < 0.001. a: TRP levels below the lower level of detection (LOD) for 9 patients with CNS infection were adjusted to
this value (0.4 μM) for calculation of the KYN/TRP ratio as the expression of IDO activity (KYN (nmol)/TRP(μmol)) urenine metabolites, and ratios in patients with encephalitis (Enc, n = 10), viral meningitis (VM, n = 12), and bacterial Fig. 4 Neopterin, kynurenine metabolites, and ratios in patients with encephalitis (Enc, n = 10), viral meningitis (VM, n = 12), and bacterial
meningitis (ABM, n = 6) in comparison with controls (Ctr, n = 22). Data shown are median with IQR, and all were significant in the analysis of
variance with the Kruskal-Wallis test. Comparisons of two groups were analyzed by Mann-Whitney U test. Discussion Asterisks above patient groups indicate
significant difference vs controls, asterisks above horizontal lines indicate significant differences between individual groups (Mann-Whitney U test):
*p < 0.05, **p < 0.01, and ***p < 0.001. a: TRP levels below the lower level of detection (LOD) for 9 patients with CNS infection were adjusted to
this value (0.4 μM) for calculation of the KYN/TRP ratio as the expression of IDO activity (KYN (nmol)/TRP(μmol)) Fig. 4 Neopterin, kynurenine metabolites, and ratios in patients with encephalitis (Enc, n = 10), viral meningitis (VM, n = 12), and bacterial
meningitis (ABM, n = 6) in comparison with controls (Ctr, n = 22). Data shown are median with IQR, and all were significant in the analysis of
variance with the Kruskal-Wallis test. Comparisons of two groups were analyzed by Mann-Whitney U test. Asterisks above patient groups indicate
significant difference vs controls, asterisks above horizontal lines indicate significant differences between individual groups (Mann-Whitney U test):
*p < 0.05, **p < 0.01, and ***p < 0.001. a: TRP levels below the lower level of detection (LOD) for 9 patients with CNS infection were adjusted to
this value (0.4 μM) for calculation of the KYN/TRP ratio as the expression of IDO activity (KYN (nmol)/TRP(μmol)) and no pleocytosis, but many of these patients suffered
from systemic infections, as reflected by elevated serum
neopterin levels. This may have camouflaged significant conclusion regarding the use of these cytokines and me-
tabolites as prognostic markers. The control group in
our study consisted of patients with similar presentation Page 11 of 14 Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 A
B
C
D
Fig. 5 Correlations of neopterin and IP-10, as markers of IFN-ƴ activity, with activation of the KP. a Neopterin vs KYN/ TRP ratio (IDO) (n = 50; Rho 0.9,
p < 0.001). b IP-10 vs KYN/TRP ratio (IDO) (n = 50; Rho 0.8, p < 0.001). c Neopterin vs KYNA/(3-HK + QA) ratio (n = 50; Rho −0.7, p < 0.001). d IP-10 vs
KYNA/(3-HK + QA) ratio (n = 50, Rho −0.5, p < 0.001). Data shown are obtained by Spearman’s rank correlation A B A B B C D C D D Fig. 5 Correlations of neopterin and IP-10, as markers of IFN-ƴ activity, with activation of the KP. a Neopterin vs KYN/ TRP ratio (IDO) (n = 50; Rho 0.9,
p < 0.001). Conclusions In conclusion, we found a marked increase in a wide range
of inflammatory mediators in CSF in aseptic and bacterial
meningitis with a more modest increase in encephalitis. The markedly higher levels in CNS than in serum for
most of the mediators indicate compartmentalization of
immune responses in the CSF. Our data may also suggest
that increased IFN-γ activity, as assessed by neopterin and
IP-10, may contribute to neurotoxicity through enhanced
TRP catabolism. In particular, dysregulation of the KP
with signs of an increased formation of neurotoxic QA in
encephalitis
should
be
explored
further
in
these
conditions. Additional file 6: Figure S2. Serum levels of KP metabolites. (PDF 81 kb) Additional file 6: Figure S2. Serum levels of KP metabolites. (PDF 81 kb) Additional file 7: Table S5. CSF/serum ratios of KP metabolites and the
correlation with CSF WBC and albumin ratio. (PDF 194 kb) Additional file 7: Table S5. CSF/serum ratios of KP metabolites and the
correlation with CSF WBC and albumin ratio. (PDF 194 kb) Additional files findings in serum profiles. On the other hand, these
controls may be more clinically relevant than healthy
controls without any disease symptoms like fever and
headache. Finally,
correlations
do
not
necessarily
mean any causal relationship and more mechanistic
studies as well as larger studies with samples also
taken during follow-up are needed to make firmer
conclusions. Additional file 1: Table S1. Case definitions of encephalitis, aseptic and
viral meningitis and bacterial meningitis. (PDF 179 kb)
Additional file 2: Figure S1. Flowchart of inclusion of patients and
overview of various analyses performed in the study population. (PDF 342 kb)
Additional file 3: Table S2. Cytokine levels in CSF and serum. (PDF 215 kb)
Additional file 4: Table S3. Correlations of cytokines in CSF with serum
levels, CSF WBC, albumin ratio and KYN/TRP ratio. (PDF 197 kb)
Additional file 5: Table S4. KP metabolites in CSF and serum. (PDF 198 kb)
Additional file 6: Figure S2. Serum levels of KP metabolites. (PDF 81 kb)
Additional file 7: Table S5. CSF/serum ratios of KP metabolites and the
l
h CSF WBC
d lb
(PDF 9 kb) Additional file 1: Table S1. Case definitions of encephalitis, aseptic and
viral meningitis and bacterial meningitis. (PDF 179 kb)
Additional file 2: Figure S1. Flowchart of inclusion of patients and
overview of various analyses performed in the study population. (PDF 342 kb)
Additional file 3: Table S2. Cytokine levels in CSF and serum. (PDF 215 kb)
Additional file 4: Table S3. Correlations of cytokines in CSF with serum
levels, CSF WBC, albumin ratio and KYN/TRP ratio. (PDF 197 kb)
Additional file 5: Table S4. KP metabolites in CSF and serum. (PDF 198 kb)
Additional file 6: Figure S2. Serum levels of KP metabolites. (PDF 81 kb)
Additional file 7: Table S5. CSF/serum ratios of KP metabolites and the Additional file 1: Table S1. Case definitions of encephalitis, aseptic and
viral meningitis and bacterial meningitis. (PDF 179 kb) Additional file 3: Table S2. Cytokine levels in CSF and serum. (PDF 215 kb) Additional file 4: Table S3. Correlations of cytokines in CSF with serum
levels, CSF WBC, albumin ratio and KYN/TRP ratio. (PDF 197 kb) Additional file 5: Table S4. KP metabolites in CSF and serum. (PDF 198 kb) Discussion b IP-10 vs KYN/TRP ratio (IDO) (n = 50; Rho 0.8, p < 0.001). c Neopterin vs KYNA/(3-HK + QA) ratio (n = 50; Rho −0.7, p < 0.001). d IP-10 vs
KYNA/(3-HK + QA) ratio (n = 50, Rho −0.5, p < 0.001). Data shown are obtained by Spearman’s rank correlation 3-HAA: 3-Hydroxyanthranilic acid; 3-HK: 3-Hydroxykynurenine; AA: Anthranilic
acid; ABM: Acute bacterial meningitis; Albumin ratio: CSF albumin/serum
albumin; ASM: Aseptic meningitis; Glucose ratio: CSF glucose/serum glucose;
HAO: 3-Hydroxyanthranilic acid oxidase; HSV1: Herpes simplex 1 virus;
IDO: Indoleamine 2,3-dioxygenase; KAT: Kynurenine aminotransferase;
KMO: Kynurenine 3-monooxygenase; KP: Kynurenine pathway of tryptophan
metabolism; KYN: Kynurenine; KYNA: Kynurenic acid; KYNU: Kynureninase;
LDL: The lowest detectable level; LOD: Lower limit of detection; NMDAr: N- Abbreviations 3-HAA: 3-Hydroxyanthranilic acid; 3-HK: 3-Hydroxykynurenine; AA: Anthranilic
acid; ABM: Acute bacterial meningitis; Albumin ratio: CSF albumin/serum
albumin; ASM: Aseptic meningitis; Glucose ratio: CSF glucose/serum glucose;
HAO: 3-Hydroxyanthranilic acid oxidase; HSV1: Herpes simplex 1 virus;
IDO: Indoleamine 2,3-dioxygenase; KAT: Kynurenine aminotransferase;
KMO: Kynurenine 3-monooxygenase; KP: Kynurenine pathway of tryptophan
metabolism; KYN: Kynurenine; KYNA: Kynurenic acid; KYNU: Kynureninase;
LDL: The lowest detectable level; LOD: Lower limit of detection; NMDAr: N- Page 12 of 14 Quist-Paulsen et al. Journal of Neuroinflammation (2018) 15:327 Received: 14 May 2018 Accepted: 11 November 2018 methyl-D-aspartate receptor; QA: Quinolinic acid; TRP: Tryptophan; VM: Viral
meningitis; VZV: Varicella-zoster virus; WBC: White blood cell count;
XA: Xanthurenic acid Received: 14 May 2018 Accepted: 11 November 2018 Received: 14 May 2018 Accepted: 11 November 2018 methyl-D-aspartate receptor; QA: Quinolinic acid; TRP: Tryptophan; VM: Viral
meningitis; VZV: Varicella-zoster virus; WBC: White blood cell count;
XA: Xanthurenic acid methyl-D-aspartate receptor; QA: Quinolinic acid; TRP: Tryptophan; VM: Viral
meningitis; VZV: Varicella-zoster virus; WBC: White blood cell count;
XA: Xanthurenic acid Funding 5. Ichiyama T, Maeba S, Suenaga N, Saito K, Matsubara T, Furukawa S. Analysis
of cytokine levels in cerebrospinal fluid in mumps meningitis: comparison
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of cytokine levels in cerebrospinal fluid in mumps meningitis: comparison
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dysregulated in inflammation, and immune activation. Front Biosci
(Landmark Ed). 2015;20:1116–43. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Competing interests Competing interests
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the corresponding author on reasonable request. 7. Grandgirard D, Gaumann R, Coulibaly B, Dangy JP, Sie A, Junghanss T,
Schudel H, Pluschke G, Leib SL. The causative pathogen determines the
inflammatory profile in cerebrospinal fluid and outcome in patients with
bacterial meningitis. Mediat Inflamm. 2013;2013:312476. 7. Grandgirard D, Gaumann R, Coulibaly B, Dangy JP, Sie A, Junghanss T,
Schudel H, Pluschke G, Leib SL. The causative pathogen determines the
inflammatory profile in cerebrospinal fluid and outcome in patients with
bacterial meningitis. Mediat Inflamm. 2013;2013:312476. Authors’ contributions VO, EQP, PA, BS, OD, AMBK, and AMDR contributed to the study design. EQP,
VO, and OD contributed to the inclusion and evaluation of patients. TEM,
ØM, and PMU contributed to the laboratory analyses. EQP, PA, TU, ØM, PMU,
TEM, BS, and AMDR contributed to the data analysis and interpretation. EQP,
TU, and TEM contributed to the statistical analyses. EQP, PA, and AMDR
contributed to the writing of the manuscript. All authors contributed to the
revision of the manuscript and approved the final version. 8. Shapiro S, Miller A, Lahat N, Sobel E, Lerner A. Expression of matrix
metalloproteinases, sICAM-1 and IL-8 in CSF from children with meningitis. J
Neurol Sci. 2003;206:43–8. 9. Lind L, Studahl M, Persson Berg L, Eriksson K. CXCL11 production in
cerebrospinal fluid distinguishes herpes simplex meningitis from herpes
simplex encephalitis. J Neuroinflammation. 2017;14:134. 10. Asaoka K, Shoji H, Nishizaka S, Ayabe M, Abe T, Ohori N, Ichiyama T, Eizuru
Y. Non-herpetic acute limbic encephalitis: cerebrospinal fluid cytokines and
magnetic resonance imaging findings. Intern Med. 2004;43:42–8. Acknowledgements 1. Asano T, Ichiki K, Koizumi S, Kaizu K, Hatori T, Fujino O, Mashiko K, Sakamoto
Y, Miyasho T, Fukunaga Y. Enhanced expression of cytokines/chemokines in
cerebrospinal fluids in mumps meningitis in children. Pediatr Int. 2011;53:
143–6. The Department of Infectious Diseases at Ullevaal Hospital represented by
head of department professor Dag Kvale (MD, PhD) provided salary and
office facilities to EQP during the study period. We would also like to thank
laboratory staff at Bevital, Bergen Norway and the Research Laboratory,
Nordland Hospital, Bodø, Norway for excellent service. The authors are
especially grateful to nurses and physicians at the Emergency Room, the
Medical Intensive Care Unit, the Department of Internal Medicine and the
Department of Neurology at Oslo University Hospital, Ullevaal for their
enthusiastic and friendly help in including patients during the study period. Finally, we thank the patients and next of kin who gave consent to participate
in this study. 2. Asano T, Ichiki K, Koizumi S, Kaizu K, Hatori T, Fujino O, Mashiko K, Sakamoto
Y, Miyasho T, Fukunaga Y. IL-17 is elevated in cerebrospinal fluids in
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East, reference number 2011/2578) and the ethical council of the hospital
approved the study protocol. Written informed consent was obtained from
all individual patients or their next of kin included in the study. The Regional Committees for Medical and Health Research Ethics (REC South
East, reference number 2011/2578) and the ethical council of the hospital
approved the study protocol. Written informed consent was obtained from
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https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.0020098&type=printable
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The Dynamics of Homologous Pairing during Mating Type Interconversion in Budding Yeast
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PLOS genetics
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cc-by
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The Dynamics of Homologous Pairing during
Mating Type Interconversion in Budding Yeast Peter L. Houston, James R. Broach*
Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America Cells repair most double-strand breaks (DSBs) that arise during replication or by environmental insults through
homologous recombination, a high-fidelity process critical for maintenance of genomic integrity. However, neither the
detailed mechanism of homologous recombination nor the specific roles of critical components of the recombination
machinery—such as Bloom and Werner syndrome proteins—have been resolved. We have taken a novel approach to
examining the mechanism of homologous recombination by tracking both a DSB and the template from which it is
repaired during the repair process in individual yeast cells. The two loci were labeled with arrays of DNA binding sites
and visualized in live cells expressing green fluorescent protein–DNA binding protein chimeras. Following induction of
an endonuclease that introduces a DSB next to one of the marked loci, live cells were imaged repeatedly to determine
the relative positions of the DSB and the template locus. We found a significant increase in persistent associations
between donor and recipient loci following formation of the DSB, demonstrating DSB-induced pairing between donor
and template. However, such associations were transient and occurred repeatedly in every cell, a result not predicted
from previous studies on populations of cells. Moreover, these associations were absent in sgs1 or srs2 mutants, yeast
homologs of the Bloom and Werner syndrome genes, but were enhanced in a rad54 mutant, whose protein product
promotes efficient strand exchange in vitro. Our results indicate that a DSB makes multiple and reversible contacts
with a template during the repair process, suggesting that repair could involve interactions with multiple templates,
potentially creating novel combinations of sequences at the repair site. Our results further suggest that both Sgs1 and
Srs2 are required for efficient completion of recombination and that Rad54 may serve to dissociate such interactions. Finally, these results demonstrate that mechanistic insights into recombination not accessible from studies of
populations of cells emerge from observations of individual cells. Citation: Houston PL, Broach JR (2006) The dynamics of homologous pairing during mating type interconversion in budding yeast. PLoS Genet 2(6): e98. DOI: 10.1371/
journal pgen 0020098 Citation: Houston PL, Broach JR (2006) The dynamics of homologous pairing during mating type interconversion in budding yeast. PLoS Genet 2(6): e98. DOI: 10.1371/
journal.pgen.0020098 2006) The dynamics of homologous pairing during mating type interconversion in budding yeast. PLoS Genet 2(6): e98. Introduction Double-stranded DNA breaks (DSBs) occur spontaneously
during replication and by exposure to certain genotoxic
chemicals or ionizing radiation. Efficient repair of these DSBs
can be accomplished non-conservatively by non-homologous
end joining (NHEJ) or with exact fidelity using homologous
recombination. Failure to repair these DSBs accurately can
perturb the normal cell cycle progression and increase
genomic instability, thereby promoting tumorigenesis. In the second step in DSB repair, the nucleoprotein
filament performs a search for homology and associates with
a donor sequence. Initial association likely occurs through a
triple-strand association between the nucleoprotein filament
with the duplex donor DNA, followed by isomerization to
form a D-loop with the single-strand end of the DSB invading
the duplex to pair with the template strand of the duplex [7]. Rad54 plays a role in strand invasion along with DNA
polymerase holoenzyme [2], which extends the invading 39
strand. In yeast, the joint between invading and template
strands is resolved 2 to 4 h after DSB formation. However, The timing, choreography, and genetic dependencies of
steps during DSB repair by homologous recombination in the
budding yeast have been examined on populations of cells
following synchronous initiation of a DSB [1–3]. Moreover,
microscopic observation of live cells expressing specific green
fluorescent protein (GFP)-tagged proteins has provided
information on the temporal sequence and dependencies in
recruitment of repair and recombination proteins to nuclear
repair foci that form following initiation of DSBs [4]. These
studies demonstrated that the repair of DSBs by homologous
recombination occurs in several steps. First, shortly after
formation of a DSB, signal transduction and nuclease
modules recognize and process the DSB. The Mre11, Rad50,
Xrs2 (MRX) module in conjunction with the Tel1 kinase
tether the ends of the newly formed DSB and promote
efficient nucleolytic processing of the break to generate 39
single-stranded ends [4–6]. The single-strand binding com-
plex, RPA, binds to the newly exposed single-stranded region. Rad51 then forms a nucleoprotein filament on the exposed Editor: Maria Pia Longhese, University of Milan–Bicocca, Italy Received February 14, 2006; Accepted May 12, 2006; Published June 23, 2006 A previous version of this article appeared as an Early Online Release on May 12,
2006 (DOI: 10.1371/journal.pgen.0020098.eor). DOI: 10.1371/journal.pgen.0020098 Copyright: 2006 Houston and Broach. The Dynamics of Homologous Pairing during
Mating Type Interconversion in Budding Yeast DOI: 10.13 single strands at the ends of the DSB. Rad52, Rad54, Rad55,
and Rad57 also bind to the exposed single-strand region and
promote formation and/or stabilization of the Rad51 nucle-
oprotein filament, in part by alleviating the inhibitory effect
of RPA on Rad51 binding [2,3]. PLoS Genetics | www.plosgenetics.org Introduction This view contradicts the accepted model of recombination as
an uninterrupted, continuous process, but explains, as has been
observed, how sequences from different regions of the genome
might arrive at a single site after recombination. Moreover, their
results show that mutants lacking the Bloom’s or Werner’s genes
have difficulty bringing the interacting strands of DNA together. Such delays could lead to errors in genome structure that could
account for the disease characteristics. In short, observing recombi-
nation in individual cells reveals unexpected but important features
of this process. most of these studies report only on the average behavior of
populations of cells. Our results described in this report
indicate that the discrete dynamics of the recombination
process in individual cells are quite different. We have created strains for observing the interaction of
MAT and its preferred donor locus during mating type
switching in single cells in order to assess the discrete
dynamics of the pairing process. The results of this analysis,
described below, indicate that the dynamics of gene con-
version during mating type interconversion are quite differ-
ent, and much more rapid, in individual cells than would be
predicted from previous studies on populations of cells. Furthermore, the dynamics of interaction of donor and
recipient loci in various mutant backgrounds suggest un-
expected roles for several components of the recombination
machinery. A variety of helicases participate in recombination. One
such helicase in Saccharomyces, Sgs1, is a member of the RecQ
helicase family that resembles the WRN and BLM helicases
defective in Werner and Bloom genome instability syndromes
in humans [8]. Deletion of SGS1 increases the frequency of
gross chromosomal rearrangements and, in conjunction with
deletion of a second related helicase gene, SRS2, causes a
severe growth defect in yeast. This growth defect can be
suppressed by mutations that inhibit initiation of homolo-
gous recombination, suggesting that Sgs1 and Srs2 prevent
formation of aberrant, irreparable complexes that could arise
during the process of homologous recombination [9]. Other
studies have indicated that, despite the synthetic lethality of
sgs1 and srs2, the two corresponding proteins act at different
steps in recombination, with Sgs1 helping to resolve
recombination intermediates and Srs2 functioning to coun-
teract initiation of recombination [10]. Introduction 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. Abbreviations: DSB, double-strand DNA break; GFP, green fluorescent protein * To whom correspondence should be addressed. E-mail: jbroach@princeton.edu 0896 0896 June 2006 | Volume 2 | Issue 6 | e98 Dynamics of Homologous Pairing in Yeast we have examined mating type switching in individual yeast
cells. Mating type switching in yeast has been extensively used
as a model system for homologous recombination [2,3,14,15]. Haploid Saccharomyces cells exist in one of two mating types, a
or a, dependent solely on the particular allele present at the
MAT locus on Chromosome III. Haploid cells can change
mating type as often as every generation (reviewed in [16,17])
through recombination initiated by introduction of a DSB at
the MAT locus, catalyzed by an endonuclease encoded by HO
[18]. Switching then occurs by a gene conversion event,
without associated crossover, that replaces the mating
information at the MAT locus with the opposite mating
information, a or a, present at either of two repository
mating loci, HML and HMR, located at the opposite ends of
Chromosome III. Mating type switching follows a precise
developmental pattern that results from the intricate mode of
transcriptional regulation of the HO gene and from a highly
regulated interaction between donor and recipient loci [19–
21]. Specifically, cell type dictates which donor locus is
selected for participation in the gene conversion event. MATa
cells predominantly select HML, which normally contains a
mating information, whereas MATa cells select HMR, which
normally contains a mating information. This selection
process insures efficient mating type switching. Synopsis Synopsis Genetic recombination not only promotes genetic diversity in a
population but also insures the integrity of an organism’s genome. Inappropriate or inefficient recombination drives tumor formation in
cancers and underlies certain premature aging diseases in humans,
such as Bloom’s and Werner’s syndromes. To gain insight into the
role of recombination in these diseases, the researchers developed a
means of observing recombination in individual cells. They
accomplish this by repeatedly monitoring the positions of the two
chromosomal segments participating in recombination. Their
observations show that interacting strands undergo repeated
association and dissociation prior to fully completing recombina-
tion. Introduction Moreover, these
proteins appear to play critical roles during replication fork
stalling, with Sgs1 promoting resolution of recombination-
dependent structures that form at damaged replication forks
and Srs2 assisting to prevent the formation of such structures
[11]. Finally, while these studies suggest that Sgs1 and Srs2
suppress recombination, sgs1 mutants are defective in DNA
damage–induced heteroallelic recombination, suggesting that
Sgs1 may promote recombination in certain contexts [9,12]. Consistent with this positive role of the yeast RecQ helicase in
recombination, WRN protein is required to complete mitotic
recombinants efficiently in unperturbed conditions in fibro-
blast cell lines [13]. Thus, we still do not have a clear
conception of the function of RecQ helicases in recombina-
tion and a full appreciation of the mechanism underlying the
pathological consequences of losing RecQ function in
mammalian cells. PLoS Genetics | www.plosgenetics.org Method for Observing Homologous Pairing in Live Yeast
Cells DOI: 10.1371/journal.pgen.0020098.g001
Dynamics of Homologous Pairing in Yeast Dynamics of Homologous Pairing in Yeast Figure 1. Visualization of Pairing between Donor and Recipient Loci during Mating Type Interconversion in Live Yeast Cells
(A) Diagram of yeast cells used in this study, indicating the relevant structure of Chromosome III, which carries arrays of LacO sites at MAT and TetO sites
at HML. The strain also constitutively expresses LacI-GFP and TetR-GFP protein fusions and contains a plasmid that carries a galactose-inducible HO gene
and CFP under control of an a-cell type–specific promoter. (B) Fluorescence micrograph of a MATa strain, Y3343, freshly transformed with plasmid B2609, 6 h after induction of HO. Green dots mark HML and MAT
loci in each cell, and uniform blue staining indicates that the cell has undergone a mating type switch to MATa. Cells are demarked by the diffuse cyan
fluorescence, and the diffuse yellow fluorescence defines the nuclei. (C) Deconvolved and compressed images showing GFP foci in isogenic strains carrying or lacking PGAL1-HO, taken 90 min following induction with
galactose. DOI: 10 1371/journal pgen 0020098 g001 Figure 1. Visualization of Pairing between Donor and Recipient Loci during Mating Type Interconversion in Live Yeast Cells
(A) Diagram of yeast cells used in this study, indicating the relevant structure of Chromosome III, which carries arrays of LacO sites at MAT and TetO sites
at HML. The strain also constitutively expresses LacI-GFP and TetR-GFP protein fusions and contains a plasmid that carries a galactose-inducible HO gene
and CFP under control of an a-cell type–specific promoter. (B) Fluorescence micrograph of a MATa strain, Y3343, freshly transformed with plasmid B2609, 6 h after induction of HO. Green dots mark HML and MAT
loci in each cell, and uniform blue staining indicates that the cell has undergone a mating type switch to MATa. Cells are demarked by the diffuse cyan
fluorescence, and the diffuse yellow fluorescence defines the nuclei. (C) Deconvolved and compressed images showing GFP foci in isogenic strains carrying or lacking PGAL1-HO, taken 90 min following induction with
galactose. DOI 10 1371/j
l
0020098 001 CFP. We found that less than 1% of MATa PMFa1-CFP cells
without HO induction accumulated CFP at the end of the
experiment, whereas approximately 50% of the cells that
were induced for HO expression accumulated CFP (Figure
1B). Method for Observing Homologous Pairing in Live Yeast
Cells Method for Observing Homologous Pairing in Live Yeast
Cells We developed strains and a method to observe the pairing
of homologous DNA loci during recombination in live yeast
cells by adopting previously described methods to visualize
discrete chromosomal loci and applying them to the process
of mating type switching [22,23]. As diagrammed in Figure
1A, we inserted an array of lac repressor binding sites
adjacent to a donor locus, HML, and an array of tet repressor
sites adjacent to the recipient locus, MAT, and then expressed
both GFP-lacI and GFP-tetR fusions in the strain [23,24]. We
performed these experiments in a cdc15–2 strain, which
arrests at the non-permissive temperature just prior to exit
from telophase. By shifting the strain from non-permissive to
permissive temperature, cells synchronously enter G1 phase
[25]. This insures that, at the beginning of our experiments,
all cells have only one marked chromosome and are at the
stage of the cell cycle in which switching normally occurs. Finally, the strain also carries the HO gene under control of
the GAL10 promoter to allow regulated induction of a DSB at
the MAT locus. To monitor cells under controlled conditions, we affixed
yeast cells to the coverslip of a thermally regulated flow cell in In order to address unresolved aspects of recombination, June 2006 | Volume 2 | Issue 6 | e98 0897 June 2006 | Volume 2 | Issue 6 | e98 Figure 1. Visualization of Pairing between Donor and Recipient Loci during Mating Type Interconversion in Live Yeast Cells
(A) Diagram of yeast cells used in this study, indicating the relevant structure of Chromosome III, which carries arrays of LacO sites at MAT and TetO sites
at HML. The strain also constitutively expresses LacI-GFP and TetR-GFP protein fusions and contains a plasmid that carries a galactose-inducible HO gene
and CFP under control of an a-cell type–specific promoter. (B) Fluorescence micrograph of a MATa strain, Y3343, freshly transformed with plasmid B2609, 6 h after induction of HO. Green dots mark HML and MAT
loci in each cell, and uniform blue staining indicates that the cell has undergone a mating type switch to MATa. Cells are demarked by the diffuse cyan
fluorescence, and the diffuse yellow fluorescence defines the nuclei. (C) Deconvolved and compressed images showing GFP foci in isogenic strains carrying or lacking PGAL1-HO, taken 90 min following induction with
galactose. PLoS Genetics | www.plosgenetics.org Method for Observing Homologous Pairing in Live Yeast
Cells The switching rate of 50% agrees well with that
determined by genetic assay with the same strain induced
for 1 h in liquid culture, indicating that manipulations in the
flow cell and observations by fluorescence microscopy did not
affect the process of switching. The rate is somewhat less than
the 85% switching rate observed with wild-type HO cells and
reflects a slight reduction in the initiation of switching under
our induction conditions, rather than any alteration in donor
preference (unpublished data). This agrees with Southern
analysis and plate-based mating assays of our test strain, from
which we concluded that under our induction conditions,
approximately half of our test cells sustain a DSB at MAT. which we could control both temperature and medium while
repeatedly interrogating by fluorescence microscopy multiple
fields of cells on the coverslip. Cells freshly transformed with
the PGAL1-HO plasmid were pregrown in non-inducing
medium at 23 8C and then shifted to 35 8C for 3 h. This
arrests all the cells just prior to entry into G1 phase of the cell
cycle. After attaching cells to the coverslip and centering the
flow cell on the microscope, we initiated each experiment by
simultaneously inducing the DSB at MAT and releasing cells
from cell cycle arrest. This was accomplished by perfusing the
flow cell with medium containing 2% galactose to induce HO
and shifting the temperature of the flow cell to 23 8C. After
DSBs were induced, we shut off HO expression to prevent
subsequent rounds of recombination, by changing the
medium flowing over the cells to one lacking galactose and
containing 2% glucose. Since HO mRNA and HO protein
have very short half-lives, no further DSB formation occurs
much beyond this medium switch [26]. Pairing between Donor and Recipient Loci during
Recombination Is Short and Reversible June 2006 | Volume 2 | Issue 6 | e98 Pairing between Donor and Recipient Loci during
Recombination Is Short and Reversible Pairing In Vivo between MAT and HML Increases following
Introduction of a DSB at MAT (A) Shown are cell histories for 11 cells each from three strains,
configured as diagrammed in Figure 1, indicating along the horizontal
time line for each cell whether at each time point the cell presented a
single dot (black bar) or two dots (white bar). Cells were interrogated at
1-min intervals for 3 h with galactose present (HO induced) for the first
hour. Upper group: Y3343 (MATa) transformed with B2609; middle
group: Y3343 (MATa) transformed with pRS415 [46]; and lower group:
Y3342 (MATa) transformed with B2608. We also noted that, despite the fact that more than half of
the cells expressing HO undergo gene conversion of MAT
using HML as donor, continuous pairing between MAT and
HML occurs for no longer than 17 min in any cell. We
evaluated the number and duration of events in which HML
and MAT appear to be in continuous association, i.e., cases in
which a cell presents a single dot in an uninterrupted series of
time points. As shown in Figure 2C, in uninduced MATa cells
or induced MATa cells, the log of the number of association
events of a given duration plotted versus the duration of
association exhibits essentially a linear relation, consistent
with a stochastic process in which the likelihood that HML
and MAT are in close proximity at any time is independent of
whether they were in close association at the previous or
subsequent time point. Only one event longer than 6 min was
observed in uninduced cells. In contrast, induced MATa cells
exhibit a statistically significant excess of events of duration
longer than 6 min. These extended associations occur for an
average of 10 min, predominantly between 80 and 160 min
postinduction (Figure 2D). Unexpectedly, each cell undergoes (B) At each time point, the fraction of cells exhibiting a single dot was
determined from the data underlying Figure 2A and plotted as a three-
point running average versus time following addition of galactose. Legends are indicated to the left of the cell histories in (A). Data were
calculated either including all 11 Y3343 (MATa) cells transformed with
B2609 (dark blue diamonds) or for only those six cells presumed to have
sustained a DSB at MAT (light blue rectangles). The horizontal axis is
aligned with the cell histories in (A). Pairing between Donor and Recipient Loci during
Recombination Is Short and Reversible We confirmed that cells attached to the coverslip and
treated as above switch efficiently and select the appropriate
donor. We can determine by fluorescence microscopy at the
single-cell level whether a cell has switched from MATa to
MATa by including in the strain a PMFa1-CFP construct, which
consists of the CFP coding region under control of the MFa1
promoter. The MFa1 promoter is active only in MATa cells. Accordingly, a MATa cell prior to switching does not express
CFP, but after it switches to MATa, it begins to accumulate To monitor pairing of MAT and HML following initiation
of a DSB at MAT, we treated MATa cells as described above
and then interrogated multiple cells every minute for 3 h. We
determined in each cell, at each time point, whether the
nucleus contained two GFP dots or a single GFP dot. In the
former case, we assume that the MAT and HML loci are
unpaired. In the latter case, we assume that the two loci are
either paired or simply too close to be resolved. Representa- 0898 0898 Dynamics of Homologous Pairing in Yeast Figure 2. Pairing In Vivo between MAT and HML Increases following
Introduction of a DSB at MAT tive images from experiments in which HO was induced or
not induced are shown in Figure 1C, demonstrating the
resolution of the two loci in the test strains and indicating
that all cells can be readily scored. In Figure 2A, we represent
the histories of association of MAT and HML as determined
by this method following HO induction for 11 wild-type
MATa cells, which normally use HML as the preferred donor. In addition, we examined 11 wild-type MATa cells following
HO induction. MATa cells normally use the untagged HMR
locus as donor and thus MAT would not be expected to pair
with HML in this strain. Finally, we examined 11 wild-type
MATa cells in the absence of HO induction, in which no
recombination should occur. In these representations, each
line describes the history of a single cell; a white bar
represents the presence of two dots in the cell’s nucleus at
that time point and a black bar represents the appearance of
only a single dot. Pairing between Donor and Recipient Loci during
Recombination Is Short and Reversible tive images from experiments in which HO was induced or
not induced are shown in Figure 1C, demonstrating the
resolution of the two loci in the test strains and indicating
that all cells can be readily scored. In Figure 2A, we represent
the histories of association of MAT and HML as determined
by this method following HO induction for 11 wild-type
MATa cells, which normally use HML as the preferred donor. In addition, we examined 11 wild-type MATa cells following
HO induction. MATa cells normally use the untagged HMR
locus as donor and thus MAT would not be expected to pair
with HML in this strain. Finally, we examined 11 wild-type
MATa cells in the absence of HO induction, in which no
recombination should occur. In these representations, each
line describes the history of a single cell; a white bar
represents the presence of two dots in the cell’s nucleus at
that time point and a black bar represents the appearance of
only a single dot. These interrogations clearly show that, relative to the
behavior of MATa cells in which HO was not induced, MATa
cells in which HO was induced exhibited a statistically
significant increase in the overall proportion of time MAT
and HML were in close proximity. Moreover, cells from the
induced culture divided into two discrete populations. In one
group, cells exhibited a pattern of association that was
indistinguishable from those of uninduced cells, whereas in
the other group, cells exhibited a succession of extended
associations between MAT and HML lasting from 7 to 15 min,
starting approximately 80 min after initiation of HO
expression. The fraction of cells exhibiting the extended
association matched the fraction of cells that ultimately
undergo gene conversion, and we believe that cells showing
extended association were those in which cleavage at MAT
had occurred. A distinction between the association patterns in induced
and uninduced cells is also evident by calculating the fraction
of cells containing a single dot at each time point. As shown
in Figure 2B, cells in which HO was not induced show a
uniform, low level of association between HML and MAT
throughout the experiment. On the other hand, MATa cells in
which HO was induced exhibit increased association of HML
and MAT starting from 80 min after initiation of induction of
HO and lasting for approximately 1 1/2 h. Figure 2. Sgs1 and Srs2 Are Required for Stable Pairing between
Donor and Recipient Loci during Switching We anticipate that assembly and resolution of recombina-
tion intermediates during switching would require the
activity of DNA helicases, and several genes encoding
helicases have been implicated in recombination and repair. In an attempt to clarify the role of various helicases in
recombination, we examined the extent of association
between MAT and HML in strains deleted for individual
helicase genes. As shown in Figure 3, the level of pairing
between HML and MAT in a MATa sgs1D (Figure 3A) or a
MATa srs2D (Figure 3B) mutant following HO induction was
significantly reduced compared to that observed in wild-type
cells. The level of pairing in the induced srs2D strain was
slightly higher than that in the same strain lacking HO but was
certainly significantly lower than that observed in the wild-
type strain. More dramatically, the level of apparent pairing
in the induced sgs1D strain was not significantly different
than that in the same strain lacking HO. Thus, association of
the DSB with the donor locus appears to be delayed in both
mutants. These results would predict that sgs1 or srs2 mutants
should be defective in mating type switching. Figure 3. HML and MAT Fail to Pair in sgs1 or srs2 Mutants Data were obtained for 17 cells under each condition and presented as
described in Figure 2 for sgs1 (A) and srs2 (B) mutant strains, with the
difference that cells were interrogated every 2 min starting 30 min after
addition of galactose. DOI: 10.1371/journal.pgen.0020098.g003 viability of mother cells in pedigrees was clearly less than that
seen with sister SGS1 spore clones (unpublished data). These
results are consistent with the assumption that srs2 mutants,
and to a lesser extent, sgs1 mutants, fail to efficiently heal DSB
generated in lineages of HO haploid cells. To confirm the roles of Sgs1 and Srs2 in repair of DSBs
during mating type switching, we examined the effect of
introducing these mutants into wild-type HO strains. A
diploid HO/HO strain, which we used previously to determine
switching frequencies under normal conditions [27], was
rendered heterozygous for deletion of SGS1 or SRS2. After
sporulation and dissection, we examined the behavior of cells
and the switching pattern during outgrowth of spore clones. As seen in Table 1, HO SGS1 and HO SRS2 clones showed
normal growth and switching patterns. Pairing between Donor and Recipient Loci during
Recombination Is Short and Reversible Rather, induced MATa cells present a pattern
indistinguishable from that obtained in uninduced cells. This
suggests that the cleaved MAT locus does not spend
significant time in association with the non-preferred donor
locus during mating type switching. more than one prolonged association event. Moreover,
during the period between two extended associations, the
average distance between the two spots (0.49 6 0.25 lm) is
equivalent to that in uninduced cells (0.51 6 0.24 lm), which
do not undergo switching. Thus, we conclude that the
multiple associations of donor and recipient in a single cell
are independent events, suggesting that the pairing of donor
and recipient loci is readily reversible during recombination. Finally, we note from the data in Figure 2 that, in contrast
to the pattern seen with MATa cells, MATa cells, which do not
use HML as the preferred donor, do not exhibit an enhanced
association between MAT and HML over the course of the
experiment. Rather, induced MATa cells present a pattern
indistinguishable from that obtained in uninduced cells. This
suggests that the cleaved MAT locus does not spend
significant time in association with the non-preferred donor
locus during mating type switching. Pairing between Donor and Recipient Loci during
Recombination Is Short and Reversible g
(C) The duration of periods of apparent continuous association between
MAT and HML. The number of continuous, uninterrupted time points in
which a cell shows only a single dot, were determined from the cell
histories in Figure 2A. The number of periods of a given duration divided
by the total number of periods (probability density) is plotted versus the
duration of apparent association for each strain shown in 2A. MATa þ HO
(light blue circles); MATa HO (green diamonds); and MATa þ HO (red
squares). Error bars are estimated by dividing the data successively into
random halves and computing the standard deviation among 20 random
halves. The dashed line shows a fit of the combined values for MATa
HO and MATaþHO cells to a normalized, exponential distribution. Under
this assumption, the p-value for the likelihood that the data for MATa þ
HO would arise by chance is less than 105. (D) Data from Figure 2A for MATa þ HO cells is replotted to show only
those pairing events of duration longer than 6 min. DOI: 10.1371/journal.pgen.0020098.g002 (D) Data from Figure 2A for MATa þ HO cells is replotted to show only
those pairing events of duration longer than 6 min. PLoS Genetics | www.plosgenetics.org 0899 June 2006 | Volume 2 | Issue 6 | e98 Dynamics of Homologous Pairing in Yeast Figure 3. HML and MAT Fail to Pair in sgs1 or srs2 Mutants
Data were obtained for 17 cells under each condition and presented as
described in Figure 2 for sgs1 (A) and srs2 (B) mutant strains, with the
difference that cells were interrogated every 2 min starting 30 min after
addition of galactose. DOI: 10.1371/journal.pgen.0020098.g003 more than one prolonged association event. Moreover,
during the period between two extended associations, the
average distance between the two spots (0.49 6 0.25 lm) is
equivalent to that in uninduced cells (0.51 6 0.24 lm), which
do not undergo switching. Thus, we conclude that the
multiple associations of donor and recipient in a single cell
are independent events, suggesting that the pairing of donor
and recipient loci is readily reversible during recombination. Finally, we note from the data in Figure 2 that, in contrast
to the pattern seen with MATa cells, MATa cells, which do not
use HML as the preferred donor, do not exhibit an enhanced
association between MAT and HML over the course of the
experiment. PLoS Genetics | www.plosgenetics.org June 2006 | Volume 2 | Issue 6 | e98 Sgs1 and Srs2 Are Required for Stable Pairing between
Donor and Recipient Loci during Switching Similarly, ho sgs1 and
ho srs2 control strains grew indistinguishably from ho SGS1
and ho SRS2 strains (unpublished data and [9]). In contrast,
HO srs2 spore clones were smaller than their sister HO SRS2
spore clones, and pedigree analysis of cells following
germination indicated that mother cells in the lineage
exhibited delayed cell cycle progression or failed to divide
at all. This growth pattern is similar to, but less severe than,
the pedigree of death that occurs in HO yeast strains
incapable of completing HO-induced switching [28], which
we observe with rad52 HO and rad54 HO spores (Table 1). Spore clones carrying an sgs1 deletion did not show as
extensive delays in growth as did srs2 spore clones, but the We also find that, although both srs2 and sgs1 mutants
exhibited normal donor selection in a MATa background,
both mutants showed essentially random selection of donor
loci following switching in a MATa background (Table 1). This observation confirms that the mutants exhibit a delay in
repairing the DSB during mating type switching. In cells in
which repair of the HO-induced DSB at MAT is delayed,
resection of the DSB into the adjacent coding region results
in deletion of the MAT locus, rendering the cell temporarily
mat-null. Since mat-null cells exhibit a MATa phenotype,
donor selection in MATa cells would be unaffected by this
delay-induced resection. However, the delay-induced resec-
tion in a MATa would render the cell phenotypically MATa,
changing the donor preference from HMR to HML. Thus, the
extent to which MATa cells select HML as donor reflects the
extent of delay in the repair of the DSB. Accordingly, our
results confirm that Srs2 and Sgs1 are both required for
timely repair of DSBs. 0900 June 2006 | Volume 2 | Issue 6 | e98 June 2006 | Volume 2 | Issue 6 | e98 Dynamics of Homologous Pairing in Yeast Table 1. Switching Efficiency in Mutant Strains Table 1. Switching Efficiency in Mutant Strains Table 1. Sgs1 and Srs2 Are Required for Stable Pairing between
Donor and Recipient Loci during Switching aSpore clones showed significantly greater inviability of mother cells than did wild-type cells, but not as great as that seen with srs2 clones. A dash (—) indicates determination of donor preference was not performed. DOI: 10.1371/journal.pgen.0020098.t001 Diploid strain Y2902 (MATa/MATa::HIS3 his3/his3 hmla1a2~Dinc/hmla1a2~Dinc hmra1Dı˜ı´nc/hmra1Dı˜ı´inc HO/HO) was made heterozygous for the indicated deletion mutations and then
sporulated and tetrads were dissected. Spores and spore clones were monitored microscopically over the first 24 h postdissection to determine the viability and growth pattern of the
emerging colony. Spores segregating a high proportion of dead cells were scored as undergoing a ‘‘pedigree of death’’ [28], whereas those segregating cells showing extended delays in
cell cycle progression were scored as ‘‘delayed division.’’ The predominant donor used in a spore clone was determined by PCR analysis as described and correlated with the MAT allele
originally present in the spore clone, as determined by the His phenotype. Those spore clones that gave essentially equivalent signals for HML and HMR were not included in the totals. aSpore clones showed significantly greater inviability of mother cells than did wild-type cells, but not as great as that seen with srs2 clones. A dash (—) indicates determination of donor preference was not performed. DOI: 10.1371/journal.pgen.0020098.t001 Rad54 Is Not Required for Stable Pairing between Donor
and Recipient Loci during Switching Rad54 Is Not Required for Stable Pairing between Donor
and Recipient Loci during Switching we examined—mating type interconversion following syn-
chronous cleavage of the MAT locus by HO induction in
Saccharomyces—has been used extensively as a model system
for homologous recombination [2,3,14,15]. Previous studies RAD54 encodes a member of the Swi2/Snf2 family of DNA-
stimulated ATPases, and loss of RAD54 function substantially
diminishes homologous recombination and renders cells
sensitive to ionizing radiation. Consistent with previous
reports that Rad54 is required for completion of DSB repair
[29], HO rad54 spore clones yield microcolonies in our donor
preference assay, resulting from extensive death of mother
cells during growth of the clone (Table 1). Previous studies on
the precise role Rad54 plays in recombination have been
inconsistent, with some evidence pointing to its involvement
in initial formation of the Rad51 presynaptic filament and
other evidence suggesting that it functions after formation of
the presynaptic complex [2,3]. To help resolve this issue, we
examined pairing of MAT and HML following HO induction
in a rad54D mutant. Figure 4. Rad54 Is Not Required for DSB-Induced Pairing of MAT and
HML
Data were obtained and presented as described in Figure 2 for MATa
rad54 cells (A) and for MATa rad54 cells (B). DOI: 10.1371/journal.pgen.0020098.g004 As evident in Figure 4A, the two loci paired as efficiently in
the rad54 mutant as in wild-type cells. Surprisingly, MAT and
HML also paired efficiently in a MATa rad54 mutant strain
(Figure 4B), despite HML not being the preferred donor for
switching in MATa cells. This result is quite distinct from that
observed with HO-induced RAD54 MATa cells, which, as
shown in Figure 2, show no prolonged association between
MAT and HML. Therefore, association between donor and
recipient loci proceeds efficiently in the absence of Rad54
function and, furthermore, association with the inappropri-
ate donor occurs as frequently as does association with the
preferred donor. PLoS Genetics | www.plosgenetics.org Sgs1 and Srs2 Are Required for Stable Pairing between
Donor and Recipient Loci during Switching Switching Efficiency in Mutant Strains
Strain
Mutation
Viability (Number of Spore Clones)
Donor Preference (%)
MATa
MATa
Normal
Delayed Division
Pedigree of Death
HML
HMR
HML
HMR
Y3446
RAD52
20
0
0
—
—
—
—
Y3446
rad52D
0
0
20
—
—
—
—
Y3443
SGS1
30
0
0
93
,3
7
71
Y3443
sgs1D
29a
0
0
76
4
28
36
Y3444
SRS2
30
0
0
79
6
,3
96
Y3444
srs2D
2
27
0
80
,3
28
36
Y3445
RAD54
18
2
0
—
—
—
—
Y3445
rad54D
0
3
17
—
—
—
—
Diploid strain Y2902 (MATa/MATa::HIS3 his3/his3 hmla1a2~Dinc/hmla1a2~Dinc hmra1Dı˜ı´nc/hmra1Dı˜ı´inc HO/HO) was made heterozygous for the indicated deletion mutations and then
sporulated and tetrads were dissected. Spores and spore clones were monitored microscopically over the first 24 h postdissection to determine the viability and growth pattern of the
emerging colony. Spores segregating a high proportion of dead cells were scored as undergoing a ‘‘pedigree of death’’ [28], whereas those segregating cells showing extended delays in
cell cycle progression were scored as ‘‘delayed division.’’ The predominant donor used in a spore clone was determined by PCR analysis as described and correlated with the MAT allele
originally present in the spore clone, as determined by the His phenotype. Those spore clones that gave essentially equivalent signals for HML and HMR were not included in the totals. aSpore clones showed significantly greater inviability of mother cells than did wild-type cells, but not as great as that seen with srs2 clones. A dash (—) indicates determination of donor preference was not performed. DOI: 10.1371/journal.pgen.0020098.t001 Diploid strain Y2902 (MATa/MATa::HIS3 his3/his3 hmla1a2~Dinc/hmla1a2~Dinc hmra1Dı˜ı´nc/hmra1Dı˜ı´inc HO/HO) was made heterozygous for the indicated deletion mutations and then
sporulated and tetrads were dissected. Spores and spore clones were monitored microscopically over the first 24 h postdissection to determine the viability and growth pattern of the
emerging colony. Spores segregating a high proportion of dead cells were scored as undergoing a ‘‘pedigree of death’’ [28], whereas those segregating cells showing extended delays in
cell cycle progression were scored as ‘‘delayed division.’’ The predominant donor used in a spore clone was determined by PCR analysis as described and correlated with the MAT allele
originally present in the spore clone, as determined by the His phenotype. Those spore clones that gave essentially equivalent signals for HML and HMR were not included in the totals. Discussion To study the dynamics of recombination, we have
developed a real-time assay for homologous pairing in
individual living cells. The results from this assay correlate
well with previous observations on the time course of the
recombination reaction obtained with populations of cells,
but also reveal unanticipated aspects of recombination not
previously accessible through population studies. The system Figure 4. Rad54 Is Not Required for DSB-Induced Pairing of MAT and
HML
Data were obtained and presented as described in Figure 2 for MATa
rad54 cells (A) and for MATa rad54 cells (B). Figure 4. Rad54 Is Not Required for DSB-Induced Pairing of MAT and
HML Data were obtained and presented as described in Figure 2 for MATa
rad54 cells (A) and for MATa rad54 cells (B). DOI: 10.1371/journal.pgen.0020098.g004 June 2006 | Volume 2 | Issue 6 | e98 0901 Dynamics of Homologous Pairing in Yeast examining products and intermediates of recombination in
vivo by PCR or Southern analysis indicate that strand
invasion begins approximately 90 min following HO induc-
tion and ends approximately 90 min later [2,14]. Further-
more, by using chromatin immunoprecipitation to quantify
in vivo association of recombination proteins with regions of
the genome, several studies determined that Rad51 initially
associates with the DSB at MAT approximately 30 min
following HO induction—coincident with DSB formation—
and with the donor locus, HML, approximately 30 min later
[2,3]. This association continues for up to 3 h. These results
suggest that synapsis between the DSB and the donor locus
begins approximately 60 min following HO induction and
that the two loci remain physically associated for up to
several hours. Our data are consistent with these population
studies in that we observe an overall increase in the average
association of MAT and HML beginning 80–90 min following
HO induction and lasting for 60–90 min. Monitoring the association of HML and MAT over time in
individual cells revealed aspects of synapsis not evident from
these population studies. First, we observed a statistically
significant increase in the number of periods of extended
association of MAT and HML in induced versus uninduced
cells, although the durations of these associations were in
general no longer than 17 min and averaged approximately
10 min. Thus, distinct periods of synapsis in individual cells
are much shorter than would be expected from population
studies. Second, individual cells exhibit several distinct
periods of extended association following HO induction. Discussion Since HO protein is not present at these later times and since
the same pattern occurs in rad54 mutants unable to complete
recombination, these sequential associations do not repre-
sent sequential rounds of resolution and re-initiation of
recombination. Also, these multiple associations are unlikely
to be a single synaptic junction punctuated by dynamic
stretching, since the distributions of distances between the
two loci in the interval between two such extended
associations are identical to those in uninduced cells. Rather,
we would interpret these observations to suggest that the
Rad51-mediated complex formed between the DSB and the
template is reversible. For example, the initial Rad51-
mediated triple-strand complex consisting of the Rad51-
coated single-strand DNA bound to the homologous double-
strand region of the donor template could be resolved in
either two ways: isomerization to yield a single-strand
invasion creating a D-loop on the template, or disassociation
to return to the pre-complex state. Alternatively, D-loop
formation could be the reversible step, either before or after
extension of the single-strand primer. Our results suggest
that either or both of these initial interactions between the
DSB and the template can occur several times before
completion of recombination. Such cycles for strand invasion
have been suggested to occur during repair of DSBs in
Drosophila [30]. The results from those studies with Drosophila
would suggest that dissociation could occur not only after D-
loop formation but also after partial extension of the primer,
as depicted in Figure 5. Finally, we note that the multiple,
stochastic associations between the DSB and the donor locus
in individual cells account for the apparent extended
association between the two loci as measured on a population
basis. Figure 5. Model for DSB-Induced Pairing of MAT with Donor Loci
Following HO-induced double-strand cleavage of MAT, Rad51 (ellipses)
binds to the resected single-strand ends of the DSB and promotes their
association with both HML and HMR via homology present at all three
loci. Blue- and pink-colored regions at the mating type loci represent
allele-specific DNA sequences, which are bracketed by sequences in
common among all three loci (shown in brown). Dissociation of these
initial complexes, suggested to be paranemic joints in this illustration, is
stimulated by Rad54-promoted removal of Rad51. Since these associa-
tions are seen only in rad54 cells, we assume that they are too short-lived
to be evident in wild-type cells, and thus are indicated in brackets. PLoS Genetics | www.plosgenetics.org Discussion This model would account for the fact that we observe
pairing of MAT and HML in the non-preferred mating
background, reflecting formation of Rad51-mediated pairing
of MAT with either donor locus via the common homology at
all three loci. We would propose that these initial pairing
events would be eliminated in a Rad54-dependent fashion,
either reversing the initial pairing or promoting progression
in the appropriate partner to productive recombination. A model for recombination during mating type switching is
presented in Figure 5. Our results suggest that following HO-
induced cleavage of MAT, the locus associates readily and
reversibly with both HML and HMR. Donor selection would
then occur at a subsequent step, for instance in the isomer-
ization from the initial synaptic intermediate to the D-loop,
upon recruitment of DNA polymerase or with extension of
the invading strand. Cell type–specific loading of factors
promoting D-loop formation and strand extension at the
preferred donor locus would then fix donor preference. Our
results also suggest that this step is stimulated by both Sgs1
and Srs2. Our results further indicate that, even in the
presence of Rad54, the association between donor and
template are reversible. This would suggest that even after
D-loop formation, the donor and recipient loci can disso-
ciate. Moreover, given recent results suggesting that a double-
strand gap requires multiple cycles of strand invasion,
synthesis, and dissociation of the nascent strand during
repair of a DSB in Drosophila [30], this reversibility could
occur after partial extension of the invading strand, as
represented in Figure 5. Further analysis should resolve
precisely at what steps recombination is reversible. However,
these results clearly indicate that the recombination process
is reversible at a much later stage than previously anticipated
and raises a novel mechanism for generating genetic
diversity. We also used our assay to examine the role of RAD54 in
pairing in vivo. Rad54, a dsDNA-dependent ATPase of the
Swi2/Snf2 family of chromatin remodeling enzymes, is
required for resistance to ionizing radiation and repair of
DSBs by homologous recombination [7,29]. Rad54 interacts
with Rad51 in vivo, and addition of Rad54 to Rad51-mediated
recombination reactions dramatically stimulates homologous
pairing in vitro. In addition, Rad54 is essential for DNA
strand invasion reactions using chromatin-packaged sub-
strates [42]. Discussion We
propose that Rad54 also stimulates conversion of the initial joint
complex into an initial D-loop structure, also by promoting removal of
Rad51, allowing subsequent extension of the heteroduplex between the
invading strand and donor locus, and elongation of the invading strand. These subsequent steps are promoted or stabilized by Srs2 and Sgs1. Our results suggest that one or more of the later-stage intermediates in
recombination, perhaps even after partial extension of the invading
strand, are also reversible prior to completion of the gene conversion
event. The dissociated, but incompletely healed, MAT locus then would
recycle through Rad51 filament formation and reassociation with the
donor locus, ultimately yielding a completely reconstituted MAT locus. DOI: 10.1371/journal.pgen.0020098.g005 homologous pairing in our system. We first examined the
role of the helicases encoded by SGS1 and SRS2 and found
that inactivation of either of these genes delayed sustained
association between MAT and HML. We confirmed this result
obtained from the physical pairing assay by showing that
repair of DSBs during mating type switching is delayed in HO
spore clones carrying sgs1 or srs2 mutations. Thus, each of
these genes appears to be independently required for
efficient homologous pairing in our system, an observation
consistent with previous studies suggesting that, despite their
synthetic lethality, Sgs1 and Srs2 play independent roles in
recombination [9,12]. Our results are somewhat unexpected since previous
genetic and biochemical studies have implicated Srs2 in
inhibiting initiation of recombination by promoting dis-
assembly of Rad51 filament complexes [31–33], whereas Sgs1
and other RecQ helicases are likely involved in resolution of
recombination intermediates [10,34–38]. Thus, we might have We explored some of the genetic requirements for 0902 June 2006 | Volume 2 | Issue 6 | e98 June 2006 | Volume 2 | Issue 6 | e98 Dynamics of Homologous Pairing in Yeast anticipated that sgs1 or srs2 mutants would exhibit prolonged
association of MAT and HML in our assay rather than
reduced association. However, previous studies have shown
that, although sgs1 mutants exhibit a hyperrecombination
phenotype under some conditions, they are hyporecombino-
genic for DNA damage–induced heteroallelic recombination
[9,12], a situation similar to DSB-induced mating type
switching. In addition, Sgs1 is required for telomere
elongation in the absence of telomerase, apparently through
recombination via a break-induced repair process that is
mechanistically similar to the DSB-induced gene conversion
we have studied here [39]. Discussion Thus, Sgs1 could be required to
stabilize the initial joint formed between a DSB and the
donor by promoting formation of an extended D-loop or
initiation of synthesis to extend the invading 39 end. In a
similar vein, the effect of inactivation of SRS2 on recombi-
nation depends on the particular assay used. Perhaps most
relevant to our observations, Aylon et al. [40] examined gene
conversion of a ura3 allele from an ectopic site of homology
following introduction of an HO-induced DSB within the ura3
allele, an assay quite similar to mating type switching. They
found that inactivation of SRS2 substantially reduced the
level of strand invasion and subsequent extension of the
invading strand and concluded that only a small subpopula-
tion of srs2 cells are able to complete recombination, whereas
the majority of cells behaved as if no homology to the DSB
were present in the cell. This is consistent with results from
Paˆques and Haber suggesting that Srs2 functions to extend
and stabilize initial joints between the ssDNA-Rad51 nucle-
ofilament and the donor locus [41]. Thus, both Sgs1 and Srs2
have been implicated independently in stabilizing initial joint
formation following DSB-induced recombination, consistent
with our current observations. ations in rad54 mutants is the same as that seen in wild-type
cells, in which no phenotypic switching occurs, we conclude
that the inappropriate associations are not the result of
phenotypic change in mating type. Rather, our results suggest
that Rad54 plays a critical role in recombination at a step
beyond initial synapsis. This conclusion is consistent with the
observations above from Sugawara et al. [2] and also with
subsequent work by Wolner and Peterson [43]. Although
Rad54 may also have a role in vivo in promoting Rad51
nucleofilament formation, our results would suggest that this
is not the rate-limiting step in recombination stimulated by
Rad54. Our results can be explained by the proposal, based
on the observation that Rad54 stimulates dissociation of
Rad51 from dsDNA, that Rad54-stimulated removal of Rad51
promotes either dissociation of the paired loci or conversion
of the initial synaptic intermediate into a D-loop and the
subsequent extension of the heteroduplex region [44]. Thus,
in the absence of Rad54, pairing between the DSB and either
donor locus should still occur, but the formation of a D-loop
and subsequent elongation of the invading strand would not. Discussion Two studies recently used chromatin immuno-
precipitation to examine the in vivo requirement of Rad54
for association of Rad51 with MAT and HML following
initiation of a DSB at MAT. Wolner et al. [3] found that Rad51
association with either locus was essentially eliminated in a
rad54D mutant background and provided evidence that
Rad54 promotes formation of the Rad51 presynaptic fila-
ment. On the other hand, Sugawara et al. [2] found that
Rad51 could, in fact, associate with MAT and HML in the
absence of Rad54, but the synaptic complex could not
proceed to a mature strand invasion complex in the absence
of Rad54. We find that MAT and HML associate as well in a rad54
mutant as they do in a wild-type strain. Moreover, we find
that MAT associates frequently with HML even in a MATa
strain, in which HML is the non-preferred donor. One
possible explanation for these associations between MATa
and HML is phenotypic switching of mating due to delay in
resolution of the MAT DSB, as we proposed for sgs1 and srs2
mutants. However, since the timing of the observed associ- PLoS Genetics | www.plosgenetics.org Materials and Methods Those cells in which only a
single intensity maximum was observed were defined as having a
single dot, i.e., the two loci were closer than could be resolved by our
microscopy. coding sequence of CFP from plasmid pDH3 (http://depts.washington. edu/;yeastrc). Both PCR fragments carried 50 base pair ends with
homology to direct recombination with the appropriate adjacent
fragments. Leuþ FAA-resistant transformants were selected and
plasmid was recovered by transformation into Escherichia coli. Plasmid
B2608 (LEU2 CEN4 ARS1 PGAL1-HO PMFa1-CFP) was constructed in an
analogous fashion using the MFa1 promoter. Plasmid structures were
confirmed by functional assays and restriction analysis. coding sequence of CFP from plasmid pDH3 (http://depts.washington. edu/;yeastrc). Both PCR fragments carried 50 base pair ends with
homology to direct recombination with the appropriate adjacent
fragments. Leuþ FAA-resistant transformants were selected and
plasmid was recovered by transformation into Escherichia coli. Plasmid
B2608 (LEU2 CEN4 ARS1 PGAL1-HO PMFa1-CFP) was constructed in an
analogous fashion using the MFa1 promoter. Plasmid structures were
confirmed by functional assays and restriction analysis. Strains used in this study are listed in Table 2 and were all obtained
from the S288C-derived haploid strain S150-2B. Construction of
strains carrying DNA binding site arrays adjacent to MAT and HML
have been described previously [45]. GFP fusions were introduced by
integrative transformation of plasmid pPJS218, selecting Hisþ trans-
formants. The cdc15–2 allele was introduced into S150-2B by first
integrating plasmid B2400 (pRS406-cdc15–2) into the CDC15 locus
and then selecting FOA-resistant revertants of selected transform-
ants. FOA-resistant, temperature-sensitive isolates were scored
microscopically for terminal arrest phenotype to confirm the
presence of the cdc15–2 allele. The cdc15–2 allele was then introduced
into the tagged strains by genetic crosses. Deletion alleles were
introduced as needed by selecting transformants resistant to G418
(200 lg/ml, CalBiochem, San Diego, California, United States)
following transformation with PCR products using DNA from the
appropriate strain from the kanMX deletion collection, and primers
500 base pairs upstream, 59, and downstream, 39, of the gene. p
p
,
,
,
,
g
Strains for donor preference were obtained by transformation of
strain Y2902 using PCR products from the deletion collection as
described above. Donor preference in spore clones of mutant strains
were determined as described previously [27]. Microscopy methods. The yeast strains were freshly transformed
with plasmid B2609 or B2608 and grown 3 d at 23 8C on SC-leu media. Materials and Methods Plasmids and strain construction. Plasmid pPJS218 (HIS3 TetR-
GFP LacI-GFP) has been previously described [45]. Plasmid B2609
(LEU2 CEN4 ARS1 PGAL1-HO PMFa1-CFP) was constructed by in vivo
recombination by co-transforming a leu2 trp1 yeast strain with
plasmid B2686 (LEU2 TRP1 CEN4 ARS1 PGAL1-HO) and two PCR
fragments, one spanning the MFa1 promoter and one spanning the 0903 0903 June 2006 | Volume 2 | Issue 6 | e98 Table 2. Strain List
Strain
Genotype
Y3343
MATa::TRP1-LacO256 HML::URA3-TetO112 leu2–3,112 ura3–52 trp1–289 his3D::HIS3::TetR-GFP,LacI-GFP gal2 cdc15–2
Y3342
MATa::TRP1-LacO256 HML::URA3-TetO112 leu2–3,112 ura3–52 trp1–289 his3D::HIS3::TetR-GFP,LacI-GFP gal2 cdc15–2
Y3344
Y3343 rad54D::kanMX
Y3345
Y3342 rad54D::kanMX
Y3346
Y3343 srs2D::kanMX
Y3348
Y3343 sgs1D::kanMX
Y2902
MATa/MATa::HIS3 hmla1a2inc/ hmla1a2inc hmra1D101inc/ hmra1D101inc ura3–52/ ura3–52 leu2–3,112/ leu2–3,112 his3D1/his3D1 trp1–289/trp1–289 HO/HO
Y3443
Y2902, sgs1D::kanMX/SGS1
Y3444
Y2902, srs2D::kanMX/SRS2
Y3445
Y2902, rad54D::kanMX/RAD54
Y3446
Y2902, rad52D::kanMX/RAD52
Dynamics of Homologous Pairing in Yeast flow cell (BioSurface Technologies, Bozeman, Montana, United
States) and perfused with SC-leu medium þ 2% raffinose as the sole
carbon source, maintained at 37 8C with a thermocouple detector and
a feedback-modulated heated stage. Cells from several fields of each
strain were repeatedly interrogated every 1 or 2 min using a Nikon
Eclipse TE200 microscope with 1003 objective (1.4 aperture) and a
planapochromatic light source (Nikon, Tokyo, Japan). Approximately
5 min after mounting the coverslip on the flow cell, the temperature
was shifted to 23 8C to release the cdc15–2–imposed cell cycle arrest,
and medium perfusing the flow cell was changed to SC-leu þ 2%
galactose. This time was set as the zero time point, and galactose
perfusion was maintained for 60 min, at which time medium was
switched to SC-leu þ 2% glucose. At each interrogation, we captured
30 0.4-lm Z-sections of a 512 3 512 pixel image (0.1 lm per pixel),
and prior to analysis, the images were processed by five cycles of
deconvolution using Deltavision SoftWoRx v. 2.5 (Applied Precision,
Issaquah, Washington, United States). The locations of GFP dots in
each cell in a field were determined from the identification of the
maximum intensity in the three-dimensional images. Distance
between the separable dots was determined by applying the
Pythagorean triangulation to the Cartesian coordinates of the
maxima using Softworx pick points function and processed with
the timedist and yeastparser programs (John Houston, Multimedia
Gaming, Austin, Texas, United States). Materials and Methods Three transformants of each strain were inoculated to 20-ml SC-leu
brothþ 2% glucose and grown overnight at 23 8C. Cultures were then
diluted into SC-leu broth þ 2% raffinose and lacking glucose to a
density of 106 cells/ml and incubated for 3 h at 37 8C to arrest growth
and render GAL1-HO readily inducible. Cells from each culture were
examined by fluorescence microscopy to confirm that most exhibited
two nuclear GFP dots and none expressed CFP. Cells from cultures
with these properties were applied to a microscope coverslip (No.1
22360 mm, Corning, Corning, New York, United States) precoated
with Concanavalin A (Sigma, St. Louis, Missouri, United States) as
described (http://www.cgr.harvard.edu/thornlab/protocols/ConA.htm). Samples of test and control cultures were applied to the same
coverslip but separated by a hydrophobic Pap pen (Daido Sangyo Co. Ltd., Tokyo, Japan) to preclude mixing. The cells were mounted on a Acknowledgments We thank Dr. Mark Rose for helpful discussions and assistance with
the Deltavision microscope, and Dr. David Tank and Don Peoples for
advice and technical help in design and construction of the flow cell
apparatus. John Houston provided methods for working with the
dataset parsing, and Erin Smith and Dr. William Bialek provided
invaluable assistance with statistical analysis of the data. Author contributions. PLH and JRB conceived and designed the
experiments. PLH performed the experiments. PLH analyzed the
data. PLH and JRB wrote the paper. Funding. This work was supported by grant GM48540 from the
National Institutes of Health. Funding. This work was supported by grant GM48540 from the
National Institutes of Health. Competing interests. The authors have declared that no competing
interests exist. Competing interests. The authors have declared that no competing
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Comprehensive identification of cell cycle-regulated genes of the yeast
Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell 9: 3273–
3297. 45. Simon P, Houston P, Broach J (2002) Directional bias during mating type
switching in Saccharomyces is independent of chromosomal architecture. EMBO J 21: 2282–2291. 26. Kaplun L, Ivantsiv Y, Kornitzer D, Raveh D (2000) Functions of the DNA
damage response pathway target Ho endonuclease of yeast for degradation
via the ubiquitin-26S proteasome system. Proc Natl Acad Sci U S A 97:
10077–10082. J
46. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host
strains designed for efficient manipulation of DNA in Saccharomyces
cerevisiae. Genetics 122: 19–27. PLoS Genetics | www.plosgenetics.org 0905 PLoS Genetics | www.plosgenetics.org June 2006 | Volume 2 | Issue 6 | e98
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Foreword: Synthesis of the Greenland Ecosystem Monitoring program
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Foreword: Synthesis of the Greenland Ecosystem Monitoring
program Torben R. Christensen, Elmer Topp-Jørgensen, Mikael K. Sejr,
Niels Martin Schmidt The Greenland Ecosystem Monitoring (GEM) program has
over the past two decades established itself firmly as an
internationally leading environmental barometer measuring
climate change impacts and ecosystem changes in the
Arctic. The program was established in 1995 at Zackenberg
Research Station in a High-Arctic ecosystem. Over the past
decade, GEM expanded to include a Low-Arctic site,
Nuuk/Kobbefjord area, and more recently initiated the
inclusion of Disko/Qeqertarsuaq at the transition between
the Low-Arctic and High-Arctic. changing Arctic. To operate extensive research and moni-
toring operations in often remote and harsh arctic envi-
ronments, close cooperation with logistics operators are
needed. Integration of monitoring, research, and logistics is
therefore fundamental to the success of GEM. The program includes a multitude of climate and
ecosystem variables being measured on a continuous basis. While these long time series are fundamental to GEM,
some degree of flexibility is also needed to continuously
implement new scientific developments (e.g., standards,
methodologies or technologies) and to address potential
changes in science agendas and policy needs. This flexi-
bility is secured through an adaptive monitoring approach
with annual reviews of sub-programs. GEM is the longest running operational ecosystem/cli-
mate-oriented monitoring program in the Arctic con-
tributing to a deeper understanding of ecosystem change
and function. The mission of GEM is threefold and
embraces the following goals: GEM is represented in numerous scientific networks,
programs, and organizations to continuously influence and
implement international protocols and standards.1 The data
generated by GEM also provide input to a variety of the-
matic, national, arctic, and international assessments,
including Arctic Councils monitoring and assessments
programs (e.g. AMAP and CAFF) and international
agreements (e.g. IPCC and CBD). All data generated
within GEM are made freely available.2 1. To contribute to a coherent and science-based descrip-
tion of the state of the environment, including its
biodiversity, in Greenland and the Arctic in relation to
climatic changes with focus on ecosystem responses
and on global impacts related to feedback processes. 2. To provide science-based input on the state of the
environment in Greenland and the Arctic for Danish,
Greenlandic and international policy development,
adaptation, and administration. Ambio 2017, 46(Suppl. 1):S1–S2
DOI 10.1007/s13280-016-0860-z The Author(s) 2017. This article is published with open access at Springerlink.com
www.kva.se/en 1 www.g-e-m.dk.
2 http://data.g-e-m.dk/. 1 www.g-e-m.dk.
2 http://data.g-e-m.d 1 www.g-e-m.dk. 2 http://data.g-e-m.dk/. Foreword: Synthesis of the Greenland Ecosystem Monitoring
program While GEM historically has focused on detailed studies
of a few locations, it holds significant potential for using
the long-term monitoring data and process understanding
for upscaling to Greenlandic scale and for strengthening
applied science components through monitoring essential 3. To provide a platform for cutting-edge inter-disci-
plinary research on the structure and function of arctic
ecosystems. To achieve this, the GEM program is composed of five
integrated sub-programs (Climate, Geo, Bio, Marine, and
Glacio) that conduct comprehensive studies of climate
change and ecosystem dynamics within the domain cov-
ered by GEM (Fig. 1). The sub-programs combine long-
term monitoring and short-term research projects to fully
understand
ecosystem
dynamics
and
processes
in
a The Author(s) 2017. This article is published with open access at Springerlink.com
www.kva.se/en 123 Ambio 2017, 46(Suppl. 1):S1–S2 S2 Fig. 1 The GEM realm: studying ecosystem patterns, dynamics, and linkages in a changing climate from the coastal zone to glaciated
environments on the fringes of the Greenlandic ice cap (from Christensen and Topp-Jørgensen 2016) Fig. 1 The GEM realm: studying ecosystem patterns, dynamics, and linkages in a changing climate from the coastal zone to glaciated
environments on the fringes of the Greenlandic ice cap (from Christensen and Topp-Jørgensen 2016) REFERENCES ecosystem components and address cumulative impacts of
climate change and societal development (see Postscript
for how GEM seek to achieve this; Christensen et al. 2017). With its publicly available data and leading role in
monitoring arctic ecosystems, GEM is an important con-
tributor to the assessment of the status and trends of
ecosystems and the organisms therein and for under-
standing ecosystem processes of relevance for providing
government advice on climate impacts, sustainability, and
adaptation. Christensen, T.R., and E. Topp-Jørgensen. (eds.). 2016. Greenland
Ecosystem
Monitoring
Strategy
2017–2021. DCE—Danish
Centre for Environment and Energy, Aarhus University, Den-
mark. 44 pp. pp
Christensen, T.R., E. Topp-Jørgensen, M.K. Sejr, and N.M. Schmidt. 2017. Postscript: The future of the Greenland Ecosystem Moni-
toring programme. Ambio. doi:10.1007/s13280-016-0871-9. Torben R. Christensen (&)
Address: Department of Earth and Ecosystem Science, Lund
University, So¨lvegatan 12, 22362 Lund, Sweden. e-mail: torben.christensen@nateko.lu.se Torben R. Christensen (&)
Address: Department of Earth and Ecosystem Science, Lund
University, So¨lvegatan 12, 22362 Lund, Sweden. This special issue presents a collection of studies that
attempts to synthesize GEM data across sites and disci-
plines. It also contains a comparison of the GEM moni-
toring sites to other arctic areas and thus put the program in
a wider geographical context. e-mail: torben.christensen@nateko.lu.se Elmer Topp-Jørgensen Address: Department of Bioscience, Aarhus University, Frederiks-
borgvej 399, 4000 Roskilde, Denmark. e-mail: jetj@bios.au.dk Address: Department of Bioscience, Aarhus University, Frederiks-
borgvej 399, 4000 Roskilde, Denmark. We trust the collected papers in this special issue will be
valuable reading for anyone interested in arctic science in
general and those sharing a concern and specific interest in
the ecosystem impacts of climate change in particular. Mikael K. Sejr
Address: Department of Bioscience, Aarhus University, Vejlsøvej 25,
Building A2.11, 8600 Silkeborg, Denmark. Address: Arctic Research Centre, Aarhus University, Ny Munkegade,
bldg 1540, 8000 Aarhus, Denmark. e-mail: mse@bios.au.dk Mikael K. Sejr
Address: Department of Bioscience, Aarhus University, Vejlsøvej 25,
Building A2.11, 8600 Silkeborg, Denmark. 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 Author(s) 2017. This article is published with open access at Springerlink.com
www.kva.se/en Niels Martin Schmidt Niels Martin Schmidt
Address: Department of Bioscience, Aarhus University, Frederiks-
borgvej 399, 4000 Roskilde, Denmark. e-mail: nms@bios.au.dk The Author(s) 2017. This article is published with open access at Springerlink.com
www.kva.se/en 3 123
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Kirghiz, Kyrgyz
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WORD ORDER IN ENGLISH AND ITS STUDY
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Zenodo (CERN European Organization for Nuclear Research)
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| 3,304
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ПОРЯДОК СЛОВ В АНГЛИЙСКОМ ЯЗЫКЕ И ЕГО ИЗУЧЕНИ Аннотация. В данной статье описывается порядок слов в английском языке и его
изучение. В ходе статьи был сравнительно изучен порядок слов в узбекском и английском
языках, а для раскрытия темы использованы различные обоснованные мнения и
соображения. Выводы и предложения даны в конце статьи. Ключевые слова: порядок слов, инверсия, притяжательный падеж, причастие,
сравнение, порядок слов. WORD ORDER IN ENGLISH AND ITS STUDY Abstract. This article describes word order in English and its study. In the course of the
article, the word order in Uzbek and English languages was studied comparatively, and various
reasonable opinions and considerations were used to reveal the topic. Conclusions and
suggestions are given at the end of the article. Key words: word order, inversion, possessive, participle, comparison, word order
placement. International scientific journal
«MODERN SCIENCE АND RESEARCH»
VOLUME 1 / ISSUE 2 ISSN:
2181-3906
2022 Annotatsiya. Mazkur maqolada ingliz tilida so’z tartibi va uning o’rganilishi yoritib
berilgan. Maqola davomida o’zbek va ingliz tillaridagi so’z tartibi qiyosan o’rganilgan,
shuningdek mavzuni ochib berish uchun turli asosli fikr va mulohazalardan foydalanilgan. Maqola so’nggida xulosa va takliflar keltirilgan. Kalit so’zlar: so’z tartibi, inversiya, ega, kesim, qiyos, so’z tartibi joylashishi. METHOD AND METHODOLOGY Shuning uchun so'z tartibi kelishiklarning aniq farqi amaliy jihatdan ega va vositali
to'ldiruvchi o'rtasidagi farqning asosiy vositadir. Yuqorida gaplarda keltirilgan, misollarda
keltirilishicha, ingliz tilida darak gaplarda so'z tartibi to'g'ridan to'g'ri xizmat qiladi. 2) Kesim 3) To’ldiruvchi
4)Hol Umuman gaplarda ikki xil so'z tartibi uchraydi. 1 )To'g'ri soz tartibi '(Direct word order) 2) Teskari so'z tartibi (Inverted order of words) Darak gapda so'z tartibi to'g'ri bo'lib ega birinchi, kesim ikkinchi, to'ldiruvchi uchinchi,
hol esa to'rtinchi o'rinda keladi. Darak gapda so'z tartibi to'g'ri bo'lib ega birinchi, kesim ikkinchi, to'ldiruvchi uchinchi,
hol esa to'rtinchi o'rinda keladi. Subject+Predicate +Object + Ad Mod Subject+Predicate +Object + Ad Mod Masalan: She was reading a book then. Jane took it yesterday. Ba'zan hol egadan oldin yoki undan keyin ham kelishi uchraydi. Masalan: Yesterday my dad bought me a doll. My dad bought me a doll yesterday. (my own sent) Predlogsiz vositali to'ldiruvchi vositasiz to'ldiruvchidan oldin keladi. Masalan: He bought me a book. Nick wrote me a letter. Predlogli vositali to'ldiruvchi vositasiz to'ldiruvchidan keyin keladi. Masalan: He bought a book for me.1 Undalma, kiritma so'z va kiritma konstruksiyalar gapning boshida. O'rtasida va oxirida
kelib, yozuvda vergul bilan ajratiladi. O'g'zaki nuqtqda esa maxsus intonatsiya bilan ajratiladi. Shuningdek bu so'zlar gap bo'laklari hisoblanmaydi. Undalma, kiritma so'z va kiritma konstruksiyalar gapning boshida. O'rtasida va oxirida
kelib, yozuvda vergul bilan ajratiladi. O'g'zaki nuqtqda esa maxsus intonatsiya bilan ajratiladi. Ingliz tilda morfologik vositalar yetarli darajada rivojlanganligi uchun so'zlar orasidagi
munosabat asosan so'z tartibi orqali ifodalanadi. Shuning uchun ham ingliz tilida, rus va o'zbek
tillarida bo'lgani kabi so'zlar o'rnini almashtirish mumkin emas. Masalan: Edward looked at Rose, and Rose looked at Edward. Masalan: Edward looked at Rose, and Rose looked at Edward. Gapdagi Edward va Rose soz'larning o'rnini o'zgartirish bilan ma'no o'zgaryapti. So'z
birikmasi bilan gap o'rtasida farq kattadir. So'z birikmasi nominatsiya (atash) funksiyasini
bajarish jihatdan so'zga yaqin turadi, va gap singari intonatsion tugalikka ega bo'lmaganligi
uchun kommunikativ birlikni tashkil qilmaydi. Ega va kesim gapning bosh bo'laklardir. Ega va kesim minimal gap strukturasining asosi,
gapning hokim bo'lagidir. 1 Bo'ronov O', Noshimov X, "Ingliz tili grammatikasi" T.304bet INTRODUCTION Ingliz tilida so'z tartibi o’zbek tiliga qaraganda biroz muhimroqdir. Gapdagi har bir
so’zning vazifasini ma'lum darajada mustaqil tuslanib ko'rsatadi. O’zbek tili so'zga so'z tartibi
tuslanishi katta hajmdaligi sababli ingliz tilida so'zlarning deyarli tuslanishi o'zgaradi va gapdagi
joylashish o'rnini bir-biriga bog'lab ko'rsatadi va ingliz tilida belgilangan so'z tartibi shakli
o'zgarmaydi. Buni namunali misollar bilan yaqqol ko'rsatiladigan bo'lsak, biz gap bo'laklarini,
asosan ega va to'ldiruvchining o'rnini o'zgartira olmaymiz. Masalan:Mrs Winter sent the little boy wishes a missage to the next village on December
day. Agar biz birinchi o'ringa vositali to'ldiruvchini qo'ysak va egani uchinchi o'ringa
joylashtirsak gapning ma'nosi umuman o'zgaradi. Chunki ega bo'lib gapning boshida
joylashtirilgan to'ldiruvchi va to'ldiruvchi bo'lib kelgan kesimdan keyin joylashtirilgan
to'ldiruvchi va to'ldiruvchi bo'lib kelgan kesimdan keyin joylashtirilgan ega bo'lib keladi. Masalan: The little boy sent Mrs. Winter wish a message to the next village on December
day. Masalan: The little boy sent Mrs. Winter wish a message to the next village on Decembe O’zbek tilida so'z tartibidagi bu kabi o'zgarishlar ko'p holatlarda uchraydi. Masalan: O’zbek tilida so'z tartibidagi bu kabi o'zgarishlar ko'p holatlarda uchraydi. Masalan: Mening opam seni opangni shaharda ko’rgan. Mening opam seni opangni shaharda ko’rgan. Моя сестра видела твою сестру в городе. Моя сестра видела твою сестру в городе. ISSN:
2181-3906
2022
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«MODERN SCIENCE АND RESEARCH»
VOLUME 1 / ISSUE 2 ISSN:
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2022 2 Bo'ronov O', Noshimov X, "Ingliz tili grammatikasi" T. 304bet RESEARCH RESULTS Ega va kesim bir-biri bilan grammatik jihatdan bog'liq. Agar gapning egasi bo'lsa, uning
kesimi ham bo'lishi kerak. Ega gapning shunday bo'lagiki u ifodalagan shaxs yoki narsaga kesim 33 International scientific journal
«MODERN SCIENCE АND RESEARCH»
VOLUME 1 / ISSUE 2 Who told you?(so’roq olmoshlari) Shaxs va predmetni ifodalovchi some, any, somebody, one, each, every, all, either,both,
other, another, something, anything kabi gumon olmoshlari bilan: Masalan: Somebody laughed, and it gave me courage. One, with a long pale face, was sitting in a chair, the other was standing in fron
of the window. The little ones todled after their mother. Everytning was still. Another was a yellow wolf of remarkable swiftness. Eslatma: One olmoshi bilan ifodalangan ega bir shaxsga emas, barcha shaxslarga
qaratilgan bo'lishi mumkin. Masalan: One wold not throw away she past like a drown tooth. International scientific journal
«MODERN SCIENCE АND RESEARCH»
VOLUME 1 / ISSUE 2 Masalan: He arrived at the club at three o'clock.(olmosh) Masalan: He arrived at the club at three o clock.(olmosh)
We stood looking at them through the window. I had a case this morning, Chris! You 'll note that I say had. Ega kishilik olmoshlar he, they, you bilan ifodalanganda, ba'zan III shaxs yoki II shaxsni
(birlik va ko'plik anglatmasdan balki umumiy bo'lishi mumkin). He laughs best who laughs last. As you make your bed, so must you lie on it. That is my opinion of you and that was all. Who told you?(so’roq olmoshlari)
What was happened? Shaxs va predmetni ifodalovchi some, any, somebody, one, each, every, all, either,both,
other, another, something, anything kabi gumon olmoshlari bilan:
Masalan: Somebody laughed, and it gave me courage. The others gave a cry of horror. They sprang all will be well with me.2
And all will be wish you too. Somebody I will help you. One, with a long pale face, was sitting in a chair, the other was standing in front
of the window. The little ones todled after their mother. Everytning was still. Another was a yellow wolf of remarkable swiftness. Eslatma: One olmoshi bilan ifodalangan ega bir shaxsga emas, barcha shaxslarga
qaratilgan bo'lishi mumkin. Masalan: One wold not throw away she past like a drown tooth. Nobody,
none
notning
no
one
kabi
bo'lishsizlik
olmoshlari
bilan:
Masalan: Nobody was absent. No one was at home. Norbody can ride him for a long time. Nothing had been taken from the dead man's pockets. No one was happy... No one know who belonged to the Avenging Angels
Egalik olmoshlarining absolyut formasi (Absolute Forms of Possisive pronouns)
bilan:
Masalan: His was a lucky lot. Ours are low islands you know. But my point of view proves to be correct theirs is wrong. Son (numerals) bilan:
2 We stood looking at them through the window. I had a case this morning, Chris! You 'll note that I say had. Ega kishilik olmoshlar he, they, you bilan ifodalanganda, ba'zan III shaxs yoki II shaxsni
(birlik va ko'plik anglatmasdan balki umumiy bo'lishi mumkin). Ega kishilik olmoshlar he, they, you bilan ifodalanganda, ba'zan III shaxs yoki II shaxsni
(birlik va ko'plik anglatmasdan balki umumiy bo'lishi mumkin). As you make your bed, so must you lie on it That is my opinion of you and that was all. International scientific journal
«MODERN SCIENCE АND RESEARCH»
VOLUME 1 / ISSUE 2 ISSN:
2181-3906
2022 ifodalangan harakat, belgi yoki holat tegishli bo'ladi. Ega mutlaqo mustaqil bo'lak bo'lib, kesim
ham egaga tob’e va ko'p hollarda ega bilan shaxs sonda moslashadi. Ega, asosan, quyidagi so'z turkumlari bilan ifodalanadi: Ega, asosan, quyidagi so z turkumlari bilan ifodalanadi:
Masalan: Bernard Higginbotham invited him to dinner.(ot) g ,
, q y
g
Masalan: Bernard Higginbotham invited him to dinner.(ot)
The man was unusual, not essentric, but unusual society opened its portals to me. Masalan: He arrived at the club at three o'clock.(olmosh)
We stood looking at them through the window. I had a case this morning, Chris! You 'll note that I say had. Ega kishilik olmoshlar he, they, you bilan ifodalanganda, ba'zan III shaxs yoki II shaxsni
(birlik va ko'plik anglatmasdan balki umumiy bo'lishi mumkin). He laughs best who laughs last. As you make your bed, so must you lie on it. That is my opinion of you and that was all. Who told you?(so’roq olmoshlari)
What was happened? Shaxs va predmetni ifodalovchi some, any, somebody, one, each, every, all, either,both,
other, another, something, anything kabi gumon olmoshlari bilan:
Masalan: Somebody laughed, and it gave me courage. The others gave a cry of horror. They sprang all will be well with me.2
And all will be wish you too. Somebody I will help you. One, with a long pale face, was sitting in a chair, the other was standing in front
of the window. The little ones todled after their mother. Everytning was still. Another was a yellow wolf of remarkable swiftness. Eslatma: One olmoshi bilan ifodalangan ega bir shaxsga emas, barcha shaxslarga
qaratilgan bo'lishi mumkin. Masalan: One wold not throw away she past like a drown tooth. Nobody,
none
notning
no
one
kabi
bo'lishsizlik
olmoshlari
bilan:
Masalan: Nobody was absent. No one was at home. Norbody can ride him for a long time. Nothing had been taken from the dead man's pockets. No one was happy... No one know who belonged to the Avenging Angels
Egalik olmoshlarining absolyut formasi (Absolute Forms of Possisive pronouns)
bilan:
Masalan: His was a lucky lot. Ours are low islands you know. But my point of view proves to be correct theirs is wrong. Son (numerals) bilan: Masalan: Bernard Higginbotham invited him to dinner.(ot) The man was unusual, not essentric, but unusual society opened its portals to me. 3 Bo'ronov O', Noshimov X, "Ingliz tili grammatikasi" T. 304bet International scientific journal
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VOLUME 1 / ISSUE 2 ISSN:
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2022 Masalan: Eighty-five is a lucky number. Masalan: Eighty-five is a lucky number. Thirty is a good age to begin all a new yet. A length the two resumed their seats at the party grew louder in their mirth. Infinitiv, infinitivli so'z birikma yoki oborot bilan:
Masalan: To come out of Wales into England was like she change fro
earthenware. To withhold the truth is sometimes wise . But never conceal the truth with lies. A wonderful shining a memorable thing to what a doctor! To get him would be a remarkable thing. For him to come was impossible. Otlashgan sifatdosh yoki-sifatdosh so'zbirikmasi bilan:
Masalan: Seated next to him was a young
Gerundiy, gerundiy so'z birikmasi yoki oboroti bilan:
Freezing is a heat evolving process. Sitting here is dangerous. Annett's being French might be upset him a little. Ayrim hollarda otlashgan ravish bilan:
The Worst had happened. Mary didn't know what had happened. Tomorrow is going to be a good day.3
Ergash gap ham ega vazifasiga kelishi mumkin:
What I want is sea air. What you say is wrong. Kontekstning talabiga ko'ra ega ba'zan boshqa so'z turkumidagi so'zlar
birikmalari, bilan ham ifodalana oladi:
DISCUSSION
B
d
' l
i
' bi ik
l
i
it’ t l
hti il
(
t ti
)
' l
' Thirty is a good age to begin all a new yet. A length the two resumed their seats at the party grew louder in their mirth. Masalan: To come out of Wales into England was like she change from China to
earthenware. To withhold the truth is sometimes wise . A wonderful shining a memorable thing to what a doctor! Masalan: Seated next to him was a young
Gerundiy, gerundiy so'z birikmasi yoki oboroti bilan:
Freezing is a heat evolving process. Sitting here is dangerous. Annett's being French might be upset him a little. Ayrim hollarda otlashgan ravish bilan:
The Worst had happened. Mary didn't know what had happened. Tomorrow is going to be a good day.3
Ergash gap ham ega vazifasiga kelishi mumkin:
What I want is sea air. What you say is wrong. Kontekstning talabiga ko'ra ega ba'zan boshqa so'z turkumidagi so'zlar, turli so'z
birikmalari, bilan ham ifodalana oladi: DISCUSSION Bunday so'zlarni, so'z birikmalarni qit’atalashtirilgan (quotation) so'zlar, so'z birikmalari
deyiladi. Eganing "It" so'zi bilan ifodalanishi. It so'zi ega bo'lib kelganda u ham predmet, ham
jonli narsalarni ifodalanishi yoki hech narsani ifodalamasligi mumkin. It so'zi predmet yoki jonli
narsani ifodalaganda shaxsli ega (shaxsni ko'rsatuvchi ega va notional subject) hech narsani
ifodalamaganda formal ega (a formal subjet) hisoblanadi. It so'zini tahlil qila oladigan bo'lsak , u
shaxsli ega. It III shaxs birlikdagi predmet yoki shaxsni bildirib, o'zbek tilidagi u olmoshiga
to'g'ri keladi. Masalan: But the master of the house wasn’t George Meadows. It was his mother. It predmet va shaxsni bildirib, o'zbek tiliga ko'rsatish olmoshi vositasida tarjima
qilinadi. Masalan: Who is knocking ? "It is me". Masalan: Who is knocking ? "It is me". ISSN:
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International scientific journal
«MODERN SCIENCE АND RESEARCH»
VOLUME 1 / ISSUE 2 ISSN:
2181-3906
2022 It formal ega bo'lib kelganda shaxs, predmetni ifodalamaydi. Formal ega quyidagi
turlarga bo'liadi: Shaxsiz ega (Impersonal subjet). It so'zi qo’yidagi holatlarda shaxs anglatmaydigan ega
tarzida qo’llanadi. a)
ob-havo, tabiat hodisalari ifodalaganda: a)
ob-havo, tabiat hodisalari ifodalaganda: Masalan: It is snowing even now. Masalan: It is snowing even now. It is drizzling now. It is drizzling now. I fraze hard It will be dark soon It was still hot. It's nippy in the mornings now. b) Vaqt ifodalaganda: Masalan: It was about 9 o'clock but felt like late afternoon. It was late in summer. It was one o'clock when we left. v) tashqi muhit, holat anglatilganda: Masalan: It was very quiet in the library. It really is nice here. It's lonely at the seaside in summer. g) masofa ifodalanganda: Masalan: It is not far from here. Kiritma ega. (Introductrory anticepatory subjet)Predmet ma'nosiga ega bo'lmagan so'z
yoki so'zlar birikmasi ega vazifasida kelganda, gap leksik ma'noga ega bo'lmagan it so'zi bilan
boshlanish mumkin. Masalan: It is impossible to go. Masalan: It is impossible to go. Bu gapda it formal ega, to go esa haqiqiy ega (real subject) hisoblanadi. Bu turdagi gaplarda haqiyqiy ega, kesimdan keyin kelib, ko'pincha infinitiv, gerundiy
bilan yoki infinitiv va gerundiyli so'z birikmalari bilan ifodalanadi. Masalan: It is necessary for him to come tonight. Masalan: It is necessary for him to come tonight. It is no use to cry over milk. It is no use to cry over milk. It is said that humans are never satisfied, that what you give them one thing and they
what sometning more. CONCLUSION Masalan: For many miles there was no fet camping-place Sostavli ot-kesim bog'lovchi fel (ko'pincha to be fe'li) va ot predikativ (a predicative) dan
tashkil topadi. You are happy today, Mrs Swarts. O'zbek tilidagi gapda -dir, bo'lmoq bog'lovchi fe'llari ko'pincha ishlatilmaydi. Ingliz tilida
esa bog'lovchi fe'lning kesim sostavida ishlatilishi shart. To be fe'lidan tashqari to look, to feel,
to turn, to remain, to grow, to come, to go, to seem, to fall, to stand, to keep kabi fe'llar ham
bog'lovchi fe'l vazifasida keladi. Masalan:You seem a little nervous. Masalan:You seem a little nervous. You look very young. DISCUSSION Emfatik ega bo'lib kelgan it so'zi predikativ rolida kelgan so’zning kommunikativ
ahamiyatini ko'chaytirish uchun xizmat qiladi. Emfatik ega bo'lib kelgan it so'zi predikativ rolida kelgan so’zning kommunikativ
ahamiyatini ko'chaytirish uchun xizmat qiladi. Qiyoslang (compare): Qiyoslang (compare):
He came to Tashkent only yesterday. He came to Tashkent only yesterday. It was only yesterday, that he came to Tashkent. Ikkinchi gapdagi it emfatik egadir. U predikativ vazifasidagi yesterday soz'ini
kommunikativ ahamiatini kuchaytiryapti. Masalan: It was not the father, however who first discovered. Masalan: It was not the father, however who first discovered. It was a Mormon law that a man might the several wives. It was a Mormon law that a man might the several wives. There+to be bilan boshlanadigan gaplar. There+to be bilan boshlanadigan gaplar. Ma'lum payt, o'rinda biror predmet yoki shaxsning mavjudligi yoki mavjud emasligini
ifodalash uchun there+tobe konsruksiyasini ishlatiladi. Ma'lum payt, o'rinda biror predmet yoki shaxsning mavjudligi yoki mavjud emasligini
ifodalash uchun there+tobe konsruksiyasini ishlatiladi. Masalan: There were no white dunes covered with reddish grass behind them: there was
notning to mark. Masalan: There were no white dunes covered with reddish grass behind them: there was
notning to mark. 36 ISSN:
2181-3906
2022
International scientific journal
«MODERN SCIENCE АND RESEARCH»
VOLUME 1 / ISSUE 2 CONCLUSION Bu gaplarda there so’zi formal ega hisoblanadi, haqiqiy ega esa to be fe'li ba'zan boshqa
fe'l bilan ifodalangan kesimdan keyin keladi. Haqiqiy ega doimo urg'uli bo'lib asosan ot bilan
ifodalagan bo'ladi. Ba'zan esa o'lishsizlik olmoshi, birikmalar bilan ham ifodalangan bo'lishi
mumkin. Kesim haqiqiy ega bilan shaxs-sonda doimo moslashadi. Kesim haqiqiy ega bilan shaxs-sonda doimo moslashadi. Masalan: There were signs of an lately winter and wish them irritating delays. Masalan: There was no keep for it. There were not more than a dozen attendants on the lectures all together. O'rin holi gapning boshida ham kelishi mumkin. Masalan: For many miles there was no feet camping-place. Gapning kesimi-to be dan boshqa fe'llar bilan ham ifodalana oladi. Masalan: In the central part of the great Noth American Continent there lies a desert. Kesim (The Predicate).Olmosh, birikmalar bilan ham ifodalangan bo'lishi mumkin. Kesim haqiqiy ega bilan shaxs-sonda doimo moslashadi. Masalan:There were signs of an early winter and wish them irrigating delays. Masalan:There was no keep for it. There were not more than a dozen attendants on the lectures all together. O'rin holi gapning boshida ham kelishi mumkin. Masalan: For many miles there was no fet camping-place
Sostavli ot-kesim bog'lovchi fel (ko'pincha to be fe'li) va ot predikativ (a predicative) dan
tashkil topadi. Masalan: The street was empty. King was young and strong. You are happy today, Mrs Swarts. O'zbek tilidagi gapda -dir, bo'lmoq bog'lovchi fe'llari ko'pincha ishlatilmaydi. Ingliz tilida
esa bog'lovchi fe'lning kesim sostavida ishlatilishi shart. To be fe'lidan tashqari to look, to feel,
to turn, to remain, to grow, to come, to go, to seem, to fall, to stand, to keep kabi fe'llar ham
bog'lovchi fe'l vazifasida keladi. Masalan:You seem a little nervous. Masalan: There were signs of an lately winter and wish them irritating delays. Masalan: There were signs of an lately winter and wish them irritating delays. There were not more than a dozen attendants on the lectures all together. O'rin holi gapning boshida ham kelishi mumkin. Masalan: For many miles there was no feet camping-place. Gapning kesimi-to be dan boshqa fe'llar bilan ham ifodalana oladi. Masalan: In the central part of the great Noth American Continent there lies a desert. Kesim (The Predicate).Olmosh, birikmalar bilan ham ifodalangan bo'lishi mumki
haqiqiy ega bilan shaxs-sonda doimo moslashadi. Masalan:There were signs of an early winter and wish them irrigating delays. There were not more than a dozen attendants on the lectures all together. REFERENCES 1. J. Bo'ronov. O' Hoshimov. X. Ismatullayev. "Ingliz tili gramatikasi" "O'qtuvchi" nashriyoti. 1996 y. 1. J. Bo'ronov. O' Hoshimov. X. Ismatullayev. "Ingliz tili gramatikasi" "O'qtuvchi" nashriyoti. 1996 y. 2. J. Bo'ronov. “Qiyosiy grammatika”.T. 1978y. 3. G'. Salomov "Tarjima nazaryasiga kirish" ,1976 y. 4. В.Фѐдоров «Искусство перевода и жизнь литературы» 1983 г. 4. В.Фѐдоров «Искусство перевода и жизнь литературы» 1983 г. 5. В.Н. Комиссаров, Я.Н. Ретскер, В. И. Тарков «Пособие по переводу с английского
языка на русский язык» Moсквa 1965 г. 6. Л. С. Баркударов, П.А. Штелинг «Грамматика английского языка» Издание «Высщая
школа» Moсквa 1965г.
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Community care: Italy's ‘U’ turn
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Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin
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Ongoing developments inpsychiatry in
Bangladesh The Institute of Postgraduate Medicine and Research
now runs a three-and-a-half
years course in psy
chiatry that parallels the British MRCPsych
and
confers the title FCPSpsych from the College of
Physicians and Surgeons in Dhaka. So far the number
of trained fellows is only of the order of two to three
per year. The country aims to have a trained psychiatrist in
each of the 64 districts to act as a referral centre
for physicians and primary care workers and to be
responsible for the development of mental health
services in his own region. g
Finally, Bangladesh is investing a great deal of its
resources in the recruitment and training of family
welfare workers who will operate at the village level
and will be involved in the identification and diag
nosis of psychiatric cases, their referral to district
specialist services and follow-up. p
y
The final MB examination now contains compul
sory psychiatry questions. It is hoped that this will
encourage medical students to investigate a career in
psychiatry and help with the current shortage of Psychiatric
Bulletin (1993), 17,494-495 494 494 Islam and Howard trained staff. A re-orientation
course for GPs who
wish to specialise in psychiatry is also now available
at the Institute of Mental Health and Research. Ongoing developments inpsychiatry in
Bangladesh Ongoing developments inpsychiatry in
Bangladesh https://doi.org/10.1192/pb.17.8.494 Published online by Cambridge University Press HENRYR. ROLLIN,Emeritus Consultant Psychiatrist, Horton Hospital, Epsom, Surrey In theory
and
in practice
the work
carried
out by the department
was governed
by a
bizarre
blend
of
Marxist
dialectic
and
Freudian
psychoanalysis
seasoned
to taste with sprinklings
of
R. D. Laing,
David
Cooper,
Thomas
Szasz and
others of that anti-psychiatry
galère. It was evident
that as a spin-off from this concoction,
genetics were
out and environmentalists
were in: it was not the
individual
who was sick, only the society in which he
had the misfortune
to live. As a corollary,
the argu
ment went on, mental disorder
must be treated in the
community
by social agents who may or may not be
doctors,
let alone psychiatrists. It was evident,
too,
that mental disorder
was considered
as a single entity
in the same
way as pears
and
pineapples
can be
lumped
together
as fruit. Classification,
it seemed,
was unimportant
as was the training (if any) of those
who leaped on the therapeutic
band-wagon. As luck would have it, we were treated to a superla
tive exposition
of the mess that psychiatry,
or, rather,
anti-psychiatry
had got itself into in this particular
neck-of-the-woods. One of our party insisted on an
answer to the sort of dilemma which arises from time
to time in any community. He described
a situation
occasioned
by a manic patient
wielding
an axe and
threatening
the lives of his family. How would
the
Crisis
Intervention
Unit
deal
with
it? The
reply
that tripped
off the tongue
like a child reciting
its
catechism
was that
the first necessity
would
be to
resolve
the
counter-transference! There
was
a
stunned
silence followed after an interval by hoots of
derisive
laughter,
echoes of which will live with me
for the rest of my days. But there
is hope. The report
from
the British
Medical
JournaPs
Rome
correspondent,
Chris
Endean
(BMJ,
306, 6 March
1993, p. 605), entitled,
'Italy retreats
from community
care for mentally
ill',
brought
tears of joy to my ageing eyes as it must have
done to other
psychiatrists,
who, like myself, have
been banging
the drum
on behalf
of the destitute,
chronic schizophrenic
for decades. HENRYR. ROLLIN,Emeritus Consultant Psychiatrist, Horton Hospital, Epsom, Surrey HENRYR. ROLLIN,Emeritus Consultant Psychiatrist, Horton Hospital, Epsom, Surrey Give or take a few months, it must be 20 years since I
led a College Study Tour Group on a tour of Italy,
or, to be more exact, of the area north of Rome,
economically privileged and steeped in the academic
tradition from the Renaissance onwards. tals, but in the same wards. There were, however,
obvious
differences in management,
particularly
marked in the use of physical restraint in patients
who, we were told, were a danger to themselves or
others. However, what struck us smartly between the eyes
was the dramatically changed concept of mental ill
ness in certain areas of Italy together
with the
changed role of the psychiatrist himself. It was all too
apparent that these changes stemmed from the politi
cal thinking of individual psychiatrists, or were a
reflection of the political climate of the area con
cerned. At at least two meetings it was in the context
of politics rather than psychiatry
that discussion
became heated, if not red-hot. At one our host was
none other than the late Professor Franco Basaglia,
an aristocrat by birth and a Marxist by conviction. He wore an air of superiority tinged with arrogance
reflecting both these threads in his make-up. But in
spite of, or, indeed, because of these traits, he was at Each and every city we visited had a magic all of its
own - Verona, Padua, Venice, Gorizia, Trieste and
Bologna. The hospitality
was unforgettable:
the
official banquets, of which there were several, were
occasions for self-indulgence on a Babylonian scale. But study tours, even Italian study tours, are not
organised in order to furnish the participants with a
Roman holiday. There was work to be done. We visited a range of psychiatric facilities under
the auspices of different university departments. From a clinical standpoint, there was little cause for
surprise. Italian schizophrenic patients, or those with
learning disability, are indistinguishable from their
British counterparts, although it came as a surprise to
see them nursed together, not only in the same hospi https://doi.org/10.1192/pb.17.8.494 Published online by Cambridge University Press 495 Community care: Italy's 'V turn mental hospitals,
with a patient
population
of some
60,000,
were
duly
closed
without,
tragically,
the
provision
of anything
like adequate
facilities in the
community. The result
was predictable:
there was
chaos. HENRYR. ROLLIN,Emeritus Consultant Psychiatrist, Horton Hospital, Epsom, Surrey Apart from a spate of suicides and deaths from
starvation,
a new class of vagrant
was created - the
abandonati. But the lunacy which inspired
the 'fiasco'
in Italy that time a most powerful,
if not, the most powerful
figure in Italian
psychiatry. He had played a central
role in persuading
his government
to introduce
Law
180, a law which caused
mental
hospitals
to close
and to discharge
their
patients
willy-nilly
into the
community. At
our
meeting. Professor
Basaglia
paraded
his Marxism
and made no bones about
the
use of Marxist
ideology in handling
the problems
of
mental disorder. that time a most powerful,
if not, the most powerful
figure in Italian
psychiatry. He had played a central
role in persuading
his government
to introduce
Law
180, a law which caused
mental
hospitals
to close
and to discharge
their
patients
willy-nilly
into the
community. At
our
meeting. Professor
Basaglia
paraded
his Marxism
and made no bones about
the
use of Marxist
ideology in handling
the problems
of
mental disorder. But the lunacy which inspired
the 'fiasco'
in Italy
had
its
equivalent
in
other
so-called
civilised
countries. In the USA, for instance, the dire situation
which ensued prompted
Dr Alan A. Stone, Professor
of Law and Psychiatry
at Harvard
(Law, Psychiatry
and Morality,
American
Psychiatric
Press, 1984) to
write: "Yet madness has not gone out of the world as
was hoped, in fact, madness is more visible than ever
in this country. One can see chronic
mental patients
in the
streets
of every
major
city in the
United
States."
Nor can this country
escape the most severe
censure. What
can be seen in New York,
Boston,
Chicago
and
Los
Angeles
can
be seen
in equal
measure
in London,
Birmingham,
Liverpool
and
Leeds. It could well be becoming
worse, to judge by
the ever-increasing
attention
of the media in recent
months
to the disgraceful
dearth of community
care
for those desperately
dependent
patients
discharged
from our disappearing
mental
hospitals. The situ
ation
has been highlighted
recently
by the much-
publicised
case of Ben Silcock, a hapless,
homeless
schizophrenic
who at the London
Zoo tried to emu
late the lion-resistant
powers
of Daniel
with near
fatal results. At
the
other
meeting
there
was
no
outright
declaration
of political affiliations,
but they obtruded
nevertheless. HENRYR. ROLLIN,Emeritus Consultant Psychiatrist, Horton Hospital, Epsom, Surrey p
Incidentally,
at great
expense,
I am
having
a
plaque cut to hang in my study on which will be writ
in letters
of gold
the inspired
words
of Guiliano
Amato,
Italy's
prime
minister,
who described
the
supporters
of Basaglia's
theory
on insanity
as "a
species close to extinction
but not worth saving." y
y
Despite
these patent
lunacies,
Law 180, passed in
1978, had
been
enacted:
the
majority
of
Italy's Are you listening, Mrs Bottomley? Are you listening, Mrs Bottomley? A gastroenterologist
friend
attending
a meeting
in
Bologna
was given a leaflet from the department
of
psychiatry
asking
foreign visitors
to take a look at
the
university's
psychiatric
clinic,
"placed
in
a
single room of 3 x 5 square metres, a crumbling
ex-
laboratory
of the Veterinary
Obstetrician
Clinic ... to illustrate
the way in which the university
and the
municipality
of Bologna consider
psychiatry."
Minerva
BMJ
15 May 1993 306 p 1354 https://doi.org/10.1192/pb.17.8.494 Published online by Cambridge University Press
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https://openalex.org/W3005875666
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https://europepmc.org/articles/pmc7048672?pdf=render
|
English
| null |
Reply to Comments on ‘A functional polymorphism rs10830963 in melatonin receptor 1B associated with the risk of gestational diabetes mellitus’
|
Bioscience reports
| 2,020
|
cc-by
| 2,422
|
To the editor, Many thanks to Professor Klara Rosta, M.D., Ph.D., G´abor Firneisz, M.D., Ph.D., et al. for their inter-
est on our recently published article, ‘A functional polymorphism rs10830963 in melatonin receptor1B
associated with the risk of gestational diabetes mellitus’ [1] and appreciate their comments [2] on it. We
believe that peer exchanges among different research groups can promote better research works. p
g
g
g
p
p
In the recent study, according to 14 reported research data on the association between a functional
polymorphism rs10830963 in MTNR1B gene and the risk of gestational diabetes mellitus, we performed
a meta-analysis by using Stata software, version 12.0 (Stata Corp LP, College Station, TX, U.S.A.) [3,4]. The false positive report probability (FPRP) analyses were adopted to confirm the positive findings [5,6]. Klara Rosta, M.D., Ph.D., et al. paid attention to one included study (good works from Rosta et al., 2017)
in this meta-analysis, then put forward some opinions and suggestions on the minor (rs10830963 G)
allele frequencies (MAF), the calculation of effect value (odds ratios, ORs) and the indication of relevant
clinical data (mean age and pre-pregnancy BMI). We are here to respond. If there are any inaccuracies in
our response, we welcome to communicate again. p
g
Since we read the original literature of Rosta et al., 2017 [7], we found that not the exact genotyping data
but an MAF of each studied SNP locus, including rs10830963 was reported. Therefore, we can not extract
the accurate sample size data of being successfully genotyped. According to the number of 287 GDM
cases meet the International Association of the Diabetes and Pregnancy Study Group (IADPSG) criteria
and 533 controls reported in the literature, we estimated the genotype data by using the Hardy–Weinberg
equilibrium (HWE) genotype distributions. The approach is recognized. As reminded by the commentary,
we have carefully verified the extraction MAF in the literature, and hereby we correct it and other relevant
research data. *These authors are considered
co-first authors. Received: 11 February 2020
Revised: 12 February 2020
Accepted: 13 February 2020
Accepted Manuscript online:
13 February 2020
Version of Record published:
28 February 2020 We recalculate the results using the new genotype data, and the association between the SNP rs10830963
and the risk of GDM is still confirmed (Figures 1–3). Further functional experimental studies are war-
ranted to explore and clarify the potential mechanism. *These authors are considered
co-first authors. Bioscience Reports (2020) 40 BSR20200370
https://doi.org/10.1042/BSR20200370
Correspondence
Reply to Comments on ‘A functional polymorphism
rs10830963 in melatonin receptor 1B associated
with the risk of gestational diabetes mellitus’
Bo Huang1,*, Yu-kun Wang2,*, Lin-yuan Qin1, Qin Wei1, Nian Liu1, Min Jiang1, Hong-ping Yu3 and
Xiang-yuan Yu1
1Department of Epidemiology and Health Statistics of Guilin Medical University, Guilin 541100, Guangxi, China; 2Department of Microbiology of Guangxi University, Nanning
530004, Guangxi, China; 3Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China
Correspondence: Xiang-yuan Yu (yhp268@163.com, guilinxiangyuan123@163.com) Bioscience Reports (2020) 40 BSR20200370
https://doi.org/10.1042/BSR20200370
Correspondence
Reply to Comments on ‘A functional polymorphism
rs10830963 in melatonin receptor 1B associated
with the risk of gestational diabetes mellitus’
Bo Huang1,*, Yu-kun Wang2,*, Lin-yuan Qin1, Qin Wei1, Nian Liu1, Min Jiang1, Hong-ping Yu3 and
Xiang-yuan Yu1
1Department of Epidemiology and Health Statistics of Guilin Medical University, Guilin 541100, Guangxi, China; 2Department of Microbiology of Guangxi University, Nanning
530004, Guangxi, China; 3Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China
Correspondence: Xiang-yuan Yu (yhp268@163.com, guilinxiangyuan123@163.com) Bioscience Reports (2020) 40 BSR20200370
https://doi.org/10.1042/BSR20200370 Th authors of ‘A functional polymorphism rs10830963 in melatonin receptor 1B associated
with the risk of gestational diabetes mellitus’ (Bioscience Reports (2019) 39, 12) have written
a reply in response to the correspondence piece by Rosta et al. (Bioscience Reports (2020)
40, 2). Reply to Comments on ‘A functional polymorphism
rs10830963 in melatonin receptor 1B associated
with the risk of gestational diabetes mellitus’
Bo Huang1 * Yu kun Wang2 * Lin yuan Qin1 Qin Wei1 Nian Liu1 Min Jiang1 Hong ping Yu3 and 1Department of Epidemiology and Health Statistics of Guilin Medical University, Guilin 541100, Guangxi, China; 2Department of Microbiology of Guangxi University, Nanning
530004, Guangxi, China; 3Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China 1Department of Epidemiology and Health Statistics of Guilin Medical University, Guilin 541100, Guangxi, China; 2Department of Microbiology of Guangxi University, Nanning
530004, Guangxi, China; 3Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China Correspondence: Xiang-yuan Yu (yhp268@163.com, guilinxiangyuan123@163.com) Correspondence: Xiang-yuan Yu (yhp268@163.com, guilinxiangyuan123@163.com) *These authors are considered
co-first authors.
Received: 11 February 2020
Revised: 12 February 2020
Accepted: 13 February 2020
Accepted Manuscript online:
13 February 2020
Version of Record published:
28 February 2020 © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY). © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4 0 (CC BY) Meanwhile, the variant rs10830963 G allele was es-
timated significantly associated with an increased GDM risk (CG vs. CC: OR = 1.44, 95% CI = 1.06−1.95;
GG vs. CC: OR = 2.06, 95% CI = 1.26−3.37; G vs. C: OR = 1.44, 95% CI = 1.16−1.78) in the meta-analysis
for Rosta et al.’s study data (Figures 1–3). There are slightly different of OR and corresponding 95% CI
from the original literature. Maybe it was caused by meta-analysis process for different algorithms with
stata software. © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4 0 (CC BY) 1 © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY). for
rols
4
3
1
2
8
9
3
8
3
1
2
12
26
89 of
trol
MAF
case/control
Mean age of
cases/controls
Mean BMI of
cases/controls
PHWE for
controls
0.52/0.41
31.8 +−4.6/29.7 +−3.5
23.6 +−3.0/21.5 +−2.4
0.84
6
0.52/0.45
33.1/32.2
23.3 +−4.0/21.4 +−2.9
0.53
9
0.46/0.43
30.0/32.0
21.5/21.7
0.81
0.41/0.28
35.4 +−4.4/31.3 +−5.2
25.8 +−5.1/26.7 +−6.2
0.02
7
0.47/0.35
32.6/29.9
26.3 +−4.7/24.1 +−3.8
0.98
0
0.45/0.40
32.4 +−4.8/31.9 +−5.2
25.3 +−5.2/24.6 +−4.6
0.79
0
0.54/0.44
28.7 +−3.1/28.1 +−2.4
NA/NA
0.43
2
0.38/0.29
34.1 +−6.1/34.8 +−15.1
24.3 +−4.9/23.7 +−4.2
0.48
5
0.42/0.45
28.2 +−3.8/27.9 +−4.1
21.2 +−1.8/20.7 +−1.4
0.53
3
0.28/0.20
32/29
32.0/25.4
0.11
4
0.51/0.44
31.6/32.1
24.4/25.2
0.02
7
0.39/0.31
31.7 +−4.5/29.2 +−5.0
25.1 +−5.5/23.0 +−4.0
0.112
9
0.35/0.31
31.8 +−4.8/29.4 +−4.8
25.7 +−5.9/22.9 +−4.5
0.426
3
0.36/0.28
Hungary:33.70/31.25;
Austria:32.04/30.51
Hungary:26.78/23.32;
Austria:28.31/23.40
0.989 http http I)
PEffect
Number of
studies
C
27)
<0.001
14
89)
<0.001
7
02)
<0.001
7 of Bioscience Reports (2020) 40 BSR20200370
https://doi.org/10.1042/BSR20200370 Figure 1. Forest plot on the risk of GDM associated with rs10830963 (CG vs. CC)
Figure 2. Forest plot on the risk of GDM associated with rs10830963 (GG vs. CC) Figure 1. Forest plot on the risk of GDM associated with rs10830963 (CG vs. CC) Figure 2. Forest plot on the risk of GDM associated with rs10830963 (GG vs. CC) 4 © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY). © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY). Bioscience Reports (2020) 40 BSR20200370
https://doi.org/10.1042/BSR20200370 Table 3 FPRP analysis for the significant associations of the MTNR1B rs10830963 C>G polymorphism and GDM risk Table 3 FPRP analysis for the significant associations of the MTNR1B rs10830963 C>G polymorphism and GDM risk
OR (95%CI)
Prior probability
0.25
0.1
0.01
0.001
0.0001
0.00001
Overall
CG vs. CC
1.29 (1.16–1.44)
0.002
0.005
0.056
0.375
0.857
0.984
GG vs. CC
1.88 (1.55–2.27)
0.002
0.007
0.070
0.433
0.884
0.987
G vs. C
1.37 (1.25–1.50)
0.001
0.004
0.038
0.286
0.800
0.976
Asian
CG vs. CC
1.15 (1.02–1.28)
0.057
0.153
0.664
0.952
0.995
1.000
GG vs. CC
1.52 (1.23–1.89)
0.003
0.009
0.092
0.506
0.911
0.990
G vs. C
1.23 (1.10–1.37)
0.003
0.010
0.097
0.519
0.915
0.991
Caucasian
CG vs. CC
1.50 (1.31–1.72)
0.002
0.007
0.074
0.446
0.889
0.988
GG vs. CC
2.45 (1.99–3.02)
0.016
0.047
0.351
0.845
0.982
0.998
G vs. C
1.55 (1.41–1.71)
0.002
0.005
0.056
0.375
0.857
0.984
Figure 3. Forest plot on the risk of GDM associated with rs10830963 (G vs. C) Table 3 FPRP analysis for the significant associations of the MTNR1B rs10830963 C>G polymorphism and GDM risk
OR (95%CI)
Prior probability
0.25
0.1
0.01
0.001
0.0001
0.00001
Overall
CG vs. CC
1.29 (1.16–1.44)
0.002
0.005
0.056
0.375
0.857
0.984
GG vs. CC
1.88 (1.55–2.27)
0.002
0.007
0.070
0.433
0.884
0.987
G vs. C
1.37 (1.25–1.50)
0.001
0.004
0.038
0.286
0.800
0.976
Asian
CG vs. CC
1.15 (1.02–1.28)
0.057
0.153
0.664
0.952
0.995
1.000
GG vs. © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY). Competing Interests Competing Interests The authors declare that there are no competing interests associated with the manuscript. Abbreviations BMI, body mass index; CI , confidence interval; FPRP , false positive report probability; GDM, gestational diabetes mellitus;
MAF , minor allele frequency; OR, odds ratio; SNP , single nucleotide polymorphism. BMI, body mass index; CI , confidence interval; FPRP , false positive report probabilit BMI, body mass index; CI , confidence interval; FPRP , false positive report probability; GDM, gestational diabe
MAF , minor allele frequency; OR, odds ratio; SNP , single nucleotide polymorphism. yi
y
g
MAF , minor allele frequency; OR, odds ratio; SNP , single nucleotide polymorphism. MAF , minor allele frequency; OR, odds ratio; SNP , single nucleotide polymorphism http CC
1.52 (1.23–1.89)
0.003
0.009
0.092
0.506
0.911
0.990
G vs. C
1.23 (1.10–1.37)
0.003
0.010
0.097
0.519
0.915
0.991
Caucasian
CG vs. CC
1.50 (1.31–1.72)
0.002
0.007
0.074
0.446
0.889
0.988
GG vs. CC
2.45 (1.99–3.02)
0.016
0.047
0.351
0.845
0.982
0.998
G vs. C
1.55 (1.41–1.71)
0.002
0.005
0.056
0.375
0.857
0.984
Figure 3. Forest plot on the risk of GDM associated with rs10830963 (G vs. C) Table 3 FPRP analysis for the significant associations of the MTNR1B rs10830963 C>G po or the significant associations of the MTNR1B rs10830963 C>G polymorphism and GDM risk gure 3. Forest plot on the risk of GDM associated with rs10830963 (G vs. C) Figure 3. Forest plot on the risk of GDM associated with rs10830963 (G vs. C) In epidemiological research, it is necessary to clarify the general demographic characteristics, and we
have also carried out extraction and display in Tables 1–3. For the mean pre-pregnancy body mass index
(BMI) and mean age values with the subjects, we have re-extracted and supplemented in the Table 1. The
mean age of cases/controls were 32.04/30.51 in subjects of Austria and 33.70/31.25 of Hungary. Mean-
while, the mean BMI of cases/controls were 28.31/23.40 in Austria and 26.78/23.32 in Hungary (Table
1). Thank you very much again for Klara Rosta, M.D., Ph.D., G´abor Firneisz, M.D., Ph.D., et al.’s thoughtfulness and
preciseness. Your comments means a great deal to us. Next, we will improve our study work together with the editors
of ‘Bioscience Reports’. 5 © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4 0 (CC BY) © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY). Bioscience Reports (2020) 40 BSR20200370
https://doi.org/10.1042/BSR20200370 Bioscience Reports (2020) 40 BSR20200370
https://doi.org/10.1042/BSR20200370 © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY). References 1 Huang, B., Wang, Y.K., Qin, L.Y., Wei, Q., Liu, N., Jiang, M. et al. (2019) A functional polymorphism rs10830963 in melatonin receptor 1B associated with
the risk of gestational diabetes mellitus. Biosci. Rep. 39, pii: BSR20190744, https://doi.org/10.1042/BSR20190744 1 Huang, B., Wang, Y.K., Qin, L.Y., Wei, Q., Liu, N., Jiang, M. et al. (2019) A functional polymorphism rs10830963 in melatonin receptor 1B associated with
the risk of gestational diabetes mellitus. Biosci. Rep. 39, pii: BSR20190744, https://doi.org/10.1042/BSR20190744 the risk of gestational diabetes mellitus. Biosci. Rep. 39, pii: BSR20190744, https://doi.org/10.1042/BSR20190744
2 Rosta, K., Harreiter, J., N´emeth, L., Kautzky-Willer, A., Somogyi, A. and Firneisz, G. (2020) Comments on “A functional polymorphism rs10830963 in
melatonin receptor 1B associated with the risk of gestational diabetes mellitus”. Biosci. Rep. pii: BSR20194316, https://doi.org/10.1042/BSR20194316
3 Lau, J., Ioannidis, J.P. and Schmid, C.H. (1997) Quantitative synthesis in systematic reviews. Ann. Intern. Med. 127, 820–826,
https://doi.org/10.7326/0003-4819-127-9-199711010-00008 2 Rosta, K., Harreiter, J., N´emeth, L., Kautzky-Willer, A., Somogyi, A. and Firneisz, G. (2020) Comments on “A functional polymorphism rs10830963 in
melatonin receptor 1B associated with the risk of gestational diabetes mellitus”. Biosci. Rep. pii: BSR20194316, https://doi.org/10.1042/BSR20194316 3 Lau, J., Ioannidis, J.P. and Schmid, C.H. (1997) Quantitative synthesis in systematic reviews. Ann. Intern. Med. 127, 820–826,
https://doi.org/10.7326/0003-4819-127-9-199711010-00008 3 Lau, J., Ioannidis, J.P. and Schmid, C.H. (1997) Quantitative synth
https://doi.org/10.7326/0003-4819-127-9-199711010-00008 4 DerSimonian, R. and Laird, N. (1986) Meta-analysis in clinical trials. Control. Clin. Trials 7, 177–188, https://doi.org/10.1016/0197-2456(86)90046-2
5 Wacholder, S., Chanock, S., Garcia-Closas, M. et al. (2004) Assessing the probability that a positive report is false: an approach for molecular
epidemiology studies. J. Natl. Cancer Inst. 96, 434–442, https://doi.org/10.1093/jnci/djh075 5 Wacholder, S., Chanock, S., Garcia-Closas, M. et al. (2004) Assessing the probability that a positive report is false: an approach for molecular
epidemiology studies. J. Natl. Cancer Inst. 96, 434–442, https://doi.org/10.1093/jnci/djh075 6 He, J., Wang, M.Y., Qiu, L.X. et al. (2013) Genetic variations of mTORC1 genes and risk of gastric cancer in an Eastern Chinese population. Mol. Carcinog. 52, E70–E79, https://doi.org/10.1002/mc.22013 7 Rosta, K., Al-Aissa, Z., Hadarits, O. et al. (2017) Association study with 77 SNPs confirms the robust role for the rs10830963/G of MTNR1B variant and
identifies two novel associations in gestational diabetes mellitus development. PLoS ONE 12, e0169781, https://doi.org/10.1371/journal.pone.0169781 6 © 2020 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution
License 4.0 (CC BY).
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Neoadjuvant PD-1 blockade: a promising nonoperative strategy for mismatch repair–deficient, locally advanced rectal cancer
|
Signal transduction and targeted therapy
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RESEARCH HIGHLIGHT
OPEN
Neoadjuvant PD-1 blockade: a promising nonoperative
strategy for mismatch repair–deficient, locally advanced rectal
cancer The work aimed to present preliminary
evidence
for
the
revolutionary
therapeutic
transition
from
neoadjuvant chemotherapy/radiotherapy followed by surgery to
immunotherapy followed by nonoperative management in this
subgroup of patients.1 g
p
p
Patients enrolled in this study were diagnosed with dMMR, stage
II or III rectal cancer. The detailed enrollment criteria included age
greater than 18 years, no signs of distant metastases, an Eastern
Cooperative Oncology Group (ECOG) performance status score of 0
or 1, and no previous exposure to immunotherapy, chemotherapy,
or radiation for the rectal tumor. The primary endpoints reported
here included the overall response to neoadjuvant dostarlimab
therapy and 1-year sustained clinical complete response (cCR) after
completion of dostarlimab therapy. Sixteen patients were recruited
and treated with dostarlimab. Twelve of these patients have
received the drug for longer than 6 months and have completed
the nine planned cycles of dostarlimab. The percentage of the 12
consecutive patients achieving a cCR was 100% (95% confidence
interval [CI], 74 to 100), and tumor eradication was observed using
endoscopy,
rectal
magnetic
resonance
imaging,
and
18F-
fluorodeoxyglucose–positron-emission tomography. During the
median follow-up period of one year, no patients required surgery,
radiotherapy or chemotherapy. To date, 4 patients have achieved
12 months of sustained cCR after the completion of dostarlimab
treatment alone. Acceptable toxicity occurred in 12 of the 16
patients (75%; 95% CI, 48 to 92) without any grade 3 or higher
adverse events. py
Lynch syndrome (LS), one of the most common hereditary
cancer syndromes, causes 3–5% of MSI CRCs.3 Controversy exists
regarding whether LS CRC is more sensitive to immunotherapy. Although some studies indicated no significant difference in the
immune response between patients with sporadic dMMR CRC and
LS CRC in small subgroup analyses,4,5 other studies showed that
this patient population tends to exhibit stronger immunological
reactions due to the increased somatic mutational load, greater
neoantigen burden and more abundant infiltrating lymphocytes
in the tumor.3 The percentages of LS CRC in the NICHE trial and
Cercek’s study were 41 and 57%, respectively. Therefore, LS CRC
might represent a confounding factor, contributing to the high
response rate of immunotherapy. Larger prospective studies
specifically assessing the response to immunotherapy of patients
with LS CRC are needed. In addition, the response of dMMR, LARCs to PD-1 inhibitors is
critically different from that of metastatic MSI-H/dMMR CRCs. Signal Transduction and Targeted Therapy Signal Transduction and Targeted Therapy Signal Transduction and Targeted Therapy www.nature.com/sigtrans Received: 30 June 2022 Revised: 30 July 2022 Accepted: 20 September 2022
1Department of Medical Oncology, Key Laboratory of Cancer Prevention and Intervention, the Second Affiliated Hospital of Zhejiang University School of Medicine, 310009
Hangzhou, Zhejiang, China and 2Cancer Center, Zhejiang University, 310058 Hangzhou, Zhejiang, China
Correspondence: Chenyang Ye (yechenyang@zju.edu.cn) or Ying Yuan (yuanying1999@zju.edu.cn) RESEARCH HIGHLIGHT
OPEN
Neoadjuvant PD-1 blockade: a promising nonoperative
strategy for mismatch repair–deficient, locally advanced rectal
cancer Mi Mi1,2, Chenyang Ye
1,2✉and Ying Yuan
1,2✉
Signal Transduction and Targeted Therapy (2022) 7:361 ; https://doi.org/10.1038/s41392-022-01216-3 ; https://doi.org/10.1038/s41392-022-01216-3 neoadjuvant immunotherapy for CRC, in which a single dose of
ipilimumab combined with two doses
of nivolumab were
administered before surgery, with a response rate of 100% and
pathological complete response (pCR) rate of 69% in 32 patients
with dMMR, stage III CRC, and all patients underwent radical
resections without delay (in 6 weeks).2 In this study, Cercek et al. reported treatment with single-agent dostarlimab (lasted for
6 months) with a 100% cCR in 12 patients with dMMR, LARC who
had not undergone radiation or surgery. The remarkable results of
these studies add to the literature about the potential efficacy of
neoadjuvant
immunotherapy. However,
the
difference
in
responses
among
these
studies
prompted
us
to
consider
establishing more appropriate subgroups that might benefit from
immunotherapy. neoadjuvant immunotherapy for CRC, in which a single dose of
ipilimumab combined with two doses
of nivolumab were
administered before surgery, with a response rate of 100% and
pathological complete response (pCR) rate of 69% in 32 patients
with dMMR, stage III CRC, and all patients underwent radical
resections without delay (in 6 weeks).2 In this study, Cercek et al. reported treatment with single-agent dostarlimab (lasted for
6 months) with a 100% cCR in 12 patients with dMMR, LARC who
had not undergone radiation or surgery. The remarkable results of
these studies add to the literature about the potential efficacy of
neoadjuvant
immunotherapy. However,
the
difference
in
responses
among
these
studies
prompted
us
to
consider
establishing more appropriate subgroups that might benefit from
immunotherapy. In a prospective phase 2 study recently published in The New
England Journal of Medicine, Cercek et al. investigated the efficacy
of the programmed death 1 (PD-1) inhibitor dostarlimab in
patients with mismatch repair–deficient (dMMR), locally advanced
rectal cancer (LARC). The work aimed to present preliminary
evidence
for
the
revolutionary
therapeutic
transition
from
neoadjuvant chemotherapy/radiotherapy followed by surgery to
immunotherapy followed by nonoperative management in this
subgroup of patients.1 In a prospective phase 2 study recently published in The New
England Journal of Medicine, Cercek et al. investigated the efficacy
of the programmed death 1 (PD-1) inhibitor dostarlimab in
patients with mismatch repair–deficient (dMMR), locally advanced
rectal cancer (LARC). Received: 30 June 2022 Revised: 30 July 2022 Accepted: 20 September 2022 RESEARCH HIGHLIGHT
OPEN
Neoadjuvant PD-1 blockade: a promising nonoperative
strategy for mismatch repair–deficient, locally advanced rectal
cancer The
results of the KEYNOTE-177 trial showed that the percentage of
patients with metastatic dMMR CRCs who had not previously
received any treatment and achieved an imaging-based full
response was 11.1%,6 which inspired us to ask why these localized
dMMR rectal tumors respond much more robustly than metastatic
MSI-H/dMMR CRCs. In addition, considering the complexity and
heterogeneity of CRC, this study also provides a new idea for
selecting immunotherapy based on the anatomic location of
colorectal tumors. Although MSI-H is more common in right-side
colon cancer than in left-side CRCs, a subgroup analysis based on
KEYNOTE-177
was
not
sufficiently
powerful
to
assess
the
differences between these two cancer types in terms of the Patients with stage II or III rectal cancer routinely undergo
neoadjuvant chemoradiotherapy followed by surgery. The quality
of life of patients is permanently impacted by both surgery and
chemoradiotherapy, affecting bowel and bladder function, as well
as fertility. Some patients with lower rectal cancer will be surgically
equipped
with
a
permanent
artificial
anus
for
life. Since
immunotherapy has been approved as a first-line treatment for
metastatic dMMR colorectal cancer (CRC), researchers are debat-
ing whether immunotherapy could be used to improve the
treatment modalities for dMMR, locally advanced CRCs. Several
studies have explored the merits of neoadjuvant immunotherapy
for dMMR, locally advanced CRCs (Fig. 1a). NICHE is the first trial of © The Author(s) 2022 Neoadjuvant PD-1 blockade: a promising nonoperative strategy for mismatch. . . Mi et al. 2 Fig. 1
PD-1 blockade as neoadjuvant therapy in DNA dismatch repair–deficient colorectal cancers. a Summary of published clinical trials of
neoadjuvant immunotherapy in dMMR, locally advanced CRCs. b The administration of anti-PD-1 antibody dostarlimab achieved clinical
complete remission in the patients with dMMR, locally advanced rectal cancer. dMMR mismatch repair-deficiency, CRC colorectal cancer, pCR
pathological complete response, cCR clinical complete response, PD-1 programmed death protein 1, PD-L1 programmed death ligand-1. Panel b was generated on Biorender.com Fi
1
PD 1 bl
k d
dj
t th
i
DNA di
t h
i
d fii
t
l
t l
S
f
bli h d li i
l t i Fig. 1
PD-1 blockade as neoadjuvant therapy in DNA dismatch repair–deficient colorectal cancers. a Summary of published clinical trials of
neoadjuvant immunotherapy in dMMR, locally advanced CRCs. b The administration of anti-PD-1 antibody dostarlimab achieved clinical
complete remission in the patients with dMMR, locally advanced rectal cancer. RESEARCH HIGHLIGHT
OPEN
Neoadjuvant PD-1 blockade: a promising nonoperative
strategy for mismatch repair–deficient, locally advanced rectal
cancer dMMR mismatch repair-deficiency, CRC colorectal cancer, pCR
pathological complete response, cCR clinical complete response, PD-1 programmed death protein 1, PD-L1 programmed death ligand-1. Panel b was generated on Biorender.com efficacy of immunotherapy. In addition, researchers have not
clearly determined whether the differences between rectal and
colon cancer caused the discrepancy in the efficacy of immu-
notherapy in these two cancer subgroups. Cercek and colleagues
speculated that in addition to tumor cell-intrinsic factors, such as
clonality, aneuploidy, and mutation class, the gut microbiome
might be a driving factor.1,6 The microbiota has been reported to
play a crucial role in determining the host immunologica
response in patients with CRC. However, Cercek and colleagues
have not analyzed the gut microbiome of patients in their study. If
possible, additional microbiome tests should be considered for
patients enrolled later. the single-agent dostarlimab, a PD-1 inhibitor, in patients with
dMMR, locally advanced rectal cancer. Although it is a small and
single-center study, its unprecedented results with a 100% clinical
complete response suggest the possibility of extending beyond
standard treatment for certain rectal cancers, such as lower rectal
cancer, with the need for organ preservation. In addition, in the
future, neoadjuvant PD-1 blockade must be evaluated in other
dMMR tumors, such as localized pancreatic cancer, gastric cancer
and prostate cancer. ADDITIONAL INFORMATION Competing interests: The authors declare no competing interests. ACKNOWLEDGEMENTS This work was supported by a research grant from the National Natural Science
Foundation of China (82103102 to C.Y.Y.), the Provincial Key R&D Program of
Zhejiang Province (2021C03125 to Y.Y.). For a number of reasons, the results from this study do not yet
affect practice. First, this study includes a small sample and was
conducted at a single center, which implies its limitations. Subgroup analyses of immunotherapy responses in patients with
LS CRCs and sporadic dMMR CRCs are not yet available. Second,
pathological complete response data are unavailable in this study,
and the radiographic response does not seem to accurately
predict the pathological complete response. Third, the median
follow-up was short, and the primary endpoint in this study was
the overall response, with no data on other clinically relevant
outcomes, such as 3-year disease-free survival. Overall, little is
known about the period needed to determine whether a clinical
full response to dostarlimab is equivalent to a clinical cure, and
immunotherapy
combined
with
chemotherapy
or
radiation
therapy may be required for patients with local or distant
recurrence. Finally, this study only reported data for dostarlimab,
and the potential clinical indications of other PD-1 and PD-L1
antibodies, such as nivolumab, pembrolizumab, and avelumab, in
patients with rectal cancer presenting dMMR are not clear. Further
studies are expected. Signal Transduction and Targeted Therapy (2022) 7:361 Neoadjuvant PD-1 blockade: a promising nonoperative strategy for mismatch. . .
Mi et al REFERENCES 1. Cercek, A. et al. PD-1 blockade in mismatch repair-deficient, locally advanced
rectal cancer. N. Engl. J. Med. 386, 2363–2376 (2022). 1. Cercek, A. et al. PD-1 blockade in mismatch repair-deficient, locally advanced
rectal cancer. N. Engl. J. Med. 386, 2363–2376 (2022). 2. Verschoor, Y. L. et al. Neoadjuvant nivolumab, ipilimumab, and celecoxib in MMR-
proficient and MMR-deficient colon cancers: final clinical analysis of the NICHE
study. J. Clin. Oncol. 40, 3511–3511 (2022). 2. Verschoor, Y. L. et al. Neoadjuvant nivolumab, ipilimumab, and celecoxib in MMR-
proficient and MMR-deficient colon cancers: final clinical analysis of the NICHE
study. J. Clin. Oncol. 40, 3511–3511 (2022). 3. Sinicrope, F. A. Lynch syndrome-associated colorectal cancer. N. Engl. J. Med. 379,
764–773 (2018). 3. Sinicrope, F. A. Lynch syndrome-associated colorectal cancer. N. Engl. J. Med. 379,
764–773 (2018). 4. Overman, M. J. et al. Durable clinical benefit with nivolumab plus ipilimumab in
DNA mismatch repair-deficient/microsatellite instability-high metastatic colorectal
cancer. J. Clin. Oncol. 36, 773–779 (2018). 5. Le, D. T. et al. Mismatch repair deficiency predicts response of solid tumors to PD-1
blockade. Science 357, 409–413 (2017). p
Improving the clinical outcomes of colorectal cancer has been a
grand challenge. Cercek et al. provide insights into the efficacy of 6. Andre, T. et al. Pembrolizumab in microsatellite-instability-high advanced color-
ectal cancer. N. Engl. J. Med. 383, 2207–2218 (2020). Signal Transduction and Targeted Therapy (2022) 7:361 Neoadjuvant PD-1 blockade: a promising nonoperative strategy for mismatch. . . Mi et al. 3 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 license, and indicate if changes were made. The images or other third party
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indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
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from
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holder. To
view
a
copy
of
this
license,
visit
http://
creativecommons.org/licenses/by/4.0/. © The Author(s) 2022
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https://risetpress.com/index.php/jimat/article/download/664/464
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Indonesian
| null |
Pengaruh Disiplin Kerja dan Pengawasan terhadap Peningkatan Produktivitas Kerja Karyawan CV. Chantika Lestari Panyabungan
|
Jurnal Riset Multidisiplin dan Inovasi Teknologi
| 2,024
|
cc-by-sa
| 2,030
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Jurnal Riset Multidisiplin dan Inovasi Teknologi
E-ISSN 3024-8582 P-ISSN 3024-9546
Volume 1 Issue 02, September 2023, Pp. 154-159
DOI: https://doi.org/10.59653/jimat.v2i02.664
Copyright by Author Jurnal Riset Multidisiplin dan Inovasi Teknologi
E-ISSN 3024-8582 P-ISSN 3024-9546
Volume 1 Issue 02, September 2023, Pp. 154-159
DOI: https://doi.org/10.59653/jimat.v2i02.664
Copyright by Author Abstract This research aims to determine and analyze the influence of work discipline and supervision
on employee work productivity at CV. Chantika Lestari Panyabungan. This research uses
quantitative methods with the main problem being whether work discipline and supervision
have a positive and significant effect on increasing employee work productivity at CV. Chantika Lestari Connection. The population in this study was the entire number of CV
employees. Chantika Lestari Panyabungan numbered 56 people and the results were analyzed
using Multiple Linear Regression. The results of the research show that work discipline does
not have a significant effect on employee work productivity, but the supervision carried out has
a positive impact and has a significant effect on CV employee work productivity. Chantika
Lestari Panyabungan. Keywords: Work Discipline, Supervision, Work Productivity Pengaruh Disiplin Kerja dan Pengawasan terhadap Peningkatan
Produktivitas Kerja Karyawan CV. Chantika Lestari
Panyabungan Ahmad Sayuti1*, Abdelina Nasution2, Makhrani3
Universitas Graha Nusantara, Indonesia1
Universitas Graha Nusantara, Indonesia2
Universitas Graha Nusantara, Indonesia3
Corresponding Email: sayutia62@gmail.com* Abstrak Penelitian ini bertujuan untuk mengetahui dan menganalisa pengaruh disiplin kerja dan
pengawasan terhadap produktivitas kerja karyawan pada CV. Chantika Lestari Panyabungan. Penelitian ini menggunakan metode kuantitatif dengan masalah utama adalah apakah disiplin
kerja dan pengawasan berpengaruh secara positif dan signifikan terhadap peningkatan
produktivitas kerja karyawan pada CV. Chantika Lestari Penyabungan. Populasi pada
penelitian ini adalah seluruh jumlah karyawan CV. Chantika Lestari Panyabungan yang
berjumlah sebanyak 56 orang dan hasilnya di analisis dengan menggunakan Regresi Linier
Berganda. Hasil penelitian menunjukkan bahwa Disiplin Kerja tidak berpengaruh signifikan
terhadap Produktivitas Kerja Karyawan tetapi Pengawasan yang dilakukan tersebut berdampak 154 positif dan berpengaruh signifikan terhadap Produktivitas Kerja Karyawan CV. Chantika
Lestari Panyabungan. Kata kunci: Disiplin Kerja, Pengawasan, Produktivitas Kerja Kata kunci: Disiplin Kerja, Pengawasan, Produktivitas Kerja Pendahuluan Lajunya pertumbuhan ekonomi mengakibatkan kian kompleknya masalah yang ada di dalam
suatu perusahaan, mendapatkan sumber daya manusia yang kompeten dan ahli di bidangnya adalah
salah satunya, banyak konsep dan cara dilakukan untuk mendapatkan karyawan yang memiliki
keahlian dan profesionalisme tinggi dalam bekerja(Sauri et al., 2023). Begitu banyak perubahan
pola pikir para ahli ekonomi mengenai pentingnya sumber daya manusia dalam suatu perusahaan,
ada yang menciptakan teori bahwa sesungguhnya karyawan hanyalah suatu bagian dari perusahaan
yang harus dioptimalkan layaknya sebuah mesin dan tak ada bedanya dengan sumber daya lain
yang memang harus dioptimalkan, namun tak sedikit pula para ahli yang menciptakan teori bahwa
karyawan adalah sebuah asset bagi perusahaan sehingga harus diperlakukan sedemikian rupa. Hal ini didasarkan bahwa karyawan adalah sumber daya yang selalu berbeda setiap
individunya dan memiliki keinginan yang tak bisa disamakan dengan sumber daya lain. Dilihat
dari tujuan perusahaan dan tujuan karyawan dalam menjalankan pekerjaannya diharapkan tidak
ada kesenjangan antara keduanya, untuk itu perusahaan harus berusaha untuk menyelesaikan
antara tujuan karyawan dengan tempat mereka bekerja apakah terdapat hubungan timbal balik
atau sebaliknya (Fathurohman, 2023). Apabila berhasil mencapai keuntungan, berarti karyawan akan terjamin kelangsungan
pendapatannya, mereka akan merasa aman dan hal ini akan dapat mendorong karyawan
melaksanakan pekerjaannya dengan penuh konsentrasi (Yanti & Pidada, 2023). Tujuan
perusahaan tidak akan tercapai tanpa peran aktif tenaga kerja yang terampil dan disiplin,
meskipun alat-alat yang dimiliki perusahaan begitu canggihnya. Mengatur karyawan sangat
sulit dan kompleks karena mereka mempunyai pikiran, perasaan, status, keraguan dan latar
belakang yang heterogen. Sehingga tenaga kerja tidak dapat diatur dan dikuasai sepenuhnya
seperti mengatur mesin, modal, dan gedung. Dalam usahanya untuk menghindari perilaku yang tidak diharapkan dan untuk
meningkatkan produktivitas kerjanya, perusahaan harus dapat menetapkan sanksi terhadap
pelanggaran peraturan, sehingga perusahaan dapat menghindari pengulangan-pengulangan
perilaku yang tidak diinginkan tersebut. Dampaknya terhadap para karyawan adalah bahwa
disiplin kerjanya dapat ditingkatkan dan dengan begitu produktivitas kerjanya juga meningkat. Untuk mendapatkan suatu hasil produk yang baik dan bermutu tinggi maka diperlukan tak
hanya dibutuhkan disiplin kerja saja, tetapi juga membutuhkan pengawasan yang baik. Pengawasan adalah kegiatan manajer yang mengusahakan agar pekerjaan-pekerjaan terlaksana
sesuai dengan rencana yang ditetapkan dan atau hasil yang dikehendaki (Lubis, 2001:154). Produktivitas kerja sering diartikan sebagai kemampuan seseorang atau kelompok orang
untuk menghasilkan barang dan jasa. Pendahuluan Seorang karyawan yang produktif adalah karyawan yang
cekatan dan mampu menghasilkan barang dan jasa sesuai mutu yang ditetapkan dan waktu 155 yang lebih singkat, sehingga akhirnya dapat tercapai tingkat produktivitas kerja karyawan yang
tinggi. Dengan demikian penting bagi seorang manajer berusaha untuk meningkatkan
produktivitas kerja karyawan, agar perusahaan dapat berkembang dan dapat mempertahankan
usahanya (Lubis, 1985:154). Faktor pengawasan dan disiplin kerja merupakan faktor yang penting bagi perusahaan
dalam mencapai tujuan yang telah ditetapkan. Oleh karena itu manajer harus melakukan
pengawasan kerja yang efektif, maka dengan sendirinya disiplin kerja karyawanpun akan baik
sehingga karyawan bisa mencapai prestasi kerja yang optimal dalam bentuk produktivitas
kerja. Fakta yang ada dilap angan menunjukkan adanya gejala-gejala yang cenderung terjadi
penurunan produktivitas kerja para karyawan bagian produksi. Hal ini disebabkan kurangnya
pengawasan yang efektif dan kurangnya sikap disiplin kerja karyawan. Untuk itu dalam
meningkatkan produktivitas kerja karyawan manajer harus melakukan pengawasan yang baik
sehingga sikap disiplin kerja dalam diri karyawan muncul (Kurniawati et al., 2023). Dengan demikian karyawan akan mengerjakan pekerjaan dengan baik dan disiplin yang
tinggi. Pengaruh pengawasan kerja dan disiplin kerja terhadap karyawan menjadi sangat
penting (Anwar & Abrar, 2023). Perusahaan perlu meningkatkan pengawasan yang efektif
sehingga sikap disiplin karyawan juga baik, untuk memacu produktivitas kerja karyawan. Karena apabila karyawan dalam bekerja ada pengawasan kerja yang efektif dari manajer maka
semangat kerja akan timbul dan para karyawan akan bekerja dengan rajin, disiplin, baik dan
bertanggung jawab sehingga produktivitas kerja akan meningkat dengan sendirinya. Berdasarkan latar belakang permasalahan yang didukung dengan teori-teori tersebut, penulis
tertarik untuk melakukan penelitian dengan judul Pengaruh Disiplin Kerja dan Pengawasan
terhadap Peningkatan Produktivitas Kerja Karyawan pada CV. Chantika Lestari Panyabungan. Instrument yang digunakan dalam pengumpulan data sebagai berikut Metode Penelitian Observasi yaitu dengan melakukan pengamatan langsung di lokasi penelitian. Metode Penelitian Jenis penelitian yang dilakukan adalah penelitian asosiatif (pengaruh), karena peneliti
ingin mengetahui pengaruh antara variabel X1 dan X2 (disiplin kerja dan pengawasan) dengan
variabel Y (produktivitas kerja). Dan hasil penelitian asosiatif berfungsi untuk menjelaskan,
meramalkan dan mengontrol suatu gejala (Sugiyono, 2006). Populasi adalah jumlah dari
keseluruhan objek penelitian yang menjadi sumber data. Dimana yang menjadi populasi dalam
penelitian ini adalah karyawan CV. Chantika Lestari Panyabungan yang berjumlah 56 orang. Dalam menetapkan sampel penulis berpedoman kepada pendapat (Suharsimi Arikunto,
2007: 125) adalah sebagai berikut Apabila subjeknya kurang dari 100 orang lebih baik diambil
semua sehingga penelitian merupakan penelitian populasi, selanjutnya jika jumlah subjeknya
besar dapat diambil antara 10%-15% atau 20%- 25% atau lebih. Sehingga penulis mengambil
sampel seluruhnya dari populasi, karena jumlah populasinya tidak sampai 100 orang, maka
sampel dalam penelitian ini sebanyak 56 orang yang diambil secara keseluruhan atau yang
disebut dengan populasi sampling. Instrument yang digunakan dalam pengumpulan data sebagai berikut: 156 1. Angket, yaitu merupakan teknik pengumpulan data yang dilakukan dengan cara
memberi seperangkat pertanyaaan atau pernyataan tertulis kepada responden untuk
dijawab. Angket yang berupa pertanyaan-pertanyaan tertulis dengan yang memiliki 5
(lima) jawaban alternatif dengan bobot nilai yang berbeda setiap jawaban akan
diberikan kepada seluruh sampel sesuai dengan pengalaman yang dirasakan oleh
seluruh sampel. Adapun bobot setiap jawaban adalah sebagai berikut: 1. Angket, yaitu merupakan teknik pengumpulan data yang dilakukan dengan cara
memberi seperangkat pertanyaaan atau pernyataan tertulis kepada responden untuk
dijawab. Angket yang berupa pertanyaan-pertanyaan tertulis dengan yang memiliki 5
(lima) jawaban alternatif dengan bobot nilai yang berbeda setiap jawaban akan
diberikan kepada seluruh sampel sesuai dengan pengalaman yang dirasakan oleh
seluruh sampel. Adapun bobot setiap jawaban adalah sebagai berikut: 1. Angket, yaitu merupakan teknik pengumpulan data yang dilakukan dengan cara
memberi seperangkat pertanyaaan atau pernyataan tertulis kepada responden untuk
dijawab. Angket yang berupa pertanyaan-pertanyaan tertulis dengan yang memiliki 5
(lima) jawaban alternatif dengan bobot nilai yang berbeda setiap jawaban akan
diberikan kepada seluruh sampel sesuai dengan pengalaman yang dirasakan oleh
seluruh sampel. Adapun bobot setiap jawaban adalah sebagai berikut: Tabel 1. Skor Pernyataan
No
Jawaban Pertanyaan
Skor
1
Sangat Setuju (SS)
5
2
Setuju (S)
4
3
Kurang Setuju (KS)
3
4
Tidak Setuju (TS)
2
5
Sangat Tidak Setuju
1 2. Wawancara, yaitu merupakan teknik pengumpulan data yang dilakukan melalui tatap
muka dan tanya jawab langsung. 2. Wawancara, yaitu merupakan teknik pengumpulan data yang dilakukan melalui tatap
muka dan tanya jawab langsung. 3. Hasil dan Pembahasan Disiplin Kerja berpengaruh positif dan memiliki peranan penting dalam meningkatkan
pengawasan pada perusahaan CV. Chantika Lestari Panyabungan, melalui disiplin kerja
tersebut kinerja para karyawan diharapkan mampu memberikan pelayanan prima dalam
peningkatan produktivitas kerja karyawan CV. Chantika Lestari Panyabungan dan melalui
disiplin kerja tersebut kita dapat mengetahui sejauh mana tingkat kinerja para karyawan atas
pengawasan yang dilakukan CV. Chantika Lestari Panyabungan. Disiplin Kerja berpengaruh
positif terhadap pengawasan maka dapat disimpulkan secara singkat bahwasanya Disiplin kerja
membantu manajemen dalam meningkatkan kualitas dan kuantitas pencapaian tujuan yang
ditetapkan oleh organisasi. Berdasarkan hasil analisis dengan mengunakan uji koefisien determinasi 1 dan uji thitung
1, menunjukkan bahwa disiplin kerja berdampak positif dan berpengaruh signifikan terhadap
pengawasan pada CV. Chantika Lestari Panyabungan, Dimana disiplin kerja mempunyai nilai
thitung sebesar 3,630 > ttabel 1,674 yang artinya signifikan. Yang dimaksud dengan signifikan
adalah Ho ditolak dan Ha diterima yakni disiplin kerja memiliki peranan penting dalam
meningkatkan pengawasan. Sedangkan pada koefisien determinasi dan uji t substruktur 2,
dapat dilihat bahwa disiplin kerja tidak memiliki pengaruh yang signifikan terhadap
produktivitas kerja dimana disiplin kerja memiliki nilai thitung sebesar 1,432 < ttabel 1,674. Tetapi
nilai pada pengawasan sebesar 4,422 > ttabel 1,674 yang berarti pengawasan yang dilakukan CV. 157 Chantika Lestari Panyabungan berpengaruh signifikan terhadap produktivitas kerja karyawan
CV. Chantika Lestari Panyabungan. Chantika Lestari Panyabungan berpengaruh signifikan terhadap produktivitas kerja karyawan
CV. Chantika Lestari Panyabungan. Kesimpulan Berdasarkan hasil analisis yang telah dilakukan dalam penelitian ini, maka penulis
mengambil kesimpulan sebagai berikut: 1. Disiplin Kerja adalah kesadaran dan kesediaan mentaati peraturan dan norma-norma sosial
yang berlaku. 2. Variabel Disiplin Kerja (X1) berpengaruh positif dan signifikan terhadap Pengawasan (X2)
Pada Perusahaan CV. Chantika Lestari Panyabungan. 3. Disiplin Kerja tidak berpengaruh signifikan terhadap Produktivitas Kerja Karyawan tetapi
Pengawasan yang dilakukan tersebut berdampak positif dan berpengaruh signifikan
terhadap Produktivitas Kerja Karyawan CV. Chantika Lestari Panyabungan. 4. Identifikasi determinan adjusted (R2) substruktur 2 sebesar 0,367 berarti 36,7 %
Produktivitas Kerja Karyawan dapat dijelaskan oleh Disiplin Kerja dan Pengawasan,
sedangkan sisanya 63,3% dapat dijelaskan oleh faktorfaktor lain yang tidak diteliti dalam
penelitian ini. Referensi Anwar, S., & Abrar, U. (2023). The Influence of Compensation and Work Motivation on
Employee Performance through Employee Discipline. International Journal of
Multidisciplinary
Approach
Research
and
Science,
1(02). https://doi.org/10.59653/ijmars.v1i02.66 Arikunto, S. 2013.Prosedur Penelitian Suatu Pendekatan Praktik. Edisi Revisi. Jakarta: PT. Rineka Cipta Dessler, Gary. 2005, Manajemen Sumber Daya Manusia, Jakarta Dessler, Gary. 2004, Human Resources Management, Upper Saddler River, New Jersey:
Prentice Hall, Inc Fathurohman, H. (2023). Strategi Pengembangan Sumber Daya Manusia di Balai Latihan Kerja
Jember Dinas Tenaga Kerja dan Transmigrasi Provinsi Jawa Timur. Journal of
Community
Service
and
Society
Empowerment,
2(01
SE-Articles),
10–18. https://doi.org/10.59653/jcsse.v2i01.359 Ghozali, Imam. 2006, Aplikasi Analisis Multivariate dengan Program SPSS. Universita
Diponegoro: Semarang Handoko, Hani,.2000, Manajemen Personalia dan Sumber Daya Manusia, BPFE: Yogyakarta Hasibuan, Malayu. 2005, Manajemen sumber Daya Manusia, PT ALexmedia Koputindo:
Jakarta 158 Kurniawati, D., Kurdi, M., & Furqani, A. (2023). Implementation of Performance Allowance
Reduction through Work Discipline on Employee Performance at the Department of
Population and Civil Registration of Sumenep Regency. International Journal of
Multidisciplinary
Approach
Research
and
Science,
1(02). https://doi.org/10.59653/ijmars.v1i02.67 Maman, Ukas. 2004, Manajemen Konsep, Prinsip dan Aplikasi,Agnini: Bandung R.Terry, George. 2006. Prinsip- Prinsip Manajemen. Jakarta: Bumi Aksara Rivao, Veithzal. 2008, Manajemen Sumber Daya Manusia: dari teori ke praktik, Rajawali Pers:
Jakarta Sauri, S., Jamaludin, A., Rosita, D., Farha, H. M., & Jaelani, J. (2023). Peranan BUMDes
Dalam Pembangunan dan Pemberdayaan Masyarakat di Desa Taringgul Landeuh
Kecamatan Kiarapedes Kabupaten Purwakrata. Jurnal Riset Multidisiplin Dan Inovasi
Teknologi, 1(02). https://doi.org/10.59653/jimat.v1i02.236 Sugiono, 2005. Metode Penelitian Bisnis, Bandung: Alfabeta. Tjiptono, Fandy. 2005. Pemasaran Jasa. Edisi Pertama. Malang, Bayu Media Umar, Husein. 2001. Riset Pemasaran dan Prilaku Konsumen. Jakarta. Gramedia Pustaka
Utama. Yanti, N. K. D. D., & Pidada, I. B. A. (2023). Analisis Yuridis Penyaluran Tenaga Kerja
Indonesia: Studi Kasus di Provinsi Bali. Jurnal Riset Multidisiplin Dan Inovasi
Teknologi, 2(01 SE-Articles), 111–118. https://doi.org/10.59653/jimat.v2i01.363 159
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FRAZEOLOGIZMLARNING O'ZIGA XOS XUSUSIYATLARI VA PRAGMATIK TAHLILI
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FRAZEOLOGIZMLARNING O’ZIGA XOS XUSUSIYATLARI VA
PRAGMATIK TAHLILI FRAZEOLOGIZMLARNING O’ZIGA XOS XUSUSIYATLARI VA
PRAGMATIK TAHLILI Toshkent shahar
Yashnobod tumani
255- sonli umumiy o’rta ta’lim maktabi
Ingliz tili fani o'qituvchisi
Tuychiyeva Manzura Xojimurodovna
aruznam88@mail.ru
Annotatsiya. Ushbu maqola frazeologiyaning o'ziga xos xususiyatlari,
lingvomadaniy tamoyillari, diniy va dunyoviy qarashlari haqida keng ma'lumot beradi. Maqolada frazeologizmlarga oid turli birikmalar batafsil tahlil qilinadi. Kalit so'zlar: frazeologoya, lingvomadaniyat, lingvokulturologiya, pragmatika,
idioma
Abstract. This article provides extensive information about the peculiarities of
phraseology, linguistic and cultural principles, religious and secular views. The article
analyzes various combinations of phraseology in detail. Key words: phraseology, linguistic culture, linguistic culture, pragmatics, idiom. Frazeologiya ma’lum bir ma’noni bo‘rttirish va yoki uni ozaytirib ko‘rsatish demakdir. Agar bu jarayonni oddiy so‘zlarda ifodalasak,
yuz – bet - aft – bashara – turq
jamol – chehra – yuz kabilar hosil qilinishi mumkin. Toshkent shahar
Yashnobod tumani
255- sonli umumiy o’rta ta’lim maktabi
Ingliz tili fani o'qituvchisi
Tuychiyeva Manzura Xojimurodovna
aruznam88@mail.ru Toshkent shahar
Yashnobod tumani
255- sonli umumiy o’rta ta’lim maktabi
Ingliz tili fani o'qituvchisi
Tuychiyeva Manzura Xojimurodovna
aruznam88@mail.ru Annotatsiya. Ushbu maqola frazeologiyaning o'ziga xos xususiyatlari,
lingvomadaniy tamoyillari, diniy va dunyoviy qarashlari haqida keng ma'lumot beradi. Maqolada frazeologizmlarga oid turli birikmalar batafsil tahlil qilinadi. Kalit so'zlar: frazeologoya, lingvomadaniyat, lingvokulturologiya, pragmatika,
idioma Abstract. This article provides extensive information about the peculiarities of
phraseology, linguistic and cultural principles, religious and secular views. The article
analyzes various combinations of phraseology in detail. Key words: phraseology, linguistic culture, linguistic culture, pragmatics, idiom. Frazeologiya ma’lum bir ma’noni bo‘rttirish va yoki uni ozaytirib ko‘rsatish demakdir. Agar bu jarayonni oddiy so‘zlarda ifodalasak,
yuz – bet - aft – bashara – turq
jamol – chehra – yuz kabilar hosil qilinishi mumkin. jamol – chehra – yuz kabilar hosil qilinishi mumkin. Diskursda qo‘llanilishiga misol keltiramiz: 263 263 263 Ququbonu basharang qursin, degandek qo‘lini siltab qaytib chiqib ketmoqchi
bo‘lganida ortidan kirib kelayotgan mahalla xotin-qizlari raisi Ma’mura opaga
urilib ketdi107. Ququbonu basharang qursin, degandek qo‘lini siltab qaytib chiqib ketmoqchi
bo‘lganida ortidan kirib kelayotgan mahalla xotin-qizlari raisi Ma’mura opaga
urilib ketdi107. Ushbu ibora “basharang qursin” pozitiv holatda qo‘llanila olmasligini o‘zi diskurs
jarayonini tashkil etadi. Shuningdek, ushbu frazemani yuzing qursin, beting qursin deb
almashtira olmaymiz ham. Chunki hikoyadagi kayfiyat, yozuvchi tabiati bizdan shuni talab
qilmoqda. Yoki quyidagi namunalarga diqqat qiling. O‘lmoq so‘zi joni uzilmoq – dunyodan ketmoq – foniy dunyodan rihlat qilmoq,
omonatini topshirmoq kabi frazeologik intensifikatorlarga ega Ingliz tilida esa: Die – pass away - depart this life - meet your end - push up the daisies - go to glory -
relinquish life ga tog‘ri keladi. E’tibor beradigan bo‘lsak, “omonatini topshirmoq” frazemasi “o‘lmoq” so‘zidan ko‘ra
anchagina keng ma’noni ifodalaydi. Millatning dinidan kelib chiqadigan bo‘lsak, inson joni – bu omonat, demak ushbu
frazema omonatni o‘z berguvchisiga, ya’ni Haqqa topshirmoq zaruratini anglatadi. “Keyingi paytlarda «xizmat vazifasini ado etayotgan paytda...» degan jumlaga ko‘p
ko‘z tushyapti. Odatda bu ta’rif Vatan sarhadlarini qo‘riqlayotgan yoki el osoyishtaligini
ta’min etayotgan askarlarga nisbatan ishlatilardi. Bugun ayni nisbat shifokorlarga
qo‘llanilayotir. Jahonni zabtiga olgan tojsimon virus ongu shuurimizni o‘zgartirib yubordi. Endilikda
shifokorlar aynan ko‘rinmas yovga birinchi qalqon vazifasini o‘tashmoqda. Ular o‘z moli
yoki mansabini emas, balki aziz jonini garovga qo‘yib, bemorlarni hayotga qaytarishga
kechayu kunduz urinmoqdalar. Taassufki, urush qurbonsiz bo‘lmaydi. Ko‘rinmas yov bilan 264
107 O‘KTАM MIRZАYOR. SOVG‘А (HIKOYА), 5-qism
https://n.ziyouz.com/portаl-hаqidа/xаritа/uzbek-nаsri/o-ktаm-mirzаyor-1956/o-ktаm-mirzаyor-sovg-а-hikoyа 107 O‘KTАM MIRZАYOR. SOVG‘А (HIKOYА), 5-qism 264 264 bo‘layotgan
muhorabalarlarda
bugunning
tom
ma’nodagi
qahramonlari
—
shifokorlarimizni ham berib qo‘ymoqdamiz. bo‘layotgan
muhorabalarlarda
bugunning
tom
ma’nodagi
qahramonlari
—
shifokorlarimizni ham berib qo‘ymoqdamiz. Kuni kecha Xiva o‘z qahramonlaridan birini Haqning huzuriga kuzatdi: tajribali va
fidoyi shifokor Baxtiyor Qilichev 66 yoshida omonatini Egasiga topshirdi. Sabab — o‘sha:
koronavirus asoratlari!” Kuni kecha Xiva o‘z qahramonlaridan birini Haqning huzuriga kuzatdi: tajribali va
fidoyi shifokor Baxtiyor Qilichev 66 yoshida omonatini Egasiga topshirdi. Sabab — o‘sha:
koronavirus asoratlari!” Kontekstda e’tibor beradigan bo‘lsak, ushbu jumla faqatgina pozitiv ma’noda, pozitiv
shaxslarga qo‘llanilayotganiga guvoh bo‘lishimiz mumkin, bu esa diskursning asosiy
mohiyatini ifoda etadi, ya’ni kontekstda uning boshqa so‘zlar bilan uyg‘un bo‘lishi ayni
muhimlikda. Big mouth frazemasini ham tahlil qilib ko‘raylik: O‘zbek tiliga katta og‘iz - og‘zi polvon – katta gapirmoq – katta ketmoq kabi tarjima
qilinadi va yuqoridagicha darajanadi. Ingliz tilida esa blow one's own trumpet – show off - blow/toot one's own horn
kabilardir. Ma’lumki, garchi “katta og‘iz” “big mouth” iboralari ikki tikda ham bir xillik kasb etsa-
da, o‘zbek xalqida polvonlik maqtashga arzigulik jarayon hisoblangani uchun “og‘zi
polvon” iborasi qo‘llanilgan bo‘lsa, ingliz xalqida “trumpet” truba musiqa asbobi juda
ommabop hisoblanadi va aynan ushbu frazemalarning intensifikatsiya jarayonida
diskursning ahamiyati hisoblanadi, ya’ni ushbu tillarning kelib chiqishi, madaniyati va
millati, urf-odat va an’analari ushbu iboralarni paydo bo‘lishiga keng zamin yaratadi va asos
bo‘ladi. Albatta, ushbu frazeologik intensifikatorlar diskursda to‘g‘ri qo‘llanilgandagina
muomala va muloqot jarayoniga mos va xos hisoblanadi. Quyida yana bir nechta frazeologik intensifikatorlarni o‘zbek tiliga muqobil tarjimasi
bilan namuna sifatida ko‘rsatamiz. Little lie - white lie – arzimas yolg‘on
young at heart – kichik ko‘ngil
black market – qor bozor
a sunny smile – yorqin tabassum
a cash cow – foydali biznes 265 265 as gentle as a lamb – qo‘ydek yuvosh
as tall as maypole - Terakdek uzun, mirzaterak
alive and well – joni temirdek
An old chestnut - siyqa gap, siyqasi chiqqan hazil
Thick skin –Terisi qalin
Thin skin –Terisi yupqa
A blind alley –Boshi berk ko`cha
Blood is thicker than water – qoni qo‘shilgan, qoni bir
“The pen is mightier than the sword” – so‘z qilichdek keskir
More power to your elbow – bilagingga kuch bersin
iboralari ham xuddi shunday tahlil qilinishi va diskurs bilan bog‘lanishi mumkin. as gentle as a lamb – qo‘ydek yuvosh
as tall as maypole - Terakdek uzun, mirzaterak
alive and well – joni temirdek
An old chestnut - siyqa gap, siyqasi chiqqan hazil
Thick skin –Terisi qalin
Thin skin –Terisi yupqa
A blind alley –Boshi berk ko`cha
Blood is thicker than water – qoni qo‘shilgan, qoni bir
“The pen is mightier than the sword” – so‘z qilichdek keskir
More power to your elbow – bilagingga kuch bersin
iboralari ham xuddi shunday tahlil qilinishi va diskurs bilan bog‘lanishi mumkin. alari ham xuddi shunday tahlil qilinishi va diskurs bilan bog‘lanishi mumkin Shu jumladan, pragmatika ham ushbu sohaga dahldor hisoblanadi Quyida frazeologik birliklarni ingliz va o‘zbek tillarida qiyoslab tahlil qilishdan avval,
pragmatik jihat nima ekanligini asoslab beramiz. Pragmatika insonning ijtimoiy faoliyatini oʻzida qamrab oluvchi nutq jarayoni
hisoblanib, muayyan muloqot jarayoni orqali namoyon boʻladi. Pragmatika aniq shakl,
tashqi koʻrinishga ega emas; uning doirasiga soʻzlovchi va tinglovchining o‘zlarigagina
tushhunarli bo‘lgan oʻzaro munosabatlari va vaziyati bilan bogʻliq ma’lum bir masala kiradi. Misol uchun: biron-bir axborot yoki fikrni yetkazishda ishlatiladigan soʻroq, buyruq, iltimos,
maslahat, vaʼda berish, uzr soʻrash, tabriklash, shikoyat qilish nutq taktikasi hamda nutq odobi turlari; suhbat, soʻzlashish qoidalari; suhbat, soʻzlashish qoidalari; soʻzlovchining maqsadi; soʻzlovchi tomonidan tinglovchining umumiy bilim jamgʻarmasi, dunyoqarashi,
qiziqishlari va xislatlariga baho berilishi; soʻzlovchi tomonidan tinglovchining umumiy bilim jamgʻarmasi, dunyoqarashi,
qiziqishlari va xislatlariga baho berilishi; soʻzlovchining oʻzi bayon qilayotgan xabarga munosabati va boshqalar kiradi. Agar frazeologizmlarni pragmatik jihatdan tahlil qiladigan bo‘lsak, ma’lum bir
vaziyatga qarata aytilgan frazeologik birlikni ham so‘zlovchi, ham tinglovchi tomonidan Agar frazeologizmlarni pragmatik jihatdan tahlil qiladigan bo‘lsak, ma’lum bir
vaziyatga qarata aytilgan frazeologik birlikni ham so‘zlovchi, ham tinglovchi tomonidan 266 to‘g‘ri tushunilib, o‘sha vaziyatga mosligi aniqlangan holda aytilgan; so‘zlovchining
maqsadi frazeologik birlik orqali to‘liq ochib berilgan va so‘zlovchi tomonidan nutqqa mos
gap ohangi aytilgan bo‘lmog‘i darkor. Endi quyidagi frazemaga e’tibor beraylik: think on one’s feet frazeologik birligi o‘zbek tiliga so‘zma so‘z tarjima qilinganda,
“kimningdir oyog‘i ustida o‘ylamoq” kabi o‘zbek tiliga tushunarsiz ma’no beradigan
so‘zlar jamlanmasini anglatadi, xolos. Biroq, uni nutqda yoki ma’lum bir asarda
shundayligicha tarjima qilib bo‘lmaydi. Shuning uchun, tilimizda unga mos bo‘lgan
iboralarni qidirib ko‘ramiz. When you’re called on in class, you have to be able to think on your feet. Ushbu inglizcha namunani tilimizga quyidagicha tarjima qilish mumkin. Dars jarayonida seni chaqirishganida, hamisha hayoling o‘zing bilan bo‘lishi lozim. Yoki: I had never heard about the company before, so I had to think on my feet. Men u kompaniya haqida avval hech qachon eshitmagandim, shuninguchun yashin
tezligida qaror qilishimga to‘g‘ri keldi. Guvohi bo‘lganimizdek, ingliz tilidagi aynan bir xil iborani o‘zbek tiliga ikki xil
tarjima qilishimizga to‘g‘ri keldi, buning sababi pragmatika. Ya’ni pragmatik ma’no nutq
jarayoniga mos bo‘lishi uchun nutqda tarjima mahoratining o‘rni beqiyos. FOYDALANILGAN ADABIYOTLAR 1. Jalilova Nilufar Dilshodovna (2019). O‘zbek va ingliz tillarida idiomalarni
taqqoslashning lingual tahlili. Vestnik nauki i obrazovaniya, (20-2 (74)) 2. Lakoff G. The Invariance Hypothesis: Is Abstract Reason Based on Image Schemata? // Cognitive Linguistics. 1990. Vol. 1,№1. P. 39−74. 2. Lakoff G. The Invariance Hypothesis: Is Abstract Reason Based on Image Schemata? // Cognitive Linguistics. 1990. Vol. 1,№1. P. 39−74. 3. Lakoff G., Johnson M. Metaphors We Live by. Chicago, 1980. 242 p. 3. Lakoff G., Johnson M. Metaphors We Live by. Chicago, 1980. 242 p. 4. Aлeхинa A.И. Ceмaнтичecкиe грyппы вo фрaзeoлoгии coврeмeннoгo aнглийcкoгo языкa. М., 1977. 4. Aлeхинa A.И. Ceмaнтичecкиe грyппы вo фрaзeoлoгии coврeмeннoгo aнглийcкoгo языкa. М., 1977. 5. Aлeхинa A.И. Фрaзeoлoгичecкaя eдиницa и cлoвo. М., 1979. (In Russ.). 5. Aлeхинa A.И. Фрaзeoлoгичecкaя eдиницa и cлoвo. М., 1979. (In Russ.). 267 267 267 6. Бyшyй Т.A., Caфaрoв Ш. Тил кyрилиши. Тaхлил мeтoдлaри вa мeтoдoлoгияcи. Тoшкeнт, Фaн, 2007. 6. Бyшyй Т.A., Caфaрoв Ш. Тил кyрилиши. Тaхлил мeтoдлaри вa мeтoдoлoгияcи. Тoшкeнт, Фaн, 2007. 6. Бyшyй Т.A., Caфaрoв Ш. Тил кyрилиши. Тaхлил мeтoдлaри вa мeтoдoлoгияcи. Тoшкeнт, Фaн, 2007. 6. Бyшyй Т.A., Caфaрoв Ш. Тил кyрилиши. Тaхлил мeтoдлaри вa мeтoдoлoгияcи. Тoшкeнт, Фaн, 2007. 268 268
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Seasonal Analysis of Microbial Communities in Precipitation in the Greater Tokyo Area, Japan
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Frontiers in microbiology
| 2,017
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cc-by
| 8,043
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Seasonal Analysis of Microbial
Communities in Precipitation in the
Greater Tokyo Area, Japan
Satoshi Hiraoka 1*, Masaya Miyahara 1, Kazushi Fujii 1, Asako Machiyama 2, 3 and
Wataru Iwasaki 1, 2, 3* 1 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of
Tokyo, Chiba, Japan, 2 Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba, Japan, 3 Department of
Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan The presence of microbes in the atmosphere and their transport over long distances
across the Earth’s surface was recently shown. Precipitation is likely a major path by
which aerial microbes fall to the ground surface, affecting its microbial ecosystems and
introducing pathogenic microbes. Understanding microbial communities in precipitation
is of multidisciplinary interest from the perspectives of microbial ecology and public
health; however, community-wide and seasonal analyses have not been conducted. Here, we carried out 16S rRNA amplicon sequencing of 30 precipitation samples that
were aseptically collected over 1 year in the Greater Tokyo Area, Japan. The precipitation
microbial communities were dominated by Proteobacteria, Firmicutes, Bacteroidetes,
and Actinobacteria and were overall consistent with those previously reported in
atmospheric aerosols and cloud water. Seasonal variations in composition were
observed; specifically, Proteobacteria abundance significantly decreased from summer
to winter. Notably, estimated ordinary habitats of precipitation microbes were dominated
by animal-associated, soil-related, and marine-related environments, and reasonably
consistent with estimated air mass backward trajectories. To our knowledge, this is
the first amplicon-sequencing study investigating precipitation microbial communities
involving sampling over the duration of a year. ORIGINAL RESEARCH
published: 11 August 2017
doi: 10.3389/fmicb.2017.01506 Edited by:
Télesphore Sime-Ngando,
CNRS, Centre National de la
Recherche, France Reviewed by:
Anne Marie Delort,
Blaise Pascal University, France
S. Venkata Mohan,
Indian Institute of Chemical
Technology (CSIR), India Reviewed by:
Anne Marie Delort,
Blaise Pascal University, France
S. Venkata Mohan,
Indian Institute of Chemical
Technology (CSIR), India *Correspondence:
Satoshi Hiraoka
hiraoka@cb.k.u-tokyo.ac.jp
Wataru Iwasaki
iwasaki@bs.s.u-tokyo.ac.jp Keywords: microbial ecology, precipitation, long-distance transportation, ice nucleation activity, habitat
estimation Specialty section:
This article was submitted to
Aquatic Microbiology,
a section of the journal
Frontiers in Microbiology Keywords: microbial ecology, precipitation, long-distance transportation, ice nucleation activity, habitat
estimation INTRODUCTION Microbes are present and move around nearly everywhere in the Earth. Aerial microbes
have received considerable attention within this context because the atmosphere not
only is an unusual habitat for microbes but also likely represents a path by which
microbes move exceptionally long distances (Kellogg and Griffin, 2006; Burrows et al.,
2009; Després et al., 2012; Smith, 2013; Fröhlich-Nowoisky et al., 2016). To date, several
studies
have
investigated
aerial
microbial
communities
on
airborne
particles
and
in
clouds
using
culture-dependent
and
independent
techniques
(Bowers
et
al.,
2011a,b,
2013; Vaïtilingom et al., 2012; Zweifel et al., 2012; DeLeon-Rodriguez et al., 2013; Woo
et al., 2013; Dong et al., 2016), and revealed that aerial microbes can originate from
terrestrial habitats, including plant surfaces (Bowers et al., 2009, 2011a,b). The long-distance Microbes are present and move around nearly everywhere in the Earth. Aerial microbes
have received considerable attention within this context because the atmosphere not
only is an unusual habitat for microbes but also likely represents a path by which
microbes move exceptionally long distances (Kellogg and Griffin, 2006; Burrows et al.,
2009; Després et al., 2012; Smith, 2013; Fröhlich-Nowoisky et al., 2016). To date, several
studies
have
investigated
aerial
microbial
communities
on
airborne
particles
and
in
clouds
using
culture-dependent
and
independent
techniques
(Bowers
et
al.,
2011a,b,
2013; Vaïtilingom et al., 2012; Zweifel et al., 2012; DeLeon-Rodriguez et al., 2013; Woo
et al., 2013; Dong et al., 2016), and revealed that aerial microbes can originate from
terrestrial habitats, including plant surfaces (Bowers et al., 2009, 2011a,b). The long-distance Received: 23 December 2016
Accepted: 27 July 2017
Published: 11 August 2017 Citation: Likewise, the outbreak
of several plant infections due to aerial microbes transported
beyond borders and seas has been hypothesized (Fitt et al., 1989;
Brown and Hovmøller, 2002). Precipitation, i.e., rainfall and snowfall, would bring aerial
microbes in the troposphere to the ground surface. Quantitative
polymerase chain reaction (PCR) has detected pathogenic
bacterial sequences in precipitation samples (Kaushik et al.,
2012), implicating that precipitation may alter microbial
ecosystems on the ground (Hervàs et al., 2009; Peter et al.,
2014). In the reverse direction, aerial microbes impact the climate
by accelerating cloud formation and precipitation, known as
“bioprecipitation” (Hamilton and Lenton, 1998; Christner et al.,
2008; Konstantinidis, 2014; Morris et al., 2014; Stopelli et al.,
2015; Hara et al., 2016). Several microbial species experimentally
exhibit ice nucleation activity (INA), which is the ability to
accelerate ice nucleation at relatively warm temperatures by
producing so-called INA proteins (Hoose and Möhler, 2012). Such INA microbes are broadly distributed among bacteria and
fungi and have been isolated from precipitation and cloud water
(Mortazavi et al., 2008; Joly et al., 2013). In addition, microbes
in clouds may affect the chemical composition of clouds via
carbon (Amato et al., 2007; Vaïtilingom et al., 2013) and nitrogen
metabolism (Hill et al., 2007). Thus, a basic understanding
of microbial communities in precipitation provides important
knowledge regarding microbial ecology, public health, and even
meteorology. To date, several cloning-based (Ahern et al., 2007;
Zweifel et al., 2012; Šantl-Temkiv et al., 2013; Peter et al.,
2014) and community-wide but short-term (Cho and Jang, 2014;
Kaushik et al., 2014) analyses of microbial communities in
precipitation have been carried out. However, community-wide
and seasonal analyses have not been conducted. We collected 25 and 5 precipitation samples containing
sufficient amounts of microbial DNA at the Kashiwa and Hongo
sites, respectively. The sampling dates spanned more than 1 year
from May 2014 to October 2015, encompassing the rainy and
typhoon seasons in Japan (Table 1; the six digits, letter, and
suffix number for each sample name represent the sampling date
(YYMMDD), the site (K for Kashiwa and H for Hongo), and
the volume (if multiple samples were collected during the same
precipitation event). A precipitation event was defined if there
was no precipitation 6 h before and after the event. The volumes
of collected and filtered precipitation ranged from 50 to 1,000 mL. DNA Extraction and PCR Amplification DNA Extraction and PCR Amplification
Microbial DNA on the Sterivex filters was retrieved using
a ChargeSwitch Forensic DNA Purification Kit (Invitrogen)
according to the supplier’s protocol with one exception: the
filters were directly suspended in the extraction solution from
the kit during the cell lysis process. The V5-V6 region
of the prokaryotic 16S rRNA gene was amplified using
a standard PCR protocol with TaKaRa Ex Taq (TaKaRa)
and the following high-performance liquid chromatography-
purified primers: 784F (5′- RGGATTAGATACCC -3′) and Citation: Hiraoka S, Miyahara M, Fujii K,
Machiyama A and Iwasaki W (2017)
Seasonal Analysis of Microbial
Communities in Precipitation in the
Greater Tokyo Area, Japan. Front. Microbiol. 8:1506. doi: 10.3389/fmicb.2017.01506 August 2017 | Volume 8 | Article 1506 Frontiers in Microbiology | www.frontiersin.org Seasonal Analysis of Precipitation Microbes Hiraoka et al. a seven-story building on the Kashiwa campus, the University
of Tokyo, Chiba, Japan, which is surrounded by residences,
farms, and woods in a suburb of Tokyo. The Hongo site was
on the roof of a five-story building on the Hongo campus, the
University of Tokyo, Tokyo, Japan, which is located in downtown
Tokyo. The sites are 25.5 km apart and neither geologically nor
meteorologically separated in the Kanto plain. The upper areas
of both sites are wide open and lack any obstructing buildings
or structures that would contaminate the precipitation samples. At the Kashiwa site, precipitation was aseptically collected using
a US-330 automatic precipitation sampler (Ogasawara Keiki,
Tokyo, Japan) following the method of Kaushik et al. (2012). This device consists of a sterile and disposable bottle inside
a 4◦C refrigerator and automatically collects precipitation by
opening the lid only when a sensor detects precipitation. At
the Hongo site, precipitation samples were manually collected
into a sterile and disposable bottle on ice and immediately
stored in a 4◦C refrigerator. At both sites, every part of
the collection equipment that potentially directly contacted
precipitation samples (e.g., disposable collection bottles and
channel tubes) was sterilized by gamma rays in advance of
each sample collection. The precipitation samples were pre-
filtered through 5-µm membrane filters, and microbial cells were
collected using 0.22-µm Sterivex filters (Millipore, USA). The
Sterivex filters were promptly moved to a −20◦C freezer and
stored until DNA extraction. Precipitation sampling required
no special permission. To prepare negative control samples,
we poured 1 L of Milli-Q purified water into the collection
equipment and carried out filtration in the same manner. transport of aerial microbes has also been reported, for example
from Chinese deserts to Japan over the east Eurasian continent
and the Sea of Japan (Echigo et al., 2005; Maki et al., 2011). Pathogens in the atmosphere may be transported over long
distances, as integrated simulation analyses of climate and
disease propagation suggest the involvement of aerial microbes in
human diseases (Rodó et al., 2011, 2014). Citation: For correlation analysis with meteorological data, we excluded
the data obtained from samples 140630K_50, 140630K _100,
140810K_50, and 140810K_100, which were retrieved as replicate
samples with different volumes. Eight negative control samples
were also collected at different dates at the Kashiwa and Hongo
sites. Here, we conducted 16S ribosomal RNA (rRNA) amplicon-
sequencing analysis of 30 precipitation samples that were
aseptically collected over 1 year in the Greater Tokyo Area, Japan. Microbial community analysis revealed seasonal variations in
their composition. Notably, the estimated original habitats
of precipitation microbes showed reasonable consistency with
estimated air mass backward trajectories. Our results support
a precipitation-mediated microbial cycle model in which soil,
oceanic, and animal-associated microbes are spread in the
atmosphere, transported for long distances, and deposited via
precipitation. Precipitation Sampling Precipitation Sampling Precipitation samples were collected at two sites in the Greater
Tokyo Area, Japan: Kashiwa (35◦54′00′′N, 139◦55′59′′E, 50 m
above sea level) and Hongo (35◦42′55”N, 139◦45’56′′E, 30 m
above sea level) (Figure 1). The Kashiwa site was on the roof of August 2017 | Volume 8 | Article 1506 Frontiers in Microbiology | www.frontiersin.org 2 Hiraoka et al. Seasonal Analysis of Precipitation Microbes FIGURE 1 | A map of the sampling sites (Kashiwa and Hongo, yellow) and meteorological observatories (Abiko and Tokyo, blue) (left panel), with photos of the
sampling sites (right panel). At the Kashiwa site, a US-330 automatic precipitation sampler (Ogasawara Keiki, Tokyo, Japan) was installed. At the Hongo site,
precipitation samples were manually collected. FIGURE 1 | A map of the sampling sites (Kashiwa and Hongo, yellow) and meteorological observatories (Abiko and Tokyo, blue) (left panel), with photos of the
sampling sites (right panel). At the Kashiwa site, a US-330 automatic precipitation sampler (Ogasawara Keiki, Tokyo, Japan) was installed. At the Hongo site,
precipitation samples were manually collected. 1064R (5′- CGACRRCCATGCANCACCT -3′) (Wang and Qian,
2009; Claesson et al., 2010). Amplified DNA was concatenated
to multiplex identifier tags that were unique to each sample,
and a mixture of 10 samples on average was sequenced in one
run on a 454 GS Junior System (Roche) after size selection
(350 ± 50 bp). Pre-packaged sterile water for injection (in lieu
of water from a laboratory water purification system) was used
throughout the DNA extraction, PCR amplification, and DNA
library preparation steps to avoid water-mediated contamination. retrieving the top hit sequence that showed e-values ≤1E-15. To estimate ordinary habitats for each 16S rRNA sequence, a
BLASTN search was performed against MetaMetaDB (Yang and
Iwasaki, 2014), and the top hit sequence with an e-value ≤1E-10
and an identity ≥90% was retrieved. Microbial habitability index
(MHI) scores were calculated as previously described (Yang and
Iwasaki, 2014). Amplicon-sequencing data of aerosol and cloud water samples
were downloaded from NCBI SRA database (the accession
numbers are shown in Supplementary Table S1). Their ordinary
habitat analyses were conducted as described above after quality
filtering. Bioinformatic Analysis For raw sequence data from both precipitation and negative
control samples, sequence regions at both ends that contained
low-quality bases (quality score < 20) were trimmed using
DynamicTrim (Cox et al., 2010), chimeric sequences were
filtered out using UCHIME with default settings (Edgar et al.,
2011), and sequences whose lengths were shorter than 150
bp were discarded. All remaining high-quality sequences were
clustered with a 97% identity threshold using CD-HIT (Fu
et al., 2012). After discarding clusters that contained negative
control sequences (Cho and Jang, 2014), each cluster was
designated as an operational taxonomic unit (OTU). For
hierarchical cluster analysis of the precipitation samples, the
Ward method was used based on Bray-Curtis dissimilarities
between their OTU compositions. Non-metric multidimensional
scaling (NMDS) analysis was conducted using Bray-Curtis
dissimilarities. The taxonomic assignment of each OTU was
performed by conducting a BLASTN search (Camacho et al.,
2009) against the SILVA database (Quast et al., 2013) and Meteorological Data Analysis g
The data on the amount of precipitation, temperature, wind
speed, and atmospheric pressure were retrieved from the website
of the Japan Meteorological Agency (http://www.jma.go.jp/jma/
menu/menureport.html), Ministry of Land, Infrastructure, and
Transport of Japan. Precipitation, wind speed, and temperature
data from the Abiko (35◦51′48′′N, 140◦06′36′′E; 16.4 km from
Kashiwa) and Tokyo (35◦41′30′′N, 139◦45′00′′E; 2.9 km from
Hongo) observatories were used for the analyses of the Kashiwa
and Hongo sites, respectively (Figure 1). Atmospheric pressure
data from the Tokyo observatory were used (this observatory
is the closest to both sites that records atmospheric pressure
data). The wind speed, temperature, and atmospheric pressure
data were averaged over the period of each precipitation event. To analyze long-range transport paths of air masses that caused
precipitation by providing water vapor, we estimated backward August 2017 | Volume 8 | Article 1506 Frontiers in Microbiology | www.frontiersin.org Frontiers in Microbiology | www.frontiersin.org 3 Seasonal Analysis of Precipitation Microbes Hiraoka et al. August 2017 | Volume 8 | Article 1506 Frontiers in Microbiology | www.frontiersin.org Meteorological Data Analysis Sample
Sampling time (YYMMDD)a
Note
Amount of
precipitation
[mm]
Temperature
[◦C]
Atmospheric
pressure
Wind speed
[m/s]
Filtration
volume [mL]
Raw
reads
Effective
reads
OTUs
Shannon’s
diversity
index
140521K
140521(01:00)–140521(18:00)
–
36
16.56
994.31
3.16
50
8,340
246
44
2.62
140630K_50
140628(01:00)–140630(05:00)
Rainy season
22
21.98
999.23
1.74
50
8,622
1,092
27
1.63
140630K_100
140628(01:00)–140630(05:00)
Rainy season
22
21.98
999.23
1.74
100
7,444
1,287
39
1.26
140630K_200
140628(01:00)–140630(05:00)
Rainy season
22
21.98
999.23
1.74
200
7,462
1,118
44
1.48
140810K_50
140810(00:00)–141810(23:00)
Typhoon
31.5
25.46
998.46
3.89
50
7,621
275
76
3.70
140810K_100
140810(00:00)–141810(23:00)
Typhoon
31.5
25.46
998.46
3.89
100
8,441
108
53
4.41
140810K_200
140810(00:00)–141810(23:00)
Typhoon
31.5
25.46
998.46
3.89
200
6,664
371
129
3.82
140926K
140925(02:00)–140926(04:00)
–
6.5
21.48
1,001.08
2.14
200
1,941
18
15
2.66
141014K
141013(13:00)–141014(07:00)
Typhoon
32.5
19.58
992.99
4.47
200
1,641
317
157
4.76
141023K
141021(05:00)–141023(18:00)
–
31.5
14.89
1,010.85
2.03
200
1,354
120
54
3.66
150107K
150106(16:00)–150106(18:00)
–
4
12.35
992.30
5.10
200
3,410
37
22
2.93
150116K
150115(11:00)–150116(00:00)
–
40.5
4.97
1,005.15
3.05
1,000
2,494
72
55
3.86
150202K
150130(05:00)–150130(19:00)
Snow
12.5
1.11
1,015.18
2.17
400
9,705
1,256
194
4.69
150409K
150407(03:00)–150408(17:00)
–
20.5
6.39
1,017.32
2.13
200
8,337
473
111
4.15
150412K
150410(17:00)–150411(14:00)
–
16
8.99
1,018.06
1.60
200
6,805
771
125
4.16
150414K
150413(11:00)–150414(17:00)
–
36.5
10.13
1,013.38
1.91
200
5,818
655
116
3.98
150513K
150512(21:00)–150513(01:00)
Typhoon
23
20.00
997.43
5.60
200
2,223
47
25
3.92
150604K
150603(08:00)–150603(13:00)
Rainy season
13
20.46
998.26
1.44
200
4,663
8
7
2.88
150628K
150626(19:00)–150627(12:00)
Rainy season
13.5
20.92
997.42
1.24
150
9,634
284
46
3.41
150711K
150708(15:00)–150709(20:00)
Rainy season
17.5
19.27
1,013.22
1.48
200
9,557
31
20
1.91
150718K
150716(04:00)–150717(13:00)
Typhoon
16.5
25.99
1,004.62
3.26
200
5,189
5
4
4.03
150816K
150814(05:00)–150814(22:00)
–
43
25.11
1,000.38
1.91
200
6,864
269
68
3.05
150827K
150826(00:00)–150826(17:00)
Typhoon
27
20.10
1,005.27
1.92
200
7,993
1,041
226
3.39
150926K
150924(19:00)–150926(06:00)
–
21
17.63
1,005.28
1.90
200
2,988
6
4
2.75
151014K
151011(01:00)–151011(11:00)
–
8
16.94
1,008.92
1.00
200
6,357
129
30
1.33
150414H
150413(07:00)–150414(12:00)
–
39.5
10.03
1,014.99
3.15
200
7,215
882
125
3.76
150513H
150512(20:00)–150513(06:00)
Typhoon
58.5
20.25
997.43
7.23
200
3,198
59
38
4.83
150627H
150626(15:00)–150627(10:00)
Rainy season
16
21.33
998.27
2.22
150
6,450
667
108
1.24
150710H
150708(10:00)–150710(00:00)
Rainy season
22
20.20
1,013.22
2.33
200
9,280
159
48
3.00
151014H
151011(02:00)–151011(10:00)
–
15
18.01
1,008.80
1.90
200
6,342
286
67
3.70
aThe six digits, letter, and suffix number in each sample name represent the sampling date (YYMMDD), the sampling site (K for Kashiwa and H for Hongo), and the filtered sample volume if prepared as a technical replicate with multiple Taxonomic Composition of Precipitation
Microbial Communities A total of 64,100 high-quality sequences 231 ± 45 bp in
length were generated from 30 precipitation and eight negative
control samples. The precipitation samples included typhoon
rain, rainy season rain, and snow. After removing sequences
exhibiting >97% similarity to the negative control samples,
12,089 “effective” sequences comprising 1,297 OTUs remained. To make our analyses based on reads that were not likely from
contamination as much as possible, we took a conservative
and strict filtering approach, whose extent of read number
reduction was similar to that in a previous study (Cho and
Jang, 2014). The number of OTUs per sample ranged from
4 to 226 (Table 1). Based on rarefaction curves, the obtained
OTUs represented their microbial communities well for some
samples, although several samples required additional sequences
(Supplementary Figure S1). Among the 12,089 effective sequences, 11,994 (99.2%) were
taxonomically assigned at the phylum level. Almost all sequences
were assigned to 24 phyla in the domain Bacteria with the
exception of 4 (0.03%) and 219 (1.7%) sequences assigned to
Archaea and mitochondria, respectively. This strong bias toward
bacterial sequences may reflect the actual composition but may
also be attributable to amplification bias introduced by primer
specificity. The top three and six most abundant bacterial phyla
accounted for >80 and >95%, respectively, of the sequence
pool of all precipitation samples (Figure 3A). Proteobacteria was
the most abundant phylum (23–88%) across all precipitation
samples with the exception of the 140630, 140926, and 150116K
samples (Firmicutes (89–94%), Actinobacteria (50%), and
Firmicutes (49%) were the most abundant phyla, respectively). A particularly exceptional microbial community dominated by
Firmicutes was observed in the 140630K sample. Firmicutes,
Bacteroidetes, and Actinobacteria were the other dominant
phyla in the total sequence pool. In principle, these results
were consistent with those of a previous study in which
Proteobacteria, Firmicutes, and Bacteroidetes were the dominant
phyla in precipitation samples captured in Seoul, Korea (Cho
and Jang, 2014), whereas comparatively greater numbers of
sequences were assigned to Actinobacteria, Planctomycetes, and
Cyanobacteria in this study. At the class level, the abundant
groups were Gammaproteobacteria, Betaproteobacteria, and
Alphaproteobacteria,
followed
by
Bacilli,
Flavobacteriia,
Clostridia, Actinobacteria, and Sphingobacteriia (Figure 3B). Data Deposition The
amplicon
sequence
data
were
deposited
in
the
DDBJ/ENA/GenBank
database
under
BioSample
IDs
SAMD00059586-SAMD00059614
and
SAMD00060461-
SAMD00060468. All data were registered under BioProject ID
PRJDB5087. FIGURE 2 | Hierarchical clustering of precipitation samples based on OTU
composition. The distance matrix was calculated based on the Bray-Curtis
dissimilarity, and clusters were calculated using Ward’s method. Open symbols
indicate samples that were collected during the same precipitation event with
different volumes. Closed symbols indicate samples that were collected on the
same day at different sites (Kashiwa and Hongo). 50
50
100
200
50
100
200
200
200
200
200
1,000
400
200
200
200
200
200
150
200
200
200
200
200
200
200
200
150
200
200 August 2017 | Volume 8 | Article 1506 4 Seasonal Analysis of Precipitation Microbes Hiraoka et al. FIGURE 2 | Hierarchical clustering of precipitation samples based on OTU
composition. The distance matrix was calculated based on the Bray-Curtis
dissimilarity, and clusters were calculated using Ward’s method. Open symbols
indicate samples that were collected during the same precipitation event with
different volumes. Closed symbols indicate samples that were collected on the
same day at different sites (Kashiwa and Hongo). trajectories of an air mass at 2,000 m altitude for 240 h prior to
all precipitation events for each sampling site. The trajectories
were calculated based on the hybrid single-particle Lagrangian
integrated trajectory (HYSPLIT) model (http://ready.arl.noaa. gov/HYSPLIT.php) provided by the Global Data Assimilation
System of National Oceanic and Atmospheric Administration,
USA (Stein et al., 2015). The HYSPLIT model uses gridded
meteorological data and considers advection and diffusion of air
parcels in calculation of their trajectories. This model has been
used in a variety of atmospheric simulations focusing on the
atmospheric transport, dispersion, and deposition of pollutants
and hazardous materials (Stein et al., 2015), while it has also
been adopted for estimation of sources of airborne microbes (e.g.,
Smith et al., 2013; Cho and Jang, 2014; Kobayashi et al., 2015; Xia
et al., 2015; Xu et al., 2017). Frontiers in Microbiology | www.frontiersin.org Taxonomic Composition of Precipitation
Microbial Communities Notably, the enrichment of these phyla and classes was
also reported in previous studies investigating aerosolized Hierarchical cluster analysis of OTU composition in the
precipitation samples indicated samples collected during the
same precipitation event with different volumes (50, 100, and
200 mL) that were highly similar to each other (Figure 2,
open symbols), suggesting that differences in volume have
little effect on analysis in the 50–200 mL range. Moreover,
microbial communities in samples that were collected on the
same day at different sampling sites (Kashiwa and Hongo) were
closely positioned in the dendrogram (Figure 2, closed symbols),
indicating that the observed OTU compositions reflect the
microbial populations in precipitation rather than those in the
atmosphere near the ground surface or equipment- or reagent-
mediated contamination at each site. NMDS analysis did not
show any clear trend, although samples of close dates tended to
be clustered together (Supplementary Figure S2). August 2017 | Volume 8 | Article 1506 5 Seasonal Analysis of Precipitation Microbes Hiraoka et al. similar trend has consistently been observed in aerosolized
microbial communities (Bowers et al., 2011b). To more
closely investigate the factors underlying changes in the
precipitation microbial communities, we performed a correlation
analysis between meteorological characteristics and microbial
composition (Figure 4). The relative abundance of the order
Bacteroidales negatively correlated with temperature (Spearman
correlation ρ = −0.70, p < 0.01 after the Bonferroni correction). Although other correlations were not statistically significant after
multiple testing correction, the amount of precipitation, wind
speed, and atmospheric pressure showed tendencies of positive
correlations with the abundance of the orders Cellvibrionales
(ρ = 0.59), Cellvibrionales (ρ = 0.58), and Pseudomonadales
(ρ = 0.57), respectively. Notably, the abundance of the order
Legionellales, which contains several known pathogens, showed
a tendency of a positive correlation with temperature (ρ = 0.47),
where aerosolized water is known to facilitate the dispersion of
Legionella (Nguyen et al., 2006) and a warm and wet climate is
associated with the incidence of Legionnaires’ disease (Fisman
et al., 2005; Fisman, 2007). Although cell numbers were not
measured except for one sample in this study, we note that
seasonal variability in cell numbers would also be important,
especially because that of atmospheric samples was reported
(Kaushik et al., 2014; Dong et al., 2016). Taxonomic Composition of Precipitation
Microbial Communities Similarly, analyses with
particulate matter density and O3 and NO3 concentrations are
also envisioned, because they would substantially affect aerial
microbes (Kaushik et al., 2012; DeLeon-Rodriguez et al., 2013;
Wei et al., 2016; Xu et al., 2017). FIGURE 3 | Relative abundances of sequences at the phylum (A) and class
(B) levels. Groups demonstrating <5% abundance were summarized as
“Others.” FIGURE 3 | Relative abundances of sequences at the phylum (A) and class
(B) levels. Groups demonstrating <5% abundance were summarized as
“Others.” Relationship between Ordinary Habitats of
Precipitation Microbes and Air Mass
Backward Trajectories Seasonal Analysis of Precipitation Microbes FIGURE 4 | Correlation analysis between relative abundances of sequences at the order level and meteorological data. The color scheme represents Spearman’s
rank correlation coefficient. FIGURE 4 | Correlation analysis between relative abundances of sequences at the order level and meteorological data. The color scheme represents Spearman’s
rank correlation coefficient. sampling methods were different from those in our study
(Supplementary Figure S5). additional sequences to reach plateaus of rarefaction curves as
mentioned already. The estimated backward trajectories of air masses that led
to the precipitation events at the Kashiwa and Hongo sites
were classified as terrestrial, oceanic, and hybrid routes. The
terrestrial route typically originated from the middle of the
Eurasian continent and passed through the East China Sea, the
Yellow Sea, and the Sea of Japan; the oceanic route typically
originated from the Pacific Ocean and passed through the East
China Sea or the Sea of Okhotsk; and the hybrid route comprised
both the terrestrial and oceanic areas. Consistent with the
typical pattern of the seasonal winds in Asia, the terrestrial and
oceanic routes dominated in winter and summer, respectively
(Figure 5, Supplementary Figure S3). The estimated ordinary
habitats of the precipitation microbes showed agreement with
the estimated air mass backward trajectories. For example,
Planctomycetes, which contains several aquatic microbes (Fuerst,
1995), was frequently found when the backward trajectories
followed oceanic routes (Figures 3A, 5). PERMANOVA analysis
showed a significant relationship between the routes and the
estimated composition of ordinary microbial habitats (p < 0.05). Notably, the ratios of marine-related environments dominated
when the air masses originated from the oceanic route, and
animal-related environments dominated when they originated
from the terrestrial route. Shannon’s diversity indices of microbes
became larger when the air masses originated from the terrestrial
route (Shannon’s diversity indices were 3.74 ± 0.68, 3.05 ±
1.00, and 3.15 ± 1.36 for the terrestrial, oceanic, and hybrid
routes, respectively. The index of each sample is shown in
Table 1); however, it should be noted that some samples required Soil, oceanic, and animal-associated microbes are spread in
the atmosphere and transported for long distances (Morris
et al., 2014; Smets et al., 2016), and precipitation may facilitate
this microbial cycle. Relationship between Ordinary Habitats of
Precipitation Microbes and Air Mass
Backward Trajectories To
estimate
the
environments
from
which
microbes
in
precipitation originated, we performed a microbial habitat index
analysis using MetaMetaDB (Yang and Iwasaki, 2014), which is
a database to estimate the ordinary habitats of microbes based
on similarity searches for 16S rRNA gene sequences against
amplicon-sequencing and shotgun metagenomic data in public
databases. In most samples, animal-associated environments,
such as gut microbiota, were estimated to be the most
dominant
ordinary
habitats
(52%
on
average)
(Figure 5,
Supplementary Figure S4), which is consistent with a previous
study in which animal feces were the dominant source of
airborne bacteria (Bowers et al., 2011b). Notably, marine-related
environments, such as marine and marine sediment, were
estimated to be relatively major ordinary habitats for several
samples (e.g., 65.1 and 63.1% in the 140810 and 141014K
samples, respectively). Soil-related environments, such as soil and
rhizosphere, were also estimated to be major ordinary habitats
(11.0% on average). For comparison, we also conducted ordinary
habitat analyses using amplicon-sequencing data from aerosol
(Xia et al., 2015) and cloud water (DeLeon-Rodriguez et al., 2013)
samples. The soil-related and animal-associated environments
were generally major ordinary habitats as consistent to the
present results, whereas marine-related environments were not
major possibly because the origins of the microbes or the FIGURE 3 | Relative abundances of sequences at the phylum (A) and class
(B) levels. Groups demonstrating <5% abundance were summarized as
“Others.” (Bowers et al., 2009, 2011a; DeLeon-Rodriguez et al., 2013)
and cloud water microbial communities (Kourtev et al., 2011;
DeLeon-Rodriguez et al., 2013). Several OTUs were assigned to genera that potentially
contain INA bacteria, i.e., Acinetobacter, Bacillus, Erwinia,
Flavobacterium, Luteimonas, Microbacterium, Pseudomonas,
Psychrobacter, Sphingomonas, and Stenotrophomonas (Després
et al., 2012). We also detected several genera containing
known pathogens, including typical human pathogens such as
Legionella, Streptococcus, Arcobacter, Rickettsia, and Clostridium,
and plant pathogens such as Erwinia, although their abundance
was low. We did not detect season-specific microbial groups in
the typhoon rain, rainy season, and snow samples with statistical
significance, probably partly due to small sample sizes. Seasonal and Meteorological Correlations
Taxonomic distribution exhibited seasonal variability (Figure 3). Notably, the abundance of Proteobacteria decreased from
summer to winter (p < 0.01, Mann-Whitney U-test), and a August 2017 | Volume 8 | Article 1506 Frontiers in Microbiology | www.frontiersin.org 6 Hiraoka et al. Relationship between Ordinary Habitats of
Precipitation Microbes and Air Mass
Backward Trajectories Sea-living microbes are emitted into the
atmosphere via the bursting of bubbles on waves (Fahlgren et al.,
2015), whereas soil-living and animal-associated microbes are
transported on soil dust (Echigo et al., 2005; Prospero et al.,
2005; Maki et al., 2011; Yamaguchi et al., 2014). In high-altitude
atmospheric environments, microbes may be under substantial
selection pressure due to harsh chemical, physical, and nutrient
conditions (Delort et al., 2010; Morris et al., 2013; Smith, 2013). INA microbes play roles in cloud formation (Morris et al.,
2013) and may facilitate the return of aerial microbes to diverse
environments. The dispersal of pathogenic microbes causes
disease epidemics that threaten public health and agricultural
plant and animal health (Brown and Hovmøller, 2002; Rodó
et al., 2011, 2014; Cao et al., 2014). Continuous long-term
monitoring and large-scale analysis of precipitation microbes
is thus envisioned to reveal the full impact of atmospheric
microbial transport on microbial ecology, microbial evolution,
public health, and climate. Frontiers in Microbiology | www.frontiersin.org ACKNOWLEDGMENTS We thank Kazuhiro Kogure, Minoru Ijichi, and Suguru Nishijima
for their helpful suggestions. FUNDING communities in precipitation samples that were collected over 1
year in the Grate Tokyo area, Japan. To our knowledge, this is
the first amplicon-sequencing study investigating precipitation
microbial communities involving sampling over the duration
of a year. Most importantly, our results suggest seasonal
variations in the microbial communities in precipitation, and
their community structures were significantly associated with
the estimated air mass trajectories. These results highlight
importance of precipitation in long-range microbial immigration
via the atmosphere, which may answer how tiny microbes can
dynamically travel around the globe. This work was supported by the Japan Science and Technology
Agency (CREST), the Japan Society for the Promotion of
Science (Grant Numbers 15J08604, 15H01725, and 16H06154),
the Ministry of Education, Culture, Sports, Science, and
Technology in Japan (221S0002 and 16H06279), and the Canon
Foundation. CONCLUSION Microbes are present nearly everywhere in the Earth, even
in precipitation from the sky. Precipitation is supposed to
make microbes in the atmosphere finally fall down to the
ground surface. In this study, we thoroughly observed microbial August 2017 | Volume 8 | Article 1506 Frontiers in Microbiology | www.frontiersin.org Frontiers in Microbiology | www.frontiersin.org 7 Seasonal Analysis of Precipitation Microbes Hiraoka et al. FIGURE 5 | Estimated ordinary habitats of precipitation microbes. Because the ordinary habitat for an individual 16S rRNA sequence cannot be conclusively
determined, the microbial habitability index (MHI) was calculated to estimate the probability of an ordinary habitat (Yang and Iwasaki, 2014). Estimated ordinary
habitats demonstrating <5% abundance were summarized as “Others.” The estimated route of the air mass before each precipitation event is indicated in the right
column. The terrestrial, oceanic, and hybrid routes are colored in orange, blue, and green, respectively. The estimated air mass backward trajectory maps are provided
in Supplementary Figure S3. FIGURE 5 | Estimated ordinary habitats of precipitation microbes. Because the ordinary habitat for an individual 16S rRNA sequence cannot be conclusively
determined, the microbial habitability index (MHI) was calculated to estimate the probability of an ordinary habitat (Yang and Iwasaki, 2014). Estimated ordinary
habitats demonstrating <5% abundance were summarized as “Others.” The estimated route of the air mass before each precipitation event is indicated in the right
column. The terrestrial, oceanic, and hybrid routes are colored in orange, blue, and green, respectively. The estimated air mass backward trajectory maps are provided
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wrote the manuscript. MM designed the experiments and
performed
the
sample
collection,
DNA
extraction,
DNA
sequencing, and bioinformatics analyses. KF and AM designed
the experiments and performed the sample collection, cell
counting, DNA extraction, and DNA sequencing. WI conceived
of
and
designed
the
study,
wrote
the
manuscript,
and
supervised the project. All authors read and approved the final
manuscript. The Supplementary Material for this article can be found
online
at:
http://journal.frontiersin.org/article/10.3389/fmicb. 2017.01506/full#supplementary-material Supplementary Figure S1 | Rarefaction curves for each precipitation sample. Supplementary Figure S1 | Rarefaction curves for each precipitation sample. Supplementary Figure S2 | Nonmetric multidimensional scaling plot for OTU
compositions. The distance matrix was calculated based on the Bray-Curtis
dissimilarity. The stress value of the final configuration was 20.46%. August 2017 | Volume 8 | Article 1506 Frontiers in Microbiology | www.frontiersin.org Seasonal Analysis of Precipitation Microbes Hiraoka et al. Supplementary Figure S3 | The estimated air mass backward trajectories 240 h
prior to precipitation events. Supplementary Figure S3 | The estimated air mass backward trajectories 240 h
prior to precipitation events. “terrestrial.” The estimated route of the air mass before each precipitation event is
indicated in the right column. “terrestrial.” The estimated route of the air mass before each precipitation event is
indicated in the right column. Supplementary Figure S4 | Estimated ordinary habitats of precipitation
microbes for three ecosystem groups. The abundance values in each ecosystem
group are summation for habitats described below. Marine-related: “aquatic”,
“marine”, “marine sediment”, “fish”, and “hot spring”; Animal-associated:
“human” “human gut” “human lung” “human nasal pharyngeal” “bovine gut” Supplementary Figure S4 | Estimated ordinary habitats of precipitation
microbes for three ecosystem groups. The abundance values in each ecosystem
group are summation for habitats described below. Marine-related: “aquatic”,
“marine”, “marine sediment”, “fish”, and “hot spring”; Animal-associated:
“human”, “human gut”, “human lung”, “human nasal pharyngeal”, “bovine gut”,
and “mouse gut”; and Soil-related: “hydrocarbon”, “rhizosphere”, “soil”, and Supplementary Figure S4 | Estimated ordinary habitats of precipitation
microbes for three ecosystem groups. The abundance values in each ecosystem
group are summation for habitats described below. Marine-related: “aquatic”, Supplementary Figure S5 | Estimated ordinary habitats of microbes in aerosol
and cloud water samples. Estimated ordinary habitats demonstrating <5%
abundance were summarized as “Others.” Supplementary Figure S5 | Estimated ordinary habitats of microbes in aerosol
and cloud water samples. SUPPLEMENTARY MATERIAL Estimated ordinary habitats demonstrating <5%
abundance were summarized as “Others.” Supplementary Table S1 | Amplicon-sequencing data of aerosol and cloud
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gks1219 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. Rodó, X., Ballester, J., Cayan, D., Melish, M. E., Nakamura, Y., Uehara, R., et al. (2011). Association of Kawasaki disease with tropospheric wind patterns. Sci. Rep. 1:152. doi: 10.1038/srep00152 Copyright © 2017 Hiraoka, Miyahara, Fujii, Machiyama and Iwasaki. 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. Copyright © 2017 Hiraoka, Miyahara, Fujii, Machiyama and Iwasaki. 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. Rodó, X., Curcoll, R., Robinson, M., Ballester, J., Burns, J. C., Cayan, D. R., et al. (2014). Tropospheric winds from northeastern China carry the etiologic agent
of Kawasaki disease from its source to Japan. Proc. Natl. Acad. Sci. U.S.A. 111,
7952–7957. doi: 10.1073/pnas.1400380111 Smets, W., Moretti, S., Denys, S., and Lebeer, S. (2016). Airborne bacteria in the
atmosphere: presence, purpose, and potential. Atmos. Environ. 139, 214–221. doi: 10.1016/j.atmosenv.2016.05.038 August 2017 | Volume 8 | Article 1506 Frontiers in Microbiology | www.frontiersin.org 10
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O Mercosul e a Nova Ordem
Econômica Internacional ALAN BARBIERO* e YVES CHALOULT** Rev. Bras. Polít. Int. 44 (1): 22-42 [2001]
*Professor do Departamento de Economia da Universidade do Tocantins.
**Professor do Departamento de Sociologia da Universidade de Brasília. ALAN BARBIERO* e YVES CHALOULT** Em 1991, ano em que surge o Mercosul, o mundo já vivia sob o impacto da
globalização e da regionalização. À época no entanto, esses dois fenômenos, embora
não fossem recentes, ainda desconheciam a intensidade com que hoje se apresentam,
menos de uma década depois. Com efeito, a partir dos anos 90 a globalização se viu impulsionada por um
cenário político internacional que não mais encontrava os antigos obstáculos
colocados pela Guerra Fria; sem falar no enorme avanço tecnológico da informática
e dos meios de comunicação e de informação, que vêm revolucionar a relação
tempo-espaço no mundo contemporâneo. Por sua vez, a regionalização tem assistido
a um processo de multiplicação dos acordos de integração regional por todo o
mundo, sendo que, somente no período de 1992 a 1996, foram registrados no Acordo
Geral sobre Tarifas Aduaneiras e Comércio (Gatt) cerca de 30 acordos bilaterais,
sub-regionais ou regionais. Uma análise do Mercosul à luz da evolução desses dois movimentos é o
que intentamos nestas linhas. Qual foi o contexto internacional que possibilitou o
surgimento do Mercosul? Que condições sócio-econômicas presentes na América
Latina favoreceram a sua formação? Como evoluíram as propostas de regionalismo
econômico? Que propósitos e influências marcaram a definição do modelo de
integração do Mercosul? Na tentativa de resposta a essas perguntas, inicialmente é debatido o
conceito de globalização com base em elementos extraídos da leitura de diversos
autores. Adiante-se que compartilhamos da idéia de que a globalização não está
formando uma sociedade homogênea, apesar de implantar, como nunca na história
da humanidade, uma ordem econômica de amplitude mundial. Na seqüência, são
expostas algumas noções básicas, mas substanciais, para a compreensão do que
estamos chamando de regionalização. Finalmente, numa abordagem histórico-
analítica que remonta à ordem econômica que emerge após a Segunda Guerra
Mundial, é desenhado o contexto internacional em que surge o Mercosul. O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 23 O objetivo principal é compreender o contexto mundial e a nova ordem
internacional que possibilitaram a criação do Mercosul, assim como as condições
que favoreceram a definição de seu modelo. No desenvolvimento da análise, a
globalização, embora seja vista como um processo multidimensional, é considerada
principalmente em sua dimensão econômica. 1. Um conceito em construção Embora o termo globalização não possa ser considerado ainda um conceito
preciso, correspondendo a uma realidade empírica inequivocamente descrita na
literatura, há um relativo consenso entre os estudiosos das ciências sociais e
econômicas de que o mesmo está associado às mudanças significativas que vêm
ocorrendo nas relações políticas, econômicas, sociais e culturais do mundo
contemporâneo. Para Giddens (1991), a globalização poderia ser melhor conceituada se os
sociólogos, em vez de darem uma importância indevida à idéia de sociedade, no
que ela significa um sistema limitado, a substituíssem por um ponto de partida que
se concentrasse em analisar como a vida social é ordenada através do tempo e do
espaço – na problemática do distanciamento tempo-espaço. Assim, a estrutura
conceitual do distanciamento tempo-espaço dirige nossa atenção às complexas
relações entre envolvimentos locais e interação à distância. “O nível de
distanciamento tempo-espaço na era moderna é muito maior do que em qualquer
outro período precedente, e as relações entre formas sociais e eventos locais e
distantes se tornam correspondentemente ‘alongadas’. A globalização se refere
essencialmente a este processo de alongamento, na medida em que as modalidades
de conexão entre diferentes regiões ou contextos sociais se enredam através da
superfície da Terra como um todo” (Giddens, 1991:69-70). Portanto, para esse
autor, a globalização pode ser definida como a intensificação das relações sociais
em escala mundial, ligando localidades distantes de tal maneira que acontecimentos
locais são modelados por eventos ocorrendo a grandes distâncias e vice-versa. Por seu turno, Santos (1997) distingue quatro constelações de relações
sociais que designa de “espaços-tempo estruturais”: o espaço-tempo doméstico, o
espaço-tempo da produção, o espaço-tempo da cidadania e o espaço-tempo mundial. Segundo ele, a problematicidade do tempo presente não advém de nenhuma dessas
constelações em separado, mas de sua conjunção. No entanto, é óbvio que o espaço-
tempo mundial, que tem uma maior influência sobre os demais, vem aumentando
sua relevância em virtude da intensificação da globalização da economia e das
interações transnacionais em geral nas duas últimas décadas. Seu problema
fundamental refere-se à crescente polarização entre o Norte e o Sul, ou seja, à
existência de desigualdades dentro do sistema mundial. 24 ALAN BARBIERO E YVES CHALOULT Apesar de coerente ao analisar as desigualdades mundiais a partir da divisão
entre Norte e Sul, a abordagem de Santos talvez não seja a mais adequada para a
conjuntura atual. 1. Um conceito em construção Dividir o mundo entre Norte rico e Sul pobre parece-nos uma
simplificação que sombreia a complexidade dos conflitos e a heterogeneidade do
mundo contemporâneo. Compreender o processo de globalização exige também
compreender o comportamento das diferentes matrizes culturais: como estas
reagem àquela? É certo que, segundo Ortiz (1996), muitos signos, símbolos, emblemas,
figuras ou ídolos circulam e flutuam pelo mundo desterritorializados. Porém, não
podemos imaginar que a sua apropriação pelas diferentes culturas se dê de forma
similar. A circulação desses signos e símbolos, produzidos geralmente pelo Ocidente
e propagados por todo o mundo através dos canais de comunicação, cria uma
situação de ambigüidade: ao mesmo tempo em que alguns valores se tornam
universais, as diversidades de valores emergem de maneira substancial. Para
Featherstone (1996), a velocidade e a expansão dos meios de comunicação, embora
não assegurem condições igualitárias de participação, permitem que novos atores
entrem no jogo e reivindiquem o direito de ser ouvidos, ainda mais com a facilidade
do transporte-transmissão de pessoas, imagens e objetos através do mundo inteiro
e com o conseqüente aumento das dificuldades dos governos para vigiar e controlar
o volume de informação e imagens que atravessam suas fronteiras. Assim, o
processo de globalização não somente favorece o aparecimento de uma cultura
global unificada, mas, sobretudo, tende a promover um campo de fragmentação,
sincretismo e hibridização das culturas. Em suma, ele revela a natureza multiforme
e a extrema complexidade dos fenômenos culturais. Por ser a identidade cultural ou étnica contrastiva, ou seja, ela se realça
quando em contato com outra, num mundo globalizado e heterogêneo, com forte
preponderância dos valores ocidentais, o contato entre as diferentes identidades
reforça as identidades em si, provocando muitas vezes uma reação de oposição
àqueles valores colocados como paradigmáticos pelo bloco que procura manter
sua hegemonia mundial. Nesse sentido, a análise de Santos deve ser enriquecida pela abordagem
desenvolvida por Huntington (1997) em sua obra O choque de civilizações. Huntington afirma que a modernização econômica e social não está produzindo
nem uma civilização universal significativa, nem a ocidentalização das sociedades
não-ocidentais. Os conflitos mais abrangentes e importantes do futuro não se
definirão entre ricos e pobres, ou grupos definidos em termos econômicos ou
ideológicos, e sim entre identidades culturais diferentes, ou seja, entre civilizações1. 1. Um conceito em construção Dentro desta perspectiva, o processo de globalização muda radicalmente
o contexto da política contemporânea, transformando suas condições, conseqüências
e atores, que por sua vez expandem o horizonte de ação – sentidos, valores, O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 25 constituição de sujeitos e de identidades, alianças e antagonismos – e interpelam
as categorias com que habitualmente são pensados seus principais problemas,
dilemas e desafios (Gómez, 1997). constituição de sujeitos e de identidades, alianças e antagonismos – e interpelam
as categorias com que habitualmente são pensados seus principais problemas,
dilemas e desafios (Gómez, 1997). É importante ressaltar no entanto que, para qualquer abordagem consistente
sobre o processo de globalização, deve-se tomar o cuidado de não cair na
ideologização ou mitificação do termo. Chesnais (1996), por exemplo, alerta-nos
sobre a popularização das expressões global e globalização no discurso político
neoliberal, muitas vezes com uma conotação ideológica, quando na verdade esses
termos são ainda vagos e ambíguos. g
g
Hirst e Thompson (1998), buscando desmistificar alguns aspectos do
processo de globalização, defendem a tese da possibilidade de governabilidade
nacional e internacional no mundo contemporâneo. Para eles, a atual economia,
altamente internacionalizada, tem precedente, sendo uma das diversas conjunturas
ou estados da economia internacional que existiram desde a segunda metade do
século XIX. Sob certos aspectos, a economia internacional contemporânea é menos
aberta e integrada do que o regime que prevaleceu de 1870 a 1914. A mobilidade
do capital não está produzindo uma transferência maciça de investimentos e de
empregos dos países avançados para os países em desenvolvimento. Contrariamente ao que pensam alguns defensores extremados da globalização, a
economia mundial está longe de ser global; os fluxos de comércio, investimento e
capital financeiro estão concentrados na Tríade formada pela Europa, Japão e
América do Norte. Os mercados globais estão fora da regulação e do controle,
ainda que o alcance atual e os objetivos da governabilidade econômica sejam
limitados pelos interesses divergentes das grandes potências e pelas doutrinas
econômicas que prevalecem entre suas elites. Em síntese, sendo as características mais visíveis da globalização a
compactação espacial, a aceleração temporal e a produção de novas
heterogeneidades, produzidas em – e dando origem a – contextos sócio-naturais
de alta incerteza (Dreifuss, 1997), os seus diversos vetores podem ser contraditórios
e abertos a vários desdobramentos, às vezes conflitantes entre si. A globalização
se mostra assim como um processo complexo e multidimensional que guarda não
poucas ambigüidades. 1. Um conceito em construção Daí em seu estudo impor-se a consideração da ambivalência
como uma importante categoria sociológica ou mesmo uma questão metodológica,
pois conforme alerta Beck (1997:22) “as categorias e os métodos da ciência social
falham diante da vastidão e da ambivalência dos fatos que devem ser apresentados
e considerados”. 2. Elementos sobre a regionalização O regionalismo econômico internacional é, junto com a globalização dos
mercados, um dos traços mais marcantes da economia mundial do Pós-guerra. ALAN BARBIERO E YVES CHALOULT 26 Percebe-se uma estreita relação entre a crise de legitimidade que atravessam
atualmente as grandes instituições econômicas internacionais e a proliferação de
acordos comerciais de várias ordens nos últimos 10 anos2. Os países são levados
a renovar suas formas de cooperação, estreitando-as em escala internacional nos
processos integrativos (Deblock e Brunelle, 1996). Isto nos faz interrogar sobre a
natureza desse fenômeno que tomou importância tanto por sua amplitude como
pelo interesse que suscita em todas as partes do mundo. Para Oman (1994), o movimento atual de regionalização responde em
parte à globalização econômica. Esses dois processos se opõem na medida em
que o primeiro é um movimento essencialmente centrípeto e político, ao passo que
o segundo é centrífugo e corresponde a um fenômeno microeconômico resultante
principalmente do comportamento e das estratégias das empresas transnacionais. Mas nem por isso um e outro são antitéticos ou antagônicos. Antes, dado que a
regionalização contribui na consolidação do jogo da concorrência, os dois processos
tendem mais a se reforçar do que a se contrapor. Se num primeiro momento as trocas comerciais predominam, a evolução
do processo de integração pode levar a sua ampliação para outros setores. Como
afirmam Deblock e Brunelle (1996), quaisquer que sejam as motivações de ordem
econômica que animam os atores estatais, os acordos regionais sempre responderam
a propósitos que vão além da esfera estritamente econômica, sobretudo quando
têm por objetivo a criação de blocos econômicos. Dizendo de outra forma, nenhum
acordo econômico regional jamais respondeu a preocupações de natureza
exclusivamente econômica. A integração regional pode ser vista como uma passagem para uma nova
estrutura organizacional dos Estados-nações, na qual novas formas de
relacionamento interno e externo surgem formalizando um novo espaço comum –
o espaço integrado. Como conseqüência, modifica-se radicalmente a concepção
do interno e do externo, chegando-se a um novo marco: as fronteiras do espaço
comum ampliado. A integração entre Estados nacionais implica um processo de
inter-relacionamento e interdependência multidimensional que obriga a ter presentes,
simultaneamente, diferentes planos da realidade social (Fernández, 1992). Vários autores assinalam as condições (Galtung, 1968), potencialidades
(Nye, 1971) ou pressupostos (Errandonea, 1977) para a integração entre Estados
nacionais. 2. Elementos sobre a regionalização Embora nenhum afirme que qualquer desses fatores seja necessário ou
suficiente para o sucesso da integração regional, eles acreditam que a presença de
determinadas condições favorecem o desenvolvimento de redes de
interdependência que facilitam a transferência de lealdade do plano nacional para
o supranacional. Algumas delas são: (1) a existência de um substrato comum de
valores e interesses e, mais importante ainda, de uma escala de preferências bem
estabelecida entre eles, de modo que conflitos e dilemas possam ser mais facilmente
resolvidos; (2) uma relativa simetria econômico-social e político-institucional, com O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 27 certo grau de complementaridade entre os Estados envolvidos, condição para que
se amplie a interdependência; (3) a complementaridade e consistência dos valores
e interesses manifestos e compartilhados pelas elites dos atores envolvidos; (4) o
apoio e o compromisso de cada Estado nacional à associação supranacional,
contando com atores políticos capazes de assumir as tarefas da integração com
continuidade, competência e flexibilidade. Existe uma dificuldade básica para qualquer tipo de integração, seja para
as organizações políticas e de segurança ou para as organizações econômicas: a
convergência e a comunhão de valores culturais. “As regiões são a base para a
cooperação entre os Estados, unicamente na medida em que a geografia coincida
com a cultura. Divorciada da cultura, a proximidade não gera por si só aspectos
em comum e pode mesmo induzir exatamente o oposto” (Huntington, 1997:161). Desta forma, as alianças militares e as associações econômicas requerem a
cooperação entre seus membros; a cooperação depende da confiança e a confiança
brota mais facilmente de valores e cultura em comum. Com várias experiências espalhadas pelo mundo, o processo de integração
regional se dá em diferentes modelos, que podem ser ou não fases sucessivas da
integração, a saber: zona de preferência tarifária, zona de livre comércio, união
aduaneira, mercado comum e união econômica. O Mercosul é um projeto de
construção de um Mercado Comum, cuja execução encontra-se na fase de União
Aduaneira parcial3. 2. Elementos sobre a regionalização No interior das Américas podemos encontrar quatro grandes tipos de
acordos comerciais: 1)
quatro uniões aduaneiras: o Mercosul (ao qual são associados o Chile
e a Bolívia), a Comunidade Andina, o Mercado Comum da América
Central (MCAC) e o Mercado Comum do Caribe (Caricom); 2)
acordos de livre comércio, como o Acordo de Livre Comércio da
América do Norte (Nafta) e o Acordo entre o Grupo dos Três
(Colômbia, México e Venezuela), além de múltiplos acordos bilaterais
assinados, por exemplo, entre o México e o Chile, o México e a Costa
Rica, o Canadá e o Chile etc.; 3)
acordos preferenciais, dentre os quais o acordo Canadá–Caraíbas
(Caribcan) e o entre os Estados Unidos (EUA) e os países da
Comunidade Andina, cujo objetivo, dentre outros, é lutar contra o
narcotráfico; 4)
acordos de caráter mais geral, como a Associação Latino-Americana
de Integração (Aladi), os acordos de complementação assinados no
interior da Aladi e os diversos acordos de cooperação em matéria de
comércio e investimento (Canadá e Estados Unidos, principalmente),
ou ainda os acordos mais setoriais ou mais técnicos. ALAN BARBIERO E YVES CHALOULT 28 3. Inter-relações entre a globalização e a regionalização Discutido o conceito de globalização e vistas algumas noções sobre
regionalização, podemos agora enfocar esses dois fenômenos e apontar as suas
inter-relações. Em nossa abordagem procuraremos sublinhar o aspecto da
historicidade e da complementaridade entre os dois movimentos, bem como
evidenciar as heterogeneidades existentes e, ainda, levar em conta as preocupações
estratégicas diferentes que podem motivar os diversos atores envolvidos. Conforme
já advertimos, a globalização, embora constituída de múltiplas dimensões inter-
relacionadas (Viola e Oliveira, 1997), será considerada sobretudo em sua dimensão
econômica, haja vista sua relação intrínseca com o surgimento do Mercosul. Segundo Brunelle e Deblock (1996)4, a idéia de globalização econômica
inscreve-se dentro de uma perspectiva histórica marcada por dois momentos
fundamentais: (1) a implantação no Pós-guerra de uma nova ordem mundial baseada
numa matriz liberal cujo principal arquiteto foi John Maynard Keynes, e (2) o fim
da Guerra Fria, que abriu às empresas multinacionais a possibilidade de um papel
mais relevante na liderança do processo de globalização econômica. O mesmo
pode-se dizer da regionalização, daí sermos levados a dissociar o regionalismo
atual, qualificado por esses autores como sendo de segunda geração, do
regionalismo que o precedeu, ou seja, de primeira geração. Desfaz-se dessa maneira a percepção de uma certa homogeneidade dentro
dos acordos estabelecidos pertencentes a uma mesma categoria. Em outras
palavras isso quer dizer que, mesmo tratando-se de dois acordos de livre comércio,
ou de duas uniões aduaneiras, eles podem diferenciar significativamente entre si:
uns podem se aproximar de um regionalismo de primeira geração e outros, de
segunda. Observa-se, assim, uma descontinuidade nos processos de regionalização
marcada principalmente pelas transformações impostas pela globalização
econômica. Para compreender melhor isto, nos reportaremos inicialmente à ordem
internacional que emerge nos anos 40. 3.1. Uma nova ordem mundial A construção da ordem do Pós-guerra se fez em dois níveis: nacional e
internacional. Os Estados afetados diretamente pela Segunda Guerra Mundial se
dedicaram à reconstrução ou à reconversão de sua economia nacional. Sobre a
influência principal de Keynes, assiste-se à transformação do modo de regulação
social e econômico dos Estados a partir da construção do que se chamou de
providencialismo5. Esta iniciativa requer uma colaboração estreita entre três atores
no plano nacional: o Estado, as organizações patronais e os sindicatos de
trabalhadores. O compromisso entre tais atores, comumente chamado de tripartismo,
foi essencial para a estabilidade da ordem do Pós-guerra. O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 29 Em nível internacional a influência teórica de Keynes (1978) foi também
marcante. O economista inglês tornou-se o grande arquiteto da nova ordem
econômica mundial que emerge na década de 1940. Inspirada em suas idéias, a
Organização das Nações Unidas (ONU) surge com um funcionamento calcado
numa concepção moderna do papel do Estado. A ONU se coloca como uma
organização compromissada em garantir uma visão universalista e pluralista da
ordem internacional. Talvez seja Shotwell (1945), em seu livro La grande décision, um dos
que mais claramente expuseram a proposta de criação da ONU. Shotwell estava
preocupado em estabelecer instrumentos capazes de evitar novos confrontos
mundiais, buscando assim organizar uma comunidade internacional. A proposta das Nações Unidas, segundo ele, tentava cobrir três grandes
problemas: segurança, bem-estar e justiça. Para cada um, dever-se-ia empregar
uma técnica diferente. Para o problema da segurança, a “action de police et l’emploi
de la force”; para o bem-estar, a criação “d’un mécanisme de coopération”; e,
para a justiça, uma expressão dentro de “une loi et une procédure internationales”
(Shotwell, 1945:37). Na verdade, estavam aí lançadas as bases para a construção
da nova ordem mundial. Essa ordem se orientava em uma matriz liberal. Entretanto, é importante
ressaltar que existiam divergências entre os liberais da época. Alguns propugnavam
o intervencionismo por parte do Estado, já outros defendiam o não intervencionismo. O encontro dos liberais em Lippmann, em 1938, foi um dos momentos culminantes
no acirramento das divergências entre os intervencionistas e os não-
intervencionistas. Liderados por Keynes, os liberais partidários da primeira
tendência formaram uma maioria. O economista Friedrich Hayek passará a ser,
nos anos 40, um forte crítico de Keynes, representando os liberais da segunda
tendência. 3.1. Uma nova ordem mundial Contudo, somente nos anos 70 é que Hayek, um dos principais
representantes do neoliberalismo, exercerá uma ascendência teórica maior, enquanto
a ordem econômica internacional seria marcada, em seus primórdios, pela influência
dos liberais intervencionistas. Os Aliados, ao formularem uma nova ordem econômica internacional,
tinham como objetivo geral colocar o mundo fora do perigo da necessidade e da
insegurança. O alcance desse objetivo passava por dois níveis. No primeiro, o
Estado aparecia como o principal ator para garantir o progresso econômico e social;
no segundo, buscava-se a criação de um sistema organizado de instituições
econômicas internacionais, oriundas do sistema geral das Nações Unidas. O sistema
implantado em nível econômico deveria ser complementar ao organizado para
assegurar a paz. Fosse de forma individual ou coletiva, ele deveria também engajar
o conjunto das nações, independentemente de seus regimes econômicos e políticos,
na construção de uma ordem na qual esperava-se garantir a segurança e a
prosperidade mutuamente. 30 ALAN BARBIERO E YVES CHALOULT As instituições internacionais deveriam ter como tripé de sustentação o
universalismo, a diferenciação e o tripartismo. Ao mesmo tempo em que deveriam
ser considerados como iguais (idéia do universalismo), os países deveriam ter
responsabilidades diferentes dentro da ordem internacional (princípio da
diferenciação). Com referência ao tripartismo, Shotwell propõe o modelo da
Organização Internacional do Trabalho (OIT) como sendo o mais indicado para
todas as organizações internacionais, visto que envolve outros atores sociais na
garantia do bem-estar. “C’est pourquoi nous avons suggéré ci-dessus que la
constitution de cet organisme (l’OIT) soit étudiée pour servir de modèle à celle des
organes autonomes qui seront nécessaires à la vie propre de l’économie
internationale” (Shotwell, 1945:222). Entretanto, o tripartismo será aplicado mais em nível nacional, pelo Estado
providência, do que em nível internacional. Buscava-se com isto desenvolver a
cooperação e a solidariedade dos principais atores na reconstrução da economia
nacional, como também servir de contrapeso às idéias socialistas que ganhavam
força na Europa do Pós-guerra. No plano internacional, a ONU criará o Conselho
Econômico e Social (Ecosoc), inspirado no tripartismo, com a incumbência de
promover a prosperidade na comunidade internacional emergente6. É importante
assinalar que houve uma forte complementaridade entre a questão nacional e a
internacional na construção dessa nova ordem mundial. 3.2. A evolução do regionalismo econômico O regionalismo econômico implantado no Pós-guerra se desenvolveu em
paralelo com o sistema multilateral do Gatt, inscrevendo-se num contexto
internacional particular marcado, de um lado, pela Guerra Fria e, de outro, pelas
frustrações em face da lentidão na edificação de uma ordem verdadeiramente
internacional. O Plano Marshall de 1947 exigia, em contrapartida à ajuda financeira
oferecida, que os países europeus destruídos pela guerra deveriam reagrupar-se e
dotar-se de instituições comuns. Impulsionada por esse plano, a Organização
Européia de Cooperação Econômica (Oece)8 foi criada em 1948, podendo ser
considerada como uma das primeiras grandes organizações econômicas regionais
do Pós-guerra. Explicitamente, ela respondia a preocupações de ordem geopolítica
e econômica, fazendo parte de uma estratégia dos EUA de conter o comunismo
que rondava a Europa, assim como de impedir que os países europeus se fechassem
sobre si mesmos. O regionalismo emergente saía do quadro multilateralista definido
em Genebra por ocasião dos encontros do Gatt. O Plano Marshall de 1947 exigia, em contrapartida à ajuda financeira
oferecida, que os países europeus destruídos pela guerra deveriam reagrupar-se e
dotar-se de instituições comuns. Impulsionada por esse plano, a Organização
Européia de Cooperação Econômica (Oece)8 foi criada em 1948, podendo ser
considerada como uma das primeiras grandes organizações econômicas regionais
do Pós-guerra. Explicitamente, ela respondia a preocupações de ordem geopolítica
e econômica, fazendo parte de uma estratégia dos EUA de conter o comunismo
que rondava a Europa, assim como de impedir que os países europeus se fechassem
sobre si mesmos. O regionalismo emergente saía do quadro multilateralista definido
em Genebra por ocasião dos encontros do Gatt. As posições entre os países europeus eram incompatíveis umas com as
outras, resultando em duas direções diferentes: um projeto mais ambicioso (1957)
de formar uma Comunidade Econômica Européia (CEE) e um mais modesto (1959)
de formar a Associação Européia de Livre Comércio (AELC). Os dois projetos se
distinguiam, principalmente, pelo fato de o primeiro ser antes de tudo político,
enquanto o segundo era um projeto econômico de inspiração mais liberal9. Embora
fossem distintos, ambos partilhavam de uma mesma preocupação: a posição da
Europa num mundo polarizado pelos EUA e União Soviética. A criação da CEE irá servir de modelo catalisador e inspirador para a
América Latina, cujo primeiro acordo de integração, a Associação Latino-
Americana de Livre Comércio (Alalc), data de 1960. 3.1. Uma nova ordem mundial A Conferência de Bretton Woods, realizada nos EUA em 1944, deu origem
ao Banco Internacional para a Reconstrução e Desenvolvimento (Bird) e ao Fundo
Monetário Internacional (FMI), além de ter desencadeado o processo de
implantação de várias outras instituições internacionais, como a Organização para
a Alimentação e Agricultura (FAO) e a Organização das Nações Unidas para a
Educação, a Ciência e a Cultura (Unesco). Foi ela importante não só para definir
o quadro internacional em seu conjunto, mas também para determinar a posição da
América Latina neste novo contexto, conforme se verá adiante Do ponto de vista comercial, a economia internacional deveria liberalizar-
se. As barreiras tarifárias teriam de ser reduzidas em favor do comércio mundial. Para normatizar a redução das barreiras (ou mesmo suprimi-las) e dar peso ao
livre comércio, criou-se, em 1947, o Acordo Geral sobre Tarifas Aduaneiras e
Comércio (Gatt), fechando o quadro da nova ordem mundial7. Contudo, a ordem mundial concebida no Pós-guerra não foi totalmente
efetivada. Conforme assinala Hobsbawm (1999:224), “a Segunda Guerra Mundial
mal terminara quando a humanidade mergulhou no que se pode encarar,
razoavelmente, como a Terceira Guerra Mundial, embora uma guerra muito
particular”. Não foi criada uma comunidade verdadeiramente internacional, mas
um mundo marcado pela bipolaridade entre os países capitalistas de um lado,
liderados pelos EUA, e os socialistas de outro, tendo a União Soviética à sua
frente. Somente em 1989, com a queda do muro de Berlim, a subseqüente O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 31 desintegração do bloco soviético e o fim do socialismo real, o quadro idealizado
seria então implantado, mas não sem alterações para se adequar à conjuntura
atual. O fim da Guerra Fria “retirou de repente os esteios que sustentavam a
estrutura internacional e ... as estruturas dos sistemas políticos internos mundiais”
(Hobsbawm, 1999:251). Se antes as empresas múlti ou transnacionais encontravam
um obstáculo para atuarem mundialmente, elas agora estão livres para liderarem o
processo de globalização econômica. Isto influenciará, sobremaneira, nos modelos
de regionalismo econômico. 3.2. A evolução do regionalismo econômico No entanto, conforme se
verá adiante, as propostas de integração latino-americanas guardarão uma certa
originalidade em relação ao modelo europeu, na medida em que se colocam
explicitamente a serviço de um projeto econômico e político de desenvolvimento. 32 ALAN BARBIERO E YVES CHALOULT Em que pesem as diferenças entre os diversos projetos de integração,
observa-se, em sua evolução, a presença de três aspectos convergentes. Primeiro,
através das discussões sobre os acordos regionais, os debates rapidamente foram
deixando o terreno da liberalização propriamente dita e passando para o da integração
econômica regional. Segundo, em todos os casos, os acordos tiveram por finalidade
última a formação de blocos econômicos homogêneos e de tamanho suficiente
para criar uma massa crítica no interior do sistema econômico internacional. Terceiro,
a linha de demarcação entre diferentes projetos integrativos se situava menos no
fato de implicar países industrializados e países em desenvolvimento, e mais nas
diferenças entre os projetos que se traduziam num projeto político explícito e os
que se revelavam mais num projeto econômico (Deblock e Brunelle, 1996). A primeira vaga de regionalismo se desenvolveu dentro de um contexto
intervencionista. O Estado e as instituições econômicas internacionais buscaram
introduzir uma certa racionalidade num mundo que passara por uma forte depressão
nos anos 30 e por uma guerra nos anos 40. Esta lógica engajava os Estados
coletivamente a produzir um bem público internacional, que seria o livre comércio. O regionalismo econômico de primeira geração ficará marcado, assim, por quatro
características fundamentais: (1) estava voltado mais para uma integração econômica
do que para um regionalismo econômico; (2) partilhava de uma visão construtivista
de integração inspirada nos parâmetros keynesianos de políticas públicas; (3) deveria
permitir a ampliação da margem de manobra dos Estados na condução de suas
políticas nacionais, dentro do contexto de liberalização10; (4) respondia a objetivos
de cunho mais político ou econômico, formando com isso um duplo movimento. Essa última característica é um dos motivos pelos quais o regionalismo de
primeira geração veio a ser ultrapassado. Em decorrência de seu duplo movimento,
houve paralelamente o desenvolvimento de instituições comuns de inspiração
federalista, por um lado, e a integração econômica e liberalização comercial, por
outro. O paralelismo entre os compromissos regionais e os engajamentos em nível
internacional acabarão provocando problemas de ordem política e econômica. 3.2. A evolução do regionalismo econômico O
convívio com esses problemas só seria possível se fosse mantida uma certa
justaposição entre os níveis nacional, regional e internacional, ou, como assinalam
Deblock e Brunelle (1996), aplicando-se as idéias de Keynes na política interna e
as de Adam Smith na política externa11. O regionalismo de primeira geração entrará,
dessa maneira, em contradição com a lógica mesma da integração dos mercados
em escala mundial. Marcados pela crise do Estado providência, os anos 70 assistirão à ascensão
dos liberais não-intervencionistas, denominados de neoliberais. Fazendo escola na
Universidade de Chicago, onde lecionou, Friedrich Hayek receberá em 1974 o
Prêmio Nobel de Economia, passando a ser um dos principais mentores do governo
Ronald Reagan. Pouco depois, principalmente a partir dos anos 80, a globalização
dos mercados virá a ser um dado incontornável, minando o compromisso histórico O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 33 sobre o qual havia sido construída a ordem do Pós-guerra. Os parâmetros da
política econômica serão, a partir de então, definidos em termos de competitividade
e não mais em termos de progresso econômico e social. É uma outra visão do
papel do Estado face à sociedade civil que se coloca. De sua parte, o perigo do
comunismo deixará de existir nos anos 90, não se justificando mais o tripartismo. David Harvey (1996) denomina essa nova situação de “acumulação
flexível”. Ela se apóia na flexibilidade dos processos e dos mercados de trabalho,
bem como dos produtos e padrões de consumo. Caracteriza-se pelo surgimento de
setores de produção inteiramente novos, maneiras novas de fornecimento de
serviços financeiros, novos mercados e, sobretudo, taxas altamente intensificadas
de inovação tecnológica e comercial12 . Esse novo contexto possibilitará a passagem para uma nova forma de
regionalismo econômico, chamada de deep integration. Agora, busca-se mais
uma integração em profundidade das atividades das empresas no interior dos espaços
cobertos pelos acordos assinados, do que simplesmente um incremento das trocas
comerciais. Trata-se menos de um projeto político do que de uma forma de permitir
aos Estados melhor realizarem seus objetivos perante a cena da economia
internacional. A segurança dentro do contexto econômico internacional passa a
ser uma preocupação maior. 3.2. A evolução do regionalismo econômico Como sublinham Deblock e Constantin (2000), o
objetivo dos Estados signatários desses novos acordos é duplamente securitário:
primeiramente para as empresas, criando um ambiente normativo propício para o
desenvolvimento de suas atividades transfronteiriças; em segundo lugar para os
Estados em si, na medida em que é também, e paralelamente ao primeiro objetivo,
uma forma de melhor coordenar o seu ambiente internacional. As características
do regionalismo de segunda geração são mais evidentes no Nafta e na proposta da
Alca (Deblock e Brunelle, 1999) do que na União Européia e no Mercosul. Sendo esses os desdobramentos verificados na ordem econômica
internacional a partir do fim da Segunda Guerra Mundial, cabe agora indagar de
que forma a América Latina se posicionou durante esse período. A resposta a
essa indagação nos ajudará a compreender melhor o contexto em que o Mercosul
surge. 4. América Latina: da substituição de importações à abertura ao mercado
mundial A Conferência de Bretton Woods ficou mundialmente conhecida pela
rivalidade entre o Plano Keynes e o Plano White13. Um representava os interesses
da Inglaterra, que perdia sua hegemonia. O outro, os interesses dos EUA, potência
emergente no cenário do Pós-guerra. A principal diferença entre os planos era
que Keynes defendia a implantação de apenas uma agência internacional e a criação
de uma nova moeda com peso internacional (bancor), enquanto White se voltava ALAN BARBIERO E YVES CHALOULT 34 para a criação de duas agências internacionais – um fundo monetário e um banco
para a reconstrução – e a fixação de paridade cambial ao dólar. Sagrando-se
vencedora a proposta americana, foram criados o FMI e o Bird. Contudo, o que quase todos os autores deixam de historiografar é que, não
apenas dois, mas três projetos estavam em disputa no encontro de Bretton Woods. O terceiro deles, o Plano Suárez, representava os interesses dos países do Terceiro
Mundo, em especial dos latino-americanos14 . É importante lembrar que dos 44
países participantes em Bretton Woods 19 eram da América Latina. O que
reivindicavam esses países? Na verdade, tanto o Plano Keynes quanto o Plano White estavam
preocupados, principalmente, com a reconstrução dos países destruídos pela guerra
e com a estabilização monetária. Os países latino-americanos não tinham sido
afetados diretamente pelo conflito, a ponto de necessitarem do benefício dos
programas de reconstrução. Portanto, para eles as novas instituições internacionais
deveriam se voltar não somente à reconstrução, mas igualmente ao desenvolvimento. Embora sua representação fosse quase a metade do total de participantes,
a única alteração lograda por esses países na conferência, ainda assim com certa
resistência, foi o compromisso de o banco recém-criado atuar na questão do
desenvolvimento. Na prática, no entanto, houve quase que somente a mudança de
nome do banco, que passou de Banco Internacional de Reconstrução e Fomento –
a proposta original – para Banco Internacional de Reconstrução e Desenvolvimento,
ficando a América Latina à margem da ordem econômica mundial que surgia15 . Ela se voltará então para dentro de si mesma e, por meio da Comissão Econômica
para a América Latina (Cepal), se concentrará em seu programa de desenvolvimento
baseado na industrialização por substituição de importações. Criada em 1948, no âmbito das Nações Unidas, a Cepal16 vai abrigar o
projeto de desenvolvimento da América Latina, o qual não havia encontrado espaço
dentro da ordem que se estabelecia no Pós-guerra. 4. América Latina: da substituição de importações à abertura ao mercado
mundial Tendo à frente o intelectual
argentino Raúl Prebisch, a Cepal se diferenciará das demais comissões econômicas
regionais da ONU pela originalidade de suas propostas17 . Prebisch defendia a
tese do nacionalismo econômico e a estratégia do desenvolvimento por substituição
de importações. A idéia central era que o livre comércio imposto aos países menos
desenvolvidos fazia crescer a sua dependência vis-à-vis a produção e exportação
dos recursos naturais não transformados. A imposição de barreiras às importações
de produtos manufaturados e o desencadeamento de um desenvolvimento industrial
endógeno colocavam-se assim como a única forma para romper o círculo vicioso. Essa estratégia prevalecerá no interior da Cepal, influenciando a maioria
dos governos latino-americanos. Os Estados vão se envolver na economia de modo
a favorecer o desenvolvimento do mercado interno e a encorajar a produção local,
fazendo uso, notadamente, do protecionismo (Cepal, 1998). O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 35 A estratégia de industrialização por substituição de importações já havia
sido adotada por diversos países nos anos 30, durante a Depressão. Todavia, ela
será retomada e intensificada nos países latino-americanos, atingindo seu auge na
década de 1950, até ser abandonada completamente na década de 1990. Essa
estratégia pode ser entendida como um processo de desenvolvimento “fechado” e
reativo às restrições do comércio exterior (Tavares, 1978), em que a dinâmica
substitutiva consiste na forma como a economia reage aos estrangulamentos
sucessivos de pagamentos. Através da compressão progressiva da lista de
importações, a industrialização passaria dos setores de instalação fácil, pouco
exigentes em matéria de tecnologia, capital e escala, a segmentos cada vez mais
sofisticados e exigentes (Bielschowsky, 1998). Não nos cabe aqui encetar uma discussão sobre os diversos motivos que
levaram à falência desse modelo, visto não ser esse nosso principal objetivo. Iremos
apenas indicar alguns pontos para demonstrar como a América Latina foi
abandonando um modelo de desenvolvimento para dentro e se aproximando de
um modelo para fora (Cepal, 1994a e 1994b). Essa transformação não se fará
sem guardar uma relação direta com as mudanças em suas propostas de integração
regional, influenciando, conseqüentemente, o surgimento do Mercosul. O regionalismo econômico fazia parte da estratégia de desenvolvimento
por substituição de importações. Era quase uma condição sine qua non ao processo
de industrialização. 4. América Latina: da substituição de importações à abertura ao mercado
mundial Os trabalhos iniciais da Cepal destacavam a necessidade de os
países da região se agruparem e desenvolverem entre eles as ligações de
complementaridade econômica necessárias à implantação de uma estratégia de
industrialização por substituição de importações. A idéia lançada nos anos 50 era
criar um mercado comum latino-americano. A integração deveria, de um lado,
assegurar a sobrevivência do processo de industrialização, contornando o obstáculo
que representava o tamanho reduzido do mercado local18 . De outro lado, deveria
diminuir a vulnerabilidade das economias locais em face dos mercados externos, a
qual, paradoxalmente, tinha se agravado com a estratégia de industrialização,
principalmente devido a um aumento no déficit externo. A integração deveria,
enfim, estabelecer, no longo prazo, uma relação mais favorável aos países da
América Latina dentro da economia mundial, permitindo-lhes modificar em seu
favor, uma vez reestruturadas suas economias, os termos das trocas internacionais
(Prebisch, 1988). O regionalismo econômico tinha, com isso, duas direções: a do
desenvolvimento através da integração “voltada para o interior”, e a da
transformação da relação centro-periferia19 (Marcoux, 1996). Foi com essas bases em mente que os governantes da América Latina
assinaram em Montevidéu, em 1960, o acordo criando a Alalc. O objetivo último
era promover a livre circulação de “bens e serviços, homens e capitais ... sem
nenhum obstáculo, dentro de um vasto mercado comum latino-americano” ALAN BARBIERO E YVES CHALOULT 36 (Marcoux, 1996:4)20. Desejava-se estabelecer uma zona de livre comércio em um
prazo de 12 anos. (Marcoux, 1996:4)20. Desejava-se estabelecer uma zona de livre comércio em um
prazo de 12 anos. Embora tenha permitido um crescimento do comércio intra-regional em
seus primeiros anos, a Alalc mostrou-se aquém das expectativas suscitadas. A
disparidade crescente das políticas econômicas dos Estados-membros e a rigidez
com que o acordo fora estabelecido estão na raiz dos principais problemas que a
conduziram ao fracasso. Ademais, ela guardava várias características do
regionalismo de primeira geração, o qual, como já vimos, havia entrado em choque
com o movimento mais geral de gobalização econômica, intensificado nos anos 80. 4. América Latina: da substituição de importações à abertura ao mercado
mundial g
g
ç
Paralelamente, inicia-se nos anos 60 um processo de autocrítica no interior
da própria Cepal que continua nos anos 70 e 80 quando, com a eclosão da crise do
endividamento externo, a paralisação das principais instituições que suportavam o
desenvolvimento e a forte queda do ritmo de crescimento, fica evidente que o
modelo de desenvolvimento para dentro, ou de substituição de importações, chegara
ao seu esgotamento. Um novo tratado será assinado em 1980, em Montevidéu, criando a
Associação Latino-Americana de Integração (Aladi). Conservando o objetivo de
longo prazo de criar um mercado comum latino-americano, a Aladi não estabelecerá
prazos precisos nem procedimentos fixos. Terá um caráter de maior flexibilidade,
se comparada com a Alalc, e tomará mais a forma de um acordo de princípios,
servindo como um guarda-chuva para outros acordos bilaterais e sub-regionais,
desde que estes estejam abertos à participação dos demais membros. Sua inserção
se fará dentro do que a Cepal (1994a) passa a chamar de regionalismo aberto21 . Enquanto isso, baseando-se nas medidas de ajustamento estrutural, os países
da América Latina vão um após o outro – logicamente que guardando certa
singularidade em cada caso – fazer uma virada fundamental, que os conduzirá a
orientar cada vez mais suas economias para o exterior. Serão elas liberadas das
regulamentações estatais percebidas como obstáculos a tal orientação. A política
de ajustamento estrutural será uma prerrogativa do FMI e do Banco Mundial para
a outorga dos apoios financeiros necessários aos países latino-americanos para
fazer frente ao aprofundamento da crise de suas dívidas externas. É bem verdade
que a crise da dívida, assim como os múltiplos problemas com que se defrontaram
os países da América Latina nos anos 80, não fizeram mais do que precipitar os
acontecimentos. No fundo, eles deveriam se preparar, ou melhor, se ajustar ao
novo contexto mundial. Desta forma, inicia-se no interior desses países um processo que busca,
entre outros objetivos, limitar o papel do Estado, desencadear um programa de
privatização, diminuir os gastos públicos, eliminar a inflação, estabilizar a moeda,
aumentar as exportações e abrir suas economias ao mercado mundial. Dentre os
principais países da América Latina, o Chile foi um dos primeiros a aplicar essa
política, sendo o Brasil um dos últimos. Estaria aí, nos anos 90, a América Latina O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 37 se inserindo na ordem econômica internacional, revigorada com o fim da Guerra
Fria. 4. América Latina: da substituição de importações à abertura ao mercado
mundial Se parece claro que a necessidade, por parte de um número crescente de
países latino-americanos, de optar por um modelo de desenvolvimento baseado em
premissas neoliberais permite explicar em boa medida a crise do modelo de
integração para dentro, a transição para um modelo de desenvolvimento aberto
permite, por sua vez, compreender por que nos anos 90 as propostas de integração
regional não poderiam ser de outra maneira que não para fora. Existe uma
coerência entre o modelo de desenvolvimento adotado pelo conjunto dos países e
suas propostas de regionalismo econômico. Mais do que isto, há uma forte
complementaridade entre os dois, bastando apenas lembrar que foi no âmbito das
negociações do Mercosul que o governo Collor se apoiou para diminuir as barreiras
tarifárias do Brasil em relação ao resto do mundo. Os acordos estabelecidos nos
moldes do regionalismo aberto vão se inscrever dentro de um processo de
liberalização paralelo e complementar ao que é seguido em nível multilateral. Sob o prisma desse contexto internacional é que o Mercosul emergirá. A
Aladi é o âmbito normativo que possibilita a assinatura do Tratado de Assunção
em 1991, o qual lhe dá origem. Em suas proposições, o Mercosul buscará agregar
os temas do desenvolvimento e da democracia, aliados à preocupação com a
modernização competitiva. Constitui-se numa visão distinta daquela derivada do
modelo de substituição de importações, de que a Alalc, na sua origem, foi exemplo. Não obstante, o Mercosul guardará aspectos do regionalismo de primeira
geração. Sua inspiração primeira será a União Européia. Embora não tenha criado
instituições supranacionais, como por exemplo o Parlamento Europeu, seu modelo
de integração atenderá a outras questões que vão além daquelas relativas à
segurança para os investimentos, tão presentes no Nafta e na proposta da Alca. No Mercosul existe uma forte motivação político-estratégica, especialmente
por parte do Brasil. Poderíamos dizer, assim, que a sua proposta se insere entre o
regionalismo de primeira geração e o de segunda geração, conjugando aspectos de
ambos esses regionalismos. Do primeiro podemos destacar a sua tendência
federalista e construtivista22 de integração, o recurso ao tripartismo23, a presença
marcante do Estado e a sua motivação político-estratégica. Do segundo destacamos,
principalmente, a idéia de um regionalismo aberto, a sua sintonia com a economia
mundial, a busca de maior competitividade sob a base de um eixo exportador e de
uma liberalização frente às trocas internacionais. 4. América Latina: da substituição de importações à abertura ao mercado
mundial Enfim, podemos afirmar que o
Mercosul é, de certa forma, um projeto original. 1
Huntington identifica sete principais civilizações contemporâneas: sínica, japonesa, hindu,
islâmica, ocidental, latino-americana e africana.
2
Aos 30 acordos notificados no Gatt de 1992 a 1996 acrescentam-se outros 57 anteriormente
existentes (Deblock e Brunelle, 1996). 5. Conclusão A globalização é um fenômeno complexo que deve ser percebido através
de suas diferentes dimensões, contradições e ambigüidades. Após apresentar nossa ALAN BARBIERO E YVES CHALOULT 38 percepção mais geral sobre a globalização e alinhar alguns elementos sobre a
regionalização, buscamos analisar a globalização do ponto de vista econômico,
numa perspectiva histórica. Vimos que a globalização econômica não é um
fenômeno novo. Suas premissas remontam à década de 1940, quando se buscou
imaginar um quadro de regulação internacional capaz de assegurar a paz e a
prosperidade num mundo que acabara de passar por um segundo conflito
generalizado. g
A regionalização econômica, por sua vez, é tão antiga quanto a globalização
e, diferentemente do que se possa pensar, não é uma conseqüência desta nem uma
resposta estrita dos Estados-nações a este movimento. Na verdade, trata-se de
dois fenômenos intimamente inter-relacionados. Do ponto de vista histórico, ambos
foram marcados pelo contexto internacional que emergiu logo após a Segunda
Guerra Mundial e, num segundo momento, pelo fim da Guerra Fria. Do ponto de
vista teórico, inspiraram-se inicialmente no keynesianismo, para posteriormente se
aproximarem das concepções neoliberais. Tanto as transformações históricas quanto
as de cunho teórico provocaram uma mutação nos modelos de regionalismo
econômico, podendo-se falar em duas gerações de regionalismo. A América Latina não deixará de receber influências dessas
transformações. Inicialmente, ao se verem à margem da ordem internacional surgida
no Pós-guerra, os países latino-americanos buscarão implantar um modelo de
desenvolvimento fechado, baseado na industrialização por substituição de
importações. A integração regional é vista como uma peça fundamental para o
sucesso dessa proposta. Com a criação da Alalc, implanta-se um regionalismo
voltado para dentro. Na elaboração dessa concepção de desenvolvimento, a Cepal
exercerá um papel fundamental, especialmente as teses defendidas por Raúl
Prebisch. Nos anos 90, com o fim da Guerra Fria e com a ascensão das idéias
neoliberais, uma nova proposta de desenvolvimento e de integração emerge, desta
vez baseada na liberalização econômica e no modelo de regionalismo voltado para
fora ou, segundo a Cepal, um regionalismo aberto. A Aladi substitui a Alalc, servindo
de âncora normativa para a criação do Mercosul. Com características do
regionalismo de primeira e de segunda geração, o Mercosul é um projeto econômico
e político-estratégico que, embora tenha se inspirado no modelo europeu, conserva
sua própria originalidade. 1
Huntington identifica sete principais civilizações contemporâneas: sínica, japonesa, hindu,
islâmica, ocidental, latino-americana e africana.
2
Aos 30 acordos notificados no Gatt de 1992 a 1996 acrescentam-se outros 57 anteriormente Notas Desta forma foram institucionalizadas as três principais preocupações,
anunciadas anteriormente, na constituição da ONU: a segurança, a justiça e o bem-estar,
respectivamente. A idéia da universalidade está representada na Assembléia Geral e a da
diferenciação, principalmente, no Conselho de Segurança (Unam, 1995). 6
A ONU é constituída de uma Assembléia Geral, um Conselho de Segurança, uma Corte
Internacional de Justiça e um Conselho Econômico e Social, além de uma Secretaria e do
Trusteeship Council. Desta forma foram institucionalizadas as três principais preocupações,
anunciadas anteriormente, na constituição da ONU: a segurança, a justiça e o bem-estar,
respectivamente. A idéia da universalidade está representada na Assembléia Geral e a da
diferenciação, principalmente, no Conselho de Segurança (Unam, 1995). 7
A proposta inicial era construir um instrumento mundial de cooperação econômica que seria
denominado Organização Internacional do Comércio. Mas, ante as tensões entre Leste e Oeste,
o projeto derivou na criação do Gatt (Tamames, 1990). Somente depois do fim da Guerra Fria
e das evoluções nas negociações da Rodada Uruguai (1986-1994) é que foi possível constituir,
em 1995, a Organização Mundial do Comércio (OMC), que veio ocupar o lugar do Gatt. 7
A proposta inicial era construir um instrumento mundial de cooperação econômica que seria
denominado Organização Internacional do Comércio. Mas, ante as tensões entre Leste e Oeste,
o projeto derivou na criação do Gatt (Tamames, 1990). Somente depois do fim da Guerra Fria
e das evoluções nas negociações da Rodada Uruguai (1986-1994) é que foi possível constituir,
em 1995, a Organização Mundial do Comércio (OMC), que veio ocupar o lugar do Gatt. 8
A Oece transformar-se-á, em 1961, num fórum mais amplo: a Organização de Cooperação e de
Desenvolvimento Econômico (OCDE). 8
A Oece transformar-se-á, em 1961, num fórum mais amplo: a Organização de Cooperação e de
Desenvolvimento Econômico (OCDE). 8
A Oece transformar-se-á, em 1961, num fórum mais amplo: a Organização de Cooperação e de
Desenvolvimento Econômico (OCDE). 9
Para os que haviam assinado o Tratado de Paris criando a Comunidade Européia do Carvão e do
Aço (Ceca), em 1951, e posteriormente o Tratado de Roma, em 1957, a integração econômica
respondia a ordens de preocupação bastante diferentes das que os EUA tinham na época. O
mais original na proposta da CEE era o objetivo de implantar instituições comuns e de fazer da
integração econômica a base sobre a qual deveria repousar o que mais tarde foi chamado de
“Maison Commune”. Notas O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 39 3
De acordo com o Protocolo de Ouro Preto de 1994, o Mercosul tornar-se-á uma União Aduaneira
plena em 2006. p
4
Esta abordagem se distancia das abordagens funcionalista e estruturalista. Os funcionalistas
abordam o regionalismo econômico numa perspectiva evolucionista. É a perspectiva que adotam
geralmente os economistas e as organizações internacionais, na qual o regionalismo econômico
participa do movimento de globalização de duas maneiras: (1) permitindo progredir mais
rapidamente uma liberalização das trocas, conduzindo os países ao mercado mundial de maneira
seqüencial e passando por fases sucessivas de integração; (2) completando e reforçando o
sistema multilateral de cooperação. Já os estruturalistas abordam o regionalismo como uma
conseqüência direta das transformações ocorridas no mapa econômico do mundo, causadas, em
parte, pela tripolarização das trocas internacionais. A primeira abordagem ignora as considerações
geoeconômicas, e a segunda minimiza o papel da articulação entre os níveis regional e mundial da
integração econômica (Deblock e Brunelle, 1996). É 4
Esta abordagem se distancia das abordagens funcionalista e estruturalista. Os funcionalistas
abordam o regionalismo econômico numa perspectiva evolucionista. É a perspectiva que adotam
geralmente os economistas e as organizações internacionais, na qual o regionalismo econômico
participa do movimento de globalização de duas maneiras: (1) permitindo progredir mais
rapidamente uma liberalização das trocas, conduzindo os países ao mercado mundial de maneira
seqüencial e passando por fases sucessivas de integração; (2) completando e reforçando o
sistema multilateral de cooperação. Já os estruturalistas abordam o regionalismo como uma
conseqüência direta das transformações ocorridas no mapa econômico do mundo, causadas, em
parte, pela tripolarização das trocas internacionais. A primeira abordagem ignora as considerações
geoeconômicas, e a segunda minimiza o papel da articulação entre os níveis regional e mundial da
integração econômica (Deblock e Brunelle, 1996). É 5
É importante também assinalar a influência de William Beveridge, tendo sido ele um dos primeiros
a teorizar a função assistencialista do Estado. Este sociólogo irá fundar sua idéia de assistência
na solidariedade nacional. 5
É importante também assinalar a influência de William Beveridge, tendo sido ele um dos primeiros
a teorizar a função assistencialista do Estado. Este sociólogo irá fundar sua idéia de assistência
na solidariedade nacional. 6
A ONU é constituída de uma Assembléia Geral, um Conselho de Segurança, uma Corte
Internacional de Justiça e um Conselho Econômico e Social, além de uma Secretaria e do
Trusteeship Council. Notas tripartismo é aplicado em alguns órgãos consultivos do Mercosul. 23
O tripartismo é aplicado em alguns órgãos consultivos do Mercosul. Notas e vinculado ao modernismo como força cultural –, implicando um encurtamento do tempo e um
encolhimento do espaço, que se processam não de modo gradual ou contínuo, mas através de
curtas e intensas implosões, durante as quais o mundo muda rapidamente, em direção incerta. Para ele, a última implosão começou em torno de 1970, tendo origem na crise de superacumulação
do sistema capitalista sob o regime fordista de produção de massa integrada e vertical. A
resposta foi a emergência de um regime flexível de acumulação. 13
Harry D. White era funcionário adjunto do Secretário da Tesouraria dos Estados Unidos. A
pedido desta Secretaria, preparou a proposta dos EUA apresentada em Bretton Woods. 13
Harry D. White era funcionário adjunto do Secretário da Tesouraria dos Estados Unidos. A
pedido desta Secretaria, preparou a proposta dos EUA apresentada em Bretton Woods. 14
Eduardo Suárez, então Secretário da Fazenda e Crédito Público do México, foi o chefe da
Delegação de seu país na Conferência de Bretton Woods. Três comissões foram constituídas
durante a conferência: uma presidida por White, outra por Keynes e uma terceira por Suárez. A
Delegação do México apresentara, em julho de 1944, uma declaração intitulada Reconstrucción
y desarrollo en pie de igualdad: propuesta de México en Bretton Woods. Esta declaração, que
continha a essência do que estamos chamando de Plano Suárez, obteve o apoio de outros países
latino-americanos, a exemplo do Brasil, Chile e Venezuela. Sobre o tema pode-se consultar
Suárez (1994) e Urquidi (1994). 15
É interessante perceber, no livro de Shotwell (1945), que a América Latina praticamente não é
citada, dando sinal de que objetivamente ela não existia na perspectiva do cenário dessa nova
ordem mundial. 16
Posteriormente incluirá o Caribe, passando a se chamar Comissão Econômica para a América
Latina e o Caribe. 16
Posteriormente incluirá o Caribe, passando a se chamar Comissão Econômica para a América
Latina e o Caribe. 16
Posteriormente incluirá o Caribe, passando a se chamar Comissão Econômica para a América
Latina e o Caribe. 17
Outros nomes importantes que devem ser citados na elaboração do pensamento cepalino em seu
início são: Celso Furtado, Osvaldo Sunkel, Aníbal Pinto, José Medina Echavarría, Regino Botti,
Juan Noyola Várquez, Jorge Ahumada, entre outros. Notas 17
Outros nomes importantes que devem ser citados na elaboração do pensamento cepalino em seu
início são: Celso Furtado, Osvaldo Sunkel, Aníbal Pinto, José Medina Echavarría, Regino Botti,
Juan Noyola Várquez, Jorge Ahumada, entre outros. 17
Outros nomes importantes que devem ser citados na elaboração do pensamento cepalino em seu
início são: Celso Furtado, Osvaldo Sunkel, Aníbal Pinto, José Medina Echavarría, Regino Botti,
Juan Noyola Várquez, Jorge Ahumada, entre outros. 18
A baixa demanda, sobretudo nos países menores, dificultava – às vezes impossibilitava – a
industrialização nos setores de bens de consumo duráveis e bens de equipamentos. 18
A baixa demanda, sobretudo nos países menores, dificultava – às vezes impossibilitava – a
industrialização nos setores de bens de consumo duráveis e bens de equipamentos. 19
Prebisch apresentava uma visão dualista do mundo marcada pela existência de um “centro”
industrializado e de uma “periferia” exportadora de matérias-primas. 19
Prebisch apresentava uma visão dualista do mundo marcada pela existência de um “centro”
industrializado e de uma “periferia” exportadora de matérias-primas. 20
O autor cita as Nações Unidas. 20
O autor cita as Nações Unidas. 21
O conceito de regionalismo aberto servirá de ponto de referência central para se relançar a 21
O conceito de regionalismo aberto servirá de ponto de referência central para se relançar a
integração regional. 21
O conceito de regionalismo aberto servirá de ponto de referência central para se relançar a
integração regional. 22
No Mercosul é marcante a perspectiva de um processo de evolução seguindo a seqüência: Zona
de Livre Comércio, União Aduaneira, Mercado Comum, podendo se chegar a uma União
Econômica. Segue a lógica do modelo de regionalismo europeu, diferentemente da proposta do
Nafta e da Alca, que não têm esse mesmo tipo de pretensão. 22
No Mercosul é marcante a perspectiva de um processo de evolução seguindo a seqüência: Zona
de Livre Comércio, União Aduaneira, Mercado Comum, podendo se chegar a uma União
Econômica. Segue a lógica do modelo de regionalismo europeu, diferentemente da proposta do
Nafta e da Alca, que não têm esse mesmo tipo de pretensão. 22
No Mercosul é marcante a perspectiva de um processo de evolução seguindo a seqüência: Zona
de Livre Comércio, União Aduaneira, Mercado Comum, podendo se chegar a uma União
Econômica. Segue a lógica do modelo de regionalismo europeu, diferentemente da proposta do
Nafta e da Alca, que não têm esse mesmo tipo de pretensão. Notas Paralelamente, a decisão da Inglaterra de não aderir a esse projeto conduziu
à criação da AELC (Deblock e Brunelle, 1996). 10 10
Num primeiro nível, essa ampliação da margem de manobra passava pela transferência de certas
prerrogativas às instituições comuns; num segundo nível, passava pela definição de políticas
comuns; e, num terceiro nível, passava para formas mais acabadas de integração decorrentes da
própria evolução desse processo. 11
O recurso a Keynes em nível da economia nacional aparecia como uma abordagem capaz de
garantir a justaposição entre o nacional, o regional e o internacional. Por outro lado, uma das
razões da existência da nova ordem econômica internacional era a de reduzir, para não dizer
eliminar, a empresa dos Estados-nações sobre o conjunto das relações econômicas internacionais,
fossem elas ligadas ao comércio, aos investimentos ou ainda ao câmbio. Todavia, no contexto da
época, isto não queria dizer de forma alguma o retorno a uma situação de laisser-faire, mas ao
contrário: pretendia construir relações mais seguras da mesma forma que poderiam ser as
relações econômicas no interior de cada uma das nações. 12
A análise de Harvey considera que houve uma mudança no regime de acumulação capitalista a
partir da intensificação do processo de compressão do espaço-tempo inerente ao capitalismo – 12
A análise de Harvey considera que houve uma mudança no regime de acumulação capitalista a
partir da intensificação do processo de compressão do espaço-tempo inerente ao capitalismo – 12
A análise de Harvey considera que houve uma mudança no regime de acumulação capitalista a
partir da intensificação do processo de compressão do espaço-tempo inerente ao capitalismo – ALAN BARBIERO E YVES CHALOULT 40 e vinculado ao modernismo como força cultural –, implicando um encurtamento do tempo e um
encolhimento do espaço, que se processam não de modo gradual ou contínuo, mas através de
curtas e intensas implosões, durante as quais o mundo muda rapidamente, em direção incerta. Para ele, a última implosão começou em torno de 1970, tendo origem na crise de superacumulação
do sistema capitalista sob o regime fordista de produção de massa integrada e vertical. A
resposta foi a emergência de um regime flexível de acumulação. Regionalismo abierto en América Latina y el Caribe. La integración económica en servicio de
transformación productiva con equidad. 1994a. www.eclac.org/espanol/textosfund/Cepal6.html Bibliografia BECK, Ulrich. A reinvenção da política: rumo a uma teoria da modernidade reflexiva. In: GIDDENS,
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Cultura, 1998. 947p. p. 9-61. CEPAL. Cincuenta anos de pensamiento en la CEPAL. Textos Seleccionados, v. 1 e 2, Santiago: Fundo
de Cultura, 1998. 947p. ______ . Regionalismo abierto en América Latina y el Caribe. La integración económica en servicio de
la transformación productiva con equidad. 1994a. www.eclac.org/espanol/textosfund/Cepal6.html O MERCOSUL E A NOVA ORDEM ECONÔMICA INTERNACIONAL 41 ______ . América Latina y Caribe: políticas para mejorar la inserción en la economia mundial, 1994b. www.eclac.org/espanol/textosfund/Cepal6.html ______ . América Latina y Caribe: políticas para mejorar la inserción en la economia mundial, 1994b. www.eclac.org/espanol/textosfund/Cepal6.html ______ . América Latina y Caribe: políticas para mejorar la inserción en la economia mundial, 1994b. www.eclac.org/espanol/textosfund/Cepal6.html g
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1996. www.unites.uqam.ca/gric ______ . Le régionalisme économique international: de la première à la deuxième génération. Montréal,
1996. www.unites.uqam.ca/gric DEBLOCK, Christian, CONSTANTIN, Christian. Intégration, régionalisme et régionalisation. Montréal, 2000. www.unites.uqam.ca/gric DREIFUSS, René Armand. A época das perplexidades. Mundialização, globalização e planetarização:
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seu modelo. Se por um lado esse contexto foi marcado pela globalização, por outro
a evolução do regionalismo econômico deixará fortes traços no formato da integração
dos países do Cone Sul. As transformações mundiais marcadas pela nova ordem
internacional do Pós-guerra e, posteriormente, pelo fim da Guerra Fria não deixarão
de influenciar a América Latina. Através de uma abordagem histórico-analítica,
os autores buscam mostrar que o Mercosul adotou características de duas gerações
distintas do regionalismo, como resultado de uma experiência histórica particular. Bibliografia Globalização, sustentabilidade e governabilidade democrática
no Brasil. In: TRINDADE, Antônio Cançado, CASTRO, Marcus Faro de (Orgs.). A sociedade
democrática no final do século. Brasília: Paralelo 15, 1997. 255 p. p. 179-254. VIOLA, Eduardo, OLIVEIRA, Alejandro. Globalização, sustentabilidade e governabilidade democrática
no Brasil. In: TRINDADE, Antônio Cançado, CASTRO, Marcus Faro de (Orgs.). A sociedade
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URQUIDI, Victor. Bretton Woods: un recorrido por el primer cincuentenario. Comercio Exterior,
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no Congresso da ANPOCS, Caxambu, 1997. mimeo, 20 p. HARVEY, David. A condição pós-moderna. São Paulo: Loyola, 1996. 333 p. HIRST, Paul, THOMPSON, Grahame. Globalização em questão. Petrópolis: Vozes, 1998. 364 p. HOBSBAWM, Eric. Era dos extremos. O breve século XX, 1914-1991. 2
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das Letras, 1999. 598 p. HUNTINGTON, Samuel. O choque de civilizações e a recomposição da ordem mundial. Rio de
Janeiro: Objetiva, 1997. 455 p. KEYNES, John Maynard. Essais sur la monnaie et l’économie. Paris: Payot, 1978. 143 p. MARCOUX, Fanny. Intégration économique et régionalisme ouvert en Amérique Latine: des
accords de Montevideo au Sommet de Miami. Continentalisation, nº 1, Montréal: UQAM/
GRIC, 1996. 24 p. NYE, Joseph. Peace in parts: integration and conflict in regional organisations. Boston: Little Brown,
1971. OMAN, Charles. Globalisation et régionalisation: quels enjeux pour les pays en développement? Paris: OCDE, 1994. 152 p. ORTIZ, Renato. Mundialização e cultura. São Paulo: Brasiliense, 1996. 234 p. PREBISCH, Raúl. Cinq étapes dans ma réflexion sur le développement. In: MEIER, Guy, SEE
Denis. Les pionniers du développement. Paris: Econômica, 1988. 376 p. p. 187-223. a ,
q
p
pp
,
y,
Denis. Les pionniers du développement. Paris: Econômica, 1988. 376 p. p. 187-223. SANTOS, Boaventura de Souza. Pela mão de Alice. O social e o político na pós-modernidade. 3
a ed. São Paulo: Cortez, 1997. 348 p. SHOTWELL, James. La grande décision. New York: Bretano’s, 1945. 436 p. Á SHOTWELL, James. La grande décision. New York: Bretano s, 1945. 436 p. SUÁREZ, Eduardo. La conferencia internacional de Bretton Woods de 1944. Comercio Exterior,
México, v. 44, nº 10, p. 848-852, 1994. TAMAMES, Ramón. Estructura económica internacional. México: CONACULTA/Alianza editorial,
1990. 256 p. TAVARES, Maria da Conceição. Da substituição de importações ao capitalismo financeiro. Ensaio
sobre a economia brasileira. 7
a ed. Rio de Janeiro: Zahar, 1978. 263 p. ALAN BARBIERO E YVES CHALOULT 42 UNAM. 50 anos de las Nacíones Unidas. Relações Internacionais, México, nº 65, p. 1-106, 1995. URQUIDI, Victor. Bretton Woods: un recorrido por el primer cincuentenario. Comercio Exterior,
México, v. 44, nº 10, p. 838-847, 1994. VIOLA Eduardo OLIVEIRA Alejandro Globalização sustentabilidadeegovernabilidadedemocrática AM. 50 anos de las Nacíones Unidas. Relações Internacionais, México, nº 65, p. 1-106, 199 VIOLA, Eduardo, OLIVEIRA, Alejandro. Abstract This article analyzes the international context which allowed the creation
of the Southern Common Market (Mercosur) and the conditions which generated
the definition of its model. If, on one hand, globalization had an impact on this
context, on the other, the evolution of economic regionalism will strongly influence
the type of integration of Mercosur countries. The world transformations, linked to
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Latin America. Through an historical and analytical approach, the authors show
that Mercosur adopted features of two different generations of regionalism, as a
result of a specific historical experience. Palavras-chave: Mercosul. Ordem econômica internacional. Globalização,
Regionalização. g
Key words: Mercosur. International economic order. Globalization. Regionalization.
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Enhanced bacterial clearance in early secondary sepsis in a porcine intensive care model
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Scientific reports
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Enhanced bacterial clearance
in early secondary sepsis
in a porcine intensive care model
Frida Wilske 1,5*, Paul Skorup 1,5, Katja Hanslin 2, Helena Janols 1, Anders Larsson 3,
Miklós Lipcsey 2,4 & Jan Sjölin 1
OPEN Early secondary sepsis (ESS), occurring after recent inflammatory activation is associated with
a reduced inflammatory response. If this attenuation also is associated with decreased bacterial
killing, the need for antibiotic efficacy might be greater than in primary sepsis (PS). This prospective,
randomised interventional study compares bacterial killing in ESS and PS in a large animal intensive
care sepsis model. 38 pigs were intravenously administered live Escherichia coli for 3 h. Before baseline
ESS was pre-exposed to endotoxin 24 h, whereas PS was not. Bacterial growth was measured in
organs immediately post-mortem, repeatedly during 6 h in blood in vivo and for blood intrinsic
bactericidal capacity ex vivo. Splenic growth was lower in ESS animals, than in PS animals (3.31 ± 0.12,
vs. 3.84 ± 0.14 log10 CFU/mL, mean ± SEM) (p < 0.01) with a similar trend in hepatic growth (p = NS). Blood bacterial count at 2 h correlated with splenic bacterial count in ESS (ESS: r = 0.71, p < 0.001) and
to blood killing capacity in PS (PS: r = 0.69, p < 0.001). Attenuated inflammation in ESS is associated
with enhanced antibacterial capacities in the spleen. In ESS blood bacterial count is related to splenic
killing and in PS to blood bactericidal capacity. The results suggest no increased need for synergistic
antibiotic combinations in ESS. Sepsis is an infection that leads to life-threatening organ dysfunctions caused by a dysregulated host response1. Current studies report simultaneously increased production of pro- and anti-inflammatory cytokines2. Most of
sepsis-related deaths are not caused by a pro-inflammatory cytokine storm but occur later during a prolonged
immunosuppressive phase, probably because of failure to control the primary infection or subsequent hospital-
acquired infections3,4. Early secondary sepsis (ESS), here defined as a sepsis that develops in a situation of recent
inflammatory activation with features of the well-studied endotoxin tolerance, is in experimental animal and
human studies associated with attenuated inflammatory responses5–8. This mitigated response is further linked to
less organ dysfunction which has been observed in an experimental porcine model and in a retrospective study
in patients in an intensive care unit (ICU)5,9. Adding an aminoglycoside or quinolone to a beta-lactam antibiotic
to achieve a synergistic effect in the initial sepsis treatment is controversial. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Material and methods Anaesthesia, preparations and intensive care settings. The animals, 38 healthy Norwegian lan-
drace-breed piglets of both sexes, were used. The preferred weight was 25 kg giving an age of 9–12 weeks, thus,
the animals were not sexually mature. They were prepared, anaesthetised and handled as previously reported20
and described in Supplemental Digital Content 1. Briefly, mechanical ventilation and intravenous (iv) general
anaesthesia were employed and the intensive care was maintained using an intensive care treatment protocol
designed to keep mean arterial pressure, cardiac output, arterial partial pressure of oxygen and carbon dioxide
and blood glucose within specified limits (Supplemental Digital Content 2). Protocol/experimental design. The experimental design is illustrated in Fig. 1. The animals were ran-
domised to either the ESS group in which animals were exposed to an endotoxin infusion and intensive care
for 24 h before baseline or the PS group, in which unexposed animals were challenged with live bacteria at
baseline. At baseline, the 6 h experiment was initiated with a 3-h iv infusion of E. coli, followed by a 3-h observa-
tion period before sacrificing the animals by a potassium chloride injection. Immediately before baseline and
repeatedly during the experiment, physiological data were recorded. Blood samples for analysis of cytokines
and Sequential Organ Failure Assessment (SOFA) were taken hourly. Organ samples were taken within 30 min
post-mortem. Endotoxin. The endotoxin in the ESS group consisted of a lipopolysaccharide from E. coli, 0111:B4; (Sigma
Chemical Co., St Louis MO, USA) and the same batch was used to all animals to minimise batch variations. The endotoxin infusion was given to establish endotoxin tolerance which has previously been reported in this
porcine model5: after a starting dose of 0.3 μg × h−1, a stepwise increase for 30 min ×followed until a final dose
of 0.063 μg × kg−1 × h−1 was achieved. This dose was then continued until completion at 24 h after the start of
the infusion. Organism. The E. coli strain B09-11822 (serotype O-rough:K1:H7; Statens Seruminstitut, Copenhagen,
Denmark) is an encapsulated and serum-resistant clinical isolate. The preparation of the bacteria is described in
Supplemental Digital Content 3. Measurements. In vivo blood and organ bacterial cultures. Bacterial investigations from arterial blood,
spleen and liver are described in Supplemental Digital Content 4. Other organs than the spleen and liver, pri- Figure 1. Schematic presentation of the study and the primary and early secondary sepsis groups. www.nature.com/scientificreports/ porcine endotoxin tolerant large animal model that has similarities to human sepsis17,18 and includes intensive
care measures known to additionally affect the inflammatory response5.ht porcine endotoxin tolerant large animal model that has similarities to human sepsis17,18 and includes intensive
care measures known to additionally affect the inflammatory response5.ht fl
Thus, the primary aim of the present study was to compare the bacterial killing in pigs with ESS after exposure
to a 24-h endotoxin challenge and intensive care treatment with that in unexposed animals with PS. Secondary
aims were to analyse the relationships between blood bacterial count during infusion and blood intrinsic bacte-
ricidal capacity before and after the bacterial challenge and bacterial growth in organs. Enhanced bacterial clearance
in early secondary sepsis
in a porcine intensive care model
Frida Wilske 1,5*, Paul Skorup 1,5, Katja Hanslin 2, Helena Janols 1, Anders Larsson 3,
Miklós Lipcsey 2,4 & Jan Sjölin 1
OPEN Clinical studies have resulted in vary-
ing outcomes10,11 and even high-quality reviews such as the Surviving Sepsis Campaign and Infectious Diseases
Society of America (IDSA) initially reached different conclusions depending on how the studies were assessed,
although their opinions now are more concordant12–14. One possible explanation for the divergent results could
be that patients with sepsis have different demands of antibiotic efficacy and synergy. If the reduced inflamma-
tory response following recent activation is also associated with decreased bacterial killing, it may be that the
effect of a synergistic antibiotic regimen will be greater in these patients than in those with primary sepsis (PS)
without such prior activation.i p
To answer this question the first issue will be to study the bacterial clearance in a model of endotoxin toler-
ance. Only a few studies exist in which sepsis caused by live bacteria represent the second challenge and there
are no data from humans or large animals. An improved bacterial clearance was demonstrated in mice6,15,16. However, there are difficulties extrapolating these results to humans because of a similarity to the human immune
system of only 20%17,18. Furthermore, contradictory results have been reported in an endotoxin-tolerant rabbit
model19. A study in humans would be challenging and unethical, which is why we explore this question in a 1Section of Infectious Diseases, Department of Medical Sciences, Uppsala University, SE 751 85 Uppsala,
Sweden. 2Section of Anaesthesiology and Intensive Care, Department of Surgical Sciences, Uppsala University,
Uppsala, Sweden. 3Section of Clinical Chemistry, Department of Medical Sciences, Uppsala University, Uppsala,
Sweden. 4Hedenstierna Laboratory, Anaesthesiology and Intensive Care, Department of Surgical Sciences,
Uppsala University, Uppsala, Sweden. 5These authors contributed equally: Frida Wilske and Paul Skorup. *email:
frida.wilske@medsci.uu.se | https://doi.org/10.1038/s41598-023-28880-x Scientific Reports | (2023) 13:1964 www.nature.com/scientificreports/ www.nature.com/scientificreports/ marily the kidney and lung were also investigated in most animals, but with too few bacteria found to allow for
a meaningful analysis. marily the kidney and lung were also investigated in most animals, but with too few bacteria found to allow for
a meaningful analysis. Blood intrinsic bactericidal capacity. The blood intrinsic bactericidal capacity was analysed by ex vivo bacte-
rial clearance. Arterial blood was obtained at baseline before bacterial infusion (ex vivoPREBACT) and 15 min
after termination (ex vivoPOSTBACT) and ex vivo inoculated with E. coli at a concentration of 105 CFU × mL−1. The ex vivoPREBACT incubation lasted for 6 h and ex vivoPOSTBACT for 3 h with bacterial quantifications at 3 and
6 h. Bacterial clearance was calculated by subtracting the log bacterial count from that of the starting inoculum
at 0 h. Due to a lack of time, ex vivoPREBACT clearance was not achieved in two ESS animals and ex vivoPOSTBACT
clearance in two PS animals. Organ parameters and laboratory tests to evaluate the sepsis reaction, maintenance of intensive care and the inflam-
matory response. Details of blood test analysis, organ dysfunction parameters and cytokines are presented in
Supplemental Digital Content 5. The sepsis reaction in the two groups was evaluated with the SOFA score1,
although no assessment of the central nervous system was possible due to sedation. The inflammatory response
was estimated by the highest levels of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in plasma ana-
lysed by commercial porcine-specific sandwich enzyme-linked immunosorbent assays. Calculations and statistics. Because the bacterial clearance from the blood has been shown to be rapid,
the primary endpoint of the study was bacterial growth in the spleen and liver, the two principal organs respon-
sible for the elimination of bacteria20. With a standard deviation in the PS group of 20%, a power of 0.8, a two-
sided α-error of 0.05 and a detectable difference of 20%, 17 animals were required. In the ESS group the standard
deviation was expected to be 25% higher and with the same power, α-error and detectable difference 21 animals
had to be included. In the analysis of the primary endpoint Student’s unpaired t-test was applied. Because ex vivo
growth in blood did not follow a normal distribution, other differences between the groups were analysed by the
Mann–Whitney U test, paired analysis by the Wilcoxon matched-paired test and correlations by the Spearman
rank test. www.nature.com/scientificreports/ Normally distributed data are expressed as mean ± SD and non-normally as median and interquartile
range (IQR). Statistica software (v13.5, StatSoft, Tulsa, OK, USA) was used to generate the calculations and
p < 0.05 was considered statistically significant. Animals and ethic statements. This study was conducted after approval from the Animal Ethical Board
in Uppsala, Sweden (permit no C250/11 and C155/14). Animals were handled in accordance with the Guide for
the Care and Use of Laboratory Animals and reported in compliance with the ARRIVE guidelines21. Results Animal experiment. The animals developed sepsis or septic shock during the bacterial infusion with SOFA
scores of 6 (3.5–8) in the ESS group and 9 (6.5–11) in the PS group. The dose of infused bacteria was 8.80 ± 0.10
in the ESS group and 8.80 ± 0.09 log10 CFU in the PS group. Body weights in the ESS and PS groups were
25.3 ± 1.9 and 25.2 ± 1.8 kg, respectively. Baseline at 0 h and the highest (peak) cytokine levels for TNF-α and
IL-6 in the two groups are shown in Table 1 with significant reductions in the ESS animals (p < 0.001). The peak
concentrations also appeared later in the ESS than in the PS group with TNF-α-peaking at 2 h compared with
1 h and IL-6 at 4 h compared with 3 h. Bacterial investigations. Organ bacterial cultures. Organ bacterial cultures are depicted in Fig. 2. The
ESS group exhibited lower growth in the spleen, than the PS group, (3.31 ± 0.56 vs 3.84 ± 0.56 log10 CFU × g−1)
(p = 0.007). Bacterial growth in the liver was also lower in the ESS group than in the PS group (1.99 ± 0.78 vs
2.44 ± 1.00 log10 CFU × g−1), although the difference did not reach statistical significance. Blood cultures in vivo. No animal exhibited E. coli growth in the blood culture before the bacterial infusion
was initiated. The ESS animals showed a tendency to fewer bacteria in cultures during the bacterial infusions Table 1. Baseline at 0 h before the bacterial challenge and the highest (peak) plasma levels of TNF-α and
IL-6 in animals with early secondary sepsis (ESS) and primary sepsis (PS). Data are presented as mean ± SD. Student’s unpaired t-test was used to compare the differences between ESS and PS. ***p < 0.001 ESS versus PS. ESS
Log10 ng × L−1 (n = 21)
PS
Log10 ng × L−1 (n = 17)
TNF-α
Baseline
1.83 ± 0.27
1.87 ± 0.22
Peak
2.43 ± 0.45***
4.80 ± 0.31
IL-6
Baseline
2.09 ± 0.35
1.85 ± 0.47
Peak
2.84 ± 0.36***
3.43 ± 0.31 3
6
|
htt
//d i
/10 1038/ 41598 023 28880
Table 1. Baseline at 0 h before the bacterial challenge and the highest (peak) plasma levels of TNF-α and
IL-6 in animals with early secondary sepsis (ESS) and primary sepsis (PS). Data are presented as mean ± SD. Material and methods ESS animals
(n = 21) were pre-exposed to 24 h endotoxin infusion and intensive care treatment before baseline, whereas PS
animals (n = 17) were unexposed at baseline when the experiment was initiated by a 3-h intravenous infusion of
live E. coli. Bars represent infusions while arrows denote time points for sampling for bacterial analyses. Figure 1. Schematic presentation of the study and the primary and early secondary sepsis groups. ESS animals
(n = 21) were pre-exposed to 24 h endotoxin infusion and intensive care treatment before baseline, whereas PS
animals (n = 17) were unexposed at baseline when the experiment was initiated by a 3-h intravenous infusion of
live E. coli. Bars represent infusions while arrows denote time points for sampling for bacterial analyses. https://doi.org/10.1038/s41598-023-28880-x Scientific Reports | (2023) 13:1964 | www.nature.com/scientificreports/ Results Student’s unpaired t-test was used to compare the differences between ESS and PS. ***p < 0.001 ESS versus PS. ESS
Log10 ng × L−1 (n = 21)
PS
Log10 ng × L−1 (n = 17)
TNF-α
Baseline
1.83 ± 0.27
1.87 ± 0.22
Peak
2.43 ± 0.45***
4.80 ± 0.31
IL-6
Baseline
2.09 ± 0.35
1.85 ± 0.47
Peak
2.84 ± 0.36***
3.43 ± 0.31 ESS
Log10 ng × L−1 (n = 21)
PS
Log10 ng × L−1 (n = 17)
TNF-α
Baseline
1.83 ± 0.27
1.87 ± 0.22
Peak
2.43 ± 0.45***
4.80 ± 0.31
IL-6
Baseline
2.09 ± 0.35
1.85 ± 0.47
Peak
2.84 ± 0.36***
3.43 ± 0.31 Table 1. Baseline at 0 h before the bacterial challenge and the highest (peak) plasma levels of TNF-α and
IL-6 in animals with early secondary sepsis (ESS) and primary sepsis (PS). Data are presented as mean ± SD. Student’s unpaired t-test was used to compare the differences between ESS and PS. ***p < 0.001 ESS versus PS. Table 1. Baseline at 0 h before the bacterial challenge and the highest (peak) plasma levels of TNF-α and
IL-6 in animals with early secondary sepsis (ESS) and primary sepsis (PS). Data are presented as mean ± SD. Student’s unpaired t-test was used to compare the differences between ESS and PS. ***p < 0.001 ESS versus PS. https://doi.org/10.1038/s41598-023-28880-x Scientific Reports | (2023) 13:1964 | www.nature.com/scientificreports/ Figure 2. Bacterial count in the spleen and liver in primary (n = 17) and early secondary sepsis (n = 21). Values
are mean ± SEM. Figure 2. Bacterial count in the spleen and liver in primary (n = 17) and early secondary sepsis (n = 21). Values
are mean ± SEM. compared with the PS animals (Table 2). After termination of the infusion, no living bacteria were found in
blood cultures. compared with the PS animals (Table 2). After termination of the infusion, no living bacteria were found in
blood cultures. Ex vivo bacterial clearance. Ex vivo results are summarised in Table 2. Blood intrinsic bactericidal capacity at
baseline, expressed as ex vivoPREBACT bacterial clearance at 6 h did not differ between the ESS and PS groups. A reduction in ex vivoPOSTBACT clearance compared with ex vivoPREBACT clearance was found in the two groups
(p = 0.004, n = 34). Results Moreover, the ex vivoPOSTBACT bacterial clearance in the PS group was lower than that in the
ESS group (p = 0.028) with some animals not killing the bacteria. Correlation analyses. A significant correlation was observed between bacterial growth in the spleen and liver
in the PS group (r = 0.69, p = 0.003), but not in the ESS group (r = 0.26, p = NS). Correlations between bacterial
in vivo count in the blood at 1–3 h and the bacterial count in the spleen and liver are depicted in Fig. 3. In the ESS
group, there was a strong correlation between growth in the blood and spleen over all 3 h (r ranging 0.58–0.71). This correlation was not seen in the PS group (r ranging 0.01–0.32) or between growth in the blood and liver
in any group (r ranging 0.03–0.38). Bacterial in vivo growth in blood was strongly and negatively correlated
with ex vivoPREBACT clearance in the PS group already at 1 h (r = − 0.81), whereas a significant correlation in the
ESS group was noted only after 3 h. There was no correlation between ex vivoPREBACT clearance and growth in
the organs in any group (data not shown). Ex vivoPOSTBACT clearance at 3 h correlated significantly with ex vivo-
PREBACT at 3 h (ESS group: r = 0.58, p = 0.009; PS group: r = 0.62, p = 0.014) and with blood bacterial count in both
groups, Fig. 3. Table 2. Bacterial growth in blood cultures obtained in vivo and blood intrinsic bactericidal capacity
expressed as bacterial clearance ex vivo in blood obtained before the bacterial challenge at 0 h (Ex vivoPrebact)
and 15 min after termination of the 3-h bacterial infusion (Ex vivoPostbact) in animals with early secondary
sepsis (ESS) and primary sepsis (PS). Bacterial clearance is calculated as the difference between growth at
0 h and the different time points. Due to a lack of time, ex vivoPREBACT analyses were not achieved in two ESS
animals and in two PS animals in the ex vivoPOSTBACT analyses. Values are median (interquartile range) log10
CFU × mL−1. NG = No growth, detection limit 5 CFU × mL−1. The Mann–Whitney U test was conducted to
determine differences between the ESS and PS groups at each time point. a p < 0.05 ESS versus PS. Results Bacterial growth
In vivo
log10 CFU × mL−1
Bacterial clearance
Ex vivoPREBACT
log10 CFU × mL−1
Bacterial clearance
Ex vivoPOSTBACT
log10 CFU ×mL−1
ESS (n = 21)
PS (n = 17)
ESS (n = 19)
PS (n = 17)
ESS (n = 21)
PS (n = 15)
Ex vivo growth 0 h
4.91 (4.86–4.97)
4.94 (4.67–4.96)
5.02 (4.99–5.12)
5.00 (4.86–5.06)
0 h
NG (NG–NG)
NG (NG–NG)
–
–
–
–
1 h
2.90 (2.48–3.02)
3.12 (2.92–3.24)
2 h
3.14 (2.77–3.41)
3.21 (3.01–3.65)
3 h
3.19 (2.88–3.55)
3.39 (3.22–3.75)
2.38 (1.52–2.99)
2.24 (1.22–3.22)
1.80 (0.91–3.16)a
0.36 (-0.64–2.52)
4 h
NG (NG–NG)
NG (NG–NG)
5 h
NG (NG–NG)
NG (NG–NG)
6 h
NG (NG–NG)
NG (NG–NG)
3.57 (2.00–4.20)
3.39 (1.35–3.98) Bacterial growth
In vivo
log10 CFU × mL−1
Bacterial clearance
Ex vivoPREBACT
log10 CFU × mL−1
Bacterial clearance
Ex vivoPOSTBACT
log10 CFU ×mL−1
ESS (n = 21)
PS (n = 17)
ESS (n = 19)
PS (n = 17)
ESS (n = 21)
PS (n = 15)
Ex vivo growth 0 h
4.91 (4.86–4.97)
4.94 (4.67–4.96)
5.02 (4.99–5.12)
5.00 (4.86–5.06)
0 h
NG (NG–NG)
NG (NG–NG)
–
–
–
–
1 h
2.90 (2.48–3.02)
3.12 (2.92–3.24)
2 h
3.14 (2.77–3.41)
3.21 (3.01–3.65)
3 h
3.19 (2.88–3.55)
3.39 (3.22–3.75)
2.38 (1.52–2.99)
2.24 (1.22–3.22)
1.80 (0.91–3.16)a
0.36 (-0.64–2.52)
4 h
NG (NG–NG)
NG (NG–NG)
5 h
NG (NG–NG)
NG (NG–NG)
6 h
NG (NG–NG)
NG (NG–NG)
3.57 (2.00–4.20)
3.39 (1.35–3.98) Table 2. Bacterial growth in blood cultures obtained in vivo and blood intrinsic bactericidal capacity
expressed as bacterial clearance ex vivo in blood obtained before the bacterial challenge at 0 h (Ex vivoPrebact)
and 15 min after termination of the 3-h bacterial infusion (Ex vivoPostbact) in animals with early secondary
sepsis (ESS) and primary sepsis (PS). Bacterial clearance is calculated as the difference between growth at
0 h and the different time points. Due to a lack of time, ex vivoPREBACT analyses were not achieved in two ESS
animals and in two PS animals in the ex vivoPOSTBACT analyses. Values are median (interquartile range) log10
CFU × mL−1. NG = No growth, detection limit 5 CFU × mL−1. The Mann–Whitney U test was conducted to
determine differences between the ESS and PS groups at each time point. a p < 0.05 ESS versus PS. Table 2. Discussion In the previous clinical study secondary sepsis was defined as a sepsis starting within 7 days after preceding
infection or trauma9. Because our animals in the ESS group had their inflammatory response activated only 24 h
before the bacterial challenge, we named secondary sepsis in the present study early secondary sepsis. Despite a
reduced inflammatory response, the spleen of animals with ESS contained fewer bacteria than animals with PS. A similar trend was seen in the liver and blood in vivo. Thus, the weakened inflammatory response in ESS was
not associated with reduced bacterial killing, neither in immune-active organs nor in the blood.h In the previous clinical study secondary sepsis was defined as a sepsis starting within 7 days after preceding
infection or trauma9. Because our animals in the ESS group had their inflammatory response activated only 24 h
before the bacterial challenge, we named secondary sepsis in the present study early secondary sepsis. Despite a
reduced inflammatory response, the spleen of animals with ESS contained fewer bacteria than animals with PS. A similar trend was seen in the liver and blood in vivo. Thus, the weakened inflammatory response in ESS was
not associated with reduced bacterial killing, neither in immune-active organs nor in the blood.h g
g
The lower splenic growth in the ESS animals might be caused by reduced splenic uptake from the blood or
increased clearance of bacteria. In this model bacteria were negligible in organs other than the spleen and liver
(data not shown). In addition, low bacterial counts in the spleen were associated with low counts in the blood,
indicating that a reduced uptake is not the mechanism, making increased splenic bacterial killing more plausible. The strong correlation between growth in the spleen and blood might also imply that the increased splenic kill-
ing contributes to the lower blood bacterial count seen in the ESS group (Fig. 3). In the spleen bacteria enter an
open blood system without an endothelial lining where macrophages reside and surviving bacteria must pass
the small endothelial slits of the splenic venous sinusoids before re-entering the circulation22. In ESS this trap-
ping mechanism may be more important than other bacterial removal processes. In the liver there is an active
uptake of bacteria from the blood23,24 and it has recently been shown that this uptake is reduced in secondary
sepsis25, which might promote a lower bacterial growth in the liver. Results Bacterial growth in blood cultures obtained in vivo and blood intrinsic bactericidal capacity
expressed as bacterial clearance ex vivo in blood obtained before the bacterial challenge at 0 h (Ex vivoPrebact)
and 15 min after termination of the 3-h bacterial infusion (Ex vivoPostbact) in animals with early secondary
sepsis (ESS) and primary sepsis (PS). Bacterial clearance is calculated as the difference between growth at
0 h and the different time points. Due to a lack of time, ex vivoPREBACT analyses were not achieved in two ESS
animals and in two PS animals in the ex vivoPOSTBACT analyses. Values are median (interquartile range) log10
CFU × mL−1. NG = No growth, detection limit 5 CFU × mL−1. The Mann–Whitney U test was conducted to
determine differences between the ESS and PS groups at each time point. a p < 0.05 ESS versus PS. Table 2. Bacterial growth in blood cultures obtained in vivo and blood intrinsic bactericidal capacity
expressed as bacterial clearance ex vivo in blood obtained before the bacterial challenge at 0 h (Ex vivoPrebact)
and 15 min after termination of the 3-h bacterial infusion (Ex vivoPostbact) in animals with early secondary
sepsis (ESS) and primary sepsis (PS). Bacterial clearance is calculated as the difference between growth at
0 h and the different time points. Due to a lack of time, ex vivoPREBACT analyses were not achieved in two ESS
animals and in two PS animals in the ex vivoPOSTBACT analyses. Values are median (interquartile range) log10
CFU × mL−1. NG = No growth, detection limit 5 CFU × mL−1. The Mann–Whitney U test was conducted to
determine differences between the ESS and PS groups at each time point. a p < 0.05 ESS versus PS. https://doi.org/10.1038/s41598-023-28880-x Scientific Reports | (2023) 13:1964 | www.nature.com/scientificreports/ w.nature.com/scientificreports/
Figure 3. Spearman rank correlation analyses between in vivo bacterial count in the blood at 1–3 h and the
bacterial counts in the spleen and liver, ex vivoPREBACT bactericidal clearance at 6 h in blood before the bacterial
challenge and ex vivoPOSTBACT bactericidal count at 3 h in blood after the bacterial challenge. The correlations
between bacterial count in the blood and the two ex vivo clearances are negative but to relate to the positive
values on the y-axis, these correlations are expressed as ex vivo bacterial count at the last determination after 6
and 3 h, respectively. Results *p < 0.05, **p < 0.01, ***p < 0.001. Figure 3. Spearman rank correlation analyses between in vivo bacterial count in the blood at 1–3 h and the
bacterial counts in the spleen and liver, ex vivoPREBACT bactericidal clearance at 6 h in blood before the bacterial
challenge and ex vivoPOSTBACT bactericidal count at 3 h in blood after the bacterial challenge. The correlations
between bacterial count in the blood and the two ex vivo clearances are negative but to relate to the positive
values on the y-axis, these correlations are expressed as ex vivo bacterial count at the last determination after 6
and 3 h, respectively. *p < 0.05, **p < 0.01, ***p < 0.001. www.nature.com/scientificreports/ Whereas our results demonstrating increased bacterial killing agree with those in mice pre-treated with
endotoxin 24 h or more before the bacterial challenge6,15,16, our results with reduced bacterial ex vivo clearance
directly after termination of the bacterial infusion seem consistent with the rabbit model19. In this rabbit model
the bacterial challenge was also given directly after a 1- or 4-h infusion of endotoxin, which could explain the
different result of the other studies.i f
Our findings, together with previous experimental studies on endotoxin tolerance induced 24–72 h before
the bacterial challenge6,15,16, indicate a decreased inflammatory response associated with increased bacterial
killing. In the studies so far, the same results have been obtained both after intravenous and intraperitoneal
induction, irrespective of the type of bacterial challenge6,15. Because trauma seems to elicit a similar response as
endotoxin and infections30,31, these results are also likely valid after trauma. Whether this augmented bacterial
killing applies later than 72 h, as shown in these studies15,16 in the immunosuppressive phase, is not known, war-
ranting further study. However, it might be speculated that there is an evolutionary benefit of protecting animals
from additional harm by invading bacteria during the early acute phase of severe trauma or infection. Although
extrapolated with caution, the PS model may show similarities to community-acquired sepsis, whereas the ESS
model is more comparable with early nosocomial infection in the ICU. Based on our findings, the increased need
for a synergistic β-lactam-aminoglycoside combination for the ESS treatment does not seem urgent. Maximum
bactericidal antibiotic therapy seems more important in PS. In this connection, a recent retrospective study,
restricting patient recruitment to community-acquired bacteraemia demonstrated a better effect by the addition
of one single dose of aminoglycoside to the β-lactam treatment than most other comparative studies investigat-
ing the effect of this combination32. f
In contrast to mice, the porcine immune system shares 80% similarity and the physiological responses
in sepsis are similar to those in humans17,18. The physiognomy is also suitable for intensive care treatment,
including sedation, vasopressors and mechanical ventilation that might additionally mitigate the inflammatory
response33–35, adding strength and clinical relevance to our model. In addition, this model complies with the
International Expert Consensus for Pre-Clinical Sepsis Studies36.h p
p
However, there are some limitations of the model. The challenge of bacteria as an intravenous infusion might
clinically resemble the administration of contaminated infusions, which is rare nowadays. Conclusion Animals with early secondary sepsis exhibit an attenuated inflammatory response as expected but show enhanced
antibacterial capacities in the major immune-active organs compared with animals with primary sepsis. During
the first hours in early secondary sepsis, blood bacterial count looks to be principally affected by splenic activity
and in primary sepsis by the intrinsic blood killing capacity. The results indicate no increased need for synergistic
antibiotic combinations in early secondary sepsis. www.nature.com/scientificreports/ The caecal ligation
puncture technique has the advantage of resembling the onset of a clinical infection37 but is difficult to standardise
because of varying numbers and species in the blood and immune-active organs, thus, significantly reducing the
power of the study. Using E. coli endotoxin followed by an E. coli bacterial challenge might be another limita-
tion. However, E. coli is the most common Gram-negative bacterial species38 and the phenomenon of endotoxin
tolerance is not species-specific8,15. Only bacterial findings from the spleen and liver were reported in our study. These organs are the major sites that clear bacteria from the bloodstream22,24,39, a finding also reported from a
porcine model on PS20. Cultures were also taken from the kidney and lung in most of our animals. Only occasion-
ally was bacterial growth observed in low numbers, too few to allow a meaningful analysis between the groups. Data availabilityh The data collected and analysed for the current study are available from the corresponding author on reasonable
request. Received: 11 October 2022; Accepted: 25 January 2023 Received: 11 October 2022; Accepted: 25 January 2023 Discussion Particularly noteworthy is that the significant
correlation between bacterial growth in the liver and spleen as demonstrated in PS, suggesting related control of
the host defence mechanisms, is not observed in ESS despite reduced growth in the spleen and a similar trend in
the liver. Dissimilar mechanisms, affected differently by the preceding activated inflammatory response, might
constitute an explanation for the lack of correlations between hepatic bacterial growth and that in the spleen
and blood in ESS.h Factors other than splenic activity seem to matter to the blood bacterial count in PS. The intrinsic blood
bactericidal capacity, as demonstrated by the ex vivoPREBACT bacterial clearance, is the sum of the bactericidal
performance of several defence actors, such as activated neutrophils, circulating antimicrobial proteins as well as
activation of the complement system, the kallikrein-kinin system and components of the coagulation systems26–29. In the PS group high ex vivoPREBACT bacterial clearance appears to be associated with lower levels of bacteraemia,
especially during the first hour when this correlation was strong. At 2 and 3 h, this correlation became weaker
though still significant. In ESS blood bacterial count was initially more related to splenic killing than blood bacte-
ricidal capacity but in contrast to PS, the latter association increased and at 3 h bacterial growth was significantly
associated with both the blood intrinsic bactericidal capacity and splenic activity. p
y
p
y
While retaining a correlation with the bactericidal capacity before the bacterial challenge, the ex vivoPOSTBACT
clearance was reduced in both groups suggesting that the components of the antibacterial systems have to some
extent been consumed or inhibited after 3 h of bacteraemia. This killing capacity was retained more in the ESS
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Open access funding provided by Uppsala University. The study was financed from the Olinder-Nielsen fund
nd R&D funds of the Uppsala County Council. Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-023-28880-x. Scientific Reports | (2023) 13:1964 | https://doi.org/10.1038/s41598-023-28880-x www.nature.com/scientificreports/ Correspondence and requests for materials should be addressed to F.W. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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The dark-side of the outside: how extracellular heat shock proteins promote cancer
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Cellular and molecular life sciences
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cc-by
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Cellular and Molecular Life Sciences (2021) 78:4069–4083
https://doi.org/10.1007/s00018-021-03764-3
Cellular and Molecular Life Sciences
REVIEW
The dark‑side of the outside: how extracellular heat shock proteins
promote cancer
Laura Seclì1
· Federica Fusella1
· Lidia Avalle1
· Mara Brancaccio1
Received: 7 October 2020 / Revised: 28 December 2020 / Accepted: 15 January 2021 / Published online: 5 February 2021
© The Author(s) 2021
Abstract
In addition to exerting several essential house-keeping activities in the cell, heat shock proteins (HSPs) are crucial players
in a well-structured molecular program activated in response to stressful challenges. Among the different activities carried
out by HSPs during emergency, they reach the extracellular milieu, from where they scout the surroundings, regulate extracellular protein activity and send autocrine and paracrine signals. Cancer cells permanently experience stress conditions
due to their altered equilibrium and behaviour, and constantly secrete heat shock proteins as a result. Other than supporting
anti-tumour immunity, extracellular heat shock proteins (eHSPs), can also exacerbate cancer cell growth and malignancy
by sustaining different cancer hallmarks. eHSPs are implicated in extracellular matrix remodelling, resistance to apoptosis,
promotion of cell migration and invasion, induction of epithelial to mesenchymal transition, angiogenesis and activation of
stromal cells, supporting ultimately, metastasis dissemination. A broader understanding of eHSP activity and contribution
to tumour development and progression is leading to new opportunities in the diagnosis and treatment of cancer.
Keywords Extracellular · Chaperones · Heat shock proteins · Tumour microenvironment · Cancer hallmarks
Introduction
“It did not matter if this interpretation was true or
false, it was a working link between imagination and
reality, like love” [1].
This is how Ferruccio Ritossa described his discovery
of the heat shock proteins, and at least 20 years later, this
perception is still alive, considering the finding of new and
unexpected roles played by these multifaceted proteins.
Heat shock proteins (HSPs) were originally identified as
stress-responsive proteins required for cell survival during
thermal stress, acting as molecular chaperones. Shortly afterwards, it became clear that HSPs are induced in response to
Laura Seclì and Federica Fusella have contributed equally to the
article.
* Laura Seclì
laura.secli@unito.it
* Mara Brancaccio
mara.brancaccio@unito.it
1
Department of Molecular Biotechnology and Health
Sciences, University of Torino, Turin, Italy
a wider variety of insults (of physical, chemical and biological origin), preventing cell death. However, despite the
name, most HSPs are ubiquitously expressed even in physiological conditions, since they are essential for housekeeping
functions inside the cell [2]. Cells possess different families
of chaperones with specific activities and functions, often
working in cooperation to fold native proteins and assist
the formation of supramolecular complexes, keep proteins
in activation-competent conformations, and stabilize them
during the conformational changes required for their activities. Chaperones are also required to re-fold denatured polypeptides, inhibit unfolded protein aggregation, and, when
proteins are defective or irreversibly misfolded, direct them
to degradation via proteasomal and autophagic pathways.
To exert their functions on their substrate proteins, named
clients, HSPs typically take part in complexes that contain
other chaperones, co-chaperones, modulators of ATPase
activity and various accessory proteins. A clear nomenclature for the HSPs and related chaperone genes was proposed
in 2009 [3]; HSPs are currently classified according to their
size into six major and broadly conserved families HSP100s,
HSP90s, HSP70s and HSP60s, that use ATP hydrolysis to
carry out their activity, HSP40s and small heat shock proteins (sHSPs), whose do not possess ATPase activity per se.
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Some HSPs are secreted
into the extracellular milieu
The year was 1986 when Tytell [4] described for the first
time the transfer of HSPs from glia to axon; almost at the
same time, another article by Hightower and Guidon [5]
showed the release of HSPs from cultured rat embryo cells
underlying their uncanonical mechanism of secretion. For
many years, these results did not convince the scientific
community and were considered as potential artefacts
caused by cell necrosis [6]. One of the issues questioning
the active release of HSPs was the absence of a secretion
leader signal in their sequence [7]. Finally, robust data
demonstrating the active secretion of HSPs by living cells
were provided and the scientific community fully accepted
this evidence [8, 9]. However, passive release from damaged or dead cells has been also observed in many cases
[8, 9]. The lack of involvement of the canonical secretory
pathway in HSP secretion was clearly demonstrated using
inhibitors of endoplasmic reticulum (ER)-Golgi vesicular trafficking. Like interleukin (IL) 1 α/β and fibroblast
growth factor (FGF), HSPs use alternative mechanisms
for secretion [10, 11] including free release, vesicles or
vesicular intermediates, derived from the autophagic membranes, endosomes and possibly secretory lysosomes [9]
(Fig. 1). Even if the molecular details at the basis of eHSP
Fig. 1 Pathways of unconventional Heat Shock Proteins
(HSPs) secretion in the extracellular space. Lysosomes or
endosomes fusion with plasma
membrane leads to the release
of HSPs in the extracellular
space. HSPs can be captured
from the cytoplasm during the
formation of endosomal internal
vesicles, which leads to the
biogenesis of multivesicular
bodies. These internal vesicles
are then released as exosomes.
HSPs can directly translocate
from the cytoplasm across the
plasma membrane facilitated
or not by ATP-binding cassette
(ABC) transporters. Microvesicle shedding from the cell surface can also lead to the release
of HSPs into the extracellular
space
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L. Seclì et al.
secretion are not completely elucidated, what is clear is
that many mechanisms could subsist and coexist for the
same HSP in the same cell. For example, HSP70 secretion
as free molecule is mediated by transmembrane proteins
like ATP-binding cassette (ABC) transporters but could
also occur via translocation through plasma membrane,
as described for FGF2 [7]. Moreover, HSP70 requires the
ABC family transporter proteins also to enter the endosomal or lysosomal vesicles, which in turn are secreted in the
extracellular compartment [7, 12, 13]. Another mechanism
is mediated by exosomes, where HSPs amount increases
when cells are under heat-shock or other stress conditions.
HSPs contained in exosomes are transferred to target cells
modifying their behaviour [14]. eHSPs localized on the
membrane of exosomes can also engage surface receptors
and trigger intracellular signalling in an autocrine or paracrine fashion [15–20]. eHSPs localizes within the lipid
bilayer of cellular plasma membranes by interacting with
membrane lipids, possibly stabilizing membranes, regulating their physical properties and organizing microdomain composition [21] (Fig. 2). For example, intracellular HSP70, recognizing phosphatidylserine (PS) moieties,
phospholipids that are normally present in the cytosolic
side of cellular membranes, undergoes an insertion into
the lipid bilayer, exposing a small region of its C-terminus
end to the extracellular environment. It has been proposed
that, during the recovery condition after a stress stimulus,
The dark‑side of the outside: how extracellular heat shock proteins promote cancer
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Fig. 2 Extracellular Heat Shock Proteins (eHSPs) induce extracellular matrix (ECM) remodelling, epithelial–mesenchymal transition
(EMT), migration and invasion. eHSPs released by cancer cells can
interact with extracellular client proteins favouring ECM remodelling
and with surface receptors, triggering signal transduction inside the
cells (HSPG heparan sulfate proteoglycans, FN fibronectin, PDPN
podoplanin)
HSP70 accumulates in respect to unfolded proteins, oligomerizes and binds to PS moieties, being embedded
in the plasma membrane. In addition, the increase in
HSP70 plasma membrane localization is associated with
the formation of ion conductance pathways, resulting in
cell death. However, some reports suggest that HSP70
interaction with lipid bilayer lies also on cholesterol-rich
microdomains, regions within the plasma membrane that
are enriched not only in cholesterol but also in glycosphingolipids, glycosylphosphatidylinositol-anchored and acetylated proteins [21–24]. Experimental data indicate that
HSP70 is exposed on the surface of tumour cells through
the binding with the ceramide-derived glycosphingolipid
globotriaoslyceramide (Gb3) that accumulates in lipid
rafts [25].
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Extracellular HSPs and cancer
HSPs levels have been found aberrantly high in human
cancers compared to normal tissues and correlated with
poor prognosis [26, 27]. HSPs overexpression in cancers is
a response to the internal stress experienced by malignant
cells, such as lack of proteostasis due to high levels of protein synthesis and to the presence of mutant proteins, and
to stresses imposed by the hostile tumour microenvironment (TME), like hypoxia, nutrient deprivation, and acidosis [28]. Cancer growth is a complex, multistep process
that requires cells to acquire some intrinsic characteristics, as genomic instability, resistance to cell death, altered
metabolism and motility and to modify the TME inducing
angiogenesis, inflammation and immune evasion [29, 30].
The role of intracellular chaperones and co-chaperones in
sustaining transformation and cancer progression is well
known, as the attitude of cancer cells to become addicted
to HSP overexpression [31]. It is now widely accepted
that HSPs are released by different types of cancers and
are associated with their plasma membranes [32]. Several
studies describe the tumour-specific immunogenicity of
these released or surface-localized HSPs, a function associated with their ability to chaperone antigenic peptides
and to activate anti-tumour innate immunity [33, 34]. The
role of eHSPs in inhibiting inflammation and in promoting
immune surveillance has been extensively investigated and
recently reviewed [35–39], and therefore is not subject of
debate in this review.
But, despite their tumour-alerting role, the surface and
extracellular localization of HSPs may not be entirely beneficial to the host and, on the contrary, can play key functions in promoting tumour progression and metastasis formation, modulating several cancer hallmarks [38, 40–42].
Different extracellular chaperones and co-chaperones have
been reported to be secreted by cancer cells; however, the
majority of data demonstrating an active role of eHSPs in
cancer refers to HSP90, HSP70 and HSP27.
HSP90 is the most abundantly expressed protein
accounting for the 2–3% of the total proteins in normal
cells and up to 7% in tumour cells [43]. HSP90 comes
in two isoforms: the inducible HSP90α and the constitutive HSP90β. While HSP90β, but not HSP90α, is critical for the cell viability, HSP90α is mainly involved in
cell responses to external stressors. Of note, cancer cells
predominantly secrete the HSP90α isoform [44]. Indeed,
analysing the sera of patients with cancer (including
esophageal squamous cell carcinoma, melanoma, lung,
breast, liver, pancreas and prostate cancer), eHSP90α
level positively correlates with tumour progression [45,
46] as well as with the metastatic lesions in distant organs
[46–48], functioning as a useful diagnostic and prognostic
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biomarker. eHSP90α translocation in the plasma membrane of different cancer cells and its subsequent release in
the medium relies on different stimulus-activated cascades
as the hypoxia-inducible factor (HIF) 1α and the epidermal
growth factor (EGF)-induced phospholipase (PLC) γ1/protein kinase C (PKC) γ signalling [49]. Moreover, eHSP90α
secretion in breast cancer cells depends also on protein
modifications, including PKA-mediated phosphorylation
[48] and acetylation [50]. The HSP70 family includes
several slightly different proteins: among them, the constitutively expressed HSC70 and the stress-inducible
HSP72, the HSP70 located in the mitochondria (GRP75)
and GRP78, resident in the endoplasmic reticulum. Cell
membrane localization of eHSP70 has been found on
different cancer cell lines and on patients’ tumours and
metastases, but not on the normal tissues counterparts
[51]. In particular, eHSP72 is described on the surface of
sarcoma, lung carcinoma and pancreatic cancer cell lines
[52–54]. eHSP70 has been found on the surface of cancer
cells in metastatic lesions in melanoma patients, together
with eHSP90 [45, 51]. Exosomes released by cancer cells
expose HSP70 on their membranes, while exosomes
derived by normal cells do not. This feature makes the
presence of HSP70 on patients’ exosomes a promising biomarker for monitoring cancer growth, relapse and appearance of metastasis, useful to guide therapeutic interventions [52, 55–58]. eHSP27, a member of the small HSPs,
has been found in the sera of patients with squamous cell
carcinoma of the tongue, breast, liver and ovarian cancer [59–64] and its secretion is associated with enhanced
tumour growth and metastasis.
After release in the extracellular compartment or inside
vesicle membranes, HSPs can chaperone and activate specific extracellular client proteins or directly interact with
surface receptors, unleashing specific intracellular signals
in target cells. In both cases, eHSPs have been described
to promote malignancy and enhance metastasis formation.
Extracellular client proteins in cancer
Extracellular matrix (ECM) is the non-cellular constituent
of tissues that provides both biochemical and structural support for the cellular component, favouring cell–cell communication, cell adhesion, and cell proliferation. In addition
to water and minerals, it is composed of collagen, proteoglycans, laminin, and fibronectin secreted by resident cells.
ECM is a highly dynamic structure, constantly undergoing
a remodelling process, in which components are degraded
and modified by different proteases. Of note, the majority
of the eHSP client proteins are ECM or enzymes involved
in ECM remodelling [65–69]. ECM remodelling is a hallmark of cancer progression, impacting on cell proliferation,
migration, and apoptosis [70, 71]. Indeed, a common feature
The dark‑side of the outside: how extracellular heat shock proteins promote cancer
of cancer malignancies is the excessive production of collagens, which are the major components of the ECM. Collagens can act as a scaffold, facilitating migration of invading
cancer cells or stromal cells. In addition, increased collagen
deposition and increased fibril cross-linking is associated
with major changes in biomechanical properties of tissues,
contributing to stiffness, which is crucial for a tumour to
displace the host tissue and grow in size [72].
Fibronectin is secreted by cells as a soluble dimer and the
subsequent binding to integrin receptors, exposed on the cell
surface, induces a fibronectin conformational changes that
expose self-association domains and promote the formation
of an insoluble matrix in the extracellular space. Increased
turnover of the extracellular fibronectin matrix has been correlated with enhanced metastatic capacity of tumour cells.
As collagen assembly relies on fibronectin, fibronectin network depends on collagen and together can favour migration
and invasion of both cancer cells and activated fibroblasts
[70, 71].
ECM modifying enzymes such as matrix metalloproteinases (MMPs), heparanase, cathepsins, plasminogen activator
(PA) and the lysyl oxidase (LOX) family are in charge of
ECM structuring and turnover and their deregulated expression in tumours, significantly contribute to cancer progression and metastasis. Of note, a number of these enzymes
depend on eHSPs for their activity and stability.
MMPs are structurally related zinc metalloproteinases,
present on the cell surface or secreted in the extracellular
compartment. At present, more than 21 mammalian MMPs
have been identified and they are historically classified
according to their substrate specificity and structural similarity in collagenases, gelatinases, stromelysins and matrilysins [73]. Due to the increasing number of proteins identified, MMPs are now divided into eight groups according
to their structure. The MMPs are synthesized as inactive
zymogens (pro-MMPs) and their activation requires proteolytic removal of the prodomain [73]. Most of the MMPs
are activated outside the cell by other activated MMPs or
serine proteinases. MMP2 and MMP9 are frequently overexpressed and highly secreted in human cancers. These soluble
gelatinases, whose preferential substrates include type IV
collagen, elastin, vitronectin, and aggrecan, possess also non
proteolytic activities, regulating signalling pathways that
control cell growth, inflammation, or angiogenesis [74, 75].
The plasminogen activator is a serine protease that
cleaves the inactive proenzyme plasminogen into active plasmin; a broad spectrum serine protease that is, in turn, able
to degrade fibronectin, laminin, vitronectin, proteoglycans,
as well as fibrin and activate latent collagenases, including
MMPs. Plasminogen activation is catalysed by urokinasetype (uPA) or tissue-type (tPA) plasminogen activators,
which are subjected to time- and space-dependent regulation. In particular, the role of PAs in tissue remodelling
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seems consistent with the finding of their overexpression
in human tumours, as well as their elevated amount in the
plasma of breast, prostate, head and colon cancer patients
[70].
Lysyl oxidases (LOX) are secreted amine oxidases. The
family includes five members (LOX and LOX-like 1–4),
whose primary function is the covalent crosslinking of collagens and/or elastin in the ECM. The aberrant expression,
secretion and activity of these proteins have been reported in
a range of human cancers. Indeed, some LOX members (in
particular LOXL2) promote tumour cell survival, regulate
cell adhesion, motility and invasion, and remodel the TME.
LOX- and LOXL2-mediated tumour progression is due primarily to ECM modifications but relies in part on intracellular signalling. Upregulation of LOXL2 has been observed
in a number of human cancers, and its expression has been
associated with cancer aggressiveness [70, 76].
eHSP receptors in cancer
In addition, eHSPs can directly interact with several cell
surface receptors influencing cell behaviour through both
autocrine and paracrine signalling [77]. The main HSPactivated receptors involved in cancer progression are Low
density lipoprotein receptor-related protein 1 (LRP1), Toll
Like Receptors (TLRs), the EGF receptor family (ERBB)
and cluster of differentiation 40 (CD40) [78, 79].
LRP1 or CD91 protein consists of a large extracellular ligand-binding subunit non-covalently associated to a
smaller subunit, containing a transmembrane domain and a
short cytoplasmic tail. LRP1 is expressed in a large panel of
cells, such as hepatocytes, fibroblasts, smooth muscle cells,
neurons and astrocytes [80]. More than forty LRP1 ligands
have been identified nowadays, including apolipoproteins,
proteinases, proteinase-inhibitor complexes, bacterial toxins, viruses, the blood coagulation factor VIII, and various
extracellular matrix proteins such as MMPs and uPA [81].
LRP1 is internalized in vesicles to deliver bound ligands to
the endosomal/lysosomal compartment and is then recycled
on the plasma membrane [82]. Beyond its ability to internalize extracellular components, LRP1 initiates and regulates
many signalling pathways, as small Rho family GTPases,
extracellular signal-regulated kinase (ERK), AKT and c-Jun
N-terminal kinase (JNK) pathways [83–85]. Indeed, LRP1
expression is often deregulated in human cancers. LRP1 also
exerts a fundamental role in cytoskeleton organization, focal
adhesion disassembly and integrin β1 maturation, regulating cell adhesion, spreading, migration and invasion. It was
proposed for the first time as the receptor of several extracellular chaperones by the group of Srivastava [86] and later
many other studies confirmed its role as eHSP receptor in
cancer cells.
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TLR family members are type I transmembrane glycoproteins structurally characterized by the presence of a
leucine-rich repeat domain in their extracellular region and
a Toll/IL-1 receptor (TIR) domain in their intracellular portion, which activates common signalling pathways via TLRspecific adaptor proteins. [87, 88]. To date, ten TLRs have
been identified in humans, the expression of which has been
demonstrated on various innate immune cells, such as macrophages, neutrophils, and dendritic cells (DCs), as well as
non-immune cells including epithelial and endothelial cells.
While, TLRs 1, 2, 4, 5 and 6 are expressed on the cell surface, TLRs 3, 7, 8 and 9 are found almost entirely within
endosomes [89]. Although individual TLRs recognize distinct ligands, the mechanisms of TLR activation and signal
transduction are highly conserved and involve MyD88dependent and -independent pathways that, in turn, activate
multiple pro-inflammatory signalling cascades including
nuclear factor kappa-light-chain-enhancer of activated B
cells (NF-κB), JNK/activator protein 1 (AP1), ERK and p38
[87, 88]. The most common ligands of TLRs are the bacterial cell-surface lipopolysaccharides (LPS), lipoproteins and
lipopeptides of bacterial origin, proteins such as flagellin
from bacterial flagella, double-stranded RNA of viruses and
the unmethylated CpG islands of bacterial and viral DNA.
It was shown that TLRs (in particular TLR2 and 4) can be
also activated by many endogenous molecules including
fibrinogen, surfactant protein-A, heparin, β-defensin 2, High
Mobility Group Box 1 (HMGB1) and HSPs. In particular,
TLR2 and 4 have been extensively validated as eHSP receptors and some indications exist also for TLR3 (when present in plasma membrane [89]) and TLR5 [87]. TLRs may
promote carcinogenesis by unleashing pro-inflammatory,
anti-apopototic, proliferative and pro-fibrogenic signals on
tumour cells and cells of the tumour microenvironment, as
fibroblasts, immune and endothelial cells [88].
ERBB receptors are a subclass of the receptor tyrosine
kinase superfamily and comprises four members: EGFR/
ERBB1, human epidermal growth factor receptor (HER)2/
ERBB2, HER3/ERBB3 and HER4/ERBB4. All members
have an extracellular ligand-binding region, a single membrane-spanning region and a cytoplasmic tyrosine-kinasecontaining domain. The ERBB receptors are expressed in
various tissues of epithelial, mesenchymal and neuronal
origin and their ligands are members of the EGF family of
growth factors. Activated ERBBs stimulate many intracellular signalling pathways including the mitogen-activated
protein kinase (MAPK) and the phosphoinositide 3-kinase
(PI3K)/AKT pathways. Despite extensive overlap in the
molecules that are recruited to the different active receptors,
different ERBBs preferentially modulate certain signalling
pathways, owing to the ability of individual ERBBs to bind
specific effector proteins. These receptors are implicated
in the development of many types of tumours favouring
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proliferation, apoptosis inhibition and cancer progression
[90].
CD40 is a member of the tumor necrosis factor (TNF)
receptor superfamily, expressed by B cells, dendritic cells,
monocytes, platelets and macrophages as well as by nonhematopoietic cells such as myofibroblasts, fibroblasts,
epithelial and endothelial cells. CD40 ligand (CD40L) is a
member of the TNF superfamily that binds to CD40 promoting the activation of signal transduction pathways thanks to
several TRAF (TNF Receptor Associated Factor) proteins,
including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6.
CD40-mediated signalling pathways include the activation of NF-κB, MAPK and signal transducer and activator
of transcription (STAT) 3, favouring the generation of an
acquired immune response. In particular, in cancer, CD40
can license DCs to promote anti-tumour T cell activation and
re-educate macrophages, from M2 state to M1 state, leading
to fibrosis degradation and tumour regression [91, 92].
eHSPs tune cancer hallmarks
Extracellular matrix remodelling
eHSPs regulate ECM remodelling and stiffness interacting and regulating several extracellular client proteins.
eHSP90α (and in some cases HSP70) has found to be crucial for the invasiveness and metastasis formation of fibrosarcoma, esophageal squamous cell carcinoma and breast
cancer cells. eHSP90α assists the proteolytic activation of
MMP2 in conjunction with HSP70 and the co-chaperones
Hop, HSP40, and p23 and protects MMP2 from inactivation, covering one of its autocatalytic cleavage site [46, 48,
65, 68]. eHSP90α acetylation seems crucial to facilitate its
association with MMP2 in the extracellular contest [50].
In addition, LOXL2 interacting with eHSP90α, reaches its
functional conformation and induces migration in breast
cancer cells [66]. Moreover, eHSP90α is found associated
with the tPA in the medium of fibrosarcoma and breast cancer cells. This interaction is essential to convert and activate
plasminogen to plasmin, favouring invasion [67]. eHSP90α
and eHSP90β are found on the surface of invasive cancer
cells, accumulated on the leading edge and then released in
the extracellular space. Invadopodial protrusion formation
is the first step in tumour invasion and eHSP localization
and activity in this site probably depend on their ability to
interact with specific ECM proteases that have found to be
essential for the matrix degradation activity of invadopodia [49, 68, 93–95]. eHSP90α and eHSP90β are found in a
common complex with fibronectin on the surface of breast
cancer cells. In particular, eHSP90β more than eHSP90α
binds to fibronectin, favouring its stability and protecting
it from degradation, thus participating in its assembly and
The dark‑side of the outside: how extracellular heat shock proteins promote cancer
turnover. eHSP90 maintains the stability of fibronectin, and
when the chaperone is inhibited, fibronectin is internalised
and degraded in lysosomes in breast cancer cells. However,
despite the observed interaction between eHSP90β and
fibronectin, it is also possible that fibronectin assembly or
turnover relies on the indirect activity of eHSP90 clients.
In addition, fibronectin and eHSP90 have been found to
co-localize in a common complex with LRP1 on the surface of breast cancer cells. After the inhibition of HSP90,
LRP1 is the putative receptor that mediates the clearance of
fibronectin, but the exact mechanism remains to be elucidated. Indeed, it has been previously reported that LRP1 is
able to mediate fibronectin internalization and degradation
following its accumulation on fibroblasts surface. It is also
possible that fibronectin internalization mediated by LRP1 is
the result of specific signalling activated by eHSP90. Indeed,
AKT and NF-κB activation (eHSP-LRP1 mediated signalling pathways) has been reported to be involved in fibronectin turnover [69, 96, 97].
Epithelial‑mesenchymal transition
The epithelial-mesenchymal transition (EMT) process is
a crucial cancer hallmark that involves the disruption of
cell–cell adhesion and cellular polarity, remodelling of the
cytoskeleton, and changes in cell–matrix adhesion, improving migratory and invasive properties. Cytokines, growth
factors and eHSPs secreted in the TME, by interacting with
cancer cell plasma membrane receptors, may induce intracellular signalling pathways that favour EMT [98]. In prostate cancer cells, eHSP90 binds to LRP1 and promotes ERK
signalling, leading to the impairment of E-cadherin function,
the loss of junctional integrity and the induction of EMT
[99, 100]. eHSP90 is found to upregulate a cohort of stemassociated markers in prostate cancer cells, promoting selfrenewal and stemness associated with metastatic propensity
[101]. In colorectal cancer (CRC) cells, eHSP90α through
LRP1, increases the levels of phosphorylated IκB kinase
(IKK) α/β and NF-κB and induces the expression of TCF12,
a class I member of the helix-loop-helix protein family
preferentially overexpressed in CRC patients with cancer
metastasis. TCF12 is responsible for eHSP90α-dependent
fibronectin expression and the repression of E-cadherin,
connexin-26, connexin-43, associated with the EMT program [102]. In liver cancer cells, eHSP70, activating p38/
MAPK signalling pathway through an unknown receptor,
causes E-cadherin reduction and alpha smooth muscle actin
(αSMA) overexpression favouring EMT, migration and
invasion [103]. An alternative mechanism by which eHSPs
may induce EMT in breast cancer relies on the ability of the
eHSP client proteins MMP2 and MMP9 to proteolytically
cleave and activate latent transforming growth factor (TGF)
β [104].
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Resistance to apoptosis
In several pathological situations, including cancer, different stimuli can induce eHSP secretion in the extracellular
milieu, where these chaperones support the response to the
stress insult protecting cells from apoptosis [6]. In particular,
eHSP90 once secreted under hypoxia, induces the activation
of AKT pathway, through the binding to LRP1 receptor, and
seems essential to protect breast cancer cells from hypoxiatriggered death [105]. In line with this, in glioblastoma
(GBM) cells, hypoxia not only amplifies eHSP90α secretion and its signalling, but also enhances LRP1 expression
resulting in a positive loop that fuels cancer survival and
progression [106]. Moreover, eHSP70 localization on the
surface of colon carcinoma cells increases after radiotherapy
and is associated with cell survival [107]. In a hepatocarcinoma model, eHSP72 binding to TLR2 and TLR4, promotes
apoptotic resistance to chemotherapy and induces proliferation. In this study eHSP72, inducing the release of HMGB1
from cancer cells, favours also a long-lasting effect of TLR4
signalling, supporting tumour growth [108]. eHSP27 is also
able to induce resistance to apoptosis. Once released from
tongue cancer cells following chemotherapy, eHSP27 binds
to TLR5 and triggers NF-κB signalling to enhance chemoresistance and promote cancer progression both in vitro and
in vivo. In addition, the treatment with neutralizing antibodies against HSP27 and HSP70 sensitizes cancer cells,
respectively to chemotherapy and radiotherapy [59, 107].
Migration and invasion
HSP90α, by binding to LRP1 and activating ERK and AKT
pathways, induce migration and invasion in breast cancer cells [43]. In GBM, the eHSP90α/LRP1 signalling is
required to sustain AKT activation and the AKT-dependent
phosphorylation of EphA2 (S897), a tyrosine kinase receptor that is overexpressed in the majority of GBM. EphA2
functions as an LRP1 co-receptor and its phosphorylation
on S897 is crucial for its interaction with LRP1. The signalling facilitates lamellipodia formation, supporting GBM cell
motility and invasion [106].
eHSP90, together with its co-chaperone CDC37, also
interacts with the family of ERBB receptors, including
EGFR or HER2, to promote cancer cell motility and invasion. The binding of eHSP90 to HER2 induces its heterodimerization with HER3, which in turn activates the MAPK
and PI3K-AKT pathways, leading to actin rearrangement
necessary for cell motility. eHSP90-dependent cancer cell
migration is impaired by a monoclonal antibody, mAb
4C5, able to disrupt the interaction between CDC37 and
HSP90 and CDC37 and ERBB [109, 110]. mAb 4C5 has
been proved effective also in inhibiting invasion and metastasis dissemination in a preclinical model of melanoma
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[111]. eHSP90α has also been described to transactivate
EGFR, another member of ERBB family, favouring GBM
cell migration. More specifically, eHSP90α triggers TLR4,
which in turn, leads to the phosphorylation and activation of EGFR in a PKCδ/proto-oncogene tyrosine-protein
kinase (c-Src)-dependent manner, favouring calcium mobilization and ATP release, events known to be associated
with cell migration [112]. Aside from eHSP90α, there are
also some reports that describe a pro-tumorigenic role of
eHSP90β on primary colon adenocarcinoma cells and on
their lymph node metastasis-derived counterpart. In this
context, eHSP90β binds to TGFβ1 forming a complex that,
instead of binding TGFβ1 canonical receptors to activate
the Smad2/3 signalling pathway, engages the integrin αvβ6
and promotes cancer cell invasion and metastasis through a
pathway still undetermined [113].
In hepatocellular carcinoma and lung cancer cells, HSP70
promotes cancer progression by binding to TLR2 and to the
receptor for advanced glycation end products (RAGE) and
inducing MyD88-dependent and -independent NF-κB activation and pro-inflammatory gene transcription [108, 114].
GRP75, the member of the HSP70 family predominantly
localized in the mitochondria, is also secreted by cancer
cells. In particular, GRP75 and podoplanin, a mucin-type
transmembrane sialoglycoprotein, are able to regulate the
activities of Rho, ezrin, and other proteins linked to the actin
cytoskeleton, co-localize on the surface of cells derived from
oral SCC patient specimens, and together regulate adhesion
and matrix remodelling [115].
Angiogenesis
Angiogenesis, the process whereby new blood vessels
develop from a pre-existing vascular network, is essential
for normal organs, as well as for tumours, to establish a
blood supply that satisfies their demand for oxygen and
nutrients and accomplishes other metabolic functions.
Hypoxia is a key driver of tumour angiogenesis and hypoxic
cancer cells secrete vascular endothelial growth factor A
(VEGFA), which initiates tumour angiogenesis binding to
VEGF receptor 2 (VEGFR2) expressed on the endothelial
cells of neighbouring blood vessels. In addition to VEGFA,
others factors participate in this process including FGFα
and β, platelet-derived growth factor (PDGF), TNFα, Angiopoietin1, MMPs, PA, TGFα and different interleukins as
IL-1, IL-6 and IL-8. VEGFA, with the help of these proangiogenic molecules, induces the motility of endothelial
cells and the remodelling of surrounding extracellular matrix
leading to a tumour vascular network that is actively growing and infiltrative [116]. Extracellular chaperones, secreted
not only by cancer cells but also by endothelial cells, may
modulate angiogenesis (Fig. 3). Indeed, eHSP90α, once
secreted in the medium, stabilizes MMP2 and favours the
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transmigration and tube formation of endothelial cells
in vitro and in vivo. In a melanoma mouse model, blocking
with neutralizing antibodies HSP90α, but not HSP90β, leads
to a dose-dependent decrease in MMP2 activity, blood vessel
density and tumour growth [117]. GRP78, the ER member
of the HSP70 family, has been found secreted by colorectal
and prostate carcinoma cells resistant to the antineoplastic
agent bortezomib, a proteasome inhibitor with antiangiogenic activity. eGRP78 potently inhibits the pro-apoptotic
activity of bortezomib on endothelial cells inducing the
ERK/AKT pro-survival pathways and sustains angiogenesis [118]. eHSP27 was shown to promote angiogenesis
through TLR3-dependent calcium entry and NF-κB activation in endothelial cells, which induce VEGF and IL-8
secretion. These factors produce autocrine and paracrine
VEGFR2 activation, causing cell migration and tubulogenesis. In vivo experiments demonstrated that the depletion of
HSP27 decreases vascularization and growth of breast and
colon cancer cells in mouse and rat animal models and that
the treatment with eHSP27 completely reverse this effect
[119], highlighting a crucial role for eHSP27 in angiogenesis
and cancer cell survival.
Stromal cell activation
Fibroblasts, under normal condition, are devoted to tissue
ECM maintenance. Once activated in the tumour microenvironment (mainly through TGFβ), fibroblasts change their
structure and function, acquiring the phenotype of cancerassociated fibroblasts (CAFs). CAFs can produce cytokines
and factors that stimulate tumour cells proliferation, migration and invasion and tumour immunosuppression, facilitating the invasive potential of cancer cells. Different reports
suggest that eHSPs released by both cancer and stroma cells
can modulate and activate fibroblasts, favouring cancer progression (Fig. 3). eHSP90α is observed to promote prostate
fibroblast cell motility and to upregulate markers associated with a CAF-like phenotype, such as vimentin, αSMA,
fibroblast activation factor (FAP) and tenascin C. eHSP90α,
likely through LRP1 and the activation of NF-κB, induces
fibroblasts to secrete inflammatory mediators as IL-6 and
IL-8 in prostate cancer [120]. It has also been observed that
the eHSP90α located on the external surface of breast cancer
cell exosomes, induces fibroblast invasion that can be inhibited by treating cells with an eHSP90α blocking antibody
[19].
Macrophages are important players in innate immunity
and can polarize in M1 or M2 phenotypes depending on
microenvironmental stimuli, such as cytokines, enzymes,
and cell surface markers. M1 macrophages are involved in
antitumor immunity and inflammatory responses characterized by the production of pro-inflammatory cytokines such
as IL-6, IL-12, and TNFα. In contrast, the M2 phenotype is
The dark‑side of the outside: how extracellular heat shock proteins promote cancer
4077
Fig. 3 Extracellular Heat Shock Proteins (eHSPs) activity in the
tumor microenvironment (TME). eHSPs can interact with different
receptors on endothelial cells and induce angiogenesis, on fibroblasts
and on macrophages and, activate them in cancer-associated fibro-
blasts (CAFs) and tumor-associated macrophages (TAMs) respectively. As a final result, eHSPs induce metastasis dissemination fuelling cancer progression
anti-inflammatory and pro-tumorigenic, and is characterized
by the production of other types of cytokines such as IL-10
and TGFβ. Cancer cells recruit macrophages at the tumour
site and activate them in tumour associated macrophages
(TAM), that acquire a phenotype similar to M2 macrophages
in the advanced stages of cancer progression. TAMs generate an immunosuppressive microenvironment and facilitate
processes such as growth, angiogenesis and metastasis, producing cytokines, chemokines, and proteases. In pancreatic
cancer cells, TAM express and secrete eHSP90α that binds
to LRP1 and activates the Janus kinase (JAK) 2-STAT3
pathway, leading to cancer progression [121]. In lung cancer
patients, a strong correlation was observed also between the
serum concentration of eHSP70 and the percentage of M2
polarized macrophages [122]. HSP110, which is HSP70related chaperone [3], activates stromal macrophages and is
present in the serum of patients with colorectal cancer. Once
secreted by colorectal cell lines, binding of HSP110 to TLR4
induces macrophages to polarize toward an M2 phenotype,
while HSP110 immunodepletion, reverts this effect [123].
eHSP27, released by cancer cells in the TME, induces
monocytes to express different cytokines, as IL-10, IL-6,
and proangiogenic factors, as prostaglandin E2, VEGFA,
IL-8, IL-1β, and TNFα. eHSP27 also promotes the expression and release of monocyte chemotactic protein-1 (MCP1), a potent chemotactic signal for monocytes. eHSP27 may
also mediate the differentiation of circulating monocytes
into TAM-like macrophages with immunosuppressive and
proangiogenic phenotypes [124]. eHSP90α exposed on the
surface of tumour released autophagosomes can induce
TLR2–MyD88–NF-κB signalling cascade and stimulate
CD4+ T cells to produce IL-6 that functions in an autocrine
manner to promote the production of IL-10 and IL-21, which
create a favourable environment to facilitate tumour growth
and metastasis in a melanoma mouse model [125] (Fig. 3).
Myeloid-derived suppressor cells (MDSCs) are an immature myeloid cell population that expands in subjects affected
by cancer and inhibits T cell-mediated anti-tumor immunity.
13
4078
HSP72 exposed on tumor-derived exosomes binds to TLR2
on MDSCs and triggers STAT3 signalling, promoting IL6
expression and immunosuppressive activity [55]. Of note, a
peptide aptamer able to interact with HSP72 [126] inhibits
MDSC activation and tumor growth and robustly potentiates
chemotherapy efficacy in cancer mouse models [57].
Even if it is not part of the topic covered by the review,
it is worth mentioning that HSPs exposed on tumor-derived
exosomes may also induce anti-tumor immunity, for instance
by recruiting and activating natural killer cells [14, 17].
Conclusions and future directions
Intracellular chaperones are essential in eliciting the initiation, the development, and the recurrence of cancer, making their overexpression and cell addiction critical requisites
in cancer evolution [127]. A number of experimental data
point to an equally important role of extracellular HSPs in
promoting and sustaining different hallmarks of cancer.
eHSPs are actively secreted by cancer cells and by other cell
populations in the tumour microenvironment in response to
stressful conditions. Once released, eHSPs interact both with
extracellular clients and with membrane receptors and tune
the behaviour of cancer cells, endothelial cells, fibroblasts
and macrophages, orchestrating a complex interaction network, ultimately fuelling cancer growth, migration, invasion,
angiogenesis and immune escape.
The targeting of HSPs may represent an attractive strategy in cancer treatment. Unfortunately, HSPs inhibition
may seriously impacts on normal cell homeostasis, causing
important side effects. Indeed, despite the big effort in testing HSPs inhibitors in cancer pre-clinical models, none of
them have yet been approved by the FDA for the treatment
of cancer patients. This is due to the severe toxicities and,
except for HSP90 inhibitors, for the lack of convincing anticancer activity because of compensatory changes in other
HSPs [27]. Instead, the possibility to block extracellular
chaperones may represent an attractive alternative. Treatments with eHSP cell‐impermeable chemical inhibitors (like
geldanamicyn beads, the geldanamicyn derivative DMAGN-oxide, the ganetespib derivative STA-12–7191) [66–68,
128] or with monoclonal antibodies blocking eHSPs [44,
48–50, 67, 111], were able to impair cancer cell invasion
and metastasis dissemination in tumour pre-clinical models,
without any effects of systemic toxicity.
Moreover, even if the eHSP pro-tumorigenic role has
been widely demonstrated, some caution is needed; eHSPs
have been first studied for their ability to facilitate antigen
presentation, stimulating anti-cancer immunity and inducing
tumour regression [41, 129, 130]. The ideal situation would
be to uncouple pro- and anti-tumorigenic effects of extracellular chaperones and selectively inhibit their pathological
13
L. Seclì et al.
activity. The growing interest in the field and the increasing
amount of data regarding extracellular chaperone function
and behaviour is depicting a complex scenario in which specific extracellular co-chaperones may drive eHSP functions,
allowing to hypothesize that a selective inhibition is feasible.
Acknowledgements We thank Federico Patella for English corrections
on the manuscript.
Author contributions LS, FF, LA and MB had the idea for the article.
LS performed the literature search and wrote the manuscript. FF, LA
and MB critically revised the work.
Funding Open Access funding provided by Università degli Studi di
Torino. This work is supported by Digital Technology For Lung Cancer
Treatment (DEFLeCT) to M.B. LS is supported by two-year fellowship
“Federica Cipolat Mis” id. 24216, from Associazione Italiana per la
Ricerca sul Cancro (AIRC).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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/.
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Publisher’s Note Springer Nature remains neutral with regard to
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Review of Alana Mann’s Global Activism in Food Politics: Power Shift.
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Review of Alana Mann’s Global Activism in Food Politics: Power Shift. Vol 1 | DOI 10 5195/JWSR 1 New articles in this journal are licensed under a Creative Commons Attribution 4.0 United States
License. This journal is published by the University Library System, University of
Pittsburgh as part of its D-Scribe Digital Publishing Program and is cosponsored
by the University of Pittsburgh Press. Alana Mann. 2014. Global Activism in Food Politics: Power Shift. New York: Palgrave
Macmillan. 205 pages, ISBN 978-1-137-34139-6 Cloth ($95.00). This journal is published by the University Library System, University of
Pittsburgh as part of its D-Scribe Digital Publishing Program and is cosponsored
by the University of Pittsburgh Press. This journal is published by the University Library System, University of
Pittsburgh as part of its D-Scribe Digital Publishing Program and is cosponsored
by the University of Pittsburgh Press Alana Mann. 2014. Global Activism in Food Politics: Power Shift. New York: Palgrave
Macmillan. 205 pages, ISBN 978-1-137-34139-6 Cloth ($95.00). As an academic with expertise in Communications, Alana Mann has angled her unique book,
Global Activism in Food Politics, to address the logistics of ‘framing’ in the global food
sovereignty movement—an approach that underlines the delicate balancing act inherent in
sustaining a transnational alliance with diverse local chapters. This particular occupational hazard
derives from the unique structure of the central player in the food sovereignty movement: La Vía
Campesina (LVC), whose operational principle depends on the autonomy of member
organizations, even as LVC is committed to member-based collective action at extra-
regional/national levels, all the way up to the United Nations Human Rights Council and the Food
and Agricultural Organization’s Committee on World Food Security. Power Shift, the book’s
subtitle, refers to the process of scaling up advocacy and discursive frames from grass-roots
organizations via “engagement with state governments, regional trade councils and supranational
bodies such as the FAO” (6). Mann notes that scale shifting can be “uneven, ambiguous and
inconsistent” (144), given that the food sovereignty movement takes the form of a network, and
that local political-economic conditions fluctuate, altering opportunities for grassroots
organizations. Nevertheless, she emphasizes that “the scope for autonomy provided by loosely knit
relationships paradoxically contributes to the durability and stability of the network” (144). And
of course the thread running through this thoughtful study is that insofar as the food regime
undermines farming cultures, exploits agricultural workers and degrades ecosystems, it provides
continuing cause for collective action. Journal of World-System Research | Vol. 22 Issue 1 | McMichael 293 Another key thread in Mann’s study concerns the nature of LVC as a transnational
movement, purportedly the largest social movement in the world, spanning over 70 countries in
North and South and involving over 200 million members (small producers and landless workers). She notes LVC’s now legendary ability to resist representation by non-governmental organizations
(NGOs) and its jealous protection of its own agenda setting, which distinguish it from other
transnational advocacy networks. This is a central theme in Mann’s nuanced characterization of
social movement logistics. Here Mann references social movement theorists’ observations of the
balancing act between local realities and transnational framing, suggesting that “the construction
of a global campaign jeopardises the diversity of subject positions of members” (152). This journal is published by the University Library System, University of
Pittsburgh as part of its D-Scribe Digital Publishing Program and is cosponsored
by the University of Pittsburgh Press She illustrates this diversity via a careful reconstruction of three case studies of domestic
organizations: the National Association of Indigenous and Rural Women (ANAMURI), a Chilean
organization uniting indigenous and non-indigenous women; the Asociación Nacional de
Empresas Comercializadoras de Productores del Campo (ANEC), a Mexican civil association of
60,000 small/medium farmers and over 200 grain producer cooperatives; and the Basque Farmers’
Union Euskal Herriko Nelazarien Elkartasuna (EHNE). As Mann notes, ANAMURI focuses on
gender, indigenous and worker solidarities, organizing around seasonal worker health, sustainable
agriculture and preservation of traditional seeds; ANEC focuses on preserving maize culture and
sustainability of rural livelihoods (competing with agribusiness in the market); and, EHNE
promotes Basque heritage, reaches out to consumer organizations, and engages in Spanish and
European Union (EU) parliamentary politics. Mann’s point is that food sovereignty provides the
ideological bridge between such local/domestic social and political particularities, given the
interpretive elasticity of the concept of ‘food sovereignty.’ This elasticity derives from the
fundamental importance of local autonomy in a transnational movement to democratize food
politics on the foundation of sustainable agriculture. As such, the food sovereignty frame is an
alternative to neoliberal conceptions of food security (via ‘free trade’) in defending local
economies, women’s rights, agrarian reform programs (rural livelihoods rather than simply land
reform, World Bank style), protection of diversity and indigenous seeds, and national food policy
autonomy. While Mann’s book is devoted to evaluating the capacities and claims of the food
sovereignty movement and its strategic alliance building, it also offers a cogent summary of the
institutional contours and social, economic, and environmental consequences of the food regime. This includes a substantive mapping of the political economy of agribusiness, from production to
retailing, and of the rise of oppositional movements (from land-based through identity-based to
alternative food networks), to the impacts of neoliberal food security policies via a
trade/investment regime that erodes local and national food provisioning as well as the integrity of jwsr.org | http://dx.doi.org/10.5195/jwsr.2016.648 Journal of World-System Research | Vol. 22 Issue 1 | Book Review 294 ecosystems. Given her discursive predilections, Mann pays close attention to representational
strategies of the food sovereignty movement writ large, emphasizing a maturing human rights
politics, including promoting “the peasant farmer as a viable economic entity” (35). This journal is published by the University Library System, University of
Pittsburgh as part of its D-Scribe Digital Publishing Program and is cosponsored
by the University of Pittsburgh Press This strategy
resonates at the United Nations (UN), where a Resolution of the Human Rights Council has created
an Intergovernmental Working Group to consider the formal recognition of the rights of peasants
and rural workers (a group constituting 40 percent of humanity), acknowledging the key role small-
scale producers play in global food provisioning (producing up to 70 percent of food). While such initiatives, together with the incorporation of ‘food sovereignty’ principles in
the national constitutions of Venezuela, Ecuador, and Bolivia, and in administrative organs of
Senegal, Mali and Nepal, represent some “reclaiming of the state in the face of globalizing forces”
(141), Mann is careful not to exaggerate the significance of these forms of ‘power shift.’ The UN
of course is composed of member states, and, as Mann points out, Northern governments’
representatives “perceive that the struggle for peasant rights is not their struggle, and shy away
from the very word ‘peasant’” (67), dismissing the mass of people who live and trade on the
margins or outside of capitalist economy. Even so, Mann notes that LVC sees promising new
openings in the international context, such as mounting resistance to U.S. ‘unilateralism,’
weakening of neoliberal institutions such as the World Bank and the IMF, growing links between
civil society and the UN, gradual recognition of food sovereignty by governments, the specter of
TNC gigantism, and a maturing climate change debate in which LVC claims small-scale
agriculture returns energy to the soil and reduces carbon emissions, effectively cooling the planet. Arguably, the value of this comprehensive book is its didacticism, attentive to the
possibilities and limits of alliance building. Mann notes that LVC is breaking out of its self-
imposed autonomy (an initial strategy to avoid NGO-style compromise, or academic influence),
and realizing the importance of reaching out to other movements and struggles, many of which are
directly or indirectly connected to what is happening on the land (from consumer movements,
through precariat struggles with race, class, and gender dimensions, and environmental
movements), and various organizations like the World Social Forum, Occupy Wall Street, People’s
Global Action, and Our World is Not for Sale. In these potential alliances Mann sees the possibility
of mass actions attracting media attention and public notice, as precursors to putting pressure on
governments, international/global governance institutions, and transnational corporations. This journal is published by the University Library System, University of
Pittsburgh as part of its D-Scribe Digital Publishing Program and is cosponsored
by the University of Pittsburgh Press Mann
challenges the food sovereignty movement to recognize the significance of projecting alternative
narratives via public media outlets to inform citizens and states of the rights and potential of farm
cultures and communities that are effectively marginalized in neoliberal discourse, and to develop
media training to understand how to gain access to media outlets and how to project alternative
narratives. jwsr.org | http://dx.doi.org/10.5195/jwsr.2016.648 Journal of World-System Research | Vol. 22 Issue 1 | McMichael 295 And, where dominant narratives circumvent movement voices, access to alternative media
outlets becomes critical. Here Mann emphasizes the importance of “providing opportunities for
social movements to produce and generate their own content, alternative platforms enable them to
avoid frame ‘traps’ that lead to ambiguity, error and misrepresentation” (160). Even there, she
maintains, there is always a danger of reducing face-to-face encounters via keyboard activism. One solution she offers is the example of farmsubsidy.org, which provides an alternative, virtual,
public sphere regarding agri-food politics in the EU. Thus: ‘While investigative journalists have
traditionally sought to reveal government money-trails, the tools to do so are now available to
citizens through cross-border data-sharing initiatives such as farmsubsidy.org’ (162). Finally,
Mann outlines some important strategic possibilities for the food sovereignty movement, such as
recognizing the livelihood dimension of biofuel production for some farming communities (even
as fuel crops displace food crops) and addressing current conceptions of ‘crisis’ in such a way as
to offer alternative ontological frameworks that preclude a market solution reflex and promote
coalition building to transform food systems. At a time of growing interest in, and adaptation of,
‘food sovereignty’ demands and claims, this book is a comprehensive, clear-headed and
challenging template for agri-food scholars and activists alike. Philip McMichael Philip McMichael Philip McMichael
Department of Development Sociology
Cornell University
pdm1@cornell.edu jwsr.org | http://dx.doi.org/10.5195/jwsr.2016.648
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https://openalex.org/W4297915555
|
https://publications.mpi-cbg.de/Stefanko_2017_6790.pdf
|
English
| null |
Lipidomic approach for stratification of Acute Myeloid Leukemia patients
|
arXiv (Cornell University)
| 2,016
|
cc-by
| 7,523
|
RESEARCH ARTICLE Adam Stefanko1, Christian Thiede2, Gerhard Ehninger2, Kai Simons1,3, Michal Grzybek4,5* Adam Stefanko1, Christian Thiede2, Gerhard Ehninger2, Kai Simons1,3, Michal Grzybek4,5* 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 2 Medical Clinic and
Polyclinic I, University Hospital TU Dresden, Dresden, Germany, 3 Lipotype GmbH, Dresden, Germany,
4 Paul Langerhans Institute Dresden of the Helmholtz Centre Munich at the University Clinic Carl Gustav
Carus, TU Dresden, Dresden, Germany, 5 German Center for Diabetes Research (DZD e.V.), Neuherberg,
Germany a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * Michal.Grzybek@tu-dresden.de * Michal.Grzybek@tu-dresden.de Editor: Maurizio Del Poeta, Stony Brook University,
UNITED STATES Editor: Maurizio Del Poeta, Stony Brook University,
UNITED STATES Received: May 28, 2016
Accepted: December 6, 2016
Published: February 16, 2017 Copyright: © 2017 Stefanko 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. OPEN ACCESS Citation: Stefanko A, Thiede C, Ehninger G, Simons
K, Grzybek M (2017) Lipidomic approach for
stratification of acute myeloid leukemia patients. PLoS ONE 12(2): e0168781. doi:10.1371/journal. pone.0168781 Citation: Stefanko A, Thiede C, Ehninger G, Simons
K, Grzybek M (2017) Lipidomic approach for
stratification of acute myeloid leukemia patients. PLoS ONE 12(2): e0168781. doi:10.1371/journal. pone.0168781 Abstract The pathogenesis and progression of many tumors, including hematologic malignancies
is highly dependent on enhanced lipogenesis. De novo fatty-acid synthesis permits
accelerated proliferation of tumor cells by providing membrane components but these
may also alter physicochemical properties of lipid bilayers, which can impact signaling or
even increase drug resistance in cancer cells. Cancer type-specific lipid profiles would
permit us to monitor and interpret actual effects of lipid changes, potential fingerprints of
individual tumors to be explored as diagnostic markers. We have used the shotgun MS
approach to identify lipid patterns in different types of acute myeloid leukemia (AML)
patients that either show no karyotype change or belong to t(8;21) or inv16 types. Differ-
ences in lipidomes of t(8;21) and inv(16) patients, as compared to AML patients without
karyotype change, presented mostly as substantial modulation of ceramide/sphingolipid
synthesis. Furthermore, between the t(8;21) and all other patients we observed signifi-
cant changes in physicochemical membrane properties. These were related to a marked
alteration in lipid saturation levels. The discovered differences in lipid profiles of various
AML types improve our understanding of the pathobiochemical pathways involved and
may serve in the development of diagnostic tools. * Michal.Grzybek@tu-dresden.de AML lipidomics malignancies is enhanced lipogenesis, arising from increased activities of fatty acid biosynthetic
enzymes (Acc1, Fasn and Scd1)[20–23], necessary for accelerated growth. Furthermore, synthe-
sis of mono-unsaturated fatty acids may have added benefit, since oleate can protect against sat-
urated fatty acid toxicity and cellular stress[18, 22, 24, 25]. Thus, Acc1, Fasn and Scd1 have been
identified as plausible targets for cancer therapy[14, 18, 20–23]. publish, or preparation of the manuscript. The
specific roles of these authors are articulated in the
‘author contributions’ section. Kai Simons receives
no salary from Lipotype although he is the CEO. We declare that the commercial affiliation
“Lipotype GmBH” does not alter our adherence to
all PLOS ONE policies on sharing data and
materials. One of the lipid classes associated with cancer pathogenesis are the sphingolipids with their
metabolites such as ceramide, hexosylceramide and sphingosine-1-phosphate, all of which act
as effector molecules[15]. In particular, ceramides are collectively involved in the regulation of
cancer-cell growth, differentiation, senescence and apoptosis[26–28]. Conversion of ceramide
to glucosylceramide contributes to multidrug resistance of several human cancers, including
leukemia, breast cancer, melanoma and neuroblastoma[17, 29–31]. Of equal importance,
sphingolipids are structural lipids that tune membrane fluidity and subdomain structure of the
lipid bilayer, especially lipid rafts[32]. These nanodomains act as regulatory platforms for a
number of growth factor receptors e.g. EGFR, c-kit, VEGFR etc. controlling the initiation of
signaling cascades in cell growth and differentiation[33, 34]. Therefore, deregulation of lipid
metabolism threatens to perturb lateral membrane organization. Mechanisms that govern
homeostasis within the enormous heterogeneity of distinct lipid species are largely unknown. Notably, metabolic transitions affecting lipid metabolism can prompt lipidome-wide ‘concen-
tration waves’, in turn triggering compensatory metabolic responses[35, 36]. There is a grow-
ing interest in lipidome changes during pathogenesis, also in the hope of establishing unique
cellular fingerprints useful as diagnostic markers. The purpose of this study was to explore the lipidomic profiles of AML patients. We focused
on three subsets of patients with specific clinical and biological AML characteristics: transloca-
tion t(8;21), inversion inv16 and patients with normal karyotype (AML-nk). Chromosomal
abnormalities are detected in approximately 60% of newly diagnosed AML patients, among
which t(8;21) and inv16 are the most common. They affect the expression of the Core Binding
Factor (CBF)—a heterodimeric transcription factor controlling key programs in cell survival
[37–39], at least in part by regulating sphingolipid metabolism [40, 41]. Both t(8;21) and inv16
are usually associated with good prognosis and survival. The ‘normal karyotype’ AML group is
very heterogeneous as it emanates from diverse factors related to aberrant signal transduction
pathways leading to uncontrolled growth and inhibition of apoptosis[2, 6, 42]. Moreover,
these changes are often associated with increased activity of multidrug resistance proteins and
the renewal of leukemia stem cells[30, 31]. Here, applying shotgun mass spectrometry we analyzed the cellular lipidomes of various
AML types and identified salient differences. Major discordancies concerned the sphingolipids
and their metabolites. We also observed pronounced shifts in membrane fluidity among the
studied AML types. The recognized lipid patterns may guide the identification of druggable
metabolic pathways for personalized anti-neoplastic therapies. publish, or preparation of the manuscript. The
specific roles of these authors are articulated in the
‘author contributions’ section. Kai Simons receives
no salary from Lipotype although he is the CEO.
We declare that the commercial affiliation
“Lipotype GmBH” does not alter our adherence to
all PLOS ONE policies on sharing data and
materials. Introduction Acute Myeloid Leukemia (AML) is a clonal bone marrow disorder resulting from diverse phe-
notypic and genetic alterations in the differentiation of hematopoietic stem cells and causing
excessive proliferation and accumulation of abnormal immature leukemic neoplastic cells,
called blasts[1–4]. The major causes of AML are deregulation in one or more of the numerous
components of signaling networks that control cell growth, either by gain-of-function mutation
or overexpression[5, 6]. Most efforts towards molecular characterization of AML have focused
on genome, transcriptome or proteome changes [7–12] while evidence is growing that neoplas-
tic pathogenesis and progression also involve, and might even be accelerated by, changes in cel-
lular lipidomes[13–19]. In fact, one of the hallmarks of many tumors including hematologic Funding: The results of this research have been
achieved using the German Federal Ministry of
Education and Research grant to the German
Center for Diabetes Research (DZD e.V.) (M.G.). Competing interests: Lipotype GmBH provided
access to their instrumentation for shotgun MS
measurements of samples included in this study,
but did not have any additional role in the study
design, data collection and analysis, decision to PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 1 / 12 Sample preparation Synthetic lipid standards: cholesterol (Chol-d6) (deuterated), sterol ester (CE 20:0), triacylgly-
cerol (TAG 17:0–17:0–17:0), diacylglycerols (DAG 17:0–17:0), phosphatidic acid (PA 17:0–
17:0), phosphatidylcholine (PC 17:0–17:0), phosphatidylethanolamine (PE 17:0–17:0), phos-
phatidylglycerol (PG 17:0–17:0), phosphatidylserines (PS 17:0–17:0), phosphatidylinositol
(PI 16:0–16:0), lysoglycerophospholipids (LPA 17:0, LPC 12:0, LPE 17:1, LPS 17:1, LPI 17:1),
sphingomyelin (SM 18:1;2–12:0) and ceramides (Cer 18:1;2–17:0, GlcCer 18:1;2–12:0, LacCer
18:1;2–12:0) were purchased from Avanti Polar Lipids. Total protein concentration of leukemic
cells was estimated using the microBCA Protein Assay Kit (Thermo Scientific). Aliquots of cells
equivalent to 20–25 μg of protein that correspond to ~250 000 cells, were transferred into 2 ml
tubes (Eppendorf AG), pelleted (1000g, 5’) washed twice with phosphate-buffered saline and
spiked with 10 μl internal lipid standards mixture dissolved in methanol. All samples were sub-
jected to two-step lipid extraction with 750 μl of 10:1 chloroform:methanol (C/M) (v/v) followed
by a 2:1 C/M mixture at 4˚C. Lipid extracts were collected from organic phases and evaporated
under vacuum. Dry lipid extracts were immediately re-dissolved in 100 μl of chloroform/metha-
nol 1:2 (v/v) and injected into the mass spectrometer Q-Exactive (Thermo Fisher Scientific)
equipped with an electrospray ion source—ESI TriVersa Nanomate (Advion Biosciences). Three
independent runs were done for each lipid extract. Sample collection All AML samples investigated in this study were bone marrow aspirates (anticoagulant lithium
heparin) collected at the time of diagnosis. The blast fraction was enriched by density gradient
centrifugation (density 1.077, BIOCOLL separating solution, Biochrom, Berlin Germany). Enriched samples were aliquoted (1–2 x106 cells/ ml), frozen viably in medium containing
DMSO and fetal calf serum (FCS) and subsequently stored until analysis in liquid nitrogen. Cytogenetic and molecular analysis followed established methods as described [43, 44]. Patients were enrolled in prospective treatment protocols of the Study Alliance Leukemia PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 2 / 12 AML lipidomics (SAL). All patients gave written informed consent making available their material and data for
scientific purposes. The studies were approved by the ethical board of the Medical Faculty TU
Dresden (EK 98032010). Full description of patients can be found in Table 1. Shotgun lipidomics analysis and data processing Lipid extracts (7.5 μl) from the first extraction step were diluted in 13 mM ammonium acetate
in propanol (10 μl) to achieve the final solvent composition of 7.5 mM ammonium acetate in
chloroform/methanol/propanol 1:2:4 (v/v). Lipid extracts from the second extraction step Table 1. Description of patients. ID
karyotype
sex
age
FLT3-ITD mutation
FLT3-
ITD
ratio
NPM1 mutation
KIT mutation
first complete remission achieved
relapse after complete remission
1
normal
f
81
N
NA
Y
NA
N
N
2
normal
m
59
Y
0.92
Y
NA
Y
Y
3
normal
f
62
Y
0.91
Y
NA
N
N
4
normal
f
72
Y
5.9
Y
NA
N
N
5
normal
f
72
N
NA
Y
NA
Y
Y
6
normal
m
63
Y
NA
Y
NA
Y
N
7
inv16
f
53
N
NA
N
N
N
N
8
inv16
f
30
N
NA
N
N
Y
Y
9
inv16
m
28
N
NA
N
Exon 8
Y
Y
10
inv16
m
36
N
NA
N
Exon 8
Y
N
11
inv16
m
41
N
NA
N
N
Y
Y
12
t(8;21)
m
37
N
NA
N
N
Y
N
13
t(8;21)
f
26
N
NA
N
D816V/H
Y
N
14
t(8;21)
m
41
N
NA
N
N
Y
N
15
t(8;21)
m
53
N
NA
N
D816V
Y
N
16
t(8;21)
f
34
N
NA
N
N
Y
Y
F—female; m—male; NA-no information; FLT3-ITD—FMS-like tyrosine kinase 3 internal tandem duplication; NPM1—Nucleophosmin 1; N—no; Y—yes
d i 10 1371/j
l
0168781 t001 Table 1. Description of patients. F—female; m—male; NA-no information; FLT3-ITD—FMS-like tyrosine kinase 3 internal tandem duplication; NPM1—Nucleophosmin 1; N—no; Y—yes PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 3 / 12 AML lipidomics were dissolved in 10 μl of 0.01% methylamine as ion modifier and subjected to negative mode
MS analysis. Lipid extracts from the 10:1 organic phase were analyzed with polarity switching in
both positive and negative ion mode, the 2:1 extracts in negative mode only. Positive ion mode
analysis was performed using FTMS with target resolution of 280,000 at m/z 200 and MS/MS
with 17,500 at m/z 200 to monitor CE, DAG and TAG species. Shotgun lipidomics analysis and data processing Negative FTMS mode was chosen
to monitor lyso-phospholipids (LPG, LPS, LPI, LPS, LPC, LPE) and combined with tandem MS/
MS for selected phospholipids (PC, PC O-, PE, PE O-, PS, PA, PI, PG), sphingolipids (SM, Cer,
Hex-Cer, diHex-Cer), globosides (Gb3 and Gb4) and ganglioside GM3. The same, targeted reso-
lution settings were used for both polarities. Cholesterol was measured separately after chemical
acetylation[45]. Briefly, 20 μl of 10:1 lipid extract was dried down and 75 μl of acetyl chloride in
1:2 C/M (v/v) added. After an hour incubation samples were dried down, re-dissolved in 50 μl
C/M 1:2 and analyzed in positive ion mode with similar settings as described above. The molar-
ity of lipid species was determined based on spiked-in, internal quantitative standards. Samples were infused into the Q-Exactive instrument TriVersa NanoMate ESI source. Data
acquisition was performed in both positive and negative mode with polarity switching where
MS was used for quantification and MS/MS for fatty acid and lipid precursor identification. Data were analyzed and de-convoluted by LipidXplorer software. Data visualization and nor-
malization (pmol, mol%) calculations were performed in Microsoft Excel, GraphPad Prism 6.0
and SIMCA 14.0. GP measurements For fluidity analysis, dried lipids were rehydrated in 150 mM NaCl, 25 mM HEPES pH 7.25. The resulting liposomes were subsequently subjected to 10 freezing/thawing cycles (liquid nitro-
gen/37˚C) and extruded through 100 nm polycarbonate filters. Finally, vesicles were stained
(15 min) with 2 μM C-laurdan. Fluorescence emission spectra were recorded (Ex. 385 nm, Em. 400–550 nm) and analyzed as described in Kaiser et al.[46]. PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 Lipidome analysis reveals significant differences among the AML
subtypes Total leukocyte fractions containing 250 000 cells, corresponding to approximately 20–25 μg
of total protein content, were subjected to two-step lipid extraction, MS lipidomics and GP
measurements. Shotgun lipidomics MS and MS/MS designated acquisition routine and Lipo-
type proprietary lipidomics software enabled profiling of more than 400 unique lipid species
of 25 analyzed lipid classes (for details see S1 Table). Lipidomics data were first normalized to internal standards and transformed to mol%. Multi-
variate statistical data analysis was applied to systematize and better understand the recorded lipi-
dome variations. Dimension reduction and data visualization followed Principal Component
Analysis (PCA) with Pareto scaling (SIMCA software). This statistical tool emphasizes variation
and highlights robust patterns in a dataset. Subsequently, a two-dimensional score plot was gener-
ated (significance of the principal components based on the explained sum of squares was 0.253
and 0.094 for the first and second principal component respectively) (Fig 1), revealing t(8;21)
samples clearly separated from inv16 and AML-nk patient samples—the latter clustered together. This suggested substantial differences between the lipidomes of t(8;21) and the other AML types. Next, lipid species differences between the AML types were analyzed. Briefly, lipid fold
change followed by multiple t-tests analysis identified unique, species with the most pro-
nounced deviations (Fig 2). The major difference between t(8;21) and AML-nk involved PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 4 / 12 AML lipidomics Fig 1. Score plots of multivariate analysis by PCA. The t(8;21) samples are separated from the inv16 and
AML-nk samples. Each patient sample was measured in triplicates and each point on the plot represents an
individual measurement. The calculated sum of squares was 0.253 and 0.094 for the first and second
component, respectively. The analysis was performed using SIMCA 14.0 software. doi:10.1371/journal.pone.0168781.g001 Fig 1. Score plots of multivariate analysis by PCA. The t(8;21) samples are separated from the inv16 and
AML-nk samples. Each patient sample was measured in triplicates and each point on the plot represents an
individual measurement. The calculated sum of squares was 0.253 and 0.094 for the first and second
component, respectively. The analysis was performed using SIMCA 14.0 software. doi:10.1371/journal.pone.0168781.g001 ceramide backbone-containing lipids: SM, Cer, Hex-Cer and GM3 ganglioside (Fig 2A). Lipidome analysis reveals significant differences among the AML
subtypes Decrease of SM in t(8;21) was correlated with an increase in the levels of ceramides (up to
3-fold), Hex-Cer (up to 5-fold), DiHex-Cer (up to 3-fold) and GM3 (up to 15-fold) suggesting
a shift towards glycosphingolipid synthesis (S1 Table). Unfortunately GM3 derivatives are cur-
rently not accessible to quantitative shotgun MS. Similarly, the comparison of groups t(8;21)
and inv16 (Fig 2B) mostly registered expression profile changes affecting sphingolipid path-
ways. We observed a decrease in SM levels and increased ceramide synthesis (Fig 2C), suggest-
ing that this type of leukemic cells might be more prone to apoptosis[26–28]. Despite the fact
that inv16 and AML-nk patients represent a mixed population in the PCA plot (Fig 1), direct ceramide backbone-containing lipids: SM, Cer, Hex-Cer and GM3 ganglioside (Fig 2A). Decrease of SM in t(8;21) was correlated with an increase in the levels of ceramides (up to
3-fold), Hex-Cer (up to 5-fold), DiHex-Cer (up to 3-fold) and GM3 (up to 15-fold) suggesting
a shift towards glycosphingolipid synthesis (S1 Table). Unfortunately GM3 derivatives are cur-
rently not accessible to quantitative shotgun MS. Similarly, the comparison of groups t(8;21)
and inv16 (Fig 2B) mostly registered expression profile changes affecting sphingolipid path-
ways. We observed a decrease in SM levels and increased ceramide synthesis (Fig 2C), suggest-
ing that this type of leukemic cells might be more prone to apoptosis[26–28]. Despite the fact
that inv16 and AML-nk patients represent a mixed population in the PCA plot (Fig 1), direct Fig 2. Comparison of lipid profiles of various AML types. Each point on the volcano graph represents a single lipid specie. The analysis was performed
using GraphPad PRISM 6.0. Fig 2. Comparison of lipid profiles of various AML types. Each point on the volcano graph represents a single lipid specie. The analysis was performed
using GraphPad PRISM 6.0. doi:10.1371/journal.pone.0168781.g002 PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 5 / 12 AML lipidomics comparison by orthogonal partial least-square displacement analysis brings to light consider-
able differences (p<0.001) between their lipidomes. The most prominent change was the
increased abundance of various Hex-Cer species in the inv16 samples (Fig 2C). These lipids
are metabolic precursors of other glycolipids, but were also ascribed an important role in mul-
tidrug resistance[17, 29, 31]. Inv16 is associated with a specific accumulation of one particular
lipid class, rather than a shift in glycosphingolipid metabolism, as in the case of t(8;21) cells. Measurement of generalized polarization index reveals dramatic
changes in membrane fluidity in t(8;21) cells Activation of the lipogenic pathway at early stages of cancer pathogenesis is critical for prolif-
eration of various types of tumors including hematologic malignancies[21, 23]. The majority
of newly synthesized fatty acids in cancer cells are converted predominantly to membrane
phospholipids. Specifically, the proportion of monounsaturated and polyunsaturated fatty
acids in the major phospholipids was reported to be significantly higher in cancer compared to
noncancerous tissues[47]. Here we compared each type of leukemia for differences in lipid sat-
uration levels. A marked difference affected t(8;21) leukemia, a significant shift towards poly-
unsaturated at the cost of monounsaturated fatty acids (Fig 3A). This observation was specific
to membrane lipids (phospholipids + sphingolipids), which comprise roughly 80% of the total
measured lipids, but not to storage lipids (TAG, DAG, SE) (S1 Fig). Fatty acid saturation and length plus cholesterol content are the three major determinants of
membrane fluidity. Having observed a significant shift of fatty acid saturation but not of choles-
terol abundance or fatty acid length (Figs 3 and S1) we expected a substantial change in membrane
fluidity of t(8;21) compared to the other AML types. Membrane fluidity (General Polarization
Index, GP) was measured on small unilamellar vesicles prepared from the extracted lipids, using
the fluorescent probe c-laurdan, as described [48]. Levels of unsaturation and GP values were
closely correlated, confirming the greater fluidity of t(8;21) membranes. These findings under-
score the importance of precisely measuring lipid and desaturation levels in cancer cells to resolve
aberrantly regulated metabolic pathways and, ultimately, therapeutic targets. Fig 3. Lipid features from extracted AML samples. (A) Saturation index of all lipids measured with shotgun MS. The analysis revealed significant changes
of the mono- and polyunsaturated fatty acids in t(8;21) samples. (B) GP index of liposomes prepared from lipid extracts of various AML samples. GP is a
measure of membrane order (higher GP equals more ordered membranes). t(8;21) samples exhibit lower GP values, indicating higher membrane fluidity. doi:10.1371/journal.pone.0168781.g003 Fig 3. Lipid features from extracted AML samples. (A) Saturation index of all lipids measured with shotgun MS. The analysis revealed significant changes
of the mono- and polyunsaturated fatty acids in t(8;21) samples. (B) GP index of liposomes prepared from lipid extracts of various AML samples. GP is a
measure of membrane order (higher GP equals more ordered membranes). t(8;21) samples exhibit lower GP values, indicating higher membrane fluidity. doi:10 1371/journal pone 0168781 g003 Fig 3. Discussion Similar to embryonic cells, cancer cells are highly dependent on de novo lipogenesis for their
proliferation, and lipogenesis is activated at a relatively early stage in various types of tumors
[18, 21, 49]. Moreover, there seems to be a correlation between fatty acid synthase expression,
which is greatly elevated in metastatic tumors, increased membrane fluidity and decreased
patient survival[50–53]. Proliferating cancer cells predominantly convert the majority of
newly synthesized fatty acids to phospholipids for membrane incorporation [18]. Changes in
lipid metabolism disequilibrating saturation levels of membrane fatty acids influence mem-
brane fluidity and structure and composition of membrane domains (rafts) where many criti-
cal signaling cascades originate. In consequence, aberrant signal transduction may enhance
the proliferation and survival of hematopoietic progenitor cells[2, 6]. An additional layer of
complexity arises as a consequence of metabolic interconversion of one lipid to another—e.g. of PI(4,5)P2 to DAG or DAG/PC to PA—that impact signaling[54, 55]. Similarly, many path-
ways of sphingolipid metabolism constitute an interconnected network wherein individual
levels and the balance of bioactive molecules can modulate cancer initiation, progression or
treatment response. For example, ceramide hydrolysis produces sphingosine, the phosphoryla-
tion of which yields sphingosine-1-phosphate that regulates cell growth and suppresses pro-
grammed cell death[56–58]. Thus the difference in lipid metabolism between tumors and
normal tissues renders lipogenic pathways attractive but so far only moderately exploited tar-
gets for the development of anti-cancer therapies [23, 59]. To evaluate the suitability of lipidomics for discriminating various types of cancer cells we
analyzed the lipid profiles of AML cells of patients with (t(8;21), inv16) and without karyotype
disorders (AML-nk). Importantly, in both t(8;21) and inv16 AML the karyotype changes result
in deregulation of CBF expression, a factor that controls the expression of genes involved in
sphingolipid metabolism[40, 41]. CBF overexpression has been shown to reduce intracellular
long-chain ceramides in NIH3T3 fibroblasts and to elevate extracellular sphingosine-1-phos-
phate levels[40]. On the other hand, CBFβ-silenced cells exhibited substantial increases in cer-
amide species and decreases in lactosylceramides that correlated with enhanced cell death[41]. Here for the first time we demonstrate quantitative lipidome changes of cells with disorders in
CBF expression. Indeed the major differences discovered between the various AML types affect
the sphingolipids and ceramides. Interestingly, although both t(8;21) and inv16 share similarities
(deregulation of CBF expression), significant differences in sphingolipid metabolism remain. Measurement of generalized polarization index reveals dramatic
changes in membrane fluidity in t(8;21) cells Lipid features from extracted AML samples. (A) Saturation index of all lipids measured with shotgun MS. The analysis revealed significant changes
of the mono- and polyunsaturated fatty acids in t(8;21) samples. (B) GP index of liposomes prepared from lipid extracts of various AML samples. GP is a
measure of membrane order (higher GP equals more ordered membranes). t(8;21) samples exhibit lower GP values, indicating higher membrane fluidity. doi:10 1371/journal pone 0168781 g003 doi:10.1371/journal.pone.0168781.g003 PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 6 / 12 AML lipidomics Discussion To evaluate the suitability of lipidomics for discriminating various types of cancer cells we
analyzed the lipid profiles of AML cells of patients with (t(8;21), inv16) and without karyotype
disorders (AML-nk). Importantly, in both t(8;21) and inv16 AML the karyotype changes result
in deregulation of CBF expression, a factor that controls the expression of genes involved in
sphingolipid metabolism[40, 41]. CBF overexpression has been shown to reduce intracellular
long-chain ceramides in NIH3T3 fibroblasts and to elevate extracellular sphingosine-1-phos-
phate levels[40]. On the other hand, CBFβ-silenced cells exhibited substantial increases in cer-
amide species and decreases in lactosylceramides that correlated with enhanced cell death[41]. Here for the first time we demonstrate quantitative lipidome changes of cells with disorders in
CBF expression. Indeed the major differences discovered between the various AML types affect
the sphingolipids and ceramides. Interestingly, although both t(8;21) and inv16 share similarities
(deregulation of CBF expression), significant differences in sphingolipid metabolism remain. Translocation t(8;21) unlike inv16 and AML-nk, is accompanied by substantial decrease of sphin-
gomyelin (Fig 2), probably, as shown by others, due to decreased expression of SGMS1 and
SGMS2[7], which detours ceramides to glycosphingolipid synthesis. In inv16 samples sphingoli-
pid metabolism appears more at equilibrium and only hexosylceramides are specifically accumu-
lated (Fig 2C), reported as a multidrug resistance phenotype in many cancer cells[17, 29–31]. The
observed pattern is particularly revealing, as in t(8;21) and inv16 patients sensitive to the drugs
ABT737 (Bcl-2 inhibitor) and BV6 (antagonist of IAP, ‘inhibitor of apoptosis proteins’), genes
responsible for ceramide synthesis were upregulated, in contrast to patients resistant to these
drugs[7]. Enzyme expression changes of sphingolipid metabolism in several cancers are consis-
tent with the emerging tumor suppressor functions of ceramide. Our data provide evidence that
ceramide levels significantly differing between patients, even those bearing similar genetic pertur-
bations, might directly correlate with these patients’ susceptibility to chemotherapy. (deregulation of CBF expression), significant differences in sphingolipid metabolism remain. Translocation t(8;21) unlike inv16 and AML-nk, is accompanied by substantial decrease of sphin-
gomyelin (Fig 2), probably, as shown by others, due to decreased expression of SGMS1 and
SGMS2[7], which detours ceramides to glycosphingolipid synthesis. In inv16 samples sphingoli-
pid metabolism appears more at equilibrium and only hexosylceramides are specifically accumu-
lated (Fig 2C), reported as a multidrug resistance phenotype in many cancer cells[17, 29–31]. PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 Supporting information S1 Fig. (A) Lipid features (fatty acid saturation and length) measured with shotgun MS for
“membrane” (PC, PC-O, PE, PE-O, PS, PI, PG, PA, SM, Cer, HexCer, DiHexCer, GM3, Gb3,
Gb4) and “storage” lipids (SE, TAG, DAG). (B) Lipid profile of sphingolipids measured in var-
ious AML samples. (PDF) S1 Table. Lipidomic analysis of AML samples. Mol% values of individual lipid species deter-
mined with shotgun MS for particular patient samples. Each sample was measured in three
independent runs. Conclusion Mass spectrometry-driven quantitative analysis of cellular lipids, especially the so-called shot-
gun lipidomics technique is increasingly being explored for its potential as a clinical diagnostic
tool. A major benefit of this technology is that hundreds of lipid species can be directly identi-
fied and accurately quantified in a relatively short analysis time[71–73]. This work demon-
strates that lipidomic profiling of acute myeloid leukemia cells supports the stratification of
hematological malignancies. The observed imbalances of lipid synthesis pathways might
directly contribute to disease progression. AML lipidomics in aberrant signaling[63–66]. On the other hand the shift towards increased levels of saturated
and monounsaturated phospholipids may protect cancer cells from oxidative damage by reduc-
ing lipid peroxidation[14, 18]. In line with this, it has been suggested of membrane active drugs
that their ability to alter membrane fluidity correlates with pharmacological activity[67, 68]. in aberrant signaling[63–66]. On the other hand the shift towards increased levels of saturated
and monounsaturated phospholipids may protect cancer cells from oxidative damage by reduc-
ing lipid peroxidation[14, 18]. In line with this, it has been suggested of membrane active drugs
that their ability to alter membrane fluidity correlates with pharmacological activity[67, 68]. The observed changes in membrane fluidity in t(8,21) samples might, to a lesser extent, be
the consequence of lower sphingomyelin concentration. Yet, this appears to be compensated
by the increase in glycosphingolipid (GSL) content (S1 Fig), making a dramatic effect on mem-
brane fluidity unlikely. Interestingly however, the changes in t(8,21) samples in the ratio of SM
and GSL might critically alter the physicochemical properties of rafts with important sequels
for signal transduction pathways. Glycosphingolipids were among the first discovered tumor-
associated antigens and are now appreciated as function modulators of several growth factor
receptors that govern cell growth and differentiation [69, 70]. Although the current approach
does not allow assaying of higher glycolipids (with more than three sugar residues), the observ-
able ones (Hex-Cer, DiHex-Cer and GM3) indicated a significant shift in sphingomyelin-gly-
cosphingolipid metabolism. Therefore, one can clearly expect significant, purely lipid-induced
responses in signal transduction pathways not brought about by changes in expression levels
or gain-of-function mutations in the components of the signaling cascades. The challenge for
the future will be to unravel how, through modulation of the growth signaling cascades, indi-
vidual differences in sphingolipid metabolism stimulate cancer development and progression. As shown here, quantification of cellular lipids, down to single species level allowed us to dis-
tinguish AML-type-specific lipid signatures. Such procedures pave the way towards new diag-
nostics and prognostics and should be integrated into a systemic approach towards the
mechanistic understanding of cancer and the optimization of therapeutical targets. Discussion The
observed pattern is particularly revealing, as in t(8;21) and inv16 patients sensitive to the drugs
ABT737 (Bcl-2 inhibitor) and BV6 (antagonist of IAP, ‘inhibitor of apoptosis proteins’), genes
responsible for ceramide synthesis were upregulated, in contrast to patients resistant to these
drugs[7]. Enzyme expression changes of sphingolipid metabolism in several cancers are consis-
tent with the emerging tumor suppressor functions of ceramide. Our data provide evidence that
ceramide levels significantly differing between patients, even those bearing similar genetic pertur-
bations, might directly correlate with these patients’ susceptibility to chemotherapy. Among all studied groups, the t(8;21) patients presented the highest membrane fluidity, pre-
dominantly due to increased unsaturation of membrane lipid fatty acid moieties. Elevated
membrane fluidity has been associated with enhanced cell growth and proliferation in many
cancers[60–62] as well as shown to modulate certain receptor tyrosine kinase proteins, resulting 7 / 12 PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 Acknowledgments We thank Christian Klose, Cornelia Schroeder and Patrycja Dubielecka for critical discussions
and reading of the manuscript. We acknowledge the support of this research by the German 8 / 12 PLOS ONE | DOI:10.1371/journal.pone.0168781
February 16, 2017 AML lipidomics Federal Ministry of Education and Research grant to the German Center for Diabetes Research
(DZD e.V.) (M.G.). Federal Ministry of Education and Research grant to the German Center for Diabetes Research
(DZD e.V.) (M.G.). Project administration: AS KS MG. Resources: CT GE KS MG. Resources: CT GE KS MG. Supervision: KS MG. Supervision: KS MG. Validation: AS KS MG. Visualization: AS MG. Writing – original draft: AS MG. Writing – original draft: AS MG. Writing – review & editing: AS CT GE KS MG. Writing – review & editing: AS CT GE KS MG. PLOS ONE | DOI:10.1371/journal.pone.0168781
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Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555
УДК 347.132.6
doi: 10.21564/2225-6555.2021.2.238013 Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555
УДК 347.132.6
doi: 10.21564/2225-6555.2021.2.238013 Теорія і практика правознавства. – 2021. – Вип. 2 (20) УДК 347.132.6 doi: 10.21564/2225-6555.2021.2 © Коробцова Н. В., 2021 РОЗПОРЯДЖЕННЯ ПАЦІЄНТА
ЯК ОКРЕМИЙ ПРОЯВ
ЙОГО ВОЛІ Коробцова Наталія Василівна,
канд. юрид. наук, доц.,
доцентка кафедри цивільного права № 1,
Національний юридичний університет
імені Ярослава Мудрого,
Україна, м. Харків
e-mail: kornv@ukr.net
ORCID 0000-0002-9997-1485 У статті розглянуто питання волевиявлення пацієнта в медичних правовідносинах. Доведено, що саме завдяки прояву волі щодо запропонованого лікування (надання згоди чи
відмова від нього) пацієнт є повноцінним активним учасником цих відносин. Однак
нездатність його до волевиявлення, тимчасова або незворотна, викликана розвитком
хвороби, особливістю її протікання, може стати перешкодою для визначення його
реального бажання щодо майбутнього лікування. Для уникнення цього в законодавстві низки
країн світу існує певний правовий інститут, за допомогою якого уможливлюється завчасне
планування свого лікування, а також відмова від нього у разі нездатності в майбутньому це
зробити. Проаналізовано зміст цього інституту, розглянуто точки зору науковців щодо
нього, проведено порівняльний аналіз з юридичною конструкцією «заповіту» та зроблено
висновок про існування суттєвих відмінних рис між цими конструкціями, що унеможливлює,
з точки зору автора, називати дане волевиявлення «заповітом». Запропоновано розглядати
таке волевиявлення як «розпорядження пацієнта», що оформлюється письмово
повнолітньою особою – пацієнтом незалежно від виду та стадії хвороби на випадок своєї
можливої в майбутньому нездатності висловити згоду на медичне обстеження, втручання
чи лікування. Ключові слова: розпорядження пацієнта; заповіт пацієнта; медичний заповіт; права
пацієнта; волевиявлення; медичні правовідносини. Постановка проблеми. Можлива нездатність пацієнта виразити в
майбутньому свою волю стосовно методів лікування і процедур, а також
бажання продовжувати або припинити лікування є суттєвою проблемою в
медичній сфері, яка безпосередньо торкається як питань захисту прав та
інтересів пацієнта, так і розвитку медицини як науки. При цьому забезпечити
належну охорону та захист своїх прав пацієнт може шляхом використання
такого правового інституту, як завчасне планування свого лікування або
відмова від нього. Висловити своє бажання продовжувати лікування чи ні,
якими методами це робити пацієнт може шляхом оформлення відповідного © Коробцова Н. В., 2021 1 1 © Korobtsova N. V. 2021 ISSN 2225-6555 Theory and Practice of Jurisprudence. – 2021. – Issue 2 (20) ISSN 2225-6555
розпорядження, адресованого лікарям або довіреній особі. Проблема, яка при
цьому виникає, полягає в тому, що законодавчого визначення даного поняття та
механізму його застосування й регулювання українське законодавство не
містить. Водночас і задовольнити всі бажання пацієнта не завжди можливо,
виходячи з імперативних положень законодавства. Тому розгляд та аналіз
даного правового інституту являє не лише теоретичний інтерес, а й має
практичне значення. Аналіз останніх досліджень і публікацій. Питання волевиявлення осіб у
сфері медичних відносин розглядали у своїх працях такі науковці, як
С. Б. Булеца (S. B. Buleca), Т. С. Комар (T. S. Komar), А. А. Литвиненко
(A. A. Lytvynenko), Р. А. Майданик (R. A. Majdanyk), Г. А. Миронова
(G. А. Myronova), О. Л. Нікітенко (O. L. Nikitenko), І. Я. Сенюта (I. Ja. Senjuta),
Г. О. Сироткина (G. O. Syrotkyna), С. Г. Стеценко (S. G. Stecenko),
В. Ю. Стеценко (V. Ju. Stecenko), Р. О. Стефанчук (R. O. Stefanchuk) та ін. Мета статті – з’ясувати сутність та зміст розпорядження пацієнта як
окремого самостійного правового інституту. Виклад основного матеріалу. Одним з основних загальновизнаних прав
є право на охорону здоров’я та медичне обслуговування. На міжнародному
рівні вже давно закріплено пріоритет інтересів і благ окремої людини над
інтересами суспільства та науки (ст. 2), захист її гідності та гарантія кожному
без виключення дотримання цілісності особистості та інших прав й основних
свобод у зв’язку із застосуванням досягнень біології та медицини (ст. 1) [1]. Конституція України визнає людину, її життя і здоров’я, недоторканність
і безпеку найвищими соціальними цінностями в державі, що спрямовує
державу на забезпечення захисту людини, її прав і законних інтересів від
протиправних посягань (статті 27, 49) [2]. Даний обов’язок держави існує й у
сфері медичного обслуговування. Першим у світі актом, який визначив і врегулював права пацієнтів, є
Другий білль про права [3], прийнятий у США, який визначив перелік прав,
одним з яких є право на медичну допомогу, що виявляється у необхідному © Korobtsova N. V. 2021 © Korobtsova N. V. 2021 2 2 © Коробцова Н. В., 2021 Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555 Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555
медичному догляді та можливості досягти й мати задоволення від міцного
здоров’я. Дані права суттєво були покращені Законом про захист пацієнтів та
доступної медицини 2010 р., який започаткував проведення наймасштабнішої в
США реформи охорони здоров’я. Україна, на жаль, не має самостійного нормативного акта про права
пацієнтів та їх захист. Загальні права в цій сфері закріплено в Конституції [2] та
ЦК України [4], Законі України «Основи законодавства України про охорону
здоров’я» [5] (далі – Основи). У країні вже декілька років точиться дискусія про
необхідність прийняття окремого документа, в якому будуть закріплені права
пацієнтів, механізм їх реалізації та захисту. Так, проєкт Закону «Про захист
прав пацієнтів» [6] передбачає існування закритого переліку цих прав, що,
враховуючи постійний розвиток медицини, інформаційних та інноваційних
технологій в цій галузі, є недоречним. Одним із фундаментальних прав у сфері медичного обслуговування, яке
знайшло відповідне закріплення на міжнародному та національному рівнях,
виступає право надати згоду на медичне втручання чи відмовитися від нього,
що є певним проявом волі пацієнта в медичних правовідносинах і впливає на
формування всіх відносин, що виникають між лікарем і пацієнтом з приводу
надання медичної послуги останньому. Особливість даного права полягає в
тому, що саме його наявність і можливість реалізації дозволяють пацієнту бути
активним учасником медичних відносин, брати безпосередню участь в
узгодженні плану лікування, запропонованих ліків і процедур. У національних і міжнародних актах «згода» та «відмова» у сфері
надання медичних послуг розглядаються як окремі права, при цьому більше
уваги законодавцем приділяється саме «згоді на медичне втручання». Так, в
Основах міститься стаття «Згода на медичне втручання», присвячена
необхідності отримання її на будь-яке медичне втручання (діагностику,
профілактику, лікування), в якій зазначаються й виключення з цього правила. При цьому зміст даної статті свідчить про можливість і доцільність розглядати
ці прояви волі пацієнта (згоду та відмову) як єдине право, оскільки декілька її © Коробцова Н. В., 2021 3 3 © Korobtsova N. V. 2021 ISSN 2225-6555 Theory and Practice of Jurisprudence. – 2021. – Issue 2 (20) ISSN 2225-6555
останніх абзаців присвячено питанням відмови [5, ст. 43]. Конвенція про права
людини та біомедицину в гл. II «Згода» містить загальне правило про те, що
медичне втручання може здійснюватися лише після того, як відповідна особа
надасть на це свою добровільну письмову згоду [1, ст. 5]. При цьому будь-яке
медичне втручання стосовно осіб, не здатних виразити волю в силу свого
психічного або фізичного стану здоров’я, має здійснюватися виключно в
інтересах таких осіб. Така підвищена увага з боку законодавця на закріплення в якості права
пацієнта надання згоди на медичне втручання безумовно є виправданою й
спрямована на усвідомлене ставлення особи до свого здоров’я, але при цьому
незначна увага приділена питанням відмови від медичного втручання відсуває
це право «на другий план». Хоча, на нашу думку, право на відмову за
юридичною силою є рівнозначним праву на надання згоди, які доцільно
розглядати в сукупності, оскільки у будь-якому випадку, вирішуючи питання
стосовно свого здоров’я, особа або погоджується на запропоноване їй лікування
й таким чином реалізує своє право на надання згоди, або відмовляється,
реалізуючи своє право відмови. У медичних правовідносинах волевиявлення особи (пацієнта) має
будуватися на певних принципах: по-перше, бути вільним, пацієнт самостійно
вирішує питання, відносно якої послуги йому укладати договір, з яким
контрагентом (виконавцем медичних послуг) та на яких безпосередньо умовах;
по-друге, бути незалежним, тобто на рішення особи-пацієнта не повинна впливати
думка чи примус інших осіб (лікарів, родичів, близьких осіб та інших суб’єктів);
по-третє, бути поінформованим. Пацієнт виражає свою згоду після отримання від
виконавця всієї інформації про стан свого здоров’я, перебіг хвороби, необхідність
отримання даної послуги та ін.; по-четверте, бути усвідомленим. Волевиявлення
особи є усвідомленим, якщо пацієнт отримав повну й достовірну інформацію про
стан свого здоров’я, характер та вид послуги, що входить до предмета даного
договору, можливі ризики, пов’язані з її наданням (ненаданням), бажані та
небажані наслідки, які можуть настати [7, с. 30]. © Korobtsova N. V. 2021 4 4 Теорія і практика правознавства. – 2021. – Вип. 2 (20) Таким чином, будь-яке медичне втручання повинно здійснюватися лише
за наявності на це вільної поінформованої належним чином оформленої згоди
пацієнта, що стало загальним правилом, закріпленим на міжнародному та
національному рівнях. На сьогодні у практиці європейських країн напрацьовано низку
ефективних юридичних конструкцій, за допомогою яких пацієнт може
висловити свою волю щодо майбутнього лікування. Такими конструкціями є: – довіреність на прийняття рішень з медичного обслуговування (Health
Care Of Attorney); – довіреність на прийняття рішень з медичного обслуговування (Health
Care Of Attorney); – медичний заповіт (Living Will) та «Не реанімувати!» (Do Not
Reanimate); дичний заповіт (Living Will) та «Не реанімувати!» (Do Not – медичний заповіт (Living Will) та «Не реанімувати!» (Do Not
Reanimate); – картка донора (Donor Card) [8]. Певним проявом волі пацієнта стосовно свого лікування або відмові від
нього можна вважати розпорядження пацієнта. У Конвенції про права людини та біомедицину міститься положення, яке
зобов’язує враховувати побажання пацієнта щодо медичного втручання
висловлені ним раніше, якщо на момент його проведення пацієнт знаходиться у
стані, що унеможливлює виразити його волю на це [1, ст. 9]. Отже, даний
міжнародний документ називає це «побажання, висловлені раніше». Цивільне уложення Німеччини в розділі «Правове піклування»
передбачає право пацієнта оформити «розпорядження» щодо медичних послуг
відносно нього на випадок своєї неможливості висловити волю в майбутньому
(§ 1901a) [9, с. 604]. Даний документ – «розпорядження пацієнта» –
оформлюється письмово повнолітньою особою – пацієнтом, незалежно від виду
та стадії хвороби, на випадок своєї можливої в майбутньому нездатності
висловити згоду на медичне обстеження, втручання чи лікування. Ним у
Німеччині визначається не тільки воля пацієнта на майбутнє (які медичні
процедури дозволяється вчиняти щодо його здоров’я), а й встановлюється сам
випадок, коли можливо його застосувати (наприклад, кома, вегетативний стан). В Уложенні розпорядження пацієнта розглядається як одна із складових © Коробцова Н. В., 2021 5 5 Theory and Practice of Jurisprudence. – 2021. – Issue 2 (20) ISSN 2225-6555
повноважень піклувальника. Так, за наявності такого волевиявлення,
піклувальник спочатку перевіряє, чи дійсно настав саме той випадок, який
прописаний пацієнтом у розпорядженні, а в подальшому «повинен проявити
вплив та авторитет по відношенню до виконання волі підопічного» (§ 1901а (1))
[9, с. 604]. Потім, разом із лікуючим лікарем, він визначає подальший прогноз
лікування пацієнта, який знаходиться вже у стані, визначеному в
розпорядженні. Прояв піклувальником «впливу та авторитету» є вкрай
важливим у спілкуванні з лікарем, адже він завжди повинен відстоювати та
захищати інтереси пацієнта, оскільки висловлене в розпорядженні бажання
пацієнта є основою для прийняття рішення про його майбутнє. Теорія і практика правознавства. – 2021. – Вип. 2 (20) Ситуація буде
дещо складнішою, якщо пацієнт не склав такого розпорядження, відмінив його
або настав не той випадок, який описаний у ньому. В таких випадках його
представник повинен самостійно визначити потенційну волю пацієнта на
майбутні дії медичного характеру щодо лікування чи його припинення. Рішення щодо цих питань він приймає на підставі попередніх розмов із
пацієнтом, якщо наявний стан його здоров’я вже унеможливлює це зробити,
усних та письмових висловлювань, релігійних переконань та ін. Однак будь-яке
рішення представника щодо медичного обстеження, лікування підопічного
вимагає в Німеччині дозволу суду у справах піклування, оскільки існує
обґрунтована загроза завдання тяжкої шкоди здоров’ю пацієнта або смерті
(§ 1904 (1)) [Там само, с. 606]. В українському законодавстві відповідне розпорядження не отримало © Korobtsova N. V. 2021 ISSN 2225-6555 В українському законодавстві відповідне розпорядження не отримало
свого законодавчого визначення й закріплення. Так, проєкт Закону України
«Про права пацієнтів» [6] серед визначеного в ньому переліку прав, право на
можливість оформлення відповідного розпорядження не визначає. Науковці
вбачають у даному розпорядженні елементи заповіту й тому в наукових
літературних джерелах його часто визначають як «медичний заповіт» або
«заповіт пацієнта». Однак розуміння даного правочину як заповіту відповідно до положень
українського законодавства викликає певні сумніви. Так, ст. 1233 ЦК України © Korobtsova N. V. 2021 6 6 Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555
заповітом вважає особисте розпорядження фізичної особи на випадок своєї
смерті [4]. Право на складання заповіту в особи виникає із досягненням
повноліття як і право пацієнта на оформлення розпорядження. Однак заповіт
укладається особою на «випадок своєї смерті» і відкривається в день смерті
особи або день, з якого вона оголошується померлою (ч. 2 ст. 1220 ЦК України)
[Там само]. Тобто воля заповідача стає відомою особам після його смерті або
оголошення померлим і спрямована на майбутні дії спадкоємців чи існуючы
вже в наявності обставини (дії). На відміну від цього, у своєму розпорядженні
пацієнт зазначає обставину або стан свого здоров’я, при існуванні якого його
воля, зазначена в розпорядженні, має бути виконана. При цьому не йдеться про
настання такого стану пацієнта як смерть для виконання волі пацієнта, навпаки,
у розпорядженні може бути висловлено бажання хворого піти з життя,
відмовитися від лікування тощо. Отже, сама суть «розпорядження пацієнта» не
має нічого спільного із «заповітом», окрім того, що вони є певним проявом волі
повнолітньої дієздатної особи і є нічим іншим, як одностороннім правочином. Тому, на нашу думку, більш правильно визначати цей документ саме як
«розпорядження пацієнта», яке спрямоване на подальше лікування чи відмову
від нього та адресоване або довіреній особі, або лікарям. При цьому «відмова
від лікування» не означає «припинення життя», оскільки згідно з положеннями
українського законодавства фізична особа не може бути позбавлена життя (ч. 2
ст. 281 ЦК України) [Там само], а медичним працівникам забороняється
здійснення евтаназії (ч. 7 ст. 52 Основ) [5]. Хоча новий клінічний Протокол, що
стосується екстреної медичної допомоги (догоспітального етапу),
затверджений наказом МОЗ України від 05.06.2019 р. № 1269, входить у
протиріччя зі змістом зазначених положень ЦК України та Основ. У ньому
наголошується на необхідності визнання та підтримки різноманітних способів,
якими пацієнти можуть висловити свої побажання щодо серцево-легеневої © Коробцова Н. В., 2021 ISSN 2225-6555 Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555
заповітом вважає особисте розпорядження фізичної особи на випадок своєї
смерті [4]. Право на складання заповіту в особи виникає із досягненням
повноліття як і право пацієнта на оформлення розпорядження. Однак заповіт
укладається особою на «випадок своєї смерті» і відкривається в день смерті
особи або день, з якого вона оголошується померлою (ч. 2 ст. 1220 ЦК України)
[Там само]. Тобто воля заповідача стає відомою особам після його смерті або
оголошення померлим і спрямована на майбутні дії спадкоємців чи існуючы
вже в наявності обставини (дії). На відміну від цього, у своєму розпорядженні
пацієнт зазначає обставину або стан свого здоров’я, при існуванні якого його
воля, зазначена в розпорядженні, має бути виконана. При цьому не йдеться про
настання такого стану пацієнта як смерть для виконання волі пацієнта, навпаки,
у розпорядженні може бути висловлено бажання хворого піти з життя,
відмовитися від лікування тощо. Отже, сама суть «розпорядження пацієнта» не
має нічого спільного із «заповітом», окрім того, що вони є певним проявом волі
повнолітньої дієздатної особи і є нічим іншим, як одностороннім правочином. Тому, на нашу думку, більш правильно визначати цей документ саме як
«розпорядження пацієнта», яке спрямоване на подальше лікування чи відмову
від нього та адресоване або довіреній особі, або лікарям. При цьому «відмова
від лікування» не означає «припинення життя», оскільки згідно з положеннями
українського законодавства фізична особа не може бути позбавлена життя (ч. 2
ст. 281 ЦК України) [Там само], а медичним працівникам забороняється
здійснення евтаназії (ч. 7 ст. 52 Основ) [5]. Хоча новий клінічний Протокол, що
стосується екстреної медичної допомоги (догоспітального етапу),
затверджений наказом МОЗ України від 05.06.2019 р. № 1269, входить у
протиріччя зі змістом зазначених положень ЦК України та Основ. У ньому
наголошується на необхідності визнання та підтримки різноманітних способів,
якими пацієнти можуть висловити свої побажання щодо серцево-легеневої
реанімації або прийняття рішень про закінчення життя [10, п. 4.4]. При цьому
пацієнт може мати при собі певний документ або ідентифікаційний браслет, на
якому зазначаються межі надання медичної допомоги. Даним Протоколом © Коробцова Н. В., 2021 7 Theory and Practice of Jurisprudence. – 2021. – Issue 2 (20) ISSN 2225-6555
передбачається також можливість існування попередніх розпоряджень
пацієнта, тобто документа, що описує дозволені для проведення процедури при
зазначених медичних станах, включно всіх або лише частково з наведених: що
робити при зупинці серця, чи дозволене штучне живлення, бажання бути
донором або ні, діаліз, а також інші параметри [10, п. 4.4]. ISSN 2225-6555 Таке розпорядження
може не застосовуватися при невідкладних або транзиторних медичних станах,
але якщо у пацієнта є дійсний один із наведених документів, в якому
зафіксована його воля стосовно подальшого лікування, це буде вважатися
критерієм для виключення надання йому допомоги. Аналіз змісту даного
Протоколу дозволяє дійти висновку, що його положення не можна вважати
повною мірою послідовними. Так, навіть «за наявності дійсного документа про
заборону розпочинати реанімацію або контроль дихальних шляхів, він не
виключає надання заспокійливої допомоги, включно введення знеболювальних
препаратів» [Там само]. Тому, враховуючи приписи Основ та цивільного
законодавства щодо евтаназії, якщо в розпорядженні пацієнта міститься
відмова від подальшого лікування або бажання припинити своє життя,
максимальне, що можуть зробити лікарі, виконуючи волю пацієнта, –
призначити йому паліативне лікування, яке являє собою комплекс заходів,
спрямованих на полегшення фізичних страждань цієї особи та підтримку її
близьких осіб. Право пацієнта на укладання розпорядження повинно виникати виключно
у дієздатної особи. Неповнолітні особи мають бути позбавлені можливості
складати відповідний документ, оскільки рівень їх розумових здібностей не
завжди дозволяє їм реально оцінювати ситуацію, стан свого здоров’я, перебіг
хвороби та наслідки, які можуть настати в результаті такого правочину. Вважаємо недопустимим і можливість наділяти батьків, близьких осіб, опікунів
та піклувальників пацієнта правом вирішувати питання подальшого його
лікування замість нього. Оскільки в такому випадку це буде прояв волі
сторонніх осіб, який не завжди збігається з волею пацієнта, дізнатися яку, як
правило, буває вже або складно, або взагалі неможливо при конкретних © Korobtsova N. V. 2021 8 Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555
обставинах. Теорія і практика правознавства. – 2021. – Вип. 2 (20) Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555 обставинах. обставинах. Цікавою є характеристика даного документа, надана А. Литвиненко
(A. Lytvynenko), який автор називає «заповітом пацієнта») [11]: 1. «Заповіт пацієнта» повинна укладати винятково дієздатна особа. 2. Оскарження його є можливим, аналогічно до звичайного заповіту, за
позовом заінтересованої особи, не очікуючи при цьому смерті чи погіршення
стану здоров’я пацієнта. 3. «Заповіт пацієнта» повинен, як і звичайний заповіт, бути завірений
нотаріусом. 4. Альтернативою його є укладення пацієнтом довіреності про
уповноваження дієздатного законного представника на ухвалення рішення
замість нього. 5. «Заповіт пацієнта» в окремих випадках може бути складений не тільки
в письмовій формі, а й у будь-якій іншій, яку суд вважатиме легітимною. 6. В окремих випадках зміст «Заповіту пацієнта» може доповнюватися
чи змінюватися, на зразок кодицилю. 7. Строк дії «Заповіту пацієнта» доволі обмежений. 8. Він є винятково добровільним. 9. Ухвалення рішення стосовно припинення життєзабезпечувального
лікування пацієнта вимагає дозволу суду, незалежно від того, є «заповіт» чи
його немає. 10. Розглядаючи справу про припинення життєзабезпечувального
лікування пацієнта, суд для ухвалення рішення має застосовувати «негативну
презумпцію». 11. Суди для надання експертної оцінки стану здоров’я пацієнта не
тільки призначають судово-психіатричну експертизу, а й залучають фахівців з
відповідних наукових установ. 12. «Заповіт пацієнта» вступає в силу лише за умови стану здоров’я
пацієнта, передбаченого у документі. 13. Право пацієнта на припинення життєзабезпечувального лікування не © Коробцова Н. В., 2021 © Коробцова Н. В., 2021
9 9 Theory and Practice of Jurisprudence. – 2021. – Issue 2 (20) ISSN 2225-6555 Theory and Practice of Jurisprudence. – 2021. – Issue 2 (20) Theory and Practice of Jurisprudence. – 2021. – Issue 2 (20) ISSN 2225-6555 можна трактувати як похідне від його права на відмову від медичних втручань,
якщо законодавство чи прецедентна практика не передбачають іншого. У свою чергу Г. Миронова (G. Myronova) вважає необхідним підписання
цього документа двома свідками, які не є родичами розпорядника, не пов’язані
з лікувальним закладом, що обслуговує хворого, і не претендують на спадок [8]. Можливість реалізації волі пацієнта у будь-якій з існуючих у світі
конструкцій вимагає насамперед її законодавчого визнання, а також
встановлення механізму реалізації відповідної можливості, яку доцільно
розглядати як відповідне право пацієнта. На жаль, в Україні сьогодні
здійснюються спроби лише тільки до оформлення волі особи щодо донорства
органів (вилучення та пересадку органів і тканих після смерті). Волевиявлення
особи щодо інших обставин (у тому числі й щодо можливості подальшого
лікування чи відмови від нього, припинення життя) в українському
законодавстві не врегульовано й не має однозначного розуміння та підтримки в
українському суспільстві. Висновки. Розпочатий в Україні процес рекодифікації цивільного
законодавства, розширення переліку об’єктів цивільних прав, переосмислення
підходів до правочинів та форми їх укладення, а також можлива легалізація в
Україні пасивної евтаназії та асистованого самогубства [12, с. 17] свідчить про
нагальну потребу законодавчого визначення та врегулювання правового
інституту «розпорядження пацієнта» й можливу його появу в оновленому
законодавстві. Список літератури р
ур
1. Конвенция о защите прав и достоинства человека в связи с применением
достижений биологии и медицины: Конвенция о правах человека и биомедицине :
международный документ. Овьедо, 04.04.1997 г. URL: https://rm.coe.int/168007d004 (дата
обращения: 11.06.2021). р
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2. Конституція України : офіц. текст. Київ : КМ, 2013. 96 с. 3. Мироненко О. Білль про права. Політична енциклопедія / редкол. : Ю. Левенець
(голова), Ю. Шаповал (заст. голови). Київ : Парлам. вид-во, 2011. С. 62. 4. Цивільний кодекс України : Закон України від 16.01.2003 р. № 435-IV. URL:
https://zakon.rada.gov.ua/laws/show/435-15#Text (дата звернення: 18.07.2021). 5. Основи законодавства України про охорону здоров’я : Закон України від
19.11.1992 р. № 2801-XII. URL: https://zakon.rada.gov.ua/laws/show/2801-12#Text (дата
звернення: 12.06.2021). © Korobtsova N. V. 2021 10 Теорія і практика правознавства. – 2021. – Вип. 2 (20) наук, доц., доцент кафедры гражданского права № 1,
Национальный юридический университет имени Ярослава Мудрого, Украина, г. Харьков. e-mail: kornv@ukr.net ; ORCID 0000-0002-9997-1485 Коробцова Н. В., канд. юрид. наук, доц., доцент кафедры гражданского права № 1,
Национальный юридический университет имени Ярослава Мудрого, Украина, г. Харьков. e-mail: kornv@ukr.net ; ORCID 0000-0002-9997-1485 Korobtsova N. V., PhD in Law, Associate Professor, Associate Professor of Department of
Civil Law № 1, Yaroslav Mudryi National Law University, Ukraine, Kharkiv.
e-mail: kornv@ukr.net ; ORCID 0000-0002-9997-1485 Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555 ISSN 2225-6555 6. Про захист прав пацієнтів : проєкт Закону України від 06.12.2007 р. № 1132. URL:
http://search.ligazakon.ua/l_doc2.nsf/link1/JF0VG00A.html (дата звернення: 12.07.2021). 6. Про захист прав пацієнтів : проєкт Закону України від 06.12.2007 р. № 1132. URL:
http://search.ligazakon.ua/l_doc2.nsf/link1/JF0VG00A.html (дата звернення: 12.07.2021). 7. Коробцова Н. В. Вади волі при укладенні договорів про надання медичних послуг. Вчені записки Таврійського національного університету імені В. І. Вернадського. 2021. № 1. С. 29–33. 7. Коробцова Н. В. Вади волі при укладенні договорів про надання медичних послуг. Вчені записки Таврійського національного університету імені В. І. Вернадського. 2021. № 1. С. 29–33. 8. Миронова Г. Пацієнт із медичним заповітом: як поводитися лікарю? URL:
https://www.vz.kiev.ua/paciyent-iz-medichnim-zapovitom-yak-povoditisya-likaryu/
(дата
звернення: 22.07.2021). 8. Миронова Г. Пацієнт із медичним заповітом: як поводитися лікарю? URL:
https://www.vz.kiev.ua/paciyent-iz-medichnim-zapovitom-yak-povoditisya-likaryu/
(дата
звернення: 22.07.2021). р
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9. Гражданское уложение Германии: Вводный закон к Гражданскому уложению /
пер. с нем. В. Бергманн ; науч. ред. Т.Ф. Яковлева. 4-е изд., перераб. Москва : Инфотропик
Медиа, 2015. 888 с. (Серия «Германские и европейские законы»; кн. 1). р
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9. Гражданское уложение Германии: Вводный закон к Гражданскому уложению /
пер. с нем. В. Бергманн ; науч. ред. Т.Ф. Яковлева. 4-е изд., перераб. Москва : Инфотропик
Медиа, 2015. 888 с. (Серия «Германские и европейские законы»; кн. 1). 10. Про затвердження та впровадження медико-технологічних документів зі
стандартизації екстреної медичної допомоги : наказ Міністерства охорони здоров’я України
від 05.06.2019 р. № 1269. URL: https://moz.gov.ua/article/ministry-mandates/nakaz-moz-ukraini- 10. Про затвердження та впровадження медико-технологічних документів зі
стандартизації екстреної медичної допомоги : наказ Міністерства охорони здоров’я України
від 05.06.2019 р. № 1269. URL: https://moz.gov.ua/article/ministry-mandates/nakaz-moz-ukraini-
id 05062019 1269
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zi-standartizacii-ekstrenoi-medichnoi-dopomogi (дата звернення: 19.07.2021). 11. Литвиненко А. Правова характеристика «Заповіту пацієнта»: доктрина і судова
практика. URL: https://ecpl.com.ua/news/pravova-kharakterystyka-zapovitu-patsiienta-doktryna-i-
sudova-praktyka/ (дата звернення: 19.07.2021). 11. Литвиненко А. Правова характеристика «Заповіту пацієнта»: доктрина і судова
практика. URL: https://ecpl.com.ua/news/pravova-kharakterystyka-zapovitu-patsiienta-doktryna-i-
sudova-praktyka/ (дата звернення: 19.07.2021). p
y
(
р
)
12. Концепція оновлення цивільного кодексу України. Київ : Вид. дім «АртЕк», 2020. 128 с. p
y
(
р
)
12. Концепція оновлення цивільного кодексу України. Київ : Вид. дім «АртЕк», 2020. 128 с. Коробцова Н. В., канд. юрид. наук, доц., доцент кафедры гражданского права № 1,
Национальный юридический университет имени Ярослава Мудрого, Украина, г. Харьков. e-mail: kornv@ukr.net ; ORCID 0000-0002-9997-1485 Коробцова Н. В., канд. юрид. Распоряжение пациента как отдельное проявление его воли р
р
В статье анализируются вопросы волеизъявления пациента в медицинских
правоотношениях, доказывается, что именно благодаря проявлению воли относительно
предложенного лечения (предоставление согласия или отказа от него) пациент является
полноценным активным участником этих отношений. Однако неспособность его к
волеизъявлению, временная или необратимая, вызванная развитием болезни, особенностью
ее протекания может стать препятствием к определению его реального желания
относительно будущего лечения. Во избежание этого в законодательстве ряда стран мира существует определенный
правовой институт, с помощью которого осуществляется возможность
заблаговременного планирования своего лечения, отказа от него, в случае неспособности в
будущем это сделать. В работе проанализировано содержание этого института,
рассмотрены точки зрения ученых относительно него, проведен сравнительный анализ с
юридической конструкцией «завещания» и сделан вывод о наличии существенных
отличительных особенностей между этими конструкциями, что делает невозможным, с
точки зрения автора, называть данное волеизъявление «завещанием». Предлагается
рассматривать такое волеизъявление как «распоряжение пациента», которое
оформляется письменно совершеннолетним лицом – пациентом независимо от вида и
стадии болезни на случай своей возможной в будущем неспособности выразить согласие на
медицинское обследование, вмешательство или лечение. Ключевые слова: распоряжение пациента; завещание пациента; медицинское
завещание; права пациента; волеизъявление; медицинские правоотношения. Korobtsova N. V., PhD in Law, Associate Professor, Associate Professor of Department of
Civil Law № 1, Yaroslav Mudryi National Law University, Ukraine, Kharkiv. e-mail: kornv@ukr.net ; ORCID 0000-0002-9997-1485 © Коробцова Н. В., 2021 11 The patient's order as a separate manifestation of his will Keywords: patient instructions; the patient's will; medical will; patient rights; expression of
will; medical legal relations. The patient's order as a separate manifestation of his will p
p
The article analyzes the issues of the patient's will in medical relations, it is proved that it is
due to the expression of will to the proposed treatment (consent or refusal) that the patient is a full
active participant in this relationship. However, his inability to express his will, temporary or
irreversible, caused by the development of the disease, the peculiarity of its course may be an
obstacle to determining his real desire for future treatment, medical intervention and jeopardize the
violation or inability to exercise the patient's right to consent or refuse medical intervention. To
avoid this, there is a certain legal institution in the legislation of a number of countries around the
world, through which it is possible to plan your treatment in advance, to refuse it, in case of
inability to do so in the future. In some legal systems, this institution has different names – "wishes made earlier", "medical
will", "patient's will", "power of attorney to make decisions on health care", "patient orders" and so
on. The paper analyzes the content of this institute, considers the views of scholars on it, made a
comparative analysis with the legal construction of the "testament" and concluded that there are
significant differences between these constructions, which makes it impossible, from the author's
point of view, to call this will "testament". It is proposed to consider such a will as one of the
patient's rights – "patient order", which is made in writing by an adult – the patient, regardless of
the type and stage of the disease in case of possible future inability to consent to medical
examination, intervention or treatment. The patient has at his disposal not only his will for the
future (list of medical procedures that are allowed to be performed in relation to his health, which
are not), but also the case when it can be used (for example, coma, autonomic state). It is
impossible to conclude it through a representative, because in this case the will of the patient is
unknown. This order is executed by proxies (relatives, close persons, representatives, doctors, etc.). Despite the fact that in Ukraine today this legal institution is absent, the main directions of
recoding of civil legislation indicate the possibility of its appearance in the updated legislation. Keywords: patient instructions; the patient's will; medical will; patient rights; expression of
will; medical legal relations. Theory and Practice of Jurisprudence. – 2021. – Issue 2 (20) ISSN 2225-6555 ISSN 2225-6555 References 1. Konvencija o zashhite prav i dostoinstva cheloveka v svjazi s primeneniem dostizhenij
biologii i mediciny: Konvencija o pravah cheloveka i biomedicine: мezhdunarodnyj document. Ov'edo, 04.04.1997. (1997). URL: https://rm.coe.int/168007d004 [in Russian]. 2. Konstytutsiia Ukrainy: ofits. tekst. (2013). Kyiv: KM. 3. Myronenko, O. (2011). Bill pro prava. Politychna entsyklopediia. Yu. Levenets, Yu. Shapoval (Eds.). Kyiv: Parlamentske vydavnytstvo, 62 [in Ukrainian]. 4. Cyvilnyj kodeks Ukrainy: Zakon Ukrainy vid 16.01.2003 r. № 435-IV. (2003). URL:
https://zakon.rada.gov.ua/laws/show/435-15#Text. 5. Osnovy zakonodavstva Ukrainy pro ohoronu zdorovja: Zakon Ukrai'ny vid 19.11.1992 r. № 2801-XII. (1992). URL: https://zakon.rada.gov.ua/laws/show/2801-12#Text. 6. Pro zahyst prav pacijentiv: proekt Zakonu Ukrainy vid 06.12.2007 r. № 1132. (2007). URL: http://search.ligazakon.ua/l_doc2.nsf/link1/JF0VG00A.html. 7. Korobcova, N.V. (2021). Vady voli pry ukladenni dogovoriv pro nadannja medychnyh
poslug. Vcheni zapysky Tavrijskogo nacionalnogo universytetu imeni V.I. Vernadskogo, 1, 29–33
[in Ukrainian]. 8. Myronova, G. Pacijent iz medychnym zapovitom: jak povodytysja likarju? URL:
https://www.vz.kiev.ua/paciyent-iz-medichnim-zapovitom-yak-povoditisya-likaryu/ [in Ukrainian]. p
p
y
p
y
p
y
y
9. Grazhdanskoe ulozhenie Germanii: Vvodnyj zakon k Grazhdanskomu ulozheniju. (2015). V. Bergmann (Transl.); T.F. Jakovleva (Ed.). Moscow: Infotropik Media [in Russian]. 9. Grazhdanskoe ulozhenie Germanii: Vvodnyj zakon k Grazhdanskomu ulozheniju. (2015). V. Bergmann (Transl.); T.F. Jakovleva (Ed.). Moscow: Infotropik Media [in Russian]. 10. Pro zatverdzhennja ta vprovadzhennja medyko-tehnologichnyh dokumentiv zi
standartyzacii ekstrenoi medychnoi dopomogy: nakaz Ministerstva ohorony zdorovja Ukrainy vid
05.06.2019 r. № 1269. (2019). URL: https://moz.gov.ua/article/ministry-mandates/nakaz-moz-
ukraini-vid-05062019--1269-pro-zatverdzhennja-ta-vprovadzhennja-mediko-tehnologichnih- © Korobtsova N. V. 2021 12 Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555 Теорія і практика правознавства. – 2021. – Вип. 2 (20) Теорія і практика правознавства. – 2021. – Вип. 2 (20) ISSN 2225-6555 ISSN 2225-6555 dokumentiv-zi-standartizacii-ekstrenoi-medichnoi-dopomogi [in Ukrainian]. dokumentiv-zi-standartizacii-ekstrenoi-medichnoi-dopomogi [in Ukrainian]. 11. Lytvynenko, A. Pravova harakterystyka «Zapovitu pacijenta»: doktryna i sudova
praktyka. URL: https://ecpl.com.ua/news/pravova-kharakterystyka-zapovitu-patsiienta-doktryna-i-
sudova-praktyka/ [in Ukrainian]. 11. Lytvynenko, A. Pravova harakterystyka «Zapovitu pacijenta»: doktryna i sudova
praktyka. URL: https://ecpl.com.ua/news/pravova-kharakterystyka-zapovitu-patsiienta-doktryna-i-
sudova-praktyka/ [in Ukrainian]. 12. Koncepcija onovlennja cyvilnogo kodeksu Ukrainy. (2020). Kyiv: Vyd-j dim «ArtEk»
[in Ukrainian]. 12. Koncepcija onovlennja cyvilnogo kodeksu Ukrainy. (2020). Kyiv: Vyd-j dim «ArtEk»
[in Ukrainian]. Надійшла до редколегії 30.07.2021 р. © Коробцова Н. В., 2021 13
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Government National Cancer Institute Qeios ID: 5Y8IRL · https://doi.org/10.32388/5Y8IRL Open Peer Review on Qeios Source National Cancer Institute. Government. NCI Thesaurus. Code C78315. The political organization by which a state or nation is ruled. Qeios ID: 5Y8IRL · https://doi.org/10.32388/5Y8IRL 1/1
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Chemical Composition and Hepatoprotective Activity of Saponaria officinalis on Paracetamol-Induced Liver Toxicity in Rats
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ABSTRACT
B
k
d ABSTRACT
Background: The present day life style causing different illness including liver diseases and
different health complications. So, there is a need to identify new chemical entities with more
efficiency in the treatment of diseases and less side effects. There were many reports in
recent times, about identifying new drugs from different medicinal plants and also precursors
for synthesis new bioactive molecules for treating various diseases. Objective: The present
study was carried out on root parts (rhizomes) of S. officinalis for phytochemical analysis
and hepatoprotective activity on Paracetamol-induced liver toxicity. Materials and Methods:
The phytochemical analysis was carried out to know biological active compounds in different
extracts of S. officinalis using standard procedures and quantified the total alkaloid and
phenolic contents. Hepatoprotective activity of the S. officinalis extracts were carried out
by using Paracetamol-induced hepatotoxicity in rats. Results: The phytochemical analysis of
S. officinalis roots’ extracts showed presence of sterols, terpenoids, glycosides, carbohy
drates, proteins, flavanoids, alkaloids, phenols, tannins and absence of saponins and oils. The
methanolic extract showed more phenolic and alkaloid contents on their quantification. The
S. officinalis roots extracts are found to be safe at 2000 mg/kg b. w. in acute toxicity study and
showed dose dependent percentage protection on liver toxicity. Methanol extract showed
more activity at 500mg/kg b. w. and is comparable with standard drug Liv 52 on altered liver
biomarker enzymes AST (SGOT), ALT (SGPT), ALP, total bilirubin and total protein with
percentage protection 56.17%, 54.53%, 61.55% 57.29% and 53.66%. Conclusion: The present
study results indicates that phytochemical constituent’s diversity in S. officinalis and those
extracts possess hepatoprotective activity. Further studies are needed and should involve the
isolation of pure, biologically active compounds.i Cite this article: Talluri MR, Gummadi VP, Battu GR. Chemical Composition and Hepatoprotective
Activity of Saponaria officinalis on Paracetamol-induced Liver Toxicity in Rats. Pharmacog J.
2018;10(6)Suppl:s129-s134. Pharmacogn J. 2018; 10(6)Suppl: s129-s134 Pharmacogn J. 2018; 10(6)Suppl: s129-s134 Pharmacogn J. 2018; 10(6)Suppl: s129-s134
A Multifaceted Journal in the field of Natural Products and Pharmacognosy
www.phcogj.com | www.journalonweb.com/pj | www.phcog.net Original Article Original Article Mallikarjuna Rao Talluri1,
Veda Priya Gummadi2,*,
Ganga Rao Battu2 1AnaCipher Clinical Research
Organization, Ramanthapur,
Hyderabad, Telangana-500013, INDIA. 2AU College of Pharmaceutical Science,
Andhra University, Visakhapatnam,
Andhra Pradesh-530003, INDIA. p
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Key words: Saponaria officinalis, Roots, Paracetamol, Liver, Toxicity. Key words: Saponaria officinalis, Roots, Paracetamol, Liver, Toxicity. History Article Available online
http://www.phcogj.com/v10/i6s Article Available online
http://www.phcogj.com/v10/i6s Mr. Veda Priya Gummadi Liver is one of the most important organ in the human
body, it performs fundamental metabolisms in the
body those include homeostasis, carbohydrates, proteins,
lipids metabolisms and mainly detoxification.1-2 It is
important to maintain healthy liver for good health
conditions. The present day life style causing different
illness including liver diseases.3 The drugs using for
the present day diseases getting resistant to them and
may cause side effects which can lead to iatrogenic
diseases. The drugs mainly inducing the vital organs
damage in the body like kidney, liver etc.4-6 The drug-
induced liver diseases are more common compared
to natural liver diseases around the world.7-8 The liver
diseases like cirrhosis, cancers, alcoholic liver diseases
and non-alcoholic liver diseases are main causes for
the liver failure and causing mortality around the
world.9 The development in modern medicine devel
oped many medication for treatment many diseases
including liver diseases.10-12 But, these treatments are
not satisfactory and as said above on the long-term
usage of drugs causing different side effects.13 So, there is a need to identify new chemical entities with more
efficiency in the treatment of diseases and less side
effects. Research Scholar, A.U College of
Pharmaceutical Sciences,
Andhra University, Visakhapatnam,
Andhra Pradesh-530 003, INDIA. Traditional medicine is mainly based herbal treatments,
which is based on knowledge, skills and practices
based on the theories, beliefs and experiences in
different indigenous cultures to maintain health. Herbal products that contain parts of plants or other
plant materials as active ingredients.14 There are
very little literature availability on plant materials as
active ingredients in herbal medicines i.e. Ayurveda
and Unani medicines and less scientific data regarding
the identity and effectiveness of these herbal products. In current times, several researchers identifying new
drugs and provide significant formulations from
different medicinal plants and those drugs are also
precursors for synthesis new bioactive molecules for
treating various diseases including liver diseases.15-18
However, there were ample of medicinal plants
available on world without their identification and
scientifically not proven about their medicinal uses. Chemical Composition and Hepatoprotective Activity of Saponaria
officinalis on Paracetamol-Induced Liver Toxicity in Rats Mallikarjuna Rao Talluri1, Veda Priya Gummadi2,*, Ganga Rao Battu2 Preparation of extractsh The plant materials Saponaria officinalis (Voucher number: 1221) were
collected from Tirupati region, Andhra Pradesh and authenticated by the
taxonomist Dr. K. Madhava Chetty, Depart of Botany, Sri Venkateswara
University. Freshly collected roots of plant material were shade dried
and then material was milled to obtain a coarse powder. The powdered
material was successively extracted with ethyl acetate, chloroform and
methanol using in a Soxhlet apparatus. Finally, collected solutions were
concentrated to dryness under vacuum by using Rota-vapor and stored
in desiccators. Copyright © 2018 Phcog.Net. This is an open-
access article distributed under the terms
of the Creative Commons Attribution 4.0
International license. S129 Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 Talluri, et al.: Hepatoprotective activity of Saponaria officinalis Talluri, et al.: Hepatoprotective activity of Saponaria officinalis libitum during experimental period. Food was withdrawn 12h before the
terminating experiment and water was allowed ad libitum. The protocols
were approved by Institutional Animal Ethics Committee and the lab
was approved by CPCSEA, Government of India (Regd. No. 516/01/A/
CPCSEA). Saponaria officinalis is one of such medicinal plant commonly called as
soapwort plant belongs to Caryophyllaceae family. The different parts
of S. officinalis has been used in traditional medicine, roots as blood
purifier, diuretic, diaphoretic; roots and leaves for scrofula and skin
diseases; sap for scabies, hepatic eruptions, to increase bile flow.19 But,
there were no scientific reports on hepatoprotective activity of root parts
of S. officinalis. In this regards, the present work carried out to evaluate
the phytochemical analysis and hepatoprotective activity of S. officinalis. Phytochemical analysis Qualitative phytochemical screening of S. officinalis extracts revealed
the presence of different phytochemical constituents like steroids,
terpenoids, flavonoids, alkaloids, glycosides, phenols, tannins and
carbohydrates. All the extracts revealed the presence of phenols, alkaloids,
steroids and glycosides, tannins and gave negative results to amino acids,
quinones and oils. The ethyl acetate and methanol extracts revealed the
presence of saponins, flavanoids but the chloroform extract gave negative
results. The chloroform and methanol extracts revealed the presence of
carbohydrates, but ethyl acetate extract gave negative results (Table 1). The quantified phenolic content of S. officinalis extracts was ranging
from 17.52±0.46 to 22.51±1.05 (mg/gm). The methanol extract has more
phenolic content 35.47±0.42 (mg/gm) than other extracts and alkaloid
content was ranging from 18.43±0.74 to 22.64±0.78 (mg/gm). The ethyl
acetate extract has more alkaloid content 22.64±0.78 (mg/gm) than
other extracts (Table 2). Phytochemical analysis Phytochemical studies were carried out for different extracts of
Saponaria officinalis to identify the presence of different phytochemical
constituents using standard procedures.20-23 Statistical analysis All the results were expressed as mean ± SEM and analysed by using
Two-way ANOVA followed by Dunnet’s multiple comparision test. All
groups were compared with Liv 52 group. ***p<0.001; **p<0.01; *p<0.05;
ns= Non significance. Chemicals and Drugs All the chemical used in the present study were analytical grade. Diagnostic kits for serum parameter analysis were purchased from
Span diagnostics Ltd, Gujarat, India. Paracetamol tablets and Live 52 was
purchased from local medical shop, Visakhapatnam, A.P, India. Total phenolic content estimation Total phenolic content was estimated using folin-ciocalteau reagent. Briefly, folin-ciocalteau colorimetry is based on a chemical reduction of
the reagent, a mixture of tungsten and molybdenum oxides. The products
of the metal oxide reduction have a blue absorption with a maximum
at 765nm.The intensity of the light absorption at that wave length is
proportional to the concentration of phenols. By using standard Gallic
acid calibration curve, measure the concentration of phenolic content in
Gallic acid total equivalents using unit’s mg/gm. (GAE).22,24 Evaluation of hepatoprotective activity using
Paracetamol-induced hepatotoxicityfi Hepatoprotective activity of the S. officinalis extracts were carried out
by using Paracetamol-induced hepatotoxicity in rats.27 The animals for
the activity, selected animals were divided into twelve groups (XII; n=6). Group I was served as control treated with only Saline (2mL/kg b.w.,
per orally) for one week. Group II was administered with saline solu
tion (2mL/kg b.w., per orally) for 7 days. The animals of group III were
administered with Liv 52 (25mg/kg per day, p. o.) for 7 days. Group IV
to XII animals were administered with ethyl acetate, chloroform and
methanol extracts of S. officinalis (125, 250 and 500 mg/kg respectively)
for 7 days. On 5th day for all groups excluding group I were treated with
paracetamol (200 mg/kg b. w., s.c.). On 7th day after dosage of 2h, the
groups were anaesthetized by chloroform and blood was collected from
retro-orbital plexus. The collected samples were centrifuged using
centrifuge at 2400rpm for 15 min and then serum was clearly separated. The collected serum was analyzed for biochemical parameters i.e. AST
(SGOT), ALT (SGPT), ALP, total bilirubin and total protein levels using
Autoanalyzer.27 Acute toxicity studies The acute toxicity study was conducted for extracts of S. officinalis
extracts as per OECD guidelines 420 and regulations.26 The albino rats of
single sex, were selected and divided into three groups (n=6). They were
maintained for one week before the experiment, under room temperature
and allowed free access to water and diet. The animals were subjected for
acute toxicity study using each extract at a dose of 2000 mg/kg orally in
3 groups at regular intervals of time for 24h. During this period, animals
were under examination to note different conditions like skin changes,
morbidity, aggressiveness, oral secretions, sensitivity to sound and pain,
respiratory movements and finally their mortality. Total alkaloid content estimationh The plant extract (1mg/mL) was dissolved in 2 N HCl and then filtered. The pH of phosphate buffer solution was adjusted to neutral with 0.1 N
NaOH. One ml of this solution was transferred to a separating funnel
and then 5 ml of Bromocresol green (BCG) solution along with 5 ml of
phosphate buffer were added. The mixture was shaken and the complex
formed was extracted with chloroform by vigorous shaking. The extracts
were collected in a 10 mL volumetric flask and diluted to volume with
chloroform. The absorbance of the complex in chloroform was measured
at 470 nm. All experiments were performed thrice; the results were
averaged and reported in the form of Mean ± S.E.M.22,25 Talluri, et al.: Hepatoprotective activity of Saponaria officinalis Table 1: Nature of phytoconstituents in different extracts of Saponaria
officinalis. + = Present, – = Absent Table 2: Total phenolic and alkaloid contents (mg/gm) of Saponaria
officinalis extracts. Name of the extract
Total Phenolic
content (mg/gm)
(Mean ± SEM)
Total alkaloidal content
(mg/gm)
(Mean ± SEM)
Ethyl acetate
21.59±0.17
22.64±0.78
Chloroform
17.52±0.46
18.43±0.74
Methanol
22.51±1.05
20.58±0.59 Table 2: Total phenolic and alkaloid contents (mg/gm) of Saponaria
officinalis extracts. Acute toxicity studieshfi The S. officinalis root extracts were found to be safe after the toxicity test
dose of 2000 mg/kg administration, because there were no sign of
mortality and no behavioral changes were observed in rats. n=6 and Mean± SD Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 Talluri, et al.: Hepatoprotective activity of Saponaria officinalis Talluri, et al.: Hepatoprotective activity of Saponaria officinalis Table 1: Nature of phytoconstituents in different extracts of Saponaria
officinalis. Name of the
Phytochemicals
Extracts of Saponaria officinalis
Chloroform
Ethyl Acetate
Methanol
Phytosterols
+
+
+
Terpenoids
+
+
+
Glycosides
+
+
+
Saponins
-
+
+
Flavonoids
-
+
+
Tannins
+
+
+
Carbohydrates
+
-
+
Alkaloids
+
+
+
Amino acids
-
-
-
Oils
-
-
-
Quinones
-
-
-
Phenols
+
+
+
+ = Present, – = Absent Table 3: Enzymes Levels of groups I to XII animals due to the effect of
different extracts of S. officinalis. Name of the
Drug
Name of Enzymes
AST
(U/L)
ALT
(U/L)
ALP
(U/L)
T. bil
(mg/dl)
T. ptn
(gm/dl)
Control
89.17±
1.11
52.33±
1.73
183.50±
0.85
0.25±
0.01
6.93±
0.06
Paracetamol
332.17±
2.18
173±
5.22
548.67±
21.60
2.17±
0.06
4.20±
0.04
Liv 52 25mg//
kg b. w. 101.83±
1.72
59.83±
1.11
195.67±
1.56
0.29±
0.01
6.67±
0.08
SOEAE 125mg/
kg b.w
308.00
±0.82
150.67
±0.76
498.67
±1.36
1.98
±0.05
4.43
±0.08
SOEAE 250mg/
kg b.w
280.67
±1.94
137.83
±0.87
445.00
±1.46
1.77
±0.06
4.73
±0.04
SOEAE 500mg/
kg b.w
230.67
±1.09
118.50
±0.96
370.67
±1.09
1.43
±0.06
5.30
±0.09
SOCE
125mg/kg b.w
292.83
±2.34
142.50
±1.06
470.33
±1.45
1.87
±0.07
4.60
±0.05
SOCE 250mg/
kg b.w
254.83
±2.26
125.83
±1.89
409.50
±3.01
1.57
±0.06
5.03
±0.06
SOCE 500mg/
kg b.w
195.67
±1.17
100.83
±2.10
316.67
±3.12
1.07
±0.04
5.67
±0.07
SOME
125 mg/kg b.w
277.17
±2.51
137.67
±0.49
445.00
±1.44
1.77
±0.06
4.77
±0.06
SOME 250mg/
kg b.w
234.00
±2.42
116.50
±1.96
375.83
±1.78
1.37
±0.06
5.27
±0.04
SOME 500mg/
kg b.w
170.17
±0.91
94.33
±1.78
301.50
±1.67
0.93
±0.07
5.87
±0.04
n=6 and Mean± SD Table 1: Nature of phytoconstituents in different extracts of Saponaria
officinalis. Name of the
Phytochemicals
Extracts of Saponaria officinalis
Chloroform
Ethyl Acetate
Methanol
Phytosterols
+
+
+
Terpenoids
+
+
+
Glycosides
+
+
+
Saponins
-
+
+
Flavonoids
-
+
+
Tannins
+
+
+
Carbohydrates
+
-
+
Alkaloids
+
+
+
Amino acids
-
-
-
Oils
-
-
-
Quinones
-
-
-
Phenols
+
+
+
+ = Present, – = Absent Table 3: Enzymes Levels of groups I to XII animals due to the effect of
different extracts of S. officinalis. Selection of Animals Albino rats of either sex weighing between 180-250 gm were obtained
from M/s. Mahaveer Enterprises, Hyderabad, India. The animals were
maintained under controlled environmental conditions (temperature of
22 ±1°C) with an alternating 12h light-dark cycle and relative humidity
of 60 ± 5%), one week before the start and also during the experimental
period. They were fed up with standard laboratory diet and water ad S130 Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 Talluri, et al.: Hepatoprotective activity of Saponaria officinalis Talluri, et al.: Hepatoprotective activity of Saponaria officinalis The phytochemical analysis of different extracts of S. officinalis root part
showed presence of bioactive compounds in them and variation in the
presence of them in different extracts. All extracts gave positive results
for terpenoids, alkaloids, steroids, glycosides and phenols and gave nega
tive results for amino acids, quinones and oils. Methanol and chloroform
extracts gave positive results for saponins and flavanoids. Carbohydrates
gave positive results for chloroform and methanol extracts, absent in
ethyl acetate extract. There were some earlier reports about the presence
of biological active compounds in S. officinalis, quillaic acid from
rhizome (root) part,39 triterpenoid saponins.40 Figure 2: Percentage protection produced by different extracts of
S. officinalis at a dose of 250mg/kg. Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(n=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. The extracts were analyzed for their hepatoprotective activity because
recently drugs induced liver damage causing one of the major mortality
problem around the world.41 In the present study, the extracts of
S. officinalis showed concentration dependent hepatoprotective activity. As the concentration of extracts increasing the protective nature of
them was also increased in restoration of the liver biomarker enzymes
like SGOT, SGPT, ALP, total bilrubin and total protein. As overall the
methanolic extract showed better activity compared to other two
extracts (Table 3) but individually different extracts showed variation on
the enzymes level protection at higher concentrations. Ethyl acetate and
chloroform extracts have more percentage protection on ALP levels but
methanolic extracts have more protection on AST levels (Figure 3). The
protective nature of S. officinalis extracts was comparable with standard
drug Liv 52. In our earlier studies, we have reported about the antioxi
dant and antibacterial activities of these extracts.42-43 As in those studies,
the methanol extract showed more antioxidant and antibacterial activity
as hepatoprotective activity in the present study. Figure 2: Percentage protection produced by different extracts of
S. officinalis at a dose of 250mg/kg. Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(n=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. Figure 2: Percentage protection produced by different extracts of
S. officinalis at a dose of 250mg/kg. Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. CONCLUSION The present research reveal that S. officinalis roots contains different
phytochemical constituents and those extracts possess hepatoprotective
activity and in our earlier research work we found these also possess
antibacterial and antioxidant activities. The liver damage will occur
mainly due to the over production of free radical and S. officinalis roots
showed free radicals inhibition. So, it may conclude that common
biological active components of them were responsible for their activities
either individually or synergistically. So, further work is needed to isolate
and characterize the responsible bioactive molecules from these extracts
as well as from different medicinal plants for development of effective
and safe medicines. and total protein levels were 16.19%, 15.47%, 17.18%, 15.63% and
14.63%, 31.82%, 31.09%, 34.74% 31.25% and 30.49%, 56.17%, 54.53%,
61.55% 57.29% and 53.66% respectively. The percentage protection produced by the methanol extract of
S. officinalis (SOME) on AST (SGOT), ALT (SGPT), ALP, total bilirubin
and total protein levels were 22.63%, 20.00%, 24.49%, 20.83% and
20.73%, 40.40%, 39.84%, 44.47%, 41.67% and 39.02%, 66.67%, 60.63%,
65.93%, 64.24% and 60.98% respectively. DISCUSSION The studies on natural products and isolation of new drugs from them
is getting more attention because of currently using drugs are becoming
inactive and micro-organisms becoming resistant to them.28-31 As
mentioned in introduction, S. officinalis one of the traditional medicinal
plant with less scientific literature, so the present work carried out on
S. officinalis root part for their phytochemical analysis using different
extracts and evaluation of hepatoprotective activity. Several studies have
been reporting on pharmacological and biological activities of medicinal
plants like anti-microbial,32-33 antioxidant and hepatoprotective,34-37 anti-
inflammatory,38 and cytotoxicity etc. Talluri, et al.: Hepatoprotective activity of Saponaria officinalis All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(n=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. Figure 3: Percentage protection produced by different extracts of
S. officinalis at a dose of 500mg/kg. Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(N=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. In recent decades, different new drugs have been isolating from natural
products.44 The plants contains wide diversity of chemical constituents
in them, but many medicinal plants are still unexplored about their
medicinal uses.45 The results of present study and previous studies indi
cating that S. officinalis roots possess different bioactive compounds with
different biological activities like antioxidant, antibacterial, hepatopro
tective and similar to us, antitumor effects,46 antimethanogenic effect47
and antimethanogenic effect48 of S. officinalis was reported. However, the
results obtained in present study are warrant for further studies on
different medicinal plants used as food and in traditional medicine. Figure 3: Percentage protection produced by different extracts of
S. officinalis at a dose of 500mg/kg. Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(N=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. Figure 3: Percentage protection produced by different extracts of
S. officinalis at a dose of 500mg/kg. fi
g
g
Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(N=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. ACKNOWLEDGMENT The authors thankful to RGNF fellowship, UGC for their financial
support, authorities of AU College of Pharmaceutical Sciences, Andhra
University for providing the necessary facilities to complete present
work. Hepatoprotective activityfi S. officinalis root extracts (ethyl acetate, chloroform and methanol) were
evaluated for their hepatoprotective activity on Paracetamol-induced
liver toxicity in rats at different concentrations (125 mg/kg b.w, 250 mg/
kg b.w and 500 mg/kg b.w) and found concentration dependent percentage
protection based on SGOT, SGPT, ALP, Total serum bilirubin and Total
protein levels on end of experiment (Table 3, Figure 1, 2 and 3). Figure 1: Percentage protection produced by different extracts of
S. officinalis at a dose of 125mg/kg. Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(n=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. The control group which was given normal saline did not showed any
significant changes but Group II which is treated with normal saline and
paracetamol on 5th day showed significant changes compared to group I,
The animals of group III was induced with paracetamol and was treated
Liv 52 (Positive) showed the significant restoration of altered biomarker
enzymes with percentage protection 94.79%, 92.97%, 96.49%, 97.57%
and 90.24% of liver (AST (SGOT), ALT (SGPT), ALP, total bilirubin and
total protein) levels. Figure 1: Percentage protection produced by different extracts of
S. officinalis at a dose of 125mg/kg. Figure 1: Percentage protection produced by different extracts of
S. officinalis at a dose of 125mg/kg. Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(n=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. The percentage protection of ethyl acetate extract of S. officinalis
(SOEAE) at 125mg/kg, 250mg/kg and 500 mg/kg b.w on AST (SGOT),
ALT (SGPT), ALP, total bilirubin and total protein levels at were 9.95%,
7.81%, 9.00% 9.55% and 8.54%, 21.19%, 19.84%, 24.49%, 20.83% and
19.51%, 41.77%, 37.97%, 45.96%, 38.19% and 40.24%. fi
Results were analysed by using Two-way ANOVA followed by
Dunnet’s multiple comparision test. All gropus were compared with
Liv 52 group. ***p<0.001; **p<0.01; *p<0.05; ns= Non significance
(n=6), T.Bil= Total Bilirubin; T.Ptn= Total Protein. The percentage protection produced by the chloroform extract of
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composed of monoclonal anti-thy 1.1 antibody and a ribosome-inactivating
protein from Saponaria officinalis: potent antitumor effects in vitro and in vivo. J Natl Cancer Inst. 1985;75(1):151-9. 21. Prashant T, Bimlesh K, Mandeep K, Gurpreet K, Harleen K. Phytochemical
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their antimicrobial activity. The Journal of Venomous Animals and Toxins Including
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Organisation for Economic Co-operation and Development; AST
(SGOT): Aspartate aminotransferase; ALT (SGPT): Alanine amino
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Fixed Dose. 2001. 27. Ramachandra SS, Quereshi AA, Viswanath SAH, Patil T, Prakash T, Prabhu K. Hepatoprotective activity of Calotropis procera flowers against paracetamol-
induced hepatic injury in rats. Fitoterapia. 2007;78(7-8):451-4. 28. Hiroshi N. Multidrug Resistance in Bacteria. Annu Rev Biochem. 2009;78:119-46. Talluri, et al.: Hepatoprotective activity of Saponaria officinalis 25. Fazel S, Hamidreza M, Rouhollah G, Verdian-rizi M. Spectrophotometric deter
mination of total alkaloids in some Iranian medicinal plants. TJPS. 2008;32:17-20. Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 Talluri, et al.: Hepatoprotective activity of Saponaria officinalis p
p
y
p
ffi
GRAPHICAL ABSTRACT
SUMMARY
• Saponaria officinalis is an traditional medicinal plants belong to the family
Caryophyllaceae grows in tropical and sub-tropical regions. There was some
eralier reports about medicinal use of different parts of it in traditional medi
cine, but there were no scientific evidence. So, phytochemical profiling of it us
ing different extracts and their hepatoprotective activity were carried out in the
present study. S. officinalis roots’ extracts showed presence of sterols, terpe
noids, glycosides, carbohydrates, proteins, flavanoids, alkaloids, phenols, tan
nins and absence of saponins and oils. The methanolic extract showed more
phenolic and alkaloid contents on their quantification. Methanol extract showed
more activity at 500mg/kg b. w. and is comparable with standard drug Liv 52
on altered liver biomarker enzymes AST (SGOT), ALT (SGPT), ALP, total bilirubin
and total protein with percentage protection 56.17%, 54.53%, 61.55% 57.29%
and 53.66%. Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 REFERENCES 47. Lu Y, Van D, Deibert L, Bishop G, Balsevich J. Antiproliferative quillaic acid
and gypsogenin saponins from Saponaria officinalis Roots. Phytochemistry. 2015;113:108-20. 22. Rao BG, Rao YV, Rao TM. Hepatoprotective and antioxidant capacity of Melochia
corchorifolia extracts. Asian Pac J Trop Med. 2013;6(7):537-43. 23. Trease G, Evans SM. Pharmacognosy. 15th ed. Bailer Tindal, London, Elsevier
Publisher. 2002;23-67. 48. Cieslak A, Zmora P, Stochmal A, Pecio L, Oleszek W, Pers-Kamczyc E, et al. Ru
men antimethanogenic effect of Saponaria officinalis L. phytochemicals in vitro. J Agric Sci. 2014;152(6):981-93. 24. Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic
acid phosphotungstic acid reagents. Am J Enol Vitic. 1965;16(3):144-58. Cite this article: Talluri MR, Gummadi VP, Battu GR. Chemical Composition and Hepatoprotective Activity of Saponaria officinalis on
Paracetamol-induced Liver Toxicity in Rats. Pharmacog J. 2018;10(6)Suppl:s129-s134. Cite this article: Talluri MR, Gummadi VP, Battu GR. Chemical Composition and Hepatoprotective Activity of Saponaria officinalis on
Paracetamol-induced Liver Toxicity in Rats. Pharmacog J. 2018;10(6)Suppl:s129-s134. Cite this article: Talluri MR, Gummadi VP, Battu GR. Chemical Composition and Hepatoprotective Activity of Saponaria officinalis on
Paracetamol-induced Liver Toxicity in Rats. Pharmacog J. 2018;10(6)Suppl:s129-s134. S133 Pharmacognosy Journal, Vol 10, Issue 6(Suppl), Nov-Dec, 2018 Talluri, et al.: Hepatoprotective activity of Saponaria officinalis Talluri, et al.: Hepatoprotective activity of Saponaria officinalis SUMMARY • Saponaria officinalis is an traditional medicinal plants belong to the family
Caryophyllaceae grows in tropical and sub-tropical regions. There was some
eralier reports about medicinal use of different parts of it in traditional medi
cine, but there were no scientific evidence. So, phytochemical profiling of it us
ing different extracts and their hepatoprotective activity were carried out in the
present study. S. officinalis roots’ extracts showed presence of sterols, terpe
noids, glycosides, carbohydrates, proteins, flavanoids, alkaloids, phenols, tan
nins and absence of saponins and oils. The methanolic extract showed more
phenolic and alkaloid contents on their quantification. Methanol extract showed
more activity at 500mg/kg b. w. and is comparable with standard drug Liv 52
on altered liver biomarker enzymes AST (SGOT), ALT (SGPT), ALP, total bilirubin
and total protein with percentage protection 56.17%, 54.53%, 61.55% 57.29%
and 53.66%. • Saponaria officinalis is an traditional medicinal plants belong to the family
Caryophyllaceae grows in tropical and sub-tropical regions. There was some
eralier reports about medicinal use of different parts of it in traditional medi
cine, but there were no scientific evidence. So, phytochemical profiling of it us
ing different extracts and their hepatoprotective activity were carried out in the
present study. S. officinalis roots’ extracts showed presence of sterols, terpe
noids, glycosides, carbohydrates, proteins, flavanoids, alkaloids, phenols, tan
nins and absence of saponins and oils. The methanolic extract showed more
phenolic and alkaloid contents on their quantification. Methanol extract showed
more activity at 500mg/kg b. w. and is comparable with standard drug Liv 52
on altered liver biomarker enzymes AST (SGOT), ALT (SGPT), ALP, total bilirubin
and total protein with percentage protection 56.17%, 54.53%, 61.55% 57.29%
and 53.66%. S134
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https://openalex.org/W4255786742
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https://www.nature.com/articles/s41467-021-23092-1.pdf
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English
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Re-evaluating the evidence for facilitation of stickleback speciation by admixture in the Lake Constance basin
|
Nature communications
| 2,021
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cc-by
| 4,001
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MATTERS ARISING MATTERS ARISING MATTERS ARISING Re-evaluating the evidence for facilitation of
stickleback speciation by admixture in the Lake
Constance basin Daniel Berner
1✉
ARISING FROM Marques et al. Nature Communications https://doi.org/10.1038/s41467-019-12182-w (2019) Daniel Berner
1✉ OM Marques et al. Nature Communications https://doi.org/10.1038/s41467-019-12182-w (2019 W than branches representing populations from other drainages in
Northern or Eastern Europe. W
here genetic variation promoting speciation originates
is a crucial question in evolutionary genomics. In a
recent article, Marques et al.1 seek to address this
question in lake and stream threespine stickleback fish from the
Lake Constance (hereafter referred to as LC) basin in Central
Europe. Based on population genetic methods, they conclude that
incipient speciation between lake and stream stickleback was
facilitated by the recent mixing of genetic variation from old
lineages evolved in isolation (i.e., admixture following secondary
contact). In this comment, I discuss conceptual and methodolo-
gical problems and unrecognized conflicts with existing evidence
that cast doubt on Marques et al.’s conclusion. p
The second insight from the phylogeny is that stickleback from
the LC basin prove closely related to populations from the
Danube drainage (Fig. 1 and Supplementary Fig. 1). The broad-
scale colonization history of Central Europe2 in mind, this close
genetic relatedness supports the possibility that stickleback in the
LC basin may originate from the natural westward colonization
by Northeastern European (but not Black Sea2,3) stickleback via
the Danube drainage. The LC basin nowadays drains into the
Atlantic via the river Rhine, so its colonization via the Danube
drainage may appear counter-intuitive. However, during the
retreat of the Pleistocene ice cover, the present-day LC basin
drained in an eastward direction via the Danube4. Even today, the
Danube and the LC drainage remain connected through a sink-
hole and a 12 km underground stream system5 inhabited by fish6. Evidence of a natural, postglacial colonization of the LC basin
also emerges from the authors’ own demographic analysis: their
deepest splitting time estimate between populations from the LC
basin is 2800 generations (a generation is 1–2 years3) before
present, a value in line with an earlier estimate for stream
populations in the LC basin (~2300 generations, Supplementary
Fig. 2 in ref. 7; note, however, that the studies differ in the
mutation rates assumed). While the authors recognize that these
estimates conflict with a very recent anthropogenic origin, they
consider shortcomings in their demographic modeling, but not
the possibility that at least the stream ecotype may have colonized
the LC basin naturally thousands of generations ago. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23092-1 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23092-1 for more complete population coverage (especially including
Danubian stickleback) for their genetic inference. analyses without carefully acknowledging potential violations of
the underlying assumptions, and ambiguity in their interpreta-
tion. A major issue is that their main methodological tools
(demographic modeling, D statistic) assume selectively neutral
evolution. However, natural selection imposed by novel local
ecological conditions profoundly re-structures genetic variation
all across the stickleback genome8. That such selection has the The origin of stickleback in the LC basin
l
h
h
kl b Marques et al. argue that threespine stickleback populations in the
LC basin result from a contact between two deeply separated
lineages from Northeastern and Western Europe, and that
anthropogenic introduction played an important role in the colo-
nization of the LC basin. I here revisit these views based on a
comprehensive nuclear phylogeny for Central European stickleback. This phylogeny (Fig. 1) confers two major insights: first, Eur-
opean stickleback populations separate deeply into a Mediterra-
nean and Black Sea lineage on the one hand, and a Central,
Eastern, and Northern European lineage on the other hand (for
evidence based on ordination see Supplementary Fig. 1). This
dichotomy
is
consistent
with
a
recent
phylogeographic
investigation2 establishing that the circum-Mediterranean and
Black Sea lineage reflects an ancient southern refugial ancestor,
whereas the more northern populations derive from a large-scale
postglacial surge in southwestward direction via an ancient Baltic
Sea. However, the phylogeny does not support Marques et al.’s
claim of the existence of an ancient, genetically distinct Western
European stickleback lineage “evolved in isolation for several
thousand generations”1: statistical support for the monophyly of
the authors’ western lineage (indicated by a gray square in Fig. 1)
is poor, and the basal branch of this western lineage is not deeper Overall, Marques et al.’s assumption of a Western European
stickleback lineage with a long history of evolution in isolation, and
the view that recent introductions were important to the estab-
lishment of stickleback in the LC region, do not appear well sup-
ported by phylogenetic evidence. Although my analyses include
new sequence data not available to the authors’ original investiga-
tion, analogous analyses considering only data contemporaneous
with their work also raise the above concerns (Supplementary
Fig. 2). In this light, it is hard to follow why the authors did not aim partment of Environmental Sciences, Zoology, University of Basel, Basel, Switzerland. ✉email: daniel.berner@unibas.ch 1 TURE COMMUNICATIONS | (2021) 12:2806 | https://doi.org/10.1038/s41467-021-23092-1 | www.nature.com/naturecommunications MATTERS ARISING NATURE COMMUNICATIONS | (2021) 12:2806 | https://doi.org/10.1038/s41467-021-23092-1 | www.nature.com/naturecommunicatio NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23092-1 10). Likewise, it is easy to
imagine that selection can bias D statistics: Marques et al. argue
that Northeastern European stickleback are phenotypically closer
to ancestral marine stickleback than are Central European
populations. However, stickleback within LC are selected for a
pelagic lifestyle resembling that of marine fish3,7, hence greater
allele sharing between the LC population and Northeastern
European populations is an expected outcome of local adaptation
potentially confounding the inference of admixture. Admixture would thus be expected to promote speciation only if
it introduced genetic variation for pelagic adaptation not pre-
viously present in the basin already. However, such variation is
unlikely to come from the authors’ western lineage, which
includes only stream-adapted populations. Admitting the possi-
bility that a stream-adapted population in one watershed may still
hold genetic variation useful to pelagic adaptation in another
watershed, this ecological ambiguity calls for a direct demon-
stration of admixture as a driver of speciation. Specifically, one
would need to (i) identify the specific haplotypes (DNA sequence
stretches) holding alleles involved in divergent adaptation, and
(ii) demonstrate that such haplotypes were initially missing in one
or the other original population. g
p p
However, none of the authors’ genetic population samples
from outside the LC basin include more than seven individuals,
thus precluding robust estimates of haplotype frequencies nee-
ded for inferring variational constraints broken by admixture. More fundamentally, the sparse marker resolution of Marques
et al. is insufficient for haplotype-level inference in the first
place13. However, for a few genome regions under divergent
lake-stream selection, genotype data at the level of phased
haplotypes have been generated previously by targeted sequen-
cing in stickleback from both the LC basin and the authors’
western lineage. These regions include the EDA locus under-
lying variation in body plating (Fig. 5b in ref. 14), and three large
inversions (Fig. 7c in ref. 7). These data demonstrate that the
genetic variants underlying lake-stream divergence within the
LC basin are not only ubiquitous across Europe, but shared
among populations on a worldwide scale. The strongest
sequence-based evidence currently available thus indicates that
lake and stream stickleback within the LC basin have diversified
just as stickleback populations do everywhere: by sorting
abundant standing genetic variation preexisting in their ances-
tors. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23092-1 Fig. 1 Phylogeny of European threespine stickleback populations. The phylogram (maximum likelihood tree) is based on DNA sequence data from 69
total stickleback individuals from the 39 freshwater populations indicated in the maps (1–2 individuals per population). The left map represents a close-up
of the Lake Constance (LC) region, located by a dark blue square in the right map, showing the precise situation of the two lake (ROM and MRH) and three
stream (GRA, NID, and OBR) sample sites. The color coding separates the populations belonging to the circum-Mediterranean and Black Sea lineage (red)
from those belonging to the Central, Northern, and Eastern European lineage (blue; populations from the LC basin are labeled in dark blue). The values next
to nodes give the strength of monophyly of the corresponding branches based on bootstrapping (500 iterations; shown only for values ≥50%). Note the
strong bootstrap support for the reciprocal monophyly of the two major (red, blue) stickleback lineages in Europe. By contrast, the basal nodes within the
blue lineage lack bootstrap support, thus challenging Marques et al.’s assumption of an old, genetically distinct Western European lineage (the basal node
of this lineage is marked by a gray square). The branch marked by the gray dot contains exclusively populations from the LC basin and the Danube river,
highlighting their close genetic relatedness. The gray triangle indicates the BRO population chosen by Marques et al. as representative of their
Northeastern European lineage. Two individuals derived from Pacific ancestors (CLU) served as the outgroup. potential to bias demographic inference has been suggested in
stickleback from the LC basin7. For fairness, it must be high-
lighted that Marques et al. attempt to reduce potential bias due to
selection in their demographic analyses by excluding markers
located in chromosome regions exhibiting a particularly low
recombination rate. While this data manipulation may increase
analytical robustness in older organismal systems in which
genomic
variation
is
shaped
by
background
selection
(a
mutation-driven process), it may be less effective in a young,
postglacial system like stickleback strongly influenced by the
rapid directional selection of standing genetic variation. This
skepticism is confirmed directly by a recent genomic analysis of
lake-stream stickleback from the LC basin based on whole-
genome marker resolution, revealing that signatures of divergent
selection are neither less common nor less extensive physically in
the chromosome peripheries exhibiting high recombination rates9
(Fig. 2 and Supplementary Fig. 1 in ref. The phylogenetic analysis involves stickleback samples from 39 localities in and
around central Europe, most of which are represented by two individuals (69
individuals in total; Supplementary Table 1). Because of strong adaptive genetic
divergence between marine and freshwater stickleback populations, all 38 Eur-
opean localities concern exclusively freshwater habitat; only a single locality
(CLU; Cluxewe Estuary, Vancouver Island, Canada) included as an outgroup
represents saltwater habitat (anadromous marine stickleback). All populations are
at least potentially natural, except for the populations CHE and SAS, which
originate from human introduction to the Lake Geneva basin ~190015,16. The Overconfidence in population genetic methods
h
bl
l
h Another problem in Marques et al. is that strong conclusions
about evolutionary history are derived from population genetic 2 2 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23092-1 Conclusively
evaluating
a
potential
contribution
of
admixture to adaptive diversification and speciation in this
system would require a methodological stringency beyond the
standard of Marques et al.’s work. Also, Marques et al. claim to evaluate several possible demo-
graphic scenarios for the colonization and subsequent divergence
of stickleback in the LC basin, and in particular to refute an
“ecological vicariance” scenario (Fig. 3 in ref. 7). An inherent
element of ecological vicariance, however, is population differ-
entiation caused by strong divergent selection, for which there is
clear experimental evidence in lake and stream fish from the LC
basin10,11. Marques et al.’s demographic analysis, assuming the
absence of selection, must therefore fail to offer an adequate
comparison of relevant evolutionary scenarios. Another source of
concern is that their demographic analysis assumes that all
populations had constant sizes during evolution. Fluctuations in
population sizes, however, can bias demographic model selection
toward secondary contact scenarios12. These methodological
caveats in mind, Marques et al.’s inference of an admixture his-
tory must be regarded as speculative and no more plausible than
alternative scenarios. To conclude, in view of the problems highlighted in this note,
Marques et al.’s claim to “have demonstrated that secondary
contact between divergent lineages and the re-assortment of
introgressed alleles […] underlie recent ecological speciation”
seems overconfident and lacking convincing empirical evidence. NATURE COMMUNICATIONS | (2021) 12:2806 | https://doi.org/10.1038/s41467-021-23092-1 | www.nature.com/naturecommunications References data underlying this analysis are SbfI or PstI enzyme restriction site-associated
DNA (RAD) sequences generated specifically for this study (four localities), or
retrieved from published investigations1,2,7,17–20. In the latter case, the two
individuals with the highest read depth were given priority when more than two
individuals were available from a given locality. The full data set is described in
detail in Supplementary Table 1. 1. Marques, D. A., Lucek, K., Sousa, V. C., Excoffier, L. & Seehausen, O. Admixture between old lineages facilitated contemporary ecological speciation
in Lake Constance stickleback. Nat. Commun. 10, 4240 (2019). 2. Fang, B., Merilä, J., Ribeiro, F., Alexandre, C. M. & Momigliano, P. Worldwide
phylogeny of three-spined sticklebacks. Mol. Phylogenet. Evol. 127, 613–625
(2018). Because some of these sequence data were not available to the investigation
published in Marques et al.1, I examined whether a phylogenetic analysis based
exclusively on data contemporaneous to that study and known to the authors
produced qualitatively similar conclusions. Specifically, I here considered the data
subset from Marques et al.1 and Fang et al.2 only, noting that the latter study was
used by Marques et al. as a source of sequence data for a few selected populations
(ALM, CHO, and KOL). This reduced analysis included 53 total individuals from
29 localities (including the same outgroup as the full analysis). 3. Moser, D., Roesti, M. & Berner, D. Repeated lake-stream divergence in stickleback
life history within a Central European lake basin. PLoS ONE 7, e50620 (2012). y
p
4. Keller, O. & Krayss, E. Die Hydrographie des Bodenseeraums in y
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4. Keller, O. & Krayss, E. Die Hydrographie des Bodenseeraums in
Vergangenheit und Gegenwart Ber St Gall Naturwiss Ges 89 39 4. Keller, O. & Krayss, E. Die Hydrographie des Bodenseeraums in
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y
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Vergangenheit und Gegenwart. Ber. St. Gall. Naturwiss. Ges. 89, 39–56 (2000). 5. Hötzl, H. Origin of the Danube-Aach system. Environ. Geol. 27, 87–96 (1996). Vergangenheit und Gegenwart. Ber. St. Gall. Naturwiss. Ges. 89, 39 g
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6. Behrmann-Godel, J., Nolte, A. W., Kreiselmaier, J., Berka, R. & Freyhof, J. The
first European cave fish. Curr. Biol. 27, R257–R258 (2017). References & Berner, D. Recombination in the threespine stickleback
genome - patterns and consequences. Mol. Ecol. 22, 3014–3027 (2013). 10. Laurentino, T. G. et al. Genomic release-recapture experiment in the wild
reveals within-generation polygenic selection in stickleback fish. Nat. Commun. 11, 1928 (2020). 11. Moser, D., Frey, A. & Berner, D. Fitness differences between parapatric lake
and stream stickleback revealed by a field transplant. J. Evol. Biol. 29, 711–719
(2016). 12. Momigliano, P., Florin, A.-B. & Merilä, J. Biases in demographic modelling
affect our understanding of recent divergence. Mol. Biol. Evol. (in the press). 13. Lowry, D. B. et al. Breaking RAD: an evaluation of the utility of restriction
site-associated DNA sequencing for genome scans of adaptation. Mol. Ecol. Res. 17, 142–152 (2017). 14. Berner, D., Roesti, M., Hendry, A. P. & Salzburger, W. Constraints on
speciation suggested by comparing lake-stream stickleback divergence across
two continents. Mol. Ecol. 19, 4963–4978 (2010). 15. Fatio, V. Faune des Vertébrés de la Suisse - Histoire naturelle des Poissons (H. Georg, 1882). 16. Bertin, L. Recherches bionomiques, biométriques et systématiques sur les
épinoches (Gastérostéidés) (Blondel La Rougery, 1925). p
g y
17. Roesti, M., Salzburger, W. & Berner, D. Uninformative polymorphisms bias
genome scans for signatures of selection. BMC Evol. Biol. 12, 94 (2012). As a robustness check, the above SNP detection and genotyping protocol was
repeated for both the complete and reduced data set by using more stringent
quality filters: for an individual, the leading haplotype at a RAD locus was accepted
only if present in at least five (as opposed to two) copies, and the minimum spacing
threshold for SNPs located on the same RAD locus was increased from 8 to 12 bp. This latter approach, yielding 797 SNPs from 563 RAD loci for the complete and
1161 SNPs from 824 loci for the reduced analysis, produced very similar phylo-
genetic tree topologies leading to the same conclusions (details not presented). 18. Roesti, M., Gavrilets, S., Hendry, A. P., Salzburger, W. & Berner, D. The
genomic signature of parallel adaptation from shared genetic variation. Mol. Ecol. 23, 3944–3956 (2014). 19. Ferchaud, A.-L. & Hansen, M. M. The impact of selection, gene flow and
demographic history on heterogeneous genomic divergence: threespine
sticklebacks in divergent environments. Mol. Ecol. 25, 238–259 (2016). 20. Marques, D. A. et al. Genomics of rapid incipient speciation in sympatric
threespine stickleback. PLoS Genet. 12, e1005887 (2016). References g
g
p
y
Using the R package ShortRead21, all raw fastq files were initially filtered for
reads starting with the exact SbfI restriction residual (TGCAGG; SbfI restriction
sites are covered by the PstI enzyme too), and the reads were trimmed to 70 base
pairs (bp). The fastq data thus obtained were aligned to the threespine stick-
leback reference genome assembly22 with Novoalign v3.00 (http://www. novocraft.com/products/novoalign), using the alignment parameters from
ref. 23 (key settings: -t180 -g40 -x15). To obtain single-nucleotide polymorph-
isms (SNPs) for phylogenetic analysis, the alignments were then converted to
BAM format and processed by using the R packages Rsamtools24 and stringr25. At each RAD locus in each individual, haploid genotyping was performed by
retrieving the leading haplotype, defined as the single sequence exhibiting the
highest read count among all unique sequences present at the RAD locus. RAD
loci at which this leading haplotype did not occur in at least two copies, or
exhibiting an excessive read depth beyond 4.5 times the expected read depth
across all genome-wide RAD loci (estimated by the total number of reads
divided by the total number of RAD loci), were excluded from the analysis. This
haploid genotyping approach was chosen because it avoids potential bias in the
identification of heterozygous positions and is therefore highly reliable. RAD
loci successfully genotyped in every single individual were then used for SNP
detection (hence, the final SNP data set contained no missing data). I accepted
SNPs along a RAD locus only if they were at least 8 bp away from the previous
polymorphic position, thus avoiding pseudo-SNPs arising from indels. For each
individual, the nucleotides present at all SNPs were concatenated to a single
string, and these strings combined across all individuals in a single fasta file. For
the analysis using the complete data set (39 localities), the fasta file contained
genotype information from 7121 SNPs from 4429 genome-wide RAD loci
shared among all individuals, while for the reduced analysis (29 localities), 7616
SNPs from 4961 RAD loci were available. 7. Roesti, M., Kueng, B., Moser, D. & Berner, D. The genomics of ecological
vicariance in threespine stickleback fish. Nat. Commun. 6, 8767 (2015). 8. Bassham, S., Catchen, J., Lescak, E., von Hippel, F. A. & Cresko, W. A. Repeated selection of alternatively adapted haplotypes creates sweeping
genomic remodeling in stickleback. Genetics 209, 921–939 (2018). g
g
9. Roesti, M., Moser, D. Data availability 26. Paradis, E. & Schliep, K. ape 5.0: an environment for modern phylogenetics
and evolutionary analyses in R. Bioinformatics 35, 526–528 (2018). and evolutionary analyses in R. Bioinformatics 35, 526–528 ( All raw Illumina sequence data newly generated for this study are available from the
NCBI Sequence Read Archive under BioProject PRJNA566094 (accession numbers:
SRX6864080, SRX6864081, SRX6864092, SRX6864103, SRX6864114, SRX6864125, 27. Schliep, K. phangorn: phylogenetic analysis in R. Bioinformatics 27, 592–593
(2011). 28. R Core Team. R: A Language and Environment for Statistical Computing (R
Foundation for Statistical Computing, 2020). SRX6864129, SRX6864130). Accession numbers of the reused sequence data are listed in
Supplementary Table 1. Supplementary Table 1. References g
p
g
g
p
Phylogenetic analysis based on the above fasta files was carried out using the R
packages ape26 and phangorn27. I first determined the most appropriate substitu-
tion model (GTR + G + I), estimated the maximum likelihood tree, and visualized
this tree as phylogram. The phylogenies were complemented by visualization of
genetic similarity among individuals using ordination. I here analyzed genetic
distance matrices derived from the fasta files using principal coordinates analysis
(PCoA). These supplementary analyses, performed for both the complete and
reduced data sets, considered the European individuals only; the two CLU indi-
viduals from Canada were excluded. Individuals were plotted along the first two
PCoA axes. All analyses were performed in R28. 21. Morgan, M. et al. ShortRead: a Bioconductor package for input, quality
assessment and exploration of high-throughput sequence data. Bioinformatics
25, 2607–2608 (2009). 22. Glazer, A. M., Killingbeck, E. E., Mitros, T., Rokhsar, D. S. & Miller, C. T. Genome assembly improvement and mapping convergently evolved skeletal
traits in sticklebacks with genotyping-by-sequencing. G3 5, 1463–1472 (2015). 23. Roesti, M., Hendry, A. P., Salzburger, W. & Berner, D. Genome divergence
during evolutionary diversification as revealed in replicate lake-stream
stickleback population pairs. Mol. Ecol. 21, 2852–2862 (2012). 24. Morgan, M., Pages, H., Obenchain, V. & Hayden, N. Rsamtools: binary
alignment (BAM), FASTA, variant call (BCF), and tabix file import. http://
bioconductor.org/packages/release/bioc/html/Rsamtools.html (2017). Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article. 25. Wickham, H. stringr: simple, consistent wrappers for common str
operations. https://CRAN.R-project.org/package=stringr (2019). ,
g
p ,
pp
g
operations. https://CRAN.R-project.org/package=stringr (2019). Facilitation of speciation? The phylogenetic analysis involves stickleback samples from 39 localities in and
around central Europe, most of which are represented by two individuals (69
individuals in total; Supplementary Table 1). Because of strong adaptive genetic
divergence between marine and freshwater stickleback populations, all 38 Eur-
opean localities concern exclusively freshwater habitat; only a single locality
(CLU; Cluxewe Estuary, Vancouver Island, Canada) included as an outgroup
represents saltwater habitat (anadromous marine stickleback). All populations are
at least potentially natural, except for the populations CHE and SAS, which
originate from human introduction to the Lake Geneva basin ~190015,16. The A final issue is that even if we assume that admixture between
distinct lineages has occurred in the LC basin, the evidence pre-
sented by Marques et al. is insufficient for demonstrating that this
has promoted divergence. A challenge is that within the LC basin,
the lake population has adapted relatively recently to the pelagic
ecological niche and represents the most derived ecotype in that
region,
while
stream-adapted
populations
are
ancestral7. 3 MATTERS ARISING NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23092-1 Author contributions A collection of all codes used for data analysis is provided as Supplementary Code 1. All the work underlying this article was performed by the author. Received: 14 May 2020; Accepted: 15 April 2021; Received: 14 May 2020; Accepted: 15 April 2021; Competing interests
The author declares no competing interests. Competing interests p
g
The author declares no competing interests. 4 NATURE COMMUNICATIONS | (2021) 12:2806 | https://doi.org/10.1038/s41467-021-23092-1 | www.nature.com/naturecommunications 4 © The Author(s) 2021 Additional information Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
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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 article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’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. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41467-021-23092-1. Correspondence and requests for materials should be addressed to D.B. Peer review information Nature Communications thanks the anonymous reviewers for
their contribution to the peer review of this work. Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. © The Author(s) 2021 NATURE COMMUNICATIONS | (2021) 12:2806 | https://doi.org/10.1038/s41467-021-23092-1 | www.nature.com/naturecommunications
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https://openalex.org/W2901508299
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https://jurnal.ugm.ac.id/jpti/article/download/25453/22572
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English
| null |
The Effect of Chitosan Application against Plant Growth and Intensity of Stunting Disease on Black Pepper (Piper nigrum L.) Seedlings
|
Jurnal perlindungan tanaman Indonesia/Jurnal Perlindungan Tanaman Indonesia
| 2,018
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cc-by-sa
| 5,763
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Pengaruh Aplikasi Kitosan terhadap Pertumbuhan dan Intensitas Penyakit Kerdil
pada Bibit Lada (Piper nigrum L.) Emerensiana Uge1)*, Sri Sulandari1), Sedyo Hartono1), & Susamto Somowiyarjo1)
1)Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada
Jln. Flora No. 1, Bulaksumur, Sleman, Yogyakarta 55281
*Corresponding author. E-mail: rensi.uge23@gmail.com Received May 30, 2017; accepted September 22, 2017 ABSTRACT Black pepper (Piper nigrum L.) is an important estate crops in Indonesia. Some pathogens that have been known
to infect black pepper plants include fungi, nematodes and viruses. The stunting disease on black pepper plants was
caused by Cucumber mosaic virus (CMV). Molecular detection using RT-PCR method showed that the samples were
positively infected by CMV which were amplified by specific primers CMV 111 with bands of 111 bp in size. This virus
can be carried by vegetative propagation material of plants. Many control strategies against this virus have been
investigated, especially inducing plant resistance with chitosan. Chitosan is a natural biopolymer that play an important
role in reducing disease incidence and severity and stimulate plant growth. The aim of this study was to figure out the
inhibiting ability of chitosan solution against infection of stunting virus on black pepper seedlings through spraying
applications. Chitosan treatments were prepared in concentrations of 0.5%, 0.75%, and 1%. The result showed that
application of chitosan at all concentrations affected the decrease of disease incidence and intensity and improved
plant growth with insignificant different amongst all treatments but significantly different with control. The highest
decrease in incidence was found at 0.75% of chitosan concentration (26.37), while the highest decrease of intensity was
expressed at 1% of chitosan (37.62). Application of chitosan also significantly affected to all parameters of plant growth
either plant height or leaf diameter. Application of 1% of chitosan increased the percentage of plant growth rather than
other treatments, with the increase of plant height 58.12 % and leaf diameter 54.74 %. Keywords: black pepper, chitosan, stunting disease ЖThe article has been presented at International Conference on Biodiversity on 20th−21st May 2017 Jurnal Perlindungan Tanaman Indonesia, Vol. 22, No. 2, 2018: 224–232
DOI: 10.22146/jpti.25453 Jurnal Perlindungan Tanaman Indonesia, Vol. 22, No. 2, 2018: 224–232
DOI: 10.22146/jpti.25453 Research Article INTRODUCTION caused by the systemic infection of virus, so the
selection of cuttings from infected plants will
produce new infected plants. The use of healthy
seedlings was one of principle technique on black
pepper cultivation in order to enhance plant growth
and production (Bhat et al., 2016). Therefore, it is
required the investigation to suppress viral infection
on cuttings and increase plant growth, primarily the
use of natural and eco-friendly products such as
chitosan. Black pepper (Piper nigrum L.) is one of important
estate crops in Indonesia. Stunting disease on black
pepper plants is one of the inhibiting factors on the
growth of black pepper plants. This disease was
reported as the result of infection from pathogenic
virus such as Cucumber mosaic virus (CMV) and
Piper yellow mottle virus (PYMoV) (de Silva et al.,
2002). Multiple infections of these viruses would
generally cause more severe symptoms and inhibit
plant growth. In addition, the recognition of visual
symptoms in the field was very difficult due to their
infection occurred at the same time (Miftakhurohmah
et al., 2016). CMV is a single-stranded RNA virus
with a broad host range and causes various symptoms. The common symptom on pepper is characterized
by shrink, curly, brittle, hardened and chlorotic
spotting leaves. In other cases, the leaves become
abnormal, narrow and shortened of segment length,
and the plant was stunting (Bhat et al., 2004). de
Silva et al. (2002) reported that CMV could be
transmitted through vegetative propagation materials
(cutting and grafting), insect vector (Aphis gosypii
Glover) and mechanically from black pepper to
other host plants. Chitosan is a natural product derived from shells
extract of crustaceans (Crustacea) such as shrimp
and crab. Chitosan has been widely used to control
plant pathogenic fungi, bacteria, viruses, pests and
insect vectors. Chitosan has been reported to control
some plant pathogenic viruses such as Potato virus
X, Tobacco mosaic virus, Tobacco necrosis virus,
Alfalfa mosaic virus, Bean stunting virus and
Cucumber mosaic virus (Pospieszny et al., 1991). Faoro (2013) reported that chitosan could reduce the
infection of Bean common mosaic virus and suppress
the vector population. This is also confirmed by
Damayanti et al. (2013) validated that the use of
chitosan on seed treatment or leaf spraying could
extend the incubation time Bean common mosaic
virus (BCMV), express the variations of milder
symptoms and significantly produce lower titer of
BCMV compared to control. INTRODUCTION Remembering to the
benefit of chitosan and systemic spread of the virus
in plant cutting, it is important to apply chitosan on
pepper cuttings which were infected by CMV. The prevalence of virus on plant could not be
only observed based on the symptom (Bhat & Siljo,
2014; Miftakhurohmah & Balfas, 2014). Therefore,
rapid detection method can determine the presence
of this virus infection. Reverse Transcription
Polymerase Chain Reaction (RT-PCR) is one of
high sensitive molecular method in detecting RNA
virus. The accurate information regarding the virus
infection on plant can assist in determining the
management strategies. The control measures for
inhibiting the spread of this virus are the selection of
healthy seeds, control of insect vector and sanitation
(Miftakhurohmah & Balfas, 2014). Meanwhile, the
development of the insect vector can be inhibited
by synthetic pesticides as one of the common applied
strategies. However, negative impacts of pesticide
application limited the implementation of this
technique. INTISARI Lada (Piper nigrum L.) merupakan salah satu tanaman perkebunan penting di Indonesia. Beberapa patogen telah
diketahui menginfeksi tanaman lada di antaranya jamur, nematoda, dan virus. Penyakit kerdil pada tanaman lada
disebabkan oleh Cucumber mosaic virus (CMV). Deteksi molekuler menggunakan metode RT-PCR menunjukan bahwa
sampel positif terinfeksi CMV yang diamplifikasi menggunakan primer spesifik CMV 111 dengan ukuran pita band target
111 bp. Virus ini dapat terbawa bahan perbanyakan tanaman secara vegetatif. Banyak strategi pengendalian virus yang
telah diuji, diantaranya induksi ketahanan tanaman dengan kitosan. Kitosan adalah biopolimer alami yang berperan
dalam menurunkan insidensi dan intensitas penyakit dan menstimulasi pertumbuhan tanaman. Tujuan dari penelitian
ini adalah untuk mengetahui kemampuan penghambatan dari larutan kitosan terhadap infeksi dari virus kerdil pada
bibit lada dengan aplikasi penyemprotan. Konsentrasi kitosan yang digunakan adalah 0,5%; 0,75%; dan 1%. Hasil
penelitian menunjukan bahwa apliksi kitosan pada semua konsentrasi berpengaruh dalam menurunkan insidensi dan
intensitas penyakit dan meningkatkan pertumbuhan tanaman dengan tidak berbeda nyata di antara perlakuan tetapi
berbeda nyata dengan kontrol. Penurunan nilai insidensi tertinggi yakni pada aplikasi kitosan 0,75% (26,37), sedangkan
penurunan nilai intensitas tertinggi yakni pada aplikasi kitosan 1% (37,62). Aplikasi kitosan juga berpengaruh signifikan
terhadap semua parameter pertumbuhan tanaman baik tinggi tanaman maupun diameter daun. Pada aplikasi kitosan
1% meningkatkan persentase tinggi tanaman lebih baik dibandingkan dengan perlakuan lainnya,yakni tinggi tanaman
sebesar 58,12 % dan diameter daun sebesar 54,74 %. Kata kunci: lada, kitosan, penyakit kerdil Uge et al.: The Effect of Chitosan Application against Stunting Disease on Black Pepper Seedlings 225 Total RNA Extraction RNA extraction was performed using Total RNA
Minikit (Plant) following provided method by
manufacturer (Geneaid). The tested samples were
plants with mosaic and stunting symptoms which
were collected from pepper plantations. Reverse Transcription PCR The synthesis of cDNA in this study was conducted
by using ReverAid First Strand cDNA synthesis kit
(Thermo Scientific, Waltham, MA USA). The RT-PCR
run templates were samples of extracted RNA. The
master mix was prepared according to the procedure
shown in the cDNA synthesis instructions. The RT-
PCR conditions were denaturation at 65°C for 5 min,
annealing at 42°C for 60 min, extension at 70°C for
5 min and final extension at 4°C for 5 min. DI (%)=
× 100 %
Total number of leaves observed
Total number of infected leaves The PCR products were electrophoresed within
a 0.8% agarose gel which was dissolved in 0.5×
Tris-Borate EDTA (TBE) at 50 Volt for 45 min. The
agarose gel was then immersed in a solution of
ethidium bromide (EtBr) for 15 min, visualized on
UV transluminator and documented with a digital
camera. The observation of disease intensity was
demonstrated by measuring the score of disease
severity on each tested plants, i.e. 0 for healthy
leaves or no symptoms of disease; 1 for mild mosaic
leaf and percentage of symptomatic leaves 5−10%;
2 for leaf mosaic clear and percentage of symptomatic
leaves 11−25%; 3 for leaf mosaic clear, shoestring
and the percentage of symptomatic leaves 26−50%;
4 for leaf mosaic clear, shoestring and the percentage
of symptomatic leaves 50−75%; and 5 for leaf mosaic
clear, shoestring, stunting and leaf percentage of
symptomatic > 75%. The observed scores were then
converted into the disease intensity with the
following formula: Application of Chitosan Chitosan was applied on viral-infected seedling
at the beginning of nursery (1st weeks) and repeated
every week (for 9times application) during 10weeks
observation, according topredetermined concentrations. It was sprayed using 500 ml of hand sprayer in the
morning as much of ± 20 ml at the upper and lower
surfaces of the leaves. MATERIALS AND METHODS The research was conducted at Virology Laboratory
and Greenhouse of Department of Crop Protection,
Faculty of Agriculture, Universitas Gadjah Mada
and at pepper plantations in Putat Village, Patuk,
Gunungkidul, Province of Daerah Istimewa Yogyakarta
(D.I.Y), in November 2016 until April 2017. The
materials were CMV-infected pepper cutting, 1% of
acetic acid solution, chitosan powder, Total RNA
Minikit (Plant) (Geneaid), ReverAid First Strand
cDNA synthesis kit and PureTaq Ready To Go PCR
beads. The primers were forward primer (CMV-F
5’-TGTGGGTGACAGTCCGTAAA-3’) and the
reverse primer (CMV-R 5’-ACGCGGCATACT
GATAAACC-3’) with expected amplicon of 111 bp. The specificity of the primers was checked using
the BLAST, and the secondary structure, intra- and Black pepper plants were generally propagated
with vegetative material from cuttings. This method
was applied due to effective and easy in obtaining
the generation with similar characteristics to par-
enting plants (Suparman et al., 1992). Cuttings from
infected plants can widely spread the virus. This is Jurnal Perlindungan Tanaman Indonesia Vol. 22 No. 2 226 inter-primer complementarities were checked using
Oligo Calc. The primers were synthesized at Integrated
DNA Technologies (Coralville, Iowa, USA) (Bhat &
Siljo, 2014). The Observed Parameters The obtained cDNA was used as template in
PCR analysis using PureTaq Ready To Go PCR beads. The procedure was based on the product instructions
by adding 20 µl of free water (dH20), 1 µl of 5 pmol
CMV111 F and CMV111 R primers and 3 µl of
cDNA template, to make the total volume of 25 µl. PCR was run under conditions of pre-denaturation
at 94°C for 3 min, denaturation at 94°C for 1 min,
annealing at 55°C for 15 s, extension at 72°C for 1
min and final extension at 72°C for 5 min. Preparation of Pepper Cutting Black pepper cuttings were collected from pepper
growing areas in Gunung kidul, Yogyakarta. These
cuttings were positively infected by CMV (confirmed
with molecular detection RT PCR) showing mosaic,
uneven surface, curling, shrink, malformations,
narrowed leaves and shoestring-like shoot leaf
symptoms. The fresh selected cuttings originated
from climbing shoot with 4 segments. Cuttings were
transferred to 5 kg in volume-polybags in green house
by adding soil and compost with a ratio 2:1. Plants
were then maintained until ready to use for treatment
about 2 months old. The Development of Stunting Disease Incidence
and Intensity Observations were conducted weekly to monitor
the development disease incidence and intensity in
the greenhouse at the plant vegetative stage as long
as 10 weeks. Weekly disease incidence was calcu-
lated by this following formula: Experimental Design and Data Analysis Experiments were arranged in completely
randomized design (CRD), consisting of 3 treatments
and 1 control and each treatment was repeated for 5
times, i.e. A = control (untreatment); B = 0.5% of
chitosan, C = 0.75% of chitosan and D = 1% of
chitosan. Data was analyzed by variance analysis
and significant different data was proceeded with
DMRT at level of α 5% using SAS version 9.13
program. The percentages of plant height and leaf
diameter were compared to control using formula; leaves appeared mild mosaic, chlorotic, curly, uneven
surface, and wrinkled leaves, as well as shoestring-
like leaf shoot and stunting plants (Figure 2). Treatments of B (Chitosan 0.5%), C (Chitosan 0.75%)
and D (Chitosan 1%) showed that the scores were
not significantly different between treatments in
weekly observation, but they were significantly
different with treatment A (control) at 3rd week
observation. The advanced of DMRT on incidence scoring of
stunting disease revealed that the average scoring
of A (control) at 10th week was 84.83%, while the
scores of decrease in disease incidence were 40.045%,
26.037% and 34.107% for treatments B, C, and D,
respectively (Figure 3). Further DMRT on intensity
scores of stunting disease documented the percent-
ages of decrease in disease intensity, i.e. 97.86%,
40.05%, 39.15% and 37.63%, for treatments of A,
B, C, and D, respectively (Figure 4). with PI = percentage increase; Original scoring =
untreatment plant scoring. Amplification of CMV RNA Using RT PCR Method The infection of CMV in black pepper plants
generated typical symptoms such as mosaic and
stunting. The use of infected plant samples in the
field successfully detected the presence of viral. The
RT-PCR method could amplify DNA band product
from symptomatic leaves with single band of 111
bp in size under annealing temperature of 55°C for
15 s and 0.8 % of agarose gel (Figure 1). The application of chitosan showed that all
concentrations of chitosan significantly affected the
decrease of stunting disease and symptoms in black
pepper seedling compared to control. Percentage of
incidence and intensity among the treatments were
not significantly different, but it was significantly
different to control. Under DMRT analysis, score of
incidence showed that treatment of 0.75% chitosan
was more able to reduce the number of infected
leaves rather than other treatments and control, while
the lower score of decrease in disease intensity was
reached by concentration of 1% chitosan. Observation Effect of Chitosan Application on
Growth of Black Pepper Seedling The plant height was weekly observed by measuring
the seedlings from above of soil surface to growing
point. The measurement was started on 1 to 10 weeks
old plants after transferring. Meanwhile, total leaf
diameter was measured at the last observation (10th
week) by measuring the diameter of 5 young leaves
for each treatment and then was averaged. Figure 1. Visualization of PCR products of mosaic leaves
and stunting samples; the DNA bands were
amplified by CMV 111 specific primer (111 bp);
lanes A−C = infected pepper samples; lane M =
100 bp DNA ladder RESULTS AND DISCUSSION Amplification of CMV RNA Using RT PCR Method Preparation of Chitosan Solution Chitosan was collected from Laboratory of
Microbiology, Department of Fisheries, Faculty of
Agriculture, Universitas Gadjah Mada. Chitosan
powder was dissolved perfectly in a solvent (1%
acetic acid). Those chitosan solutions were prepared
by dissolving 5, 7.5, and 10 gram of powdered
chitosan in 1000 ml of acetic acid solvent for getting
concentrations of 0.5 %, 0.75%, and 1%, respectively. DI (%) =
×100%
(Z×N)
∑(n×v) 227 Uge et al.: The Effect of Chitosan Application against Stunting Disease on Black Pepper Seedlings With : DI = disease intensity, n = number of infected
leaves with certain grade, v = disease score; Z =
maximum disease score, N = total number of leaves
observed Figure 1. Visualization of PCR products of mosaic leaves
and stunting samples; the DNA bands were
amplified by CMV 111 specific primer (111 bp);
lanes A−C = infected pepper samples; lane M =
100 bp DNA ladder Observation Effect of Chitosan Application on
Growth of Black Pepper Seedling The Effect of Chitosan Application on the Growth
of Black Pepper Seedling explained that the plant growth were higher under
treatment of 1% concentration than other treatments
and control (Figure 6). The Application of chitosan on black pepper
seedling showed a significant effect on the growth
and development of plants (Table 1). Observation
of plant height and leaf diameter showed that
application of chitosan was able to increase the
percentage of plant height and
leaf diameter
compared to control. The percentage of increase in
plant height were obtained i.e. 20.40%, 28.45 %,
38.44% and 58.12%, for treatments A, B, C, and D,
respectively (Figure 5). Stunting Disease Progression The observations on disease incidence and
intensity showed that all concentration treatments
affected decrease of disease incidence and intensity. Variation of the symptoms on the black pepper Jurnal Perlindungan Tanaman Indonesia
228 Vol. 22 No. 2 Jurnal Perlindungan Tanaman Indonesia Jurnal Perlindungan Tanaman Indonesia Vol. 22 No. 2 228 Figure 2. Stunting symptoms on Black Pepper leaves; healthy leaves (a), mild mosaic and uneven surface leaf (b), mosaic
and curly leaf (c), mosaic and shrink leaf (d), malformation leaf (e), mosaic leaf buds and narrowed leaf (f),
mosaic and shoestring-like leaf shoot (g)
a
b
c
d
e
f
g Figure 2. Stunting symptoms on Black Pepper leaves; healthy leaves (a), mild mosaic and uneven surface leaf (b), mosaic
and curly leaf (c), mosaic and shrink leaf (d), malformation leaf (e), mosaic leaf buds and narrowed leaf (f),
mosaic and shoestring-like leaf shoot (g) Figure 3. Effect of chitosan application on the incidence
of stunting disease on black pepper seedling
(*: significant difference between the chitosan
treatments and control with a DMRT further
test level α 0.05). Disease Incidence (%)
Figure 4. Effect of chitosan application on the intensity of
the stunting disease on black pepper seedling
(*: significant difference between the chitosan
treatments and control in a DMRT further test
level α 0.05) Figure 3. Effect of chitosan application on the incidence
of stunting disease on black pepper seedling
(*: significant difference between the chitosan
treatments and control with a DMRT further
test level α 0.05). Disease Incidence (%) Disease Incidence (%) Figure 4. Effect of chitosan application on the intensity of
the stunting disease on black pepper seedling
(*: significant difference between the chitosan
treatments and control in a DMRT further test
level α 0.05) Figure 4. Effect of chitosan application on the intensity of
the stunting disease on black pepper seedling
(*: significant difference between the chitosan
treatments and control in a DMRT further test
level α 0.05) Figure 3. Effect of chitosan application on the incidence
of stunting disease on black pepper seedling
(*: significant difference between the chitosan
treatments and control with a DMRT further
test level α 0.05). Disscussion CMV is one of the virus which infecting black
pepper plants with typical symptoms mosaic and
stunting. However, the types of symptoms caused
by virus infection are very diverse and systemic. Further infection will produce more severe leaves
symptoms such as mottle, vein banding, shrink,
surface uneven, curly and shoestring leaves, while
flower or short fruits cannot produce seeds. High
variation in symptoms is very important for method
of detection and characterization of this virus. Virus
detection is generally performed by molecular
methods such as Polymerase Chain Reaction (PCR)
and Reverse Transcriptation Polymerase Chain
Reaction (RT-PCR). Both methods will give rapid
and high sensitivity results. The availability of The decline intensity of pepper stunting mosaic
disease was also indicated by narrowing the leaf size
which was observed in leaf diameter. Application of
chitosan showed significant effect on leaf diameter
rather than the control. The increasing percentages in
the leaf diameter were 29.92%, 30.65% and 54.74%
for treatments B, C, and D, respectively. Data Uge et al.: The Effect of Chitosan Application against Stunting Disease on Black Pepper Seedlings 229 Parameter
Treatment
Percentage of increase (%)
Plant height
Chitosan 0.05 %
26.230a
28.45
Chitosan 0.75 %
28.270a
38.44
Chitosan 1 %
32.290a
58.12
Control (Untreatment plant)
20.420b
Leaf diameter
Chitosan 0.05 %
3.5600a
29.92
Chitosan 0.75 %
3.5800a
30.65
Chitosan 1 %
4.2400a
54.74
Control (Untreatment plant)
2.7400b
Table 1. The effect of chitosan application on the growth of black pepper seedling
Remark: The data followed by the same letter in the same column did not significantly different according to the Duncan test at the
95% confidence level. Table 1. The effect of chitosan application on the growth of black pepper seedling ata followed by the same letter in the same column did not significantly different according to the Duncan test at the
confidence level. Figure 5. Effect of chitosan application on plant height; untreated plant (A), treated plants: B (0.5% of chitosan), C
(0.75% of chitosan), and D (1% of chitosan) Figure 5. Effect of chitosan application on plant height; untreated plant (A), treated plants: B (0.5% of chitosan), C
(0.75% of chitosan), and D (1% of chitosan) Figure 6. Disscussion In Indonesia, stunting
disease has been reported since 1987 (Firdausil et al.,
1992), while serological detection with ELISA method
on pepper seedlings in Sukabumi and Purbalingga
has demonstrated the infection of CMV and PYMoV
on all tested varieties (Mariana & Miftakhurohmah,
2016). Meanwhile the reports about the presence of
CMV infection in Yogyakarta have been known
before in banana (Sumardiyono et al., 1996) and
cucurbit plants (Daryono & Natsuaki, 2009). All these
revealed that the infection of CMV has been widely
distributed in Indonesia. However, the possibility of
PYMoV infection on pepper in this research was not
confirmed. molecular detection with RT PCR will help to detect
the virus quickly and accurately as a first step in
determining a management strategy for target virus. The pepper plant showing the presence of mosaic and
stunting symptoms from Yogyakarta were identified
infected by CMV. This was successfully indicated
by the amplification of bands at 111 bp in size using
a specific primer CMV 111. This research was not
applied negative and positive control, due to the
unavailability of samples as a comparison (Figure 1)
and the research was finished. The detection of
CMV in black pepper was also reported by Bath and
Siljo (2014) using the same primer. The similar
infection in pepper plants has also been reported by
Siju et al. (2007) in India. In Indonesia, stunting
disease has been reported since 1987 (Firdausil et al.,
1992), while serological detection with ELISA method
on pepper seedlings in Sukabumi and Purbalingga
has demonstrated the infection of CMV and PYMoV
on all tested varieties (Mariana & Miftakhurohmah,
2016). Meanwhile the reports about the presence of
CMV infection in Yogyakarta have been known
before in banana (Sumardiyono et al., 1996) and
cucurbit plants (Daryono & Natsuaki, 2009). All these
revealed that the infection of CMV has been widely
distributed in Indonesia. However, the possibility of
PYMoV infection on pepper in this research was not
confirmed. In this research, DMRT also showed that treatment
of chitosan at all concentrations affected plant growth
parameters such as plant height and leaf diameter. Scoring indicated that 1% chitosan treatment had a
greater impact on all plant growth parameters, i. e.,
plant height and leaf diameter than other treatments
and control (Table 1). In addition, the chitosan-treated
plant demonstrated better growth than untreated
ones. This was supported by research of Noiket et
al. Disscussion The observation on leaf diameter of black pepper: (1) mosaic and stunted leaf (untreatment plant); (2) mosaic,
chlorotic, narrow leaf and uneven leaf surface (treatment of chitosan 0.5%); (3) mosaic and narrow leaf (treatment
of chitosan 0.75%); (4) mild mosaic leaf (treatment of chitosan 1%)
1
2
3
4 1
2
3
4 1 4 3 2 4 Figure 6. The observation on leaf diameter of black pepper: (1) mosaic and stunted leaf (untreatment plant); (2) mosaic,
chlorotic, narrow leaf and uneven leaf surface (treatment of chitosan 0.5%); (3) mosaic and narrow leaf (treatment
of chitosan 0.75%); (4) mild mosaic leaf (treatment of chitosan 1%) Jurnal Perlindungan Tanaman Indonesia 230 Vol. 22 No. 2 oxide and activity of the enzyme phenylalanine
ammonia-lyase (PAL) (Iriti et al., 2006; Zhao et al.,
2007). Damayanti et al. (2013) confirmed that the
treatment of chitosan could increase plant resistance
under biotic stress (pathogen infection) and increases
tolerance of abiotic factors. They also explained that
long bean plants treated with chitosan showed a
longer time of virus incubation and the variation of
symptom was lower than the untreated control. This
was confirmed by the presence of peroxidase activity
and the lower titers of BCMV. The chitosan
mechanism in inhibiting the virus movement was
initiated by inducing the inhibitor enzymes, for
example is peroxidase. Peroxidase enzymes could
enhance inducing the plant resistance through physical
barriers such as lignification. Lignification of cell wall
was capable of inhibiting penetration of insect vector
and preventing the virus movement (Goodman et al.,
1986). molecular detection with RT-PCR will help to detect
the virus quickly and accurately as a first step in
determining a management strategy for target virus. The pepper plant showing the presence of mosaic and
stunting symptoms from Yogyakarta were identified
infected by CMV. This was successfully indicated
by the amplification of bands at 111 bp in size using
a specific primer CMV 111. This research was not
applied negative and positive control, due to the
unavailability of samples as a comparison (Figure 1)
and the research was finished. The detection of
CMV in black pepper was also reported by Bath and
Siljo (2014) using the same primer. The similar
infection in pepper plants has also been reported by
Siju et al. (2007) in India. LITERATURE CITED Bhat, A.I, T.H. Faisal, R. Madhubala, P.S. Hareesh
& R.P. Pant. 2004. Purification, Production of
Antiserum and Development of Enzyme Linked
Immunosorbent Assay-Based Diagnosis for
Cucumber mosaic virus Infecting Black Pepper
(Piper nigrum L.). Journal of Spices Aromatic
Crops 13: 6−21. Bhat, A.I. & A. Siljo. 2014. Detection of Virus
Infecting Black Pepper by SYBR Green-Based
Real Time PCR Assay. Journal of Plant Pathology
96: 105−109. Based on all above explanations, it is known that
chitosan is a natural product that can induce the
decrease of disease in infected plants, although the
plants remain infected by virus with lower intensity. It is recognized that the decrease in disease incidence
and intensity could indirectly reach up to 100% in
which there is still virus accumulation in plant tissue
which displays as light mosaic symptom. Therefore,
further investigation regarding the use of chitosan
in reducing the CMV infection on black pepper is
required, particularly pre-application of chitosan
prior to infection and increasing the concentration of
chitosan application. Some reports also suggest that
stunting disease in black pepper was known has two
viruses infection, those viruses were PYMoV and
CMV (de Silva et al., 2002; Miftakhurohmah et al.,
2016). The use of infected cuttings from the field in
this study is likely carry multiple infections. Therefore
it is also suspected that chitosan can also inhibit the
development of PYMoV infection. However, accurate
research is needed to ensure this inhibitory ability. Bhat,A.I., T.Hohn&R.Selvarajan. 2016.Badnaviruses:
The Current Global Scenario. Viruses. 8: 1−29. Boonlertnirun, S, C. Boonraung & R. Suvanasara. 2008. Application of Chitosan in Rice Production. Journal of Metal, Materials and Mineral 18:
47−52. Damayanti, T.A., Haryanto & S. Wiyono. 2013. Pemanfaatan Kitosan untuk Pengendalian Bean
common mosaic virus (BCMV) pada Kacang
Panjang [Utilization of Chitosan to Control Bean
commom mosaic virus (BCMV) on Yard Long
Bean]. Jurnal Hama dan Penyakit Tumbuhan
Tropika 13: 110–116. Daryono, B.S, & K.T. Natsuaki. 2009. Survey on
the Occurrence of Viruses Infecting Cucurbits in
Yogyakarta and Central Java. Jurnal Perlindungan
Tanaman Indonesia 15: 83−89. de Silva , D.P.P, P. Jones & M.W. Shaw. 2002. Identification and Transmission of Piper yellow
mottle virus and Cucumber mosaic virus
Infecting Black Pepper (Piper nigrum) in Sri
Langka. Plant Pathology 51: 537−545. Firdausil, A.B, S. Rusmilah & D. Sitepu. 1992. Stunted Disease of Black Pepper, p. 220−226. In
P. Wahid, D. Sitepu, S. Deciyanto, & U. Disscussion (2014) which stated that chitosan was a natural
compound that could improve plant growth, induce
plant resistance against diseases and reduce damage
from plant pathogens. The results of this experiment
proved that the application of chitosan at 10−20
ppm revealed lower scores of disease severity than
the applications at 60 ppm. However, the concen-
tration of 60 ppm was able to increase the height of
tomato Sridathip 3 from Thailand compared to other
treatments. The investigation of Mishra et al. (2014)
also supported that the co-application of chitosan
and beneficial microbes such as Pseudomonas sp. could reduce the severity of disease and stimulate
the growth of tomato plants compared to control. Chitosan is known as good inducer to stimulate the
resilience of the plant. Suptijah et al. (2010) stated that
the combination of dipping and spraying treatments
of 25 ppm chitosan gave the best effect on each
parameter of plant growth such as height, number
of branches and leaves, length and width of leaf as
well as wet and dry weight of plants. CMV has a broad host range with diverse
symptoms. Such insights suggested that the control
of selected pathogen should emphasize the general
defense response. The use of chitosan has been known
to boost the plant resistance against various types of
plant pathogens and increase plant growth. Therefore,
this research used chitosan as one alternative in
controlling CMV on black pepper.The results of this
research showed that the application of chitosan
0.5%, 0.75%, and 1% significantly influenced the
inhibition of virus on black pepper plants. It was due
to the ability of chitosan in inhibiting virus growth
directly or indirectly. Direct inhibition is conducted
by blocking the spread of the virus systemically
throughout the plant and indirectly by increasing the
hypersensitive response of the host to infection through
inducing plant resistance. Induction mechanism of
plant resistance with chitosan applayed through the
formation of enzymes, protein and structure
inhibitor such as callus, micro-oxidativeburst, micro
hypersensitive response, the production of nitric 231 Uge et al.: The Effect of Chitosan Application against Stunting Disease on Black Pepper Seedlings ACKNOWLEDGEMENTS The ability of chitosan in inducing the growth of
plants was regulated by the signal boosters to produce
plant growth hormones such as gibberellins and also
to enhance the signaling pathways to auxin bio-
synthesis (Uthairatanakij et al., 2007). Wanichpongpan
et al. (2001) reported that chitosan treatment on
Gerbera plants could increase the average number
of buds, length of stem, number, width and length of
leaf and number of flower per clump. The use of
single chitosan without any chemical fertilizer was
able to increase the population of microbes and fasten
the transformation process of nutrient from organic
to be inorganic compounds, so that they were easily
absorbed by plant roots (Boonlertnirun et al., 2008). Meanwhile, Borkowski et al. (2007) cit. Suptijah et
al. (2010) reported that the spraying of 25 ppm of
chitosan on tomato plant could increase its harvested
yield. Highly appreciation and thanks to LPDP (Lembaga
Pengelola Dana Pendidikan) Finance Ministry of
Republic Indonesia for financial support in this
research. This paper is part of thesis. LITERATURE CITED Superman
(eds.), Proceeding of the International Workshop
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affected the reduction of disease incidence and
intensity and improved the growth of black pepper
seedling compared to control. The highest decrease
percentage in incidence was found at treatment 0.75%,
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Retrospective Analysis of Spectrum of Presentation and Treatment Outcome in Extremity Sarcomas: A Single-Centre Experience
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Sarcoma
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Saurabh Bansal,1 Kunal Das
,2 Navneet Jain,3 Vipul Nautiyal,1 Meenu Gupta,1
Nadia Shirazi,4 Sanjiv Verma,5 Mushtaq Ahmad,1 and Sunil Saini3 Saurabh Bansal,1 Kunal Das
,2 Navneet Jain,3 Vipul Nautiyal,1 Meenu Gupta,1
Nadia Shirazi,4 Sanjiv Verma,5 Mushtaq Ahmad,1 and Sunil Saini3 Saurabh Bansal,1 Kunal Das
,2 Navneet Jain,3 Vipul Nautiyal,1 Meenu Gupta,1
Nadia Shirazi,4 Sanjiv Verma,5 Mushtaq Ahmad,1 and Sunil Saini3
1Department of Radiotherapy, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
2Department of Medical Oncology & Hematology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
3Department of Surgical Oncology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
4Department of Pathology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
5Department of Medical Oncology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India 1Department of Radiotherapy, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
2Department of Medical Oncology & Hematology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
3Department of Surgical Oncology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
4Department of Pathology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
5Department of Medical Oncology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India 1Department of Radiotherapy, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
2Department of Medical Oncology & Hematology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
3Department of Surgical Oncology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
4Department of Pathology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India
5Department of Medical Oncology, Cancer Research Institute, SRHU, Dehradun, Uttarakhand, India orrespondence should be addressed to Kunal Das; drkunaloncology@gmail.com Correspondence should be addressed to Kunal Das; drkunaloncology@gmail.com Received 27 October 2017; Revised 3 January 2018; Accepted 6 March 2018; Published 1 April 2018 Academic Editor: R. Lor Randall Copyright © 2018 Saurabh Bansal et al. Tis 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. Introduction. Te most common site for soft tissue sarcoma is extremity. As complete surgical resection is possible in majority,
outcome of this subset is relatively better. Tere is paucity of data regarding extremity soft tissue sarcoma (STS) from sub-
Himalayan and hilly geographical regions. Materials and Methods. Retrospective analysis was done for extremity STS visiting the
study center over a period of 5 years. Data were collected and analyzed for demography, disease characteristics, treatment
modalities, and outcome. Result. Extremity STS constituted 32.8% of all STS enlisted. Most common subtype noted was
pleomorphic STS. Metastatic disease at presentation was noted among 7/43 cases with lung being the most common metastasis
site. Saurabh Bansal,1 Kunal Das
,2 Navneet Jain,3 Vipul Nautiyal,1 Meenu Gupta,1
Nadia Shirazi,4 Sanjiv Verma,5 Mushtaq Ahmad,1 and Sunil Saini3 Wide local excision was done in 37 cases while amputation was required in 5 cases. Adjuvant radiotherapy was given in 27
cases while 18 cases received adjuvant chemotherapy. At median follow-up of 47 months, the overall survival and event-free
survival were noted as 47.64% and 41.49%, respectively. Conclusion. This study depicts single-center experience of extremity STS. Te population analyzed was from sub-Himalayan region with significant lost to follow-up. Pooling of data from different centers
has been advocated to derive conclusive results. Te Cancer Research Institute, Dehradun, is the largest
and single referral tertiary cancer center in the state of
Uttarakhand, India. We present our experience of soft tissue
sarcoma occurring primarily in extremities. 1. Introduction Soft tissue sarcoma (STS) group of tumors are heteroge-
neous histologically distinct entities. It comprises around 1%
of all malignancies in adult [1]. Extremities STS are the most
common anatomical presentation of STS. Te possibility of
wide local excision and good local control makes it different
from intraabdominal and head and neck STS. Surgery re-
mains the mainstay of treatment supplemented by radiation
therapy. While adjuvant radiotherapy is recommended for
intermediate- and high-grade sarcomas, the role of adjuvant
chemotherapy is still debatable. Te data regarding STS
clinicobiological behavior and outcome is sparse from sub-
Himalayan region and similar high-altitude geographical
regions. Even Indian data are sparse and limited to indi-
vidual center experiences. Hindawi
Sarcoma
Volume 2018, Article ID 4350634, 5 pages
https://doi.org/10.1155/2018/4350634 Hindawi
Sarcoma
Volume 2018, Article ID 4350634, 5 pages
https://doi.org/10.1155/2018/4350634 Research Article
RetrospectiveAnalysisofSpectrumofPresentationandTreatment
Outcome in Extremity Sarcomas: A Single-Centre Experience aurabh Bansal,1 Kunal Das
,2 Navneet Jain,3 Vipul Nautiyal,1 Meenu Gupta,1
Nadia Shirazi,4 Sanjiv Verma,5 Mushtaq Ahmad,1 and Sunil Saini3 2. Materials and Methods A retrospective analysis was done for the patients diagnosed
with extremities soft tissue sarcomas from January 2011 to
December 2015. Electronic database of the hospital was
searched for the patient details (maintained by the Uttar-
akhand State Council for Science and Technology support)
and paper records were retrieved from medical record de-
partment. Institutional ethical committee clearance was
taken beforehand. Te details regarding the demographic
profile of patients, anatomical and histological characteristics 2 Sarcoma of STS, and treatment modalities and follow-up were col-
lected. Records with insufficient or incomplete information
regarding histology or treatment were excluded. Patients
enrolled for second opinion were excluded, as no treatment
modality was used. Te outcome was assessed by electronic
record of last visits and telephonic confirmation of survival or
demise. Subsets that were not reachable telephonically were
tried to contact by post. Table 1: Tumor characteristics. Table 1: Tumor characteristics. Tumor histology
Numbers
Pleomorphic sarcoma
9
Synovial sarcoma
7
Spindle cell sarcoma
5
Liposarcoma
6
Leiomyosarcoma
4
Fibrosarcoma
4
Dermatofibrosarcoma
3
Malignant peripheral nerve sheath tumor
2
Unclassified/undifferentiated
2
Epitheloid sarcoma
1
Grade
Low grade
10
Intermediate grade
4
High grade
29
Location
Upper limb
12
Lower limb
31
Size
T1 (<5 cm)
9
T2 (>5 cm)
34
Metastasis
M0
36
M1
7
Stage
Stage I
10
Stage II
6
Stage III
20
Stage IV
7 Table 1: Tumor characteristics. Tumor histology
Numbers
Pleomorphic sarcoma
9
Synovial sarcoma
7
Spindle cell sarcoma
5
Liposarcoma
6
Leiomyosarcoma
4
Fibrosarcoma
4
Dermatofibrosarcoma
3
Malignant peripheral nerve sheath tumor
2
Unclassified/undifferentiated
2
Epitheloid sarcoma
1
Grade
Low grade
10
Intermediate grade
4
High grade
29
Location
Upper limb
12
Lower limb
31
Size
T1 (<5 cm)
9
T2 (>5 cm)
34
Metastasis
M0
36
M1
7
Stage
Stage I
10
Stage II
6
Stage III
20
Stage IV
7 y
Confirmation of diagnosis was done by core needle
biopsy or incisional biopsy in all cases. All cases underwent
staging workup with computer tomography (CT) of chest
and involved limb magnetic resonance imaging. Te 2013
WHO Classification for soft tissue sarcoma was used for
tumor grouping. Sarcomas lacking characteristic histology,
immunohistochemistry, and/or genetic features were cate-
gorized as unclassified/undifferentiated sarcoma [2]. Tumor
grade was decided based on differentiation and mitotic
figures and necrosis according to the Federation Nationale
des Centers de Lutte Contre le Cancer (FNCLCC) system. 2. Materials and Methods Size, nodal status, and metastasis were noted as per AJCC
staging system [3]. Surgical margin status was noted as clear
or involved (microscopic or gross residual). A 6-week
follow-up for initial two visits followed by 3 monthly visits
for next 2 years was advocated to all cases as per institutional
protocol. After 2 years of completion of therapy, a 6-month
follow-up was planned for each case. Locoregional clinical
examination was done at each visit, and annual imaging with
CT chest was done. A relapse was confirmed histologically,
and local as well as metastatic workup was done to define the
pattern of relapse. Treatment-related toxicities requiring
admission or intervention were recorded. liposarcoma and synovial sarcoma and one case of leio-
myosarcoma, malignant peripheral nerve sheath tumor, and
pleomorphic STS each were metastatic at presentation. Te
median time lag of onset/observation of swelling or pain and
diagnosis were 8.5 months. Postoperative grading and TNM
was done in all cases. Nine cases were with tumor size <5 cm
in maximal dimension. Majority (29/43) were high-grade
histology while 4 were intermediate and 10 were low-grade
sarcoma (Table 1). 2.1. Statistical Analysis. Te collected data was analyzed for
demographic characteristics, tumor characteristics, and
treatment modalities. Statistical analysis was done using
SPSS version 17.0 (SPSS Inc., Chicago, IL). Time interval to
local recurrence or distant failure was calculated from the
date of start of treatment to date of recurrence detection,
with censoring at date of death or last contact. Overall
survival was calculated from treatment onset to date of death
irrespective of the cause of death, with censoring at the date
of last contact for the patient alive. Actuarial survival rates
were calculated using the Kaplan–Meier method. Of 36 localized sarcomas, all underwent upfront surgical
resection. Wide local excision was a preferred modality and
was performed in 33 patients while 3 underwent limb
amputation. Of 7 metastatic sarcomas, surgical resection was
performed amongst 6 cases, all after first-line chemotherapy
while 1 case opted for palliative care (Table 2). Tree cases
responded well to chemotherapy with disappearance of
pulmonary metastasis, and two of them underwent ampu-
tation of limb while wide local excision was done in one. One
case showed good partial response and underwent lung
metastatectomy alone with wide local excision. Two cases
showed mild reduction in size and underwent excision in
view of local fungating mass over variable period of time. 2. Materials and Methods A
total of 5 patients underwent amputation. Tree were with
neurovascular involvement rendering limb salvage difficult
while two were with skip bone metastasis at the same limb. Wide local excision was performed in total of 37 cases (33
localized and 4 primary metastatic). Margin positivity was
noted among 3 cases, only one underwent re-resection to
achieve negative margin. One of them died later with 3. Result During study period, a total of 250 cases of STS were en-
listed. Out of which, 82 were STS of extremities. A cohort of
43 patients was analyzed as 39 cases either visited for opinion
with diagnosis done outside or did not opt for surgical
treatment in this center. Median age of presentation was 48
years (range 11–75 years), and there was noticeable male
dominance (male : female 28 :15). Commonest histology
noted was pleomorphic sarcoma (9/43), followed by synovial
(7/43), and liposarcoma (6/43). Lower limb showed pref-
erential site of occurrence in comparison to upper limb. Seven cases presented with metastatic disease with lung
being the most common site (5/7). Two cases of each Sarcoma 3 Table 2: Treatment modalities. Modalities
Nonmetastatic disease
Metastatic disease
Surgery
Wide local excision
33
4
Amputation
3
2
Margin positivity
3
—
Chemotherapy
First line
—
6
Adjuvant
18
4
Palliative
—
1
Radiotherapy
27
3
Relapse of disease/progressive disease
4
4 Modalities
Nonmetastatic disease
Metastatic disease
Surgery
Wide local excision
33
4
Amputation
3
2
Margin positivity
3
—
Chemotherapy
First line
—
6
Adjuvant
18
4
Palliative
—
1
Radiotherapy
27
3
Relapse of disease/progressive disease
4
4 Nonmetastatic disease Metastatic disease Cum survival
Time
0.00
0.0
0.2
0.4
0.6
0.8
1.0
20.00
40.00
60.00
80.00
Censored
Survival function
Survival function
Figure 1: Kaplan–Meier curve showing overall survival (OS). Cum survival
Time
0.00
0.0
0.2
0.4
0.6
0.8
1.0
20.00
40.00
60.00
80.00
Censored
Survival function
Survival function
Figure 1: Kaplan–Meier curve showing overall survival (OS). recurrence of disease while other is alive till analysis. All 3
received adjuvant chemotherapy and radiotherapy to the
local site. Postoperative complications noted amongst 3
cases in the form of wound dehiscence in two and partial
graft necrosis in one case. g
Radiotherapy was administered to 27 cases in adjuvant
setting. Indication of radiotherapy involved surgical margin
and/or intermediate to high grade of tumor. One case re-
ceived upfront radiotherapy to primary and metastatic site
as palliative care. In metastatic STS cohort, adjuvant ra-
diotherapy was administered to 3 cases at primary site and 2
cases at metastasis site after good response to chemotherapy. Te dose of radiotherapy was uniform to all 60–64 Gy in
2 Gy/day fractionation (external beam radiotherapy using
linear accelerator with CT-based three dimensional con-
formal treatment planning), given in two phases. 3. Result Phase I
included tumor bed, postoperative scar, all drainage sites,
and 3-4 cm margin in longitudinal plane and 1.5–2 cm
margin in transverse plane with a dose of 44–46 Gy. Phase II
was delivered for remaining dose on reduced field (tumor
bed + 2 cm margin). Chemotherapy was administered to 6
metastatic cases upfront and 18 as adjuvant (ifosphamide-
Adriamycin). One patient received low-dose chemotherapy
with palliative goal (etoposide-Adriamycin). Figure 1: Kaplan–Meier curve showing overall survival (OS). 18 cases did not turn up for routine follow-up. Tele-
phonic and postal communications resulted in noting
outcomes among 5 cases; 13 cases could not be reached and
were censored at last point of contact. Among 36 localized
sarcomas, recurrence of disease was noted among 4 cases. Recurrence at primary site alone was noted among 3 cases
and at lung alone in 1 case. Good locoregional control mea-
sures (surgery±radiotherapy) resulted in low local recurrence. Of 7 primary metastatic cases, 1 opted for palliative care and
died at 4 months. Of remaining 6 cases, three were surviving at
the time of analysis, and 3 got recurrences (lung) and died later. With a median follow-up of 47 months (range 7–68 months),
the overall survival and event-free survival were noted as 47.64%
and 41.49%, respectively (Figures 1 and 2). extremities [4, 5]. With reason unexplained, lower limb is
a more preferred site of occurrence. Probably, large bulk of
soft tissue at thigh and leg compartments is the reason. An
unusual trend of early age presentation was noted in this
study. Male sex has been reported more frequently with STS
although this result is not consistent [6–8] Te study group
also noted 72% of STS occurrence in lower extremity and
male predominance. However, in comparison to all sarco-
mas reported in center, limb sarcoma constituted only 32%. Cohort noted pleomorphic sarcoma as the most common
variant followed by synovial sarcoma. Te sample size is
small to comment on incidence in general. Diagnosis of STS of extremities is often delayed. Studies
have tried to devise predictive clinical features for occur-
rence of STS. Swelling exceeding 5 cm, increase in size, pain,
and deep location confer more chances of STS in the swelling
[9]. Such swelling must be promptly examined by histo-
pathology for STS. Fine-needle aspiration is not a preferred
modality, and an excisional biopsy or punch biopsy should
be done [10]. 3. Result In this study, as standard protocol, all diagnosis 4. Discussion Soft tissue sarcoma of adult encompasses wide histological
variants. Te most frequent sarcomas noted are liposarcoma,
fibrosarcoma, and pleomorphic sarcoma. It can occur
throughout the body; however, majority (60%) occurs in Sarcoma 4 effects to nearby structures [19]. A neoadjuvant chemo-
radiotherapy followed by surgery and postoperative che-
motherapy has shown comparable disease control and
practically no negative impact of delay in surgery due to
intensive neoadjuvant modality [20, 21]. Study cohort noted
postoperative radiotherapy in 27 cases. It was given to either
patient with involved margin or with intermediate-/high-
grade feature. Radiotherapy was not given among low-grade
tumors that underwent wide local excision, and margins
were negative. Chemotherapy has been noted to have limited
role except rhabdomyosarcoma and Ewing’s group of sar-
coma. Various trials have noted conflicting benefit of
neoadjuvant/adjuvant chemotherapy with no benefit in
overall survival [22, 23]. For locally advanced STS, reports of
regional hyperthermia in combination of chemotherapy
have shown better local control and outcome as well. Similarly, option of isolated limb perfusion of affected ex-
tremity with chemotherapeutic agents has been reported for
primary inoperable limb sarcomas [24–26]. Survival function Cum survival
Time
0.00
0.0
0.2
0.4
0.6
0.8
1.0
20.00
40.00
60.00
80.00
Censored
Survival function
Survival function
Figure 2: Kaplan–Meier curve showing event-free survival (EFS). Extremities sarcoma has been noted to have better out-
come, and the most likely explanation is complete surgical
excision. Various studies have noted survival at 5 years as
65–75% with local recurrence rate of about 10% [27–29]. SEER data showed 3-year overall survival of 73% if radio-
therapy was administered in adjuvant setting in high-risk STS
[30]. Present study noted inferior overall and event-free
survival. Censoring of cases at last follow-up and large
number of lost to follow-up might be affecting the actual
survival result. As this study analyzed population of hilly
terrain, lost to follow-up was significant. In this study, tele-
phonic and postal communication was used to ensure proper
follow-up; however, a robust use of telemedicine and mes-
saging by social networking would have been more useful. Study noted large number of high-risk STS which might have
contributed to inferior outcome as well. Tis study presents
the experience of extremity STS from a tertiary referral cancer
center at sub-Himalayan region. Te number of cases is small
to draw any definitive conclusion regarding treatment efficacy
or environmental effect on outcome. Disclosure Earlier version of part of this work was presented as an
abstract at the 2nd Indian Cancer Congress 08–12 November,
2017, Bengaluru, and published as an abstract in Journal of
Cancer Research & Terapeutics, 2017 supplement, vol. 13,
pS369, 1/4p. Role of adjuvant radiotherapy after a wide local excision
is well established in high-grade and intermediate-grade
STS. Tis multimodality achieves about 95% of local con-
trol; however, benefit in terms of overall survival is uncertain
[14, 15]. Radiotherapy alone after a marginal resection is
again inferior to reresection. Te timing of radiotherapy is
also controversial with no superiority of preoperative, or
postoperative radiotherapy over another [16–18]. While
preoperative radiotherapy makes tumor resectable, a chance
of increased wound complications cannot be negated. Trials
of perioperative radiotherapy with brachytherapy have been
shown comparable to local control with limited radiation Conflicts of Interest Te authors declare that they have no conflicts of interest
regarding this study. 4. Discussion Tere is a need of
pooling data from Himalayan/sub-Himalayan region to
delineate effects of this geographical factor. Figure 2: Kaplan–Meier curve showing event-free survival (EFS). was made on histopathology on tissues retrieved from in-
cision or core needle biopsy and confirmed with immu-
nohistochemistry in majority. Surgical excision of tumor with wide margin, 4-5 cm to
the sides and 1-2 cm deep to the tumor, is desired. Curative
chances are majorly guided by a complete excision. If his-
topathology
result
shows
narrow
or
positive
margin,
a resurgery to achieve a wide margin has clearly shown
survival benefit over radiotherapy to margin [11, 12]. A re-
resection was advised to cases with positive margin in this
cohort; however, it was done in one case only. Re-resection
was not technically feasible in one, and resurgery was declined
by another. Amputation surgery has a very limited role in
current era. In 90% of extremity STS, limb-sparing excision
has been found feasible with local failure rate not exceeding
10% [13]. An infiltration of major blood vessel or nerve of
limb practically makes limb salvage impossible. A plan for
reconstruction with plastic surgery team is mandatory for
amputation. Study cohort noted five cases of amputation,
which happened in either nonsalvageable neurovascular in-
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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. * Correspondence: Domenico.Mangino@ulss12.ve.it
3Department of Cardiac Surgery, Ospedale dell’Angelo, Venezia-Mestre, Italy
Full list of author information is available at the end of the article Background Papillary fibroelastoma is the third most common pri-
mary cardiac benign tumor with an incidence of up to
0.33% in autopsy series; it accounts for approximately
75% of all cardiac valvular tumors and affects men and
women equally with a mean age of 60 years at diagnosis. Despite the benign nature of this tumor, it carries very
high risk of embolic complications. Here we described a
case of stroke in a 28-year-old man due to cerebral
embolization
originated
from
a
cardiac
papillary
fibroelastoma. Nothing of significant emerged in the past medical his-
tory. Physical examination confirmed the neurological
deficit, blood pressure was 120/80, heart rate 90 bpm,
no carotid bruit detected. Blood tests (blood count, kid-
ney and liver function, coagulation and electrolytes)
were normal. Electrocardiography (ECG) showed normal
sinus rhythm. Computer Tomography (CT) of the brain,
performed in urgency, was normal. A Magnetic Reson-
ance (MRI) of the brain showed areas of recent ischemia
in the left cerebral hemisphere with more extensive in-
volvement of the putamen, globus pallidus and temporal
and parietal cortical-subcortical posterior lobes (Fig. 1
panel a and b). Smaller areas of similar meaning were
detectable always on the left, adjacent parietal-occipital
sulcus and on the right in the frontal and cerebellar
regions. Some small areas compatible with vascular
outcomes were found in the cerebellum. Magnetic
Resonance Angiography (MRA) showed thrombosis of Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33
DOI 10.1186/s13019-017-0592-6 Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33
DOI 10.1186/s13019-017-0592-6 Abstract Background: Papillary fibroelastoma is the third most common primary benign tumor with an incidence of up to
0.33% in autopsy series; it accounts for approximately 75% of all cardiac valvular tumors. Case presentation: We describe a rare case of a 28-Year-old man that while playing football, had a sudden onset
of neurological deficit: aphasia, right hemiparesis and right facial numbness. Transthoracic echocardiography (TTE)
showed a 10x10 mm mass attached to the anterior mitral valve leaflet. The patient was treated surgically for the
prevention of further embolic complications. Histologic examination of the resected mass revealed a papillary
fibroelastoma. It is the third most frequent primary cardiac tumor, after myxoma and fibroma, and the most
common primary tumor of heart valves. Despite the benign nature of this tumor, it carries very high risk of embolic
complications. The successful complete resection of the papillary fibroelastoma is curative and the long-term
postoperative prognosis is excellent. Conclusions: Differential diagnosis of cardiac masses requires clinical informations, laboratory tests, blood cultures
and appropriate use of imaging modalities. Papillary fibroelastoma is a potential cause of embolic stroke in the
young. The prompt surgical excision of papillary fibroelastoma is curative and the long-term postoperative
prognosis is excellent. Keywords: Papillary fibroelastoma, Cardiac tumors, Cardioembolic stroke, Cerebrovascular disease, Case report Keywords: Papillary fibroelastoma, Cardiac tumors, Cardioembolic stroke, Cerebrovasc while playing football. He was not an athlete and never
performed a medical examination for sports fitness. Case presentation A 28-years-old man was admitted to the Emergency
Department of our Hospital for the sudden onset of
aphasia, right facial numbness and right hemiparesis, Page 2 of 5 Page 2 of 5 Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33 mechanical thrombectomy was not considered as an
adjuntive treatment either. Fig. 1 Brain Magnetic Resonance shows areas of recent ischemia in
the left cerebral hemisphere with more extensive involvement of the
putamen, globus pallidus and temporal and parietal cortical-subcortical
posterior lobes Transthoracic echocardiography (TTE) showed a 10 ×
10 mm mass attached to the anterior mitral valve leaflet,
fluttering into the cardiac chambers. (Fig. 3 panel a and b). There were no evidence of valvular stenosis, regurgitation,
or left ventricular outflow tract obstruction and left ven-
tricular function was normal. A transesophageal echocardi-
ography (TEE) was also performed: it confirmed the
presence of a hyper echogenic nodular and mobile mass,
attached to the atrial side of A3 scallop of the anterior
mitral leaflet; it also allowed to rule out other causes of em-
bolism such as patent foramen oval or cardiac thrombosis
(Fig. 4). A cardiac MRI confirmed the presence of the
lesion, with a maximum size equal to about 1 cm (Fig. 5). ECG monitoring during hospitalization in Stroke Unit and
the Holter ECG revealed no arrhythmias. Fig. 1 Brain Magnetic Resonance shows areas of recent ischemia in
the left cerebral hemisphere with more extensive involvement of the
putamen, globus pallidus and temporal and parietal cortical-subcortical
posterior lobes The decision was to perform the excision of the mass. We considered the possibility to remove it in the cardiac
catheterization laboratory but the mass was apparently
too big and indented to be removed with out risk of
embolization. To avoid it we decided to perform an
open heart surgical excision. The operation was per-
formed under normothermic cardiopulmonary bypass the left middle cerebral artery (Fig. 2). The patient was
hospitalized in Stroke Unit and in accordance with the
guidelines was treated with thrombolysis with r-TPA
(0.9 mg/kg). Mechanical thrombectomy was not consid-
ered as the first choice of treatment since the patient did
not have contraindications to thrombolysis; given the
good
clinical
response
to
thrombolytic
treatment, Fig. 3 Transthoracic echocardiogram. Case presentation The long axis (panel a) and
short axis views (panel b) show the hyperechogenous mass (white
arrow) attached to atrial side of A3 scallop of the anterior
mitral leaflet Fig. 2 Epiaortic Magnetic Resonance Angiography shows an
occlusion at the origin of the left middle cerebral artery Fig. 2 Epiaortic Magnetic Resonance Angiography shows an
occlusion at the origin of the left middle cerebral artery Fig. 3 Transthoracic echocardiogram. The long axis (panel a) and
short axis views (panel b) show the hyperechogenous mass (white
arrow) attached to atrial side of A3 scallop of the anterior
mitral leaflet Fig. 2 Epiaortic Magnetic Resonance Angiography shows an
occlusion at the origin of the left middle cerebral artery Page 3 of 5 Page 3 of 5 Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33 using ascending aortic and bicaval cannulation. After
cardiac arrest with antegrade cardioplegia, left atrium
was opened A 1-cm, gelatinous and solid looking mass
was found attached to the anterior leaflet of mitral valve
near
the
posteromedial
mitral
commisure. It
was
resected with its stalk (Fig. 6). Post-operative TEE con-
firmed normal valvular functions and absence of residual
left atrial mass. The histological examination of the
tissue revealed a papillary fibroelastoma. The early post-
operative period was uncomplicated, and the patient was
discharged on postoperative day-10. Actually remains a
permanent nuanced right hemisyndrome. A 1-month
postoperative echocardiography control showed perfect
valve function with no residual mitral regurgitation or
stenosis. Fig. 4 Transesophageal echocardiogram. The intercommissural view
(76°) shows a mobile nodular mass, measuring 1 × 1 cm, on the
anterior leaflet of the mitral valve Fig. 4 Transesophageal echocardiogram. The intercommissural view
(76°) shows a mobile nodular mass, measuring 1 × 1 cm, on the
anterior leaflet of the mitral valve Discussion Here we described a rare and very special case of stroke
in a 28-year-old man due to cerebral embolization origi-
nated from a cardiac papillary fibroelastoma. Stroke in
the young, with no obvious risk factors, usually requires
extensive evaluation: it is important to look for second-
ary
cause
of
cerebral
ischemia,
searching
carefully
especially diseases with potential cardioembolic. Fig. 5 The Cardiac MRI shows a round mass adhering to the mitral valve Papillary fibroelastoma is the third most common pri-
mary benign tumor with an incidence of up to 0.33% in
autopsy series; it accounts for approximately 75% of all
cardiac valvular tumors and affects men and women
equally with a mean age of 60 years at diagnosis. According to epidemiological data described cases rarely
involve young patients [1, 2]. Althought papillary fibroe-
lastomas are histologically benign neoplasms, they may
result in life-threatening complications if valve obstruc-
tion or systemic embolization occurs, as described in
our patient. Fig. 6 In the intraoperative view the lesion appears as a white round
mass, without a peduncle. The gross specimen of the mass
demonstrated gelatinous appearance and smooth surface, with many
white papillary fragments with frond-like projections, typical of
papillary fibroelastoma Fig. 6 In the intraoperative view the lesion appears as a white round
mass, without a peduncle. The gross specimen of the mass
demonstrated gelatinous appearance and smooth surface, with many
white papillary fragments with frond-like projections, typical of
papillary fibroelastoma Fig. 6 In the intraoperative view the lesion appears as a white round
mass, without a peduncle. The gross specimen of the mass
demonstrated gelatinous appearance and smooth surface, with many
white papillary fragments with frond-like projections, typical of
papillary fibroelastoma Fig. 5 The Cardiac MRI shows a round mass adhering to the mitral valve Page 4 of 5 Page 4 of 5 Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33 Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33 Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33 Most patients are asymptomatic, but some patients
may experience cerebral embolic symptoms, such as
stroke or transient ischemic attack or angina, acute
coronary syndrome, myocardial infarction or death from
coronary ostial obstruction [3–25]: transient ischemic
attack/cerebrovascular accident is considered by far the
most common presentation of papillary fibroelastoma
[26]. Ethics approval and consent to participate Ethics approval and consent to participate Ethics approval and consent to participate Ethics approval and consent to participate
Not applicable (This is a Clinical Case Report and not a Study). Funding y
All data are in the medical record of the patient. References 1. Smajlović D. Strokes in young adults: epidemiology and prevention. Vasc
Health Risk Manag. 2015;11:157–64. 1. Smajlović D. Strokes in young adults: epidemiology and prevention. Vasc
Health Risk Manag. 2015;11:157–64. 2. Edwards FH, Hale D, Cohen A, Thompson L, Pezzella AT, Virmani R. Primary
cardiac valve tumors. Ann Thorac Surg. 1991;52:1127–31. 2. Edwards FH, Hale D, Cohen A, Thompson L, Pezzella AT, Virmani R. Primary
cardiac valve tumors. Ann Thorac Surg. 1991;52:1127–31. 3. Abbasi AS, Da Costa M, Hennessy T, Kiernan TJ. Cardiac papillary
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published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Discussion This tumor has a predilection for the left side of
the heart: the aortic valve is the predominant site in-
volved, followed by mitral leaflets. Grossly, papillary
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usually has a gelatinous membrane on the surface and a
stalk with multiple delicate papillary projections, best
appreciated by immersing the specimen in water [27]. Microscopically, it is characterized by a collection of
avascular fronds of dense connettive tissue lined by
endothelium and may arise from any endocardial sur-
face. Embolic fragments may originate from the tumor
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texture, or from surface formation of platelet and fibrin
thrombi [28]. Most are solitary and small, some are mo-
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of papillary fibroelastomas [30]. Due to their small size
generally 0.5 to 2.0 cm in diameter and their valvular in-
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distinguish from valvular vegetation. For this reason,
clinical informations, laboratory tests and blood cultures
are extremely important for differential diagnosis. The
differential diagnosis includes the presence of mixoma,
lipoma, rhabdomyoma or amorphous tumors. Since
symptomatic papillary fibroelastoma carries a definite
risk of severe complications, aggressive surgical manage-
ment is recommended, irrespective of the tumor’s size
or the patient’s symptoms [26], the successful complete
resection of the papillary fibroelastoma is curative and
the long-term postoperative prognosis is excellent. The
patients who are not surgical candidates could be offered
long-term oral anticoagulation, although non random-
ized controlled data are available on its efficacy. Funding
This study received no external funding. 9.
Zhang M, Liu X, Song Z, Zou L, Xiang B. Cardiac papillary fibroelastom
retrospect of four cases. J Cardiothorac Surg. 2013;8:65. Authors’ contributions All the authors have made substantial contributions in data interpretation,
and revised the manuscript critically. All authors read and approved the final
manuscript. Competing interests
The authors declare that they have no competing interest. Competing interests
The authors declare that they have no competing interest. 8.
Santos AF, Pinho J, Ramos V, Pardal J, Rocha J, Ferreira C. Stroke and cardiac
papillary fibroelastoma: mechanical thrombectomy after thrombolytic
therapy. J Stroke Cerebrovasc Dis. 2014;23:1262–4. Competing interests Consent for publication
Written informed consent for publication of his clinical details and clinical
images was obtained from the patient. Author details
1 1Department of Cardiology, Ospedale dell’Angelo, Venezia-Mestre, Italy. 2Department of Internal Medicine, Angiology Unit, Ospedale dell’Angelo,
Venezia-Mestre, Italy. 3Department of Cardiac Surgery, Ospedale dell’Angelo,
Venezia-Mestre, Italy. 4Department of Neurology, Ospedale dell’Angelo,
Venezia-Mestre, Italy. Received: 29 November 2016 Accepted: 10 May 2017 Received: 29 November 2016 Accepted: 10 May 2017 7.
Floria M, Gerard M, Louagie Y, Weynand B, Schroeder E. Double papillary
fibroelastoma: beautiful, innocent flowers in the left heart. J Clin Ultrasound.
2014;42:574–5. Consent for publication Ethics approval and consent to participate
Not applicable (This is a Clinical Case Report and not a Study). Conclusions 8. Santos AF, Pinho J, Ramos V, Pardal J, Rocha J, Ferreira C. Stroke and cardiac
papillary fibroelastoma: mechanical thrombectomy after thrombolytic
therapy. J Stroke Cerebrovasc Dis. 2014;23:1262–4. Echocardiography transthoracic and transesophageal are
essential investigations in young patients with stroke. Differential diagnosis of cardiac masses requires clinical
informations, laboratory tests, blood cultures and appro-
priate use of imaging modalities. Papillary fibroelastoma
is a potential cause of embolic stroke in the young. The
prompt surgical excision of papillary fibroelastoma is
curative and the long-term postoperative prognosis is
excellent. 9. Zhang M, Liu X, Song Z, Zou L, Xiang B. Cardiac papillary fibroelastoma: a
retrospect of four cases. J Cardiothorac Surg. 2013;8:65. 10. Val-Bernal JF, Mayorga M, Garijo MF, Val D, Nistal JF. Cardiac papillary
fibroelastoma: retrospective clinicopathologic study of 17 tumors with
resection at a single institution and literature review. Pathol Res Pract. 2013;
209:208–14. 11. Kim SY, Park TH, Lee DY, Lee DH, Cho YR, Kim MH, et al. Papillary
fibroelastoma mimicking vegetation of the mitral valve. J Cardiovasc
Ultrason. 2012;20:213–5. Page 5 of 5 Page 5 of 5 Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33 12. Ljevak J, Mišmaš A, Bazina A, Matijević V, Alvir D, Supe S, et al. An infrequent
type of stroke with an unusual cause and successful therapy: basilar artery
occlusion caused by a cardiac papillary fibroelastoma recanalized 12 h after
onset. Intern Med. 2013;52:277–9. 13. van der Meulen TA, Budde RP, Randjgari A, de Heer LM. Multimodality
imaging of a papillary fibroelastoma of the mitral valve. Eur J Cardiothorac
Surg. 2012;42:747–8. 14. Gallanagh S, Walters M, Mallon E, Sonnecki P. Fibroelastoma of the mitral
valve as a cause of embolic cerebral infarction. BMJ Case Rep. 2011. doi:10. 1136/bcr.02.2011.3855. 15. Huang H, Falik R. Papillary fibroelastoma of the subvalvular apparatus of the
mitral valve found on echocardiography after the clinical presentation of
embolic CVA. Am J Med. 2010;123:e7–8. 16. Corrado G, Panisi P, Checcarelli N, Ambrosiani L. An unusual cause of
ischemic stroke with successful thrombolysis. J Stroke Cerebrovasc Dis. 2013;22:e691–2. 17. Gagliardi RJ, Franken RA, Protti GG. Cardiac papillary fibroelastoma and stroke
in a young man—etiology and treatment. Cerebrovasc Dis. 2008;25:185–7. 18. Liebeskind DS, Buljubasic N, Saver JL. Cardioembolic stroke due to papillary
fibroelastoma. J Stroke Cerebrovasc Dis. 2001;10(2):94–5. fibroelastoma. J Stroke Cerebrovasc Dis. 2001;10(2):94–5 19. Dehnee AE, Brizendine S, Herrera CJ. Grolla et al. Journal of Cardiothoracic Surgery (2017) 12:33 Conclusions Recurrent strokes in a young patient
with papillary fibroelastoma: a case report and literature review. Echocardiography. 2006;23:592–5. 20. Saw W, Nicholls S, Trim G, Thomson D, Hughes C, Mitchell S, et al. Papillary
fibroelastoma, a rare but potentially treatable cause of embolic stroke:
report of three cases. Heart Lung Circ. 2001;10:105–7. 21. Baba Y, Tsuboi Y, Sakiyama K, Nakajima M, Fjino Y, Meschia JF, et al. Cardiac
papillary fibroelastoma as a cause of recurrent ischemic strokes: the
diagnostic value of serial transesophageal echocardiography. Cerebrovasc
Dis. 2002;14:256–9. 22. Burri H, Vuille C, Sierra J. Papillary fibroelastoma as a cause of cardioembolic
stroke. Heart. 2002;88:216. 22. Burri H, Vuille C, Sierra J. Papillary fibroelastoma as a cause of cardioembolic
stroke. Heart. 2002;88:216. 23. Karaeren H, Ilgenli TF, Celik T, Deveci S, Kuralay E, Barçin C, et al. Papillary
fibroelastoma of the mitral valve with systemic embolization. Echocardiography. 2000;17:165–7. 23. Karaeren H, Ilgenli TF, Celik T, Deveci S, Kuralay E, Barçin C, et al. Papillary
fibroelastoma of the mitral valve with systemic embolization. Echocardiography. 2000;17:165–7. 24. Burke A, Virmani R. Tumors of the heart and great vessels. In Atlas of Tumor
Pathology. Third series. Washington, DC: Armed Forces Institute of
Pathology; 1996. p. 47–54. 25. Kassop D, Donovan MS, Cheezum MK, Nguyen BT, Gambill NB, Blankstein R,
et al. Cardiac masses on cardiac CT: a review. Curr Cardiovasc Imaging Rep. 2014;7:9281. 26. Howard RA, Aldea GS, Shapira OM, Kasznica JM, Davidoff R. Papillary
fibroelastoma: increasing recognition of a surgical disease. Ann Thorac Surg. 1999;68:1881–5. 27. Ragland MM, Tak T. The role of echocardiography in diagnosing space-
occupying lesions of the heart. Clin Med Res. 2006;4:22–32. 28. Kim EY, Choe YH, Sung K, Park SW, Kim JH, Ko YH. Multidetect 28. Kim EY, Choe YH, Sung K, Park SW, Kim JH, Ko YH. Multidetector CT and MR
imaging of cardiac tumors. Korean J Radiol. 2009;10:164–75. imaging of cardiac tumors. Korean J Radiol. 2009;10:164–75. 29. Shanian DM. Papillary fibroelastomas. Semin Thorac Cardiovasc Surg. 2000;
1103:167–9. 30. Gowda RM, Khan IA, Nair CK, Mehta NJ, Vasavada BC, Sacchi TJ. Cardiac
papillary fibroelastoma: a comprehensive analysis of 725 cases. Am Heart J. 2003;146:404–10. Submit your next manuscript to BioMed Central
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English
| null |
Peer Review #1 of "Could hand-eye laterality profiles affect sport performance? A systematic review (v0.1)"
| null | 2,022
|
cc-by
| 21,151
|
Background. Laterality effects on sports performance have been a field of interest for the sports sciences, especially in
asymmetrical sports, which require the preferential use of one side of the body. Some sports in particular
involve the visual system and ocular laterality, due to the need to clearly focus on a dynamic object (ball,
opponent, projectile, etc.). The relationship between manual and ocular laterality results in two
perceptual-motor profiles, one where the dominant hand and eye are ipsilateral (uncrossed hand-eye
laterality profile, UC-HELP), and the other where they are contralateral (crossed hand-eye laterality
profile, C-HELP). Miquel Moreno
1, 2 , Lluis Capdevila
Corresp., 1, 3 , Josep-Maria Losilla
3, 4 1 Laboratory of Sport Psychology, Department of Basic Psychology., Universitat Autónoma de Barcelona, Bellaterra, Catalunya, Spain
2 Department of Sport Sciences, Universitat de Vic, Vic, Barcelona, Catalonia, Spain
3 Sport Research Institute, Universitat Autónoma de Barcelona, Bellaterra, Catalunya, Spain 1 Laboratory of Sport Psychology, Department of Basic Psychology., Universitat Autónoma de Barcelona, Bellaterra, Catalunya, Spain
2 Department of Sport Sciences, Universitat de Vic, Vic, Barcelona, Catalonia, Spain
3 Sport Research Institute, Universitat Autónoma de Barcelona, Bellaterra, Catalunya, Spain
4 1 Laboratory of Sport Psychology, Department of Basic Psychology., Universitat Autónoma de Barcelo
2 Department of Sport Sciences, Universitat de Vic, Vic, Barcelona, Catalonia, Spain
3 Sport Research Institute, Universitat Autónoma de Barcelona, Bellaterra, Catalunya, Spain
4 1 Laboratory of Sport Psychology, Department of Basic Psychology., Universitat Autónoma de Barcelona, Bellaterra, Catalunya, Spain
2 Department of Sport Sciences, Universitat de Vic, Vic, Barcelona, Catalonia, Spain 2 Department of Sport Sciences, Universitat de Vic, Vic, Barcelona, Catalonia, Spain
3 Sport Research Institute, Universitat Autónoma de Barcelona, Bellaterra, Catalunya, Spain 4 Departament of Psychobiology and Methodology of Health Science, Universitat Autónoma de Barcelona, Bellater 4 Departament of Psychobiology and Methodology of Health Science, Universitat Autónoma de Barcelona, Bellaterra, Catalunya, Spain Corresponding Author: Lluis Capdevila
Email address: lluis.capdevila@uab.cat Corresponding Author: Lluis Capdevila
Email address: lluis.capdevila@uab.cat Methodology. A systematic reviewof the literaturewas carried out to determine the prevalence of hand-eye laterality
profiles inthe differentsports modalities and their relationship with psychological factors and sports
performance. Searches of PsycInfo, Medline, Scopus and grey literature identified 14 studies (2759
participants) regarding hand-eye laterality in sports that met the eligibility criteria. Results. Previous studies have estimated that between 10-30% of the general population exhibit a C-HELP, and
70-90% have an UC-HELP. The results of the reviewed studies indicate that in some sports the
percentage of C-HELP is higher in amateur and high-level athletes than in the normal population: golf
(52.55%), soccer (53%), tennis (42%) and team sports (50.7%). In target sports (archery and shooting)
athletes with an UC-HELP seem to have an advantage given the significant concentration of this profile in
the highest performing populations (82.3%). In basketball, cricket and golf, the literature reviewed also
reported biomechanical differences in the execution of some techniques between the two profiles. We did
not find any study in our review that related hand-eye laterality with cognitive, tactical, or psychological
aspects of athletes. Manuscript to be reviewed Conclusions. These results should be taken with great caution due to the potential bias linked to the methodologies
used in the investigations, the heterogeneity in the assessment of hand-eye laterality, the few studies
available on the subject and the indirect nature of many of the observed relationships between
PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) 1 Could hand-eye laterality profiles affect sport performance?
2 A systematic review. 19 Manuscript to be reviewed 1 Could hand-eye laterality profiles affect sport performance? 2 A systematic review. 3
4
Miquel Moreno 12, Lluis Capdevila 1,3, Josep-Maria Losilla 3,4
5
6
1 Laboratory of Sport Psychology, Department of Basic Psychology, Universitat Autònoma de
7
Barcelona (UAB), Bellaterra, Spain. 8
2Department of Sport Science, Universitat de Vic, Vic, Barcelona. 9
3 Sport Research Institute, Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain. 10
4 Departament of Psychobiology and Methodology of Health Science, Universitat Autònoma de
11
Barcelona (UAB), Bellaterra, Spain. 12
13
14
Corresponding Author:
15
Lluis Capdevila
16
Sport Research Institute, Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain. 17
Email address: lluis.capdevila@uab.cat
18
19 Manuscript to be reviewed performance and laterality. For further investigation, we propose a standardized terminology and
protocol of hand-eye laterality assessment in sports. The advancement in knowledge about hand-eye
laterality profiles, along with the study of the relationship with psychological or tactical-sports patterns,
can contribute to more effective development plans for athletes and can be a complement to talent
detection. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed 21
Abstract 21
Abstract
22
Background. Laterality effects on sports performance have been a field of interest for the spor
23
sciences, especially in asymmetrical sports, which require the preferential use of one side of the
24
body. Some sports in particular involve the visual system and ocular laterality, due to the need
25
clearly focus on a dynamic object (ball, opponent, projectile, etc.). The relationship between
26
manual and ocular laterality results in two perceptual-motor profiles, one where the dominant
27
hand and eye are ipsilateral (uncrossed hand-eye laterality profile, UC-HELP), and the other
28
where they are contralateral (crossed hand-eye laterality profile, C-HELP). 29
Methodology. A systematic review of the literature was carried out to determine the prevalenc
30
of hand-eye laterality profiles in the different sports modalities and their relationship with
31
psychological factors and sports performance. Searches of PsycInfo, Medline, Scopus and grey
32
literature identified 14 studies (2759 participants) regarding hand-eye laterality in sports that m
33
the eligibility criteria. 34
Results. Previous studies have estimated that between 10-30% of the general population exhib
35
a C-HELP, and 70-90% have an UC-HELP. The results of the reviewed studies indicate that in
36
some sports the percentage of C-HELP is higher in amateur and high-level athletes than in the
37
normal population: golf (52.55%), soccer (53%), tennis (42%) and team sports (50.7%). In targ
38
sports (archery and shooting) athletes with an UC-HELP seem to have an advantage given the
39
significant concentration of this profile in the highest performing populations (82.3%). In
40
basketball, cricket and golf, the literature reviewed also reported biomechanical differences in
41
the execution of some techniques between the two profiles. We did not find any study in our
42
review that related hand-eye laterality with cognitive, tactical, or psychological aspects of
43
athletes. 44
Conclusions. These results should be taken with great caution due to the potential bias linked t
45
the methodologies used in the investigations, the heterogeneity in the assessment of hand-eye
46
laterality, the few studies available on the subject and the indirect nature of many of the observ
47
relationships between performance and laterality. For further investigation, we propose a
48
standardized terminology and protocol of hand-eye laterality assessment in sports. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) 21
Abstract In
40
basketball, cricket and golf, the literature reviewed also reported biomechanical differences in
41
the execution of some techniques between the two profiles. We did not find any study in our
42
review that related hand-eye laterality with cognitive, tactical, or psychological aspects of
43
athletes. 44
Conclusions. These results should be taken with great caution due to the potential bias linked to
45
the methodologies used in the investigations, the heterogeneity in the assessment of hand-eye
46
laterality, the few studies available on the subject and the indirect nature of many of the observed
47
relationships between performance and laterality. For further investigation, we propose a
48
standardized terminology and protocol of hand-eye laterality assessment in sports. The
49
advancement in knowledge about hand-eye laterality profiles, along with the study of the
50
relationship with psychological or tactical-sports patterns, can contribute to more effective
51
development plans for athletes and can be a complement to talent detection. 52
53
54
Key words: hand-eye laterality, crossed laterality, sports performance, handedness, eyedness,
55
systematic review. 56
57 34
Results. Previous studies have estimated that between 10-30% of the general population exhibit
35
a C-HELP, and 70-90% have an UC-HELP. The results of the reviewed studies indicate that in
36
some sports the percentage of C-HELP is higher in amateur and high-level athletes than in the
37
normal population: golf (52.55%), soccer (53%), tennis (42%) and team sports (50.7%). In target
38
sports (archery and shooting) athletes with an UC-HELP seem to have an advantage given the
39
significant concentration of this profile in the highest performing populations (82.3%). In
40
basketball, cricket and golf, the literature reviewed also reported biomechanical differences in
41
the execution of some techniques between the two profiles. We did not find any study in our
42
review that related hand-eye laterality with cognitive, tactical, or psychological aspects of
43
athletes. 44
Conclusions. These results should be taken with great caution due to the potential bias linked to
45
the methodologies used in the investigations, the heterogeneity in the assessment of hand-eye
46
laterality, the few studies available on the subject and the indirect nature of many of the observed
47
relationships between performance and laterality. For further investigation, we propose a
48
standardized terminology and protocol of hand-eye laterality assessment in sports. 21
Abstract The
49
advancement in knowledge about hand-eye laterality profiles, along with the study of the
50
relationship with psychological or tactical-sports patterns, can contribute to more effective
51
development plans for athletes and can be a complement to talent detection. 52
53
54
Key words: hand-eye laterality, crossed laterality, sports performance, handedness, eyedness,
55
systematic review. 56 22
Background. Laterality effects on sports performance have been a field of interest for the sports
23
sciences, especially in asymmetrical sports, which require the preferential use of one side of the
24
body. Some sports in particular involve the visual system and ocular laterality, due to the need to
25
clearly focus on a dynamic object (ball, opponent, projectile, etc.). The relationship between
26
manual and ocular laterality results in two perceptual-motor profiles, one where the dominant
27
hand and eye are ipsilateral (uncrossed hand-eye laterality profile, UC-HELP), and the other
28
where they are contralateral (crossed hand-eye laterality profile, C-HELP). 29
Methodology. A systematic review of the literature was carried out to determine the prevalence
30
of hand-eye laterality profiles in the different sports modalities and their relationship with
31
psychological factors and sports performance. Searches of PsycInfo, Medline, Scopus and grey
32
literature identified 14 studies (2759 participants) regarding hand-eye laterality in sports that met
33
the eligibility criteria. 29
Methodology. A systematic review of the literature was carried out to determine the prevalence
30
of hand-eye laterality profiles in the different sports modalities and their relationship with
31
psychological factors and sports performance. Searches of PsycInfo, Medline, Scopus and grey
32
literature identified 14 studies (2759 participants) regarding hand-eye laterality in sports that met
33
the eligibility criteria. 32
literature identified 14 studies (2759 participants) regarding hand-eye laterality in sports that met
33
the eligibility criteria. 34
Results. Previous studies have estimated that between 10-30% of the general population exhibit
35
a C-HELP, and 70-90% have an UC-HELP. The results of the reviewed studies indicate that in
36
some sports the percentage of C-HELP is higher in amateur and high-level athletes than in the
37
normal population: golf (52.55%), soccer (53%), tennis (42%) and team sports (50.7%). In target
38
sports (archery and shooting) athletes with an UC-HELP seem to have an advantage given the
39
significant concentration of this profile in the highest performing populations (82.3%). 58
Introduction For example, Porac & Coren (1976) found that the C-HELP was more prevalent in
83
individuals manifesting a variety of behavioral disorders, and Nagae (1983) showed that C-
84
HELP children performed significantly worse at verbal self-regulation of motor behavior,
85
supporting the view that the functions of cerebral hemispheres in C-HELP children were more
86
immature and linked with learning disabilities. However, a meta-analysis by Bourassa, Bryden &
87
MacManus (1996) with 54087 participants from 47 studies on hand-eye laterality did not find
88
enough evidence to associate hand-eye laterality with learning and indicated the necessity of
89
conducting more research in the field. In a more recent systematic review, Ferrero, Vadillo &
90
West (2017) also found a lack of scientific evidence on the relationship between C-HELPs,
91
academic achievement, and intelligence. 92
Determining the prevalence of C-HELPs in the general population has also been the subject of
93
various studies. Robinson, Jacobsen & Heintz (1997) compiled a multi-site sample of 1005
94
participants and reported a C-HELP prevalence of 41.4%. The above cited meta-analysis by
95
Bourassa et al. (1996) found a 34.8% prevalence of C-HELPs. In another meta-analysis with
96
10635 participants from 14 studies, MacManus et al. (1999) used the throwing hand and the
97
writing hand as criteria to assess handedness and observed a C-HELP prevalence of 25.4% with
98
respect to the throwing hand and of 25.8% with respect to the writing hand. 99
Sports that are considered asymmetric have been more deeply studied since they imply the
100
preferential use of one of the two sides of the body to throw, hit or use implements. These
101
include tennis (Ziagkas, Mavvidis & Georgios, 2018), golf (Dalton, Guillon & Naroo, 2015; 59
Laterality is the preferential use of one part of the body with respect to its symmetrical side. This
60
phenomenon has been a subject of scientific interest and it’s been researched in fields like
61
biology and psychology (e.g., MacManus, 2002; Rogers, Vallortigara & Andrew, 2013). The
62
relationship between two types of laterality is examined here: handedness, commonly defined as
63
the preference of one hand over the other in unimanual tasks (Scharoun & Byden, 2014); and
64
eyedness or eye-dominance, the preference for visual input from one eye over the other. 21
Abstract The
49
advancement in knowledge about hand-eye laterality profiles, along with the study of the
50
relationship with psychological or tactical-sports patterns, can contribute to more effective
51
development plans for athletes and can be a complement to talent detection. 57 PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) 58
Introduction 59
Laterality is the preferential use of one part of the body with respect to its symmetrical side. This
60
phenomenon has been a subject of scientific interest and it’s been researched in fields like
61
biology and psychology (e.g., MacManus, 2002; Rogers, Vallortigara & Andrew, 2013). The
62
relationship between two types of laterality is examined here: handedness, commonly defined as
63
the preference of one hand over the other in unimanual tasks (Scharoun & Byden, 2014); and
64
eyedness or eye-dominance, the preference for visual input from one eye over the other. The
65
dominant eye provides more input to the visual cortex and relays information more accurately,
66
such as the location of objects, and it is observed when monocular images cannot be fused or
67
when monocular viewing is required (Valle-Inclan et al., 2008). The first publication regarding
68
the relationship between handedness and eyedness dates back to the 16th century, when Porta
69
(1593) defined hand-eye laterality profiles and introduced the first eyedness measurement test. 70
This relationship is significant for activities that require coordination of the eyes (as receptor
71
organs) and the limbs (as effector organs) for accurate response. In this kind of task, manual
72
responses are lateralized in the contralateral hemisphere while the dominant eye is functionally
73
connected to the ipsilateral hemisphere (Azémar, Stein & Ripoll, 2008). There are two types of
74
hand-eye laterality profiles: one results from having the same side of preference for both hand
75
and eye (uncrossed hand-eye laterality profile, UC-HELP), and the other from having eye and
76
hand preference on different sides of the body (crossed hand-eye laterality profile, C-HELP). 77
Ever since Orton (1925) pointed out a relationship between C-HELPs and reading difficulties in
78
children, crossed laterality has received considerable study in the field of literacy which supports
79
the association between C-HELPs and neurological problems that may result in poor reading
80
performance (e.g., Orton, 1937; Vernon, 1971; Kershner, 1975; Abigail & Johnson, 1976;
81
Richardson & Firlej, 1979). Some studies have linked C-HELPs with specific cognitive
82
disorders. Manuscript to be reviewed The present systematic review aims to analyze the literature available to date on
128
hand-eye laterality profiles in the different sports modalities, with three specific objectives: a) to
129
estimate the prevalence of hand-eye laterality profiles, b) to examine the relationship between
130
hand-eye laterality profiles, psychological factors and sports performance, and c) to propose a
131
methodological and terminological consensus. 132
133
Methods
134
The protocol for this systematic review was registered with the International Platform of
135
Registered Systematic Review and Meta-Analysis Protocols (INPLASY) on 28 November 2020
136
(registration number INPLASY2020110127; doi:10.37766/inplasy2020.11.0127). The study was
137
undertaken in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-
138
Analyses (PRISMA 2020) statement (Page et al, 2021a, 2021b). The Ethics Commission for
139
Human Experimentation of The Universitat Autònoma de Barcelona granted Ethical approval to
140
carry out the study (protocol code CEEAH-5745)
141
142
Search strategy
143
Literature searches were performed using the following databases: PsycInfo by EBSCOhost,
144
Scopus by Elsevier, Medline by PubMed, and Dissertations & Theses Global by ProQuest. To 102
Sugiyama & Lee, 2005), baseball (Laby et al., 1998; Classe et al., 1996; Portal & Romano,
103
1998), cricket (Thomas, Harden & Rogers, 2005) and basketball (Shick, 1971, 1977; Lopez-Diaz
104
et al., 2015). Several studies have analyzed the relation between the distribution of laterality
105
profiles and their effects on sports performance. Azemar (2003), in a survey of 1707 participants
106
(including 229 normal controls, 1126 sports students and 352 elite athletes), observed that the
107
prevalence of C-HELP was significantly higher in tennis, fencing, boxing and gymnastics, and
108
significantly lower in archery, when compared to normal population values. These authors also
109
pointed to a significantly higher percentage of C-HELPs in duel or adversary sports (47.8% in
110
tennis, fencing and boxing) compared with non-adversary (35% C-HELPs in gymnastics and
111
archery). Significant differences between sports modalities have also been reported in a study
112
from Quevedo et al. (2014) with a sample of 536 elite multi-sport athletes, where a C-HELP
113
prevalence of 55% (95% CI: 44.03%, 65.97%) was observed in golf, compared to a prevalence
114
of 9% (95% CI: 2.69%, 15.31%) in shooting. Some authors have hypothesized about specific
115
physiological advantages for the performance of certain tasks in C-HELP subjects. Manuscript to be reviewed 102
Sugiyama & Lee, 2005), baseball (Laby et al., 1998; Classe et al., 1996; Portal & Romano,
103
1998), cricket (Thomas, Harden & Rogers, 2005) and basketball (Shick, 1971, 1977; Lopez-Diaz
104
et al., 2015). Several studies have analyzed the relation between the distribution of laterality
105
profiles and their effects on sports performance. Azemar (2003), in a survey of 1707 participants
106
(including 229 normal controls, 1126 sports students and 352 elite athletes), observed that the
107
prevalence of C-HELP was significantly higher in tennis, fencing, boxing and gymnastics, and
108
significantly lower in archery, when compared to normal population values. These authors also
109
pointed to a significantly higher percentage of C-HELPs in duel or adversary sports (47.8% in
110
tennis, fencing and boxing) compared with non-adversary (35% C-HELPs in gymnastics and
111
archery). Significant differences between sports modalities have also been reported in a study
112
from Quevedo et al. (2014) with a sample of 536 elite multi-sport athletes, where a C-HELP
113
prevalence of 55% (95% CI: 44.03%, 65.97%) was observed in golf, compared to a prevalence
114
of 9% (95% CI: 2.69%, 15.31%) in shooting. Some authors have hypothesized about specific
115
physiological advantages for the performance of certain tasks in C-HELP subjects. For example,
116
Azemar and Ripoll (1987) observed a visuo-motor advantage in response time for C-HELP
117
subjects compared to UC-HELPs in laboratory experiments with spatio-temporal tasks. 118
Dorochenko (2009) also raised the possibility of the existence of differences in personality and
119
mental performance to explain a hypothetical over-representation of C-HELPs in the sport of
120
tennis. In this same sense, Laborde et al. (2009) reported that knowledge of hand-eye laterality
121
could be reliably used to advise sports training to enable more efficient adaptations in talent
122
detection, learning skills and in achieving better levels of coordination. Nevertheless, Laby and
123
Kirschen (2011) have warned about the lack of consensus among researchers on whether C-
124
HELPs or UC-HELPs could be advantageous in various sports. 125
More research is needed to determine the practical applications of hand-eye laterality in training
126
and to clarify the differences in hand-eye laterality profiles reported so far between sports
127
modalities. 58
Introduction The
65
dominant eye provides more input to the visual cortex and relays information more accurately,
66
such as the location of objects, and it is observed when monocular images cannot be fused or
67
when monocular viewing is required (Valle-Inclan et al., 2008). The first publication regarding
68
the relationship between handedness and eyedness dates back to the 16th century, when Porta
69
(1593) defined hand-eye laterality profiles and introduced the first eyedness measurement test. 70
This relationship is significant for activities that require coordination of the eyes (as receptor
71
organs) and the limbs (as effector organs) for accurate response. In this kind of task, manual
72
responses are lateralized in the contralateral hemisphere while the dominant eye is functionally
73
connected to the ipsilateral hemisphere (Azémar, Stein & Ripoll, 2008). There are two types of
74
hand-eye laterality profiles: one results from having the same side of preference for both hand
75
and eye (uncrossed hand-eye laterality profile, UC-HELP), and the other from having eye and
76
hand preference on different sides of the body (crossed hand-eye laterality profile, C-HELP). 77
Ever since Orton (1925) pointed out a relationship between C-HELPs and reading difficulties in
78
children, crossed laterality has received considerable study in the field of literacy which supports
79
the association between C-HELPs and neurological problems that may result in poor reading
80
performance (e.g., Orton, 1937; Vernon, 1971; Kershner, 1975; Abigail & Johnson, 1976;
81
Richardson & Firlej, 1979). Some studies have linked C-HELPs with specific cognitive
82
disorders. For example, Porac & Coren (1976) found that the C-HELP was more prevalent in
83
individuals manifesting a variety of behavioral disorders, and Nagae (1983) showed that C-
84
HELP children performed significantly worse at verbal self-regulation of motor behavior,
85
supporting the view that the functions of cerebral hemispheres in C-HELP children were more
86
immature and linked with learning disabilities. However, a meta-analysis by Bourassa, Bryden &
87
MacManus (1996) with 54087 participants from 47 studies on hand-eye laterality did not find
88
enough evidence to associate hand-eye laterality with learning and indicated the necessity of
89
conducting more research in the field. In a more recent systematic review, Ferrero, Vadillo &
90
West (2017) also found a lack of scientific evidence on the relationship between C-HELPs,
91
academic achievement, and intelligence. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed For example,
116
Azemar and Ripoll (1987) observed a visuo-motor advantage in response time for C-HELP
117
subjects compared to UC-HELPs in laboratory experiments with spatio-temporal tasks. 118
Dorochenko (2009) also raised the possibility of the existence of differences in personality and
119
mental performance to explain a hypothetical over-representation of C-HELPs in the sport of
120
tennis. In this same sense, Laborde et al. (2009) reported that knowledge of hand-eye laterality
121
could be reliably used to advise sports training to enable more efficient adaptations in talent
122
detection, learning skills and in achieving better levels of coordination. Nevertheless, Laby and
123
Kirschen (2011) have warned about the lack of consensus among researchers on whether C-
124
HELPs or UC-HELPs could be advantageous in various sports. 25
More research is needed to determine the practical applications of hand-eye laterality in training
26
and to clarify the differences in hand-eye laterality profiles reported so far between sports
27
modalities. The present systematic review aims to analyze the literature available to date on
28
hand-eye laterality profiles in the different sports modalities, with three specific objectives: a) to
29
estimate the prevalence of hand-eye laterality profiles, b) to examine the relationship between
30
hand-eye laterality profiles, psychological factors and sports performance, and c) to propose a
31
methodological and terminological consensus. Manuscript to be reviewed 145
include grey literature, we also searched in
146
search alerts in PsycINFO and Scopus were
147
The search strategy followed the recommen
148
Strategies (PRESS) guidelines (McGowan e
149
hand-eye laterality and sports and due to the
150
domain of knowledge, the search strategy in
151
The search was limited by population (hum
152
by publication type (peer reviewed journals
153
can be found in Appendix 1. 154
155
Eligibility criteria and study selection
156
Eligible studies had to fulfill the criteria of b
157
experimental, observational, or single-case
158
laterality (distribution, predictiveness and in
159
with psychological factors). 160
No exclusion criteria were applied by gende
161
there is currently great interest in hand-eye
162
traditional sports, so studies referring to e-sp
163
our review. 164
One reviewer (MM) applied the inclusion/e
165
meeting the eligibility criteria were selected
166
the inclusion/exclusion criteria were also pr
167
pre-selected papers were checked independe
168
Discrepancies were resolved through discus
169
reaching consensus. 170
171
Data extraction
172
A data extraction template was previously d
173
Extracted information included: study chara
174
design); sample information (size, mean age
175
population/country, etc.), and hand-eye late
176
and UC-HELP distribution by sports modal
177
analysed, relationships between HEL and p
178
out independently by two reviewers (MM, L
179
discussion with a third author (JML) where
180
181
Strategy for data synthesis
182
This review provides a narrative and tabular
183
studies, structured around the research desig
184
main information is shown in tables. In the 145
include grey literature, we also searched in Google and reviewed up to 100 links. In addition,
146
search alerts in PsycINFO and Scopus were set until December 2020. 147
The search strategy followed the recommendations of the Peer Review of Electronic Search
148
Strategies (PRESS) guidelines (McGowan et al., 2016). With the aim of identifying studies about
149
hand-eye laterality and sports and due to the lack of consensus in the use of the terms for this
150
domain of knowledge, the search strategy included a long string of synonyms and related terms. 151
The search was limited by population (humans), by language (English, French or Spanish) and
152
by publication type (peer reviewed journals). The specific search syntax used for each database
153
can be found in Appendix 1. Manuscript to be reviewed 154
155
Eligibility criteria and study selection
156
Eligible studies had to fulfill the criteria of being original empirical studies (experimental, quasi-
157
experimental, observational, or single-case designs) providing direct information on hand-eye
158
laterality (distribution, predictiveness and influence on sports performance, or any correlation
159
with psychological factors). 160
No exclusion criteria were applied by gender, age, or temporal limit of the publication. Although
161
there is currently great interest in hand-eye coordination in electronic games, our focus was on
162
traditional sports, so studies referring to e-sports, virtual reality or gaming were excluded from
163
our review. 164
One reviewer (MM) applied the inclusion/exclusion criteria to all titles and abstracts. Studies
165
meeting the eligibility criteria were selected and studies that could cause controversy regarding
166
the inclusion/exclusion criteria were also pre-selected and the full text was retrieved as well. The
167
pre-selected papers were checked independently by two review authors (MM, LC). 168
Discrepancies were resolved through discussion with a third author (JML) where necessary until
169
reaching consensus. 170
171
Data extraction
172
A data extraction template was previously designed to extract data from the included studies. 173
Extracted information included: study characteristics (authors, title, year, journal, research
174
design); sample information (size, mean age, sex distribution, sports disciplines,
175
population/country, etc.), and hand-eye laterality data (handedness test, eyedness test, C-HELP
176
and UC-HELP distribution by sports modalities and sex, effects of HEL on performance, skills
177
analysed, relationships between HEL and psychological traits, etc.). Data extraction was carried
178
out independently by two reviewers (MM, LC) and discrepancies were resolved through
179
discussion with a third author (JML) where necessary. 180
181
Strategy for data synthesis
182
This review provides a narrative and tabular synthesis of the data extracted from the included
183
studies, structured around the research design, sport discipline and other factors of interest. The
184
main information is shown in tables. In the discussion, some information about the findings of
185
the review and how these findings may guide further research is reported. 145
include grey literature, we also searched in Google and reviewed up to 100 links. In addition,
146
search alerts in PsycINFO and Scopus were set until December 2020. 142
Search strategy 143
Literature searches were performed using the following databases: PsycInfo by EBSCOhost,
144
Scopus by Elsevier, Medline by PubMed, and Dissertations & Theses Global by ProQuest. To 143
Literature searches were performed using the following databases: PsycInfo by EBSCOhost,
144
Scopus by Elsevier, Medline by PubMed, and Dissertations & Theses Global by ProQuest. To 144
Scopus by Elsevier, Medline by PubMed, and Dissertations & Theses Global by ProQuest. To PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed 156
Eligible studies had to fulfill the criteria of being original empirical studies (experimental, quasi-
157
experimental, observational, or single-case designs) providing direct information on hand-eye
158
laterality (distribution, predictiveness and influence on sports performance, or any correlation
159
with psychological factors). 168
Discrepancies were resolved through discussion with a third author (JML) where necessary until
169
reaching consensus. Manuscript to be reviewed 186
187
Risk of bias assessment
188
The critical appraisal checklist for analytical cross-sectional, prevalence and quasi experimental
189
studies proposed by the Joanna Briggs Institute (JBI) (Moola et. al, 2020) was applied to assess
190
the risk of bias of the selected studies (Appendix 2). No studies will be excluded due to high risk
191
of bias because the amount of risk of bias is a relevant result in and of itself in our review. 192
The risk of bias was evaluated independently by two review authors (MM, JML). Discrepancies
193
were resolved through discussion with a third author (LC) where necessary. 194
195
Results
196
Literature Search
197
Figure 1 shows the flow diagram for systematic reviews of scientific literature proposed by
198
PRISMA. After duplicate records in the databases were excluded, a total of 1297 potential
199
studies regarding hand-eye laterality in sports were identified. There was 100% agreement
200
during the selection phase without the need for the participation of the third reviewer. In the end,
201
14 studies were considered for this review for the qualitative synthesis of the data. 202
203
*** Include Figure 1 here ***
204
205
The demographic data extracted from the reviewed studies is shown in Table 1, and the main
206
results found in the reviewed studies are shown in Table 2. 207
208
*** Include Table 1 and Table 2 here ***
209
210
Distribution of the age, gender, and geographical origin of the participants in the selected
211
studies
212
A total number of 2759 participants have been studied in the selected studies. Considering the
213
distribution by age, we have a 2.5% of children (up to 12 years), 19.4% of teenagers (13-18
214
years), and 78.1% of adult population (older of 18 years). Only two studies (14.2%) were carried
215
out with children and adolescents (9-17 years); five studies (35.7%) were carried out with
216
college students, but not all of them reported the participants ages; five other studies (35.7%)
217
selected samples of high performance athletes with ages ranged between 16 and 35 years old;
218
five studies (35.7%) used amateur athletes or sports practitioners (16.9-31.3 years); and finally,
219
three of those studies (21.4%) compared data between professional (16-35.2 years) and amateur
220
athletes (16.9-31.3 years). 171
Data extraction 182
This review provides a narrative and tabular synthesis of the data extracted from the included
183
studies, structured around the research design, sport discipline and other factors of interest. The
184
main information is shown in tables. In the discussion, some information about the findings of
185
the review and how these findings may guide further research is reported. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) In the section on the terminology used, we detail the methodology
221
used to determine the level of sports practice. 222
Geographical analysis of the selected studies revealed that eight of them (57%) were performed
223
in Europe (including two in France, two in Spain, one in Greece, one in the Czech Republic and 192
The risk of bias was evaluated independently by two review authors (MM, JML). Discrepancies
193
were resolved through discussion with a third author (LC) where necessary. 210
Distribution of the age, gender, and geographical origin of the participants in the selected
211
studies
212
A total number of 2759 participants have been studied in the selected studies. Considering the
213
distribution by age, we have a 2.5% of children (up to 12 years), 19.4% of teenagers (13-18
214
years), and 78.1% of adult population (older of 18 years). Only two studies (14.2%) were carried
215
out with children and adolescents (9-17 years); five studies (35.7%) were carried out with
216
college students, but not all of them reported the participants ages; five other studies (35.7%)
217
selected samples of high performance athletes with ages ranged between 16 and 35 years old;
218
five studies (35.7%) used amateur athletes or sports practitioners (16.9-31.3 years); and finally,
219
three of those studies (21.4%) compared data between professional (16-35.2 years) and amateur
220
athletes (16.9-31.3 years). In the section on the terminology used, we detail the methodology
221
used to determine the level of sports practice. 222
G
hi
l
l
i
f th
l
t d t di
l d th t i ht f th
(57%)
f
d 222
Geographical analysis of the selected studies revealed that eight of them (57%) were performed
223
in Europe (including two in France, two in Spain, one in Greece, one in the Czech Republic and 222
Geographical analysis of the selected studies revealed that eight of them (57%) were performed
223
in Europe (including two in France, two in Spain, one in Greece, one in the Czech Republic and PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) 245
Sports studied 246
Only one study (Quevedo et al., 2014) analyzed hand-eye laterality in a multisport perspective
247
including acrobatics (gymnastics and synchro), combat (taekwondo, wrestling, and judo), team
248
sports (soccer, volleyball, handball, basketball, hockey, softball, and water polo), skiing,
249
motorsport, modern pentathlon, golf, shooting, swimming, athletics, weightlifting and racket
250
sports (tennis and table tennis). Three studies (21.4%) were focused on basketball, two of the
251
studies (14.3%) were focused on golf, and for the rest of the studies the relationship between
252
hand-eye laterality and performance was studied in tennis, baseball, soccer, cricket, archery,
253
biathlon, motorsports and darts, with one article for each discipline. 254
255
Hand-Eye laterality Assessment
256
In laterality research, a wide range of assessment methods are continuously altered and developed. 257
As it is a multidimensional phenomenon, many different tools try to measure the underlying
258
variables. In the studies selected for this review, diverse and varied strategies for the evaluation of
259
eyedness and handedness have been identified. The following assessment types have been
260
proposed by Faurie, Raymond & Uomini (2016) to better classify and identify the predominant
261
methods used in the current literature:
262 246
Only one study (Quevedo et al., 2014) analyzed hand-eye laterality in a multisport perspective
247
including acrobatics (gymnastics and synchro), combat (taekwondo, wrestling, and judo), team
248
sports (soccer, volleyball, handball, basketball, hockey, softball, and water polo), skiing,
249
motorsport, modern pentathlon, golf, shooting, swimming, athletics, weightlifting and racket
250
sports (tennis and table tennis). Three studies (21.4%) were focused on basketball, two of the
251
studies (14.3%) were focused on golf, and for the rest of the studies the relationship between
252
hand-eye laterality and performance was studied in tennis, baseball, soccer, cricket, archery,
253
biathlon, motorsports and darts, with one article for each discipline. Manuscript to be reviewed 237
The application of the risk of bias assessment tools proposed by the JBI for the research design
238
of the selected studies shows a moderate or high presence of bias in most of them (Appendix 2
239
Nine of the cross-sectional studies do not clearly define the criteria for inclusion in the sample
240
(Q1), and also 9 of them don’t identify or treat potential confounding factors (Q5, Q6). Half of
241
these studies also do not measure the exposure (Q3) and the condition studied (Q4) in a valid a
242
reliable way. The prevalence study that was included in the review fails 4 of the 5 risks of bias
243
assessed, and the two quasi-experimental studies fail a third. 244
245
Sports studied
246
Only one study (Quevedo et al., 2014) analyzed hand-eye laterality in a multisport perspective
247
including acrobatics (gymnastics and synchro), combat (taekwondo, wrestling, and judo), team
248
sports (soccer, volleyball, handball, basketball, hockey, softball, and water polo), skiing,
249
motorsport, modern pentathlon, golf, shooting, swimming, athletics, weightlifting and racket
250
sports (tennis and table tennis). Three studies (21.4%) were focused on basketball, two of the
251
studies (14.3%) were focused on golf, and for the rest of the studies the relationship between
252
hand-eye laterality and performance was studied in tennis, baseball, soccer, cricket, archery,
253
biathlon, motorsports and darts, with one article for each discipline. 254
255
Hand-Eye laterality Assessment
256
In laterality research, a wide range of assessment methods are continuously altered and develop
257
As it is a multidimensional phenomenon, many different tools try to measure the underly
258
variables. In the studies selected for this review, diverse and varied strategies for the evaluation
259
eyedness and handedness have been identified. The following assessment types have be
260
proposed by Faurie, Raymond & Uomini (2016) to better classify and identify the predomin
261
methods used in the current literature:
262 224
two in the United Kingdom), four (29%) studies were performed in the United States and two
225
studies (14%) in Asia (Japan and Iran). Manuscript to be reviewed 224
two in the United Kingdom), four (29%) studies were performed in the United States and two
225
studies (14%) in Asia (Japan and Iran). 226
227
Study publication dates
228
Study publication dates ranged between 1971 and 2020, skewed heavily towards the last two
229
decades (Figure 2), with more than half of the studies in this period (57% between 2010-2022). 230
231
*** Include Figure 2 here ***
232
233
Risk of bias of the selected studies
234
The vast majority (78.5%) of studies have implemented cross-sectional designs, two studies
235
(14.4%) used a quasi-experimental pre-post design without a control group, and one was a
236
prevalence study (7.1%). None of the studies were implemented with an experimental design. 237
The application of the risk of bias assessment tools proposed by the JBI for the research designs
238
of the selected studies shows a moderate or high presence of bias in most of them (Appendix 2). 239
Nine of the cross-sectional studies do not clearly define the criteria for inclusion in the sample
240
(Q1), and also 9 of them don’t identify or treat potential confounding factors (Q5, Q6). Half of
241
these studies also do not measure the exposure (Q3) and the condition studied (Q4) in a valid and
242
reliable way. The prevalence study that was included in the review fails 4 of the 5 risks of bias
243
assessed, and the two quasi-experimental studies fail a third. 244
245
S
t
t di d 224
two in the United Kingdom), four (29%) studies were performed in the United States and two
225
studies (14%) in Asia (Japan and Iran). 226
227
Study publication dates
228
Study publication dates ranged between 1971 and 2020, skewed heavily towards the last two
229
decades (Figure 2), with more than half of the studies in this period (57% between 2010-2022)
230
231
*** Include Figure 2 here ***
232
233
Risk of bias of the selected studies
234
The vast majority (78.5%) of studies have implemented cross-sectional designs, two studies
235
(14.4%) used a quasi-experimental pre-post design without a control group, and one was a
236
prevalence study (7.1%). None of the studies were implemented with an experimental design. 255
Hand-Eye laterality Assessment 256
In laterality research, a wide range of assessment methods are continuously altered and developed. 257
As it is a multidimensional phenomenon, many different tools try to measure the underlying
258
variables. In the studies selected for this review, diverse and varied strategies for the evaluation of
259
eyedness and handedness have been identified. The following assessment types have been
260
proposed by Faurie, Raymond & Uomini (2016) to better classify and identify the predominant
261
methods used in the current literature:
262 PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed (2009), following Buxton & Crosland’s (1937) protocol, specified a minimum
313
distance of 2 meters between target and subject. Dalton, Guillon & Naroo (2015) used a specific
314
chart developed at the Michel Guillon vision clinic, which is scalable at any distance. Suyigama
315
& Lee (2005) used the examiner’s nose as a target with no indications of the distance. Finally,
316
Mann, Runswick & Allen (2016) implemented the pointing test using a camera as a target at a
317
distance of 3 meters. 318
Regarding variations in the pointing technique, three of the studies (Suyigama & Lee, 2005;
319
Dalton, Guillon & Naroo, 2015; Mann, Runswick & Allen, 2016;) used a finger (index or thumb)
320
on both arms alternately to reduce the interference with handedness; this procedure was reported
321
by Porac & Coren (1976). The studies from Razegui (2012) and Laborde et al. (2009), both
322
studying precision sports (darts and archery), used the index finger of a single hand to point; this
323
single-handed procedure was validated by Lora, Heilman & Roth (2002). 324
Finally, regarding the variations in the identification of the dominant eye, three different
325
procedures were found: in two studies (Laborde et al., 2009; Razegui, 2012) the subject actively
326
closed each eye to determine which eye was aligned with the target. Dalton, Guillon & Naroo
327
(2015) used a passive measurement to identify the dominant eye in which the examiner covered
328
one eye of the participant, followed by the other eye, and asked participants to report the
329
resulting deflection from the center of the target. In two studies, the examiners observed which
330
eye was dominant, using a photograph aligning the finger/thumb with the focus of the camera
331
and observing in the photograph which eye was aligned (dominant) and which was covered by
332
the hand (non-dominant) (Mann, Runswick & Allen 2015). In one study they used direct
333
observation while sighting (Suyigama & Lee 2015). 334
335
*** Include Table 4 here ***
336
337
Sighting test 302
The pointing test is the most frequently used procedure; six of the selected studies in this review
303
applied this method. Is also known as the Porta test because the earliest known reference dates
304
back to Porta (1593). The pointing test tries to create a situation in which the two eyes cannot be
305
used simultaneously. Manuscript to be reviewed The subjects must align 3 points: the dominant eye, the finger and a distant
306
target. The test starts by keeping both eyes open and proceeds by closing one eye at a time,
307
which reveals the dominant eye (the eye that is aligned with the finger). Variations and modified
308
versions have been found in the application of the target (type and distance), the pointing
309
technique and the identification method of the dominant eye (Table 4). 309
technique and the identification method of the dominant eye (Table 4). 310
Regarding the variations in the target, two studies indicated that any object could be used as a
311
target, and while Razeghi (2012) did not indicate the distance between the target and the subject,
312
Laborde et al. (2009), following Buxton & Crosland’s (1937) protocol, specified a minimum
313
distance of 2 meters between target and subject. Dalton, Guillon & Naroo (2015) used a specific
314
chart developed at the Michel Guillon vision clinic, which is scalable at any distance. Suyigama
315
& Lee (2005) used the examiner’s nose as a target with no indications of the distance. Finally,
316
Mann, Runswick & Allen (2016) implemented the pointing test using a camera as a target at a
317
distance of 3 meters. 318
Regarding variations in the pointing technique, three of the studies (Suyigama & Lee, 2005;
319
Dalton, Guillon & Naroo, 2015; Mann, Runswick & Allen, 2016;) used a finger (index or thumb)
320
on both arms alternately to reduce the interference with handedness; this procedure was reported
321
by Porac & Coren (1976). The studies from Razegui (2012) and Laborde et al. (2009), both
322
studying precision sports (darts and archery), used the index finger of a single hand to point; this
323
single-handed procedure was validated by Lora, Heilman & Roth (2002). 324
Finally, regarding the variations in the identification of the dominant eye, three different
325
procedures were found: in two studies (Laborde et al., 2009; Razegui, 2012) the subject actively
326
closed each eye to determine which eye was aligned with the target. Dalton, Guillon & Naroo
327
(2015) used a passive measurement to identify the dominant eye in which the examiner covered
328
one eye of the participant, followed by the other eye, and asked participants to report the
329
resulting deflection from the center of the target. Manuscript to be reviewed 302
The pointing test is the most frequently used procedure; six of the selected studies in this review
303
applied this method. Is also known as the Porta test because the earliest known reference dates
304
back to Porta (1593). The pointing test tries to create a situation in which the two eyes cannot be
305
used simultaneously. The subjects must align 3 points: the dominant eye, the finger and a distant
306
target. The test starts by keeping both eyes open and proceeds by closing one eye at a time,
307
which reveals the dominant eye (the eye that is aligned with the finger). Variations and modified
308
versions have been found in the application of the target (type and distance), the pointing
309
technique and the identification method of the dominant eye (Table 4). 310
Regarding the variations in the target, two studies indicated that any object could be used as a
311
target, and while Razeghi (2012) did not indicate the distance between the target and the subject,
312
Laborde et al. (2009), following Buxton & Crosland’s (1937) protocol, specified a minimum
313
distance of 2 meters between target and subject. Dalton, Guillon & Naroo (2015) used a specific
314
chart developed at the Michel Guillon vision clinic, which is scalable at any distance. Suyigama
315
& Lee (2005) used the examiner’s nose as a target with no indications of the distance. Finally,
316
Mann, Runswick & Allen (2016) implemented the pointing test using a camera as a target at a
317
distance of 3 meters. 318
Regarding variations in the pointing technique, three of the studies (Suyigama & Lee, 2005;
319
Dalton, Guillon & Naroo, 2015; Mann, Runswick & Allen, 2016;) used a finger (index or thumb)
320
on both arms alternately to reduce the interference with handedness; this procedure was reported
321
by Porac & Coren (1976). The studies from Razegui (2012) and Laborde et al. (2009), both
322
studying precision sports (darts and archery), used the index finger of a single hand to point; this
323
single-handed procedure was validated by Lora, Heilman & Roth (2002). 324
Finally, regarding the variations in the identification of the dominant eye, three different
325
procedures were found: in two studies (Laborde et al., 2009; Razegui, 2012) the subject actively
326
closed each eye to determine which eye was aligned with the target. Manuscript to be reviewed 263
1. Performance tasks: activities designed to induce actions from which a degree or level of
264
laterality can be deduced. 265
2. Preference tasks: activities designed to induce direct spontaneous actions of a preferred
266
side of the body. 267
3. Self-report questionnaire: questionnaires where the subjects decide whether they prefer one
268
side or the other for different contexts and actions. 269
4. Other author assessment measures, including interviews and active observation by
270
evaluators. 271
The measurement methods used in the selected studies have been classified in the next two
272
subsections according to the variable they measure and the type of evaluation (Table 3). 273
274
Handedness assessment
275
Handedness measurement is further divided into measures of preference and performance. While
276
hand preference identifies the preferred hand for completing a task, hand performance
277
differentiates between the ability or proficiency of one hand over the other in a particular task
278
(MacManus & Bryden, 1992). There is debate over whether performance and preference
279
measures are indicators of common underlying factors, or separate dimensions of behavior with
280
different causes (Byshop, 1989). 281
Eight different methods have been used to identify hand preference. 62% of the studies used self-
282
reported questionnaires, approximately 23% of the studies used direct observation as a method to
283
determine preference, and 15% of the studies used other methods including performance tasks
284
and interviews. 285
286
Questionnaires
287
Three studies included a self-reported author questionnaire as an instrument to assess the
288
handedness (Daltlon, Guillon & Naroo, 2015; Pointer, 2008; Quevedo et al. 2014)
289
Zouhal et al. (2018) also used an author questionnaire validated with a sample of 1500 athletes
290
(Azemar, 2003). In this case the researcher filled out the questionnaire based on the direct
291
observation of 11 performance tasks. 292
293
Eyedness assessment
294
Different tests have been described to measure eyedness, and there is controversy in determining
295
whether commonly used tests report an accurate evaluation of this phenomenon (Laby &
296
Kirschen, 2011). Subsequently, we have described the different methods used and the variations
297
incorporated by the authors in the selected studies. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Dalton, Guillon & Naroo
327
(2015) used a passive measurement to identify the dominant eye in which the examiner covered
328
one eye of the participant, followed by the other eye, and asked participants to report the
329
resulting deflection from the center of the target. In two studies, the examiners observed which
330
eye was dominant, using a photograph aligning the finger/thumb with the focus of the camera
331
and observing in the photograph which eye was aligned (dominant) and which was covered by
332
the hand (non-dominant) (Mann, Runswick & Allen 2015). In one study they used direct
333
observation while sighting (Suyigama & Lee 2015). 334
335
*** Include Table 4 here ***
336
337
Sighting test
338
The sighting test, also known as the Miles Test, was initially introduced by Zazzo (1960). 339
According to Laby & Kirschen (2011) is one of the most common and easy behavioral tests to
340
determine eye dominance. The procedure responds to a similar mechanism as the pointing test,
341
aligning an object with a reference from our hands. In this test, instead of using a finger or a pen,
342
the subjects are asked to hold their hands together, with their palms facing away at arm’s length, 302
The pointing test is the most frequently used procedure; six of the selected studies in this review
303
applied this method. Is also known as the Porta test because the earliest known reference dates
304
back to Porta (1593). The pointing test tries to create a situation in which the two eyes cannot be
305
used simultaneously. The subjects must align 3 points: the dominant eye, the finger and a distant
306
target. The test starts by keeping both eyes open and proceeds by closing one eye at a time,
307
which reveals the dominant eye (the eye that is aligned with the finger). Variations and modified
308
versions have been found in the application of the target (type and distance), the pointing
309
technique and the identification method of the dominant eye (Table 4). 310
Regarding the variations in the target, two studies indicated that any object could be used as a
311
target, and while Razeghi (2012) did not indicate the distance between the target and the subject,
312
Laborde et al. Manuscript to be reviewed In two studies, the examiners observed which
330
eye was dominant, using a photograph aligning the finger/thumb with the focus of the camera
331
and observing in the photograph which eye was aligned (dominant) and which was covered by
332
the hand (non-dominant) (Mann, Runswick & Allen 2015). In one study they used direct
333
observation while sighting (Suyigama & Lee 2015). 334
335
*** Include Table 4 here ***
336
337
Sighting test
338
The sighting test, also known as the Miles Test, was initially introduced by Zazzo (1960). 339
According to Laby & Kirschen (2011) is one of the most common and easy behavioral tests to
340
determine eye dominance. The procedure responds to a similar mechanism as the pointing test,
341
aligning an object with a reference from our hands. In this test, instead of using a finger or a pen,
342
the subjects are asked to hold their hands together, with their palms facing away at arm’s length,
343
in such a way that a small space remains between the thumbs and fingers of the two hands. We
344
found two different procedures to determine the dominant eye: passive or active measurement. 310
Regarding the variations in the target, two studies indicated that any object could be used as a
311
target, and while Razeghi (2012) did not indicate the distance between the target and the subject,
312
Laborde et al. (2009), following Buxton & Crosland’s (1937) protocol, specified a minimum
313
distance of 2 meters between target and subject. Dalton, Guillon & Naroo (2015) used a specific
314
chart developed at the Michel Guillon vision clinic, which is scalable at any distance. Suyigama
315
& Lee (2005) used the examiner’s nose as a target with no indications of the distance. Finally,
316
Mann, Runswick & Allen (2016) implemented the pointing test using a camera as a target at a
317
distance of 3 meters. 318
Regarding variations in the pointing technique, three of the studies (Suyigama & Lee, 2005;
319
Dalton, Guillon & Naroo, 2015; Mann, Runswick & Allen, 2016;) used a finger (index or thumb)
320
on both arms alternately to reduce the interference with handedness; this procedure was reported
321
by Porac & Coren (1976). The studies from Razegui (2012) and Laborde et al. Manuscript to be reviewed 345
For the passive measurement, the examiner covered one eye of the subject, followed by the other
346
eye, and asked with which eye the target was no longer seen (Dorochenko, 2009; Laby &
347
Kirschen, 2011). 348
For the active measurement, the subject brings their hands to their face quickly keeping the
349
object in view, moving the hole in the hands to their dominant eye, thus indicating the dominant
350
eye (Knudson & Kluka, 1997). Quevedo et al. (2014) used a sighting test without giving any
351
further procedural information. Finally, Lopez-Diaz et al. (2015) applied both active and passive
352
procedures. 353
354
Hole-in-the-card Test
355
The hole-in-the-card test is also a behavioural-preference-sighting test. In this case, the subject
356
holds a card with a hole in the middle at arm’s length, he is instructed to focus both eyes on an
357
object through the hole, then without taking his focus off he will bring the card closer to his face,
358
directing the hole at the dominant eye (Crider, 1944; Coren & Kaplan, 1973). This measurement
359
has been applied in 4 studies (Shick, 1971, 1977; Razegui, 2012; Pointer, 2008). The
360
involvement of both hands in the card test and the sighting test allows handedness interference to
361
be avoided. 362
363
Observation of pictures
364
One of the studies (Ziagkas, Mavvidis & Georgios., 2018) used observation of photographs of
365
the athletes playing found on the web to evaluate the hand and eye dominance. This is a non-
366
validated method that is based on a subjective assessment by the examiner. 367
368
Terminology
369
Terminological dispersion has been observed among the studies when classifying hand-eye
370
laterality profiles. The terms “crossed” vs. “uncrossed”, also seen as “crossed” vs. “noncrossed”
371
were the most common and were used in five studies (35.71%). The terms “crosslateral” vs. 372
“unilateral” or vs. “homolateral” were used in two studies (14.29%). The terms “contralateral”
373
vs. “unilateral” were used in 2 studies (14.29%), both by the same author. Other terms used were
374
“crossed” vs. “identical”, “crossed” vs. “homogeneous”, “crossed” vs. “pure”, and
375
“contralateral” vs. “ipsilateral”, each of them in a single article. 376
We have also found very diverse terminology regarding the categorization of the different skill
377
levels. Manuscript to be reviewed (2009), both
322
studying precision sports (darts and archery), used the index finger of a single hand to point; this
323
single-handed procedure was validated by Lora, Heilman & Roth (2002). PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed One study by Portal & Romano (1998) indicated
406
central ocular dominance (cyclopean eye) in baseball players, where the athletes’ eye preference
407
is balanced, using a modified version of the pointing test. Four of the selected studies do not
408
show information on laterality distributions, as they directly study differences in performance
409
between hand-eye laterality profiles using different indicators. 410
411
*** Include Figure 3 here ***
412
413
Effects on performance
414
The last columns of Table 2 also classify the results of the studies as follows to assess the effects
415
on performance of both hand-eye laterality profiles: 1) direct effects, when performance
416
indicators have been assessed; and 2) indirect effects, when a relative advantage is observed
417
because of the over-representation of one profile over the other in the most skilled athletes. 418
Six different studies confirm performance enhancements of C-HELPs over UC-HELPs,
419
including both direct and indirect effects. The results for baseball (Portal & Romano, 1998)
420
showed an increase in the UC-HELP prevalence for the group of non-athletes (65%), in relation
421
to the group of amateur athletes (39%). We observed an indirect effect on golf performance in
422
two different studies (Quevedo et al. 2014; and Dalton, Guillon & Naroo, 2015) due to the
423
enhanced distributions of C-HELPs in the HPA sample (55.1% and 50% respectively for the two
424
studies). We also observed an increased percentage of C-HELPs (42%) in the top 50 tennis
425
players in the world (Ziagkas, Mavvidis & Georgios, 2018). We consider an indirect effect on
426
performance for soccer players because 53% of the HPA are C-HELPs (Zouhal et al., 2018). The
427
results for basketball, however, are inconsistent; while Shick (1971) found a favorable direct
428
effect that was later refuted (Shick, 1975), Lopez-Diaz et al. (2015) reported some distributions
PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed 387
considered in the studies in relation to a specific sports skill; and d) a non-athlete (NA) group,
388
where we included random subjects without any relation to the sport studied. 389 387
considered in the studies in relation to a specific sports skill; and d) a non-athlete (NA) group,
388
where we included random subjects without any relation to the sport studied. 389 387
considered in the studies in relation to a specific sports skill; and d) a non-athlete (NA) group,
388
where we included random subjects without any relation to the sport studied. 389 387
considered in the studies in relation to a specific sports skill; and d) a non-athlete (NA) group,
388
where we included random subjects without any relation to the sport studied. 387
considered in the studies in relation to a specific sports skill; and d) a non-athlete (NA) group,
388
where we included random subjects without any relation to the sport studied. 389
390
Distribution of laterality profiles
391
Assuming the distribution of 10-30% for C-HELPs in the general population reported by
392
Robinson, Jacobsen & Heintz (1997), or the 34.8% reported by Bourassa et al. (1996), we found
393
a significant C-HELP overrepresentation in high-performance athletes for four different
394
modalities: golf (52.55%), soccer (53%), tennis (42%) and team sports (50.7%) (Table 2; Figure
395
3). However, the results also show a UC-HELP overrepresentation for some target sports: high-
396
performance shooters (93,1%) and amateur archers (82.3%). In that sense, Erickson (2007)
397
noticed that in aiming sports such as target shooting or archery, the UC-HELP offers advantages
398
in acquiring the skills required for success due to the specific homolateral demands of this sport
399
(riffle and eye must be aligned on the same side to aim properly. 400
For the amateur athlete sample, the only remarkable result is the overrepresentation of C-HELPs
401
in the sport of golf. This was observed among a sample of golfers from the Challenge Tour, one
402
step below the European Tour, who are still dedicated athletes with an advanced skill level
403
(Dalton, Guillon & Naroo, 2015). 404
The results for the AA, BA and NA samples for the other sports are coincident with the
405
distribution reported for the general population. Manuscript to be reviewed In the present study we have unified the terms and categorized 4 different groups: a) a
378
high-performance athlete (HPA) group, where we included all samples related with professional
379
athletes who train full-time, like the best 50 tennis players in the world, the first division of
380
soccer (league one in France), elite multisport athletes awarded with national grants at the
381
national high performance center in Spain and golf players from the European Tour and Ryder 345
For the passive measurement, the examiner covered one eye of the subject, followed by the other
346
eye, and asked with which eye the target was no longer seen (Dorochenko, 2009; Laby &
347
Kirschen, 2011). PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) 390
Distribution of laterality profiles p
(
)
(
)
(
)
397
noticed that in aiming sports such as target shooting or archery, the UC-HELP offers advantages
398
in acquiring the skills required for success due to the specific homolateral demands of this sport
399
(riffle and eye must be aligned on the same side to aim properly. 400
For the amateur athlete sample, the only remarkable result is the overrepresentation of C-HELPs
401
in the sport of golf. This was observed among a sample of golfers from the Challenge Tour, one
402
step below the European Tour, who are still dedicated athletes with an advanced skill level
403
(Dalton, Guillon & Naroo, 2015). 402
step below the European Tour, who are still dedicated athletes with an advanced skill level
403
(Dalton, Guillon & Naroo, 2015). 404
The results for the AA, BA and NA samples for the other sports are coincident with the
405
distribution reported for the general population. One study by Portal & Romano (1998) indicated
406
central ocular dominance (cyclopean eye) in baseball players, where the athletes’ eye preference
407
is balanced, using a modified version of the pointing test. Four of the selected studies do not
408
show information on laterality distributions, as they directly study differences in performance
409
between hand-eye laterality profiles using different indicators. 410
411
*** Include Figure 3 here ***
412
413
Effects on performance
414
The last columns of Table 2 also classify the results of the studies as follows to assess the effects
415
on performance of both hand-eye laterality profiles: 1) direct effects, when performance Manuscript to be reviewed 429
in amateur athletes that are congruent with those of the normal population. Even though over-
430
representations of C-HELPs are observed in the highest-level athletes, no study has shown direct
431
effects on performance for the crossed profiles. 432
Three different studies confirm advantages of UC-HELPs in target sports (archery, biathlon and
433
shooting) over C-HELPs, including both direct and indirect evidence. Archery and shooting are
434
the only samples that show an overrepresentation of UC-HELPs in relation to the distributions in
435
the normal population. 436
Four studies have found no relevant effects on performance related to hand-eye laterality
437
profiles. Shick (1975) refuted the relationship found above in basketball players (Shick, 1971)
438
between UC-HELPs and lateral throwing errors, where UC-HELPs seemed to make more
439
mistakes than C-HELPs. Razegui (2012) did not observe differences in accuracy between darts
440
players, conflicting with other reported results on the advantage of UC-HELPs in precision or
441
target sports (Erikson, 2007). In motorsports (Pointer, 2008), we observed congruent distribution
442
of laterality profiles between athletes and the normal population. Suyigama (2005) concluded
443
that more research is needed to confirm possible effects of hand-eye laterality profiles on golf
444
putting stance. 445
Finally, one study shows biomechanical differences concerning hand-eye laterality profiles and a
446
specific technique, which were unable to be categorized as positive or negative. The results on
447
Lopez-Diaz et al. (2015) support that an alternative basketball shot technique for UC-HELP
448
(rotating their body position 45º) will help them to obtain higher shot percentages. 449
Figure 4 summarizes the findings as to whether there is a favorable effect on performance for C-
450
HELP over UC-HELP, for UC-HELP over C-HELP, if there is a biomechanical effect reported
451
or if no effect is found. We observed reports of 11 performance effects that were related to hand-
452
eye laterality profiles in selected studies, while 4 studies didn’t report any effect. 453
454
*** Include Figure 4 here ***
455
456
Psychological traits and hand-eye laterality
457
458
None of the reviewed studies provided data on the relationship between psychological traits and
459
hand-eye laterality. 461
Discussion 462
The aim of this study was to systematically review the scientific publications on hand-eye laterality
463
in sports, to estimate the prevalence of C-HELPs and UC-HELPs in different sports modalities and
464
to examine their association with sports performance and psychological traits. We would like to
465
lay the groundwork for future research into the study of hand-eye laterality profiles in sports,
466
considering the growing number of publications about this topic. 467 413
Effects on performance PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed 460
461
Discussion
462
The aim of this study was to systematically review the scientific publications on hand-eye laterality
463
in sports, to estimate the prevalence of C-HELPs and UC-HELPs in different sports modalities and
464
to examine their association with sports performance and psychological traits. We would like to
465
lay the groundwork for future research into the study of hand-eye laterality profiles in sports,
466
considering the growing number of publications about this topic. 467
468
Distribution of laterality profiles and effects on performance 429
in amateur athletes that are congruent with those of the normal population. Even though over-
430
representations of C-HELPs are observed in the highest-level athletes, no study has shown direct
431
effects on performance for the crossed profiles. 432
Three different studies confirm advantages of UC-HELPs in target sports (archery, biathlon and
433
shooting) over C-HELPs, including both direct and indirect evidence. Archery and shooting are
434
the only samples that show an overrepresentation of UC-HELPs in relation to the distributions in
435
the normal population. 458
None of the reviewed studies provided data on the relationship between psychological traits and
459
hand-eye laterality. 467
468
Distribution of laterality profiles and effects on performance PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed 469
The results referring to the distribution of the hand-eye laterality profiles according to level of
470
practice, as well as the direct and indirect results found on performance, indicate that hand-eye
471
laterality profiles could be considered as a valid performance indicator. 472
We used the figure from Bourassa et al. (1996), who found that 34.8% of the general population
473
exhibited a C-HELP, as a control value to compare against sporting profiles, since they obtained
474
a larger sample in their meta-analysis. In the studies included in our review, we observed that
475
certain sports have different incidences of hand-eye laterality profiles than the normal population
476
depending on the level of practice. These results mostly refer to the distribution of hand-eye
477
laterality profiles in different sports and levels of practice, but they do not allow us to conclude
478
that there is a direct relationship between these profiles and sports performance. Even so,
479
relevant patterns have been found in this regard, which we discuss below. 480
The C-HELP percentage reported for amateur and high-level athletes of certain sports is higher
481
than in the normal population, 52.5% in golf (Dalton, 2015; Quevedo, 2014), 42% in tennis
482
(Ziagkas, Mavvidis & Georgios, 2018), and between 50.7% and 53% in soccer, volleyball,
483
handball, basketball, hockey, softball, and water polo (Quevedo et al. 2014; Zouhal et al., 2018). 484
As these data indicate, C-HELP subjects seem to have performance advantages in these sports
485
modalities. The explanation for the overrepresentation of C-HELPs in some sports seems to be
486
complex. Some publications (Siefer et al., 2003) point to specific advantages for C-HELPs
487
(especially those with left eyedness) in asymmetrical ball sports (tennis, soccer and basketball). 488
Some literature focuses on the biomechanical effects of hand-eye laterality profiles, which
489
modify and influence the specific movement, position, and technique of some asymmetric sports. 490
For example, Lopez-Diaz et al. (2015) pointed out distinct technical adaptations in the basketball
491
shot for the two profiles; while Suyigama & Lee (2005) analyzed the differences in golf putting
492
stances for the two profiles. There are also informative publications about tennis that reported
493
accommodations of the hitting technique depending on the hand-eye laterality profile (Garipuy &
494
Wolff, 1999). Manuscript to be reviewed For instance, a right-handed player who predominantly perceived the ball with the
495
right eye hit the forehand and served in a more frontal position than a right-handed player who
496
was left eye dominant. This is because the sight from the dominant eye (perceptive input) and the
497
racket on the dominant hand (motor output) must coincide at the point of impact of the ball and
498
the player will naturally adjust his position based on both. For his part, Dorochenko (2013) also
499
considered the advantage of the C-HELP for tennis performance in an informative, non-scientific
500
publication. Bache & Orellana (2014) collected Dorochenko’s (2013) observations, pointing out
501
that most of the Top 10 ATP tennis players are C-HELPs. We should also be cautious with the
502
results for tennis given by Ziagkas, Mavvidis & Georgios (2015), who reported an
503
overrepresentation of C-HELPs (42%) in the world’s top fifty tennis players, as we found
504
methodological inadequacies in eyedness assessment with indirect and non-standardized
505
measurements (observation of images from the internet). Another hypothesis reported by
506
Azemar, Stein & Ripoll (2008) tries to explain the advantage of the C-HELP in dual sports as the
507
result of a shorter reaction time for C-HELP subjects. 508 469
The results referring to the distribution of the hand-eye laterality profiles according to level of
470
practice, as well as the direct and indirect results found on performance, indicate that hand-eye
471
laterality profiles could be considered as a valid performance indicator. 472
We used the figure from Bourassa et al. (1996), who found that 34.8% of the general population
473
exhibited a C-HELP, as a control value to compare against sporting profiles, since they obtained
474
a larger sample in their meta-analysis. In the studies included in our review, we observed that
475
certain sports have different incidences of hand-eye laterality profiles than the normal population
476
depending on the level of practice. These results mostly refer to the distribution of hand-eye
477
laterality profiles in different sports and levels of practice, but they do not allow us to conclude
478
that there is a direct relationship between these profiles and sports performance. Even so,
479
relevant patterns have been found in this regard, which we discuss below. Manuscript to be reviewed 480
The C-HELP percentage reported for amateur and high-level athletes of certain sports is higher
481
than in the normal population, 52.5% in golf (Dalton, 2015; Quevedo, 2014), 42% in tennis
482
(Ziagkas, Mavvidis & Georgios, 2018), and between 50.7% and 53% in soccer, volleyball,
483
handball, basketball, hockey, softball, and water polo (Quevedo et al. 2014; Zouhal et al., 2018). 484
As these data indicate, C-HELP subjects seem to have performance advantages in these sports
485
modalities. The explanation for the overrepresentation of C-HELPs in some sports seems to be
486
complex. Some publications (Siefer et al., 2003) point to specific advantages for C-HELPs
487
(especially those with left eyedness) in asymmetrical ball sports (tennis, soccer and basketball). 488
Some literature focuses on the biomechanical effects of hand-eye laterality profiles, which
489
modify and influence the specific movement, position, and technique of some asymmetric sports. 490
For example, Lopez-Diaz et al. (2015) pointed out distinct technical adaptations in the basketball
491
shot for the two profiles; while Suyigama & Lee (2005) analyzed the differences in golf putting
492
stances for the two profiles. There are also informative publications about tennis that reported
493
accommodations of the hitting technique depending on the hand-eye laterality profile (Garipuy &
494
Wolff, 1999). For instance, a right-handed player who predominantly perceived the ball with the
495
right eye hit the forehand and served in a more frontal position than a right-handed player who
496
was left eye dominant. This is because the sight from the dominant eye (perceptive input) and the
497
racket on the dominant hand (motor output) must coincide at the point of impact of the ball and
498
the player will naturally adjust his position based on both. For his part, Dorochenko (2013) also
499
considered the advantage of the C-HELP for tennis performance in an informative, non-scientific
500
publication. Bache & Orellana (2014) collected Dorochenko’s (2013) observations, pointing out
501
that most of the Top 10 ATP tennis players are C-HELPs. We should also be cautious with the
502
results for tennis given by Ziagkas, Mavvidis & Georgios (2015), who reported an
503
overrepresentation of C-HELPs (42%) in the world’s top fifty tennis players, as we found
504
methodological inadequacies in eyedness assessment with indirect and non-standardized
505
measurements (observation of images from the internet). Manuscript to be reviewed 514
while aiming (Jones, Classe, Hester & Harris, 1996). In addition, the literature under review
515
reported performance effects based on biomechanical differences in technical execution between
516
the two profiles in some sports, such as basketball (Lopez-Diaz et al., 2015), cricket (Mann,
517
Runswick & Allen, 2016), and golf (Suyigama, 2005). In conclusion, it seems that laterality
518
patterns may influence performance depending on the sport modality and that awareness of them
519
could be a complement to talent detection and coaching development. 521
Methodological and terminological consensus 522
In reference to the assessment of laterality, there is no homogeneity regarding the instruments
523
used in the reviewed studies. Three different methods have been used to identify hand
524
preference, 62% of the studies used self-reported questionnaires, approximately 23% of the
525
studies relied on direct observation, and 15% of the studies used other methods including
526
performance tasks and interviews. This lack of coherence stems from the different orientations
527
of each study and each modality. We consider that to determine the handedness of asymmetric
528
implement sports, such as tennis or fencing, direct observation is sufficient since the hand
529
holding the racket or implement will reliably give us the hand preference information. Other
530
asymmetric sports modalities that do not involve the grasp of an implement, such as basketball or
531
soccer, or where the implement is wielded with two hands (golf, cricket, or baseball) may require
532
more specific assessment types, like self-reported questionnaires or even performance tasks. On
533
the other hand, the study of manual laterality in symmetric sports, such as cycling or swimming,
534
would not have a special interest given the equivalent use of both body hemispheres. 522
In reference to the assessment of laterality, there is no homogeneity regarding the instruments
523
used in the reviewed studies. Three different methods have been used to identify hand
524
preference, 62% of the studies used self-reported questionnaires, approximately 23% of the
525
studies relied on direct observation, and 15% of the studies used other methods including
526
performance tasks and interviews. This lack of coherence stems from the different orientations
527
of each study and each modality. We consider that to determine the handedness of asymmetric
528
implement sports, such as tennis or fencing, direct observation is sufficient since the hand
529
holding the racket or implement will reliably give us the hand preference information. Other
530
asymmetric sports modalities that do not involve the grasp of an implement, such as basketball or
531
soccer, or where the implement is wielded with two hands (golf, cricket, or baseball) may require
532
more specific assessment types, like self-reported questionnaires or even performance tasks. On
533
the other hand, the study of manual laterality in symmetric sports, such as cycling or swimming,
534
would not have a special interest given the equivalent use of both body hemispheres. Manuscript to be reviewed Another hypothesis reported by
506
Azemar, Stein & Ripoll (2008) tries to explain the advantage of the C-HELP in dual sports as the
507
result of a shorter reaction time for C-HELP subjects. 509
In contrast, the C-HELP distribution recorded in target sports is extremely low, with 6.9% in
510
high performance shooters (Quevedo et al., 2014), and 17.1% in amateur archers (Laborde et al.,
511
2009). Due to this data, UC-HELP subjects seem to have performance advantages in target sports
512
modalities. The explanation for this phenomenon relies on a biomechanical argument, given that
513
shooters and archers prefer to hold the weapon on the same side of the body as the dominant eye PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) 521
Methodological and terminological consensus 535
To identify the dominant eye, four different methods have been used in the reviewed studies. The
536
pointing test, the hole-in-the-card test and the sighting test are the most widespread protocols and
537
have been used in 92% of the selected studies. These methods are all preference, behavioral and
538
sighting tests, based on the mechanism of aligning an object with a reference from one's hands. 539
The pointing test involves aligning a target with one finger, or a pen held with only one hand, a
540
fact that may cause handedness interference (Porac & Coren, 1976). The hole-in-the-card test
541
avoids handedness interference by holding a card with two hands, and finally, the sighting test
542
seems to be equally reliable and more practical since no material is needed as it is implemented
543
with two hands (avoiding handedness interference). It is remarkable that only one of the studies
544
(Portal & Romano, 1988) considered a type of neutral or central ocular dominance. This form of
545
laterality should be considered when the subject sees from the bridge of the nose like a cyclopean
546
eye in passive measurement, or when the test is repeated and the subject brings their hands once
547
to each eye inconsistently in active measurement. On that topic, Laby et al. (1998) concluded
548
that although the one-handed pointing test does provide the possibility of detecting central
549
dominance, it appears to be highly dependent on which hand is used for testing due to
550
handedness interference. 551
After analyzing the results of this study, we consider that it would be necessary to establish a
552
single and universal method for the measurement of hand-eye laterality that would avoid
553
dispersion between methods. In our opinion, given the previous explanations, the most complete
554
protocol would be made up of the combination of the assessment of handedness with direct 535
To identify the dominant eye, four different methods have been used in the reviewed studies. The
536
pointing test, the hole-in-the-card test and the sighting test are the most widespread protocols and
537
have been used in 92% of the selected studies. These methods are all preference, behavioral and
538
sighting tests, based on the mechanism of aligning an object with a reference from one's hands. Manuscript to be reviewed Although this relationship and its applications seem to be very widespread in some
596
specific areas such as professional tennis training, we did not find any study in our review that
597
related laterality with psychological aspects of athletes. Research is needed on this possible
598
association in the field of sports, through the application of behavioral and cognitive style
599
questionnaires in conjunction with the application of consensual laterality tests, such as the Sport
600
Orientation Questionnaire (SOQ) from Gill and Deeter (1988), the Sport Competition Anxiety
601
Test (SCAT) developed by Martens (1977), the Coaching Behavior Assessment System (CBAS) 569
Limitations and future lines of research 569
Limitations and future lines of research
570
Concerning laterality profile distribution, one of the biggest limitations that we find is that the
571
distribution of C-HELPs and UC-HELPs in the normal population (non-athletes) is not yet clear,
572
and therefore we cannot compare sports values with a standard value. While the hand-eye
573
laterality meta-analysis of Bourassa et al. (1996) compiled a 34.8% prevalence of C-HELPs,
574
MacManus et al. (1999) reported a range between 24% and 27% for C-HELP prevalence, and
575
other studies reported a range between 10% and 30% crossed (Robinson, Jacobsen & Heintz,
576
1997). Further investigation is needed to clarify and determine more objective and recent data
577
about hand-eye laterality profiles in the general population. 578
It is clear that the study of laterality profiles is not as relevant in sports with “symmetrical”
579
laterality, such as swimming, cycling or athletics (footraces), as it is in sports which require
580
asymmetrical actions for throwing, hitting or shooting. In that sense, it would be necessary to
581
corroborate the results and hypotheses about the effects of laterality profiles on performance in
582
“asymmetrical” sports such as soccer, tennis, basketball, or hockey, and in target sports such as
583
archery or shooting. 584
The methods or measurements used to establish the favorable C-HELP distribution in some
585
studies are unknown and not published, as in the studies of Dorochenko (2013) or Bache &
586
Orellana (2014), while in other studies they are improper and subjective, as in the work of
587
Ziagkas, Mavvidis & Georgios (2018). This could lead to hasty conclusions in sports like tennis,
588
where more data is needed. In addition, to clarify these possible relationships between
589
performance and hand-eye laterality, studies on specific performance indicators would be
590
convenient, in addition to the standardization of the methods for assessing laterality. 591
Another goal we had set ourselves in this review was to relate the hand-eye laterality profile with
592
psychological traits of the athletes. Some recognized experts from different disciplines point to a
593
relationship between the different hand-eye laterality profiles and certain behavioral models,
594
associating the dominance of the eye with the corresponding cerebral hemisphere (Dorochenko,
595
2009). 521
Methodological and terminological consensus 539
The pointing test involves aligning a target with one finger, or a pen held with only one hand, a
540
fact that may cause handedness interference (Porac & Coren, 1976). The hole-in-the-card test
541
avoids handedness interference by holding a card with two hands, and finally, the sighting test
542
seems to be equally reliable and more practical since no material is needed as it is implemented
543
with two hands (avoiding handedness interference). It is remarkable that only one of the studies
544
(Portal & Romano, 1988) considered a type of neutral or central ocular dominance. This form of
545
laterality should be considered when the subject sees from the bridge of the nose like a cyclopean
546
eye in passive measurement, or when the test is repeated and the subject brings their hands once
547
to each eye inconsistently in active measurement. On that topic, Laby et al. (1998) concluded
548
that although the one-handed pointing test does provide the possibility of detecting central
549
dominance, it appears to be highly dependent on which hand is used for testing due to
550
handedness interference. 551
After analyzing the results of this study, we consider that it would be necessary to establish a
552
single and universal method for the measurement of hand-eye laterality that would avoid
553
dispersion between methods. In our opinion, given the previous explanations, the most complete
554
protocol would be made up of the combination of the assessment of handedness with direct
555
observation (for asymmetric sports with implements like tennis, fencing, table tennis etc.) and
556
the Edinburgh Handedness Inventory for other sports, and the application of the sighting test for
557
ocular dominance, considering the active measurement protocol (bringing hands from arms PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed 558
length to the eyes), and using the examiner's nose or a camera lens at three meters of distance as
559
a target, and also considering its repetition for detecting possible central dominance cases. 560
From our review, we have noticed an important terminological dispersion between the studies
561
when referring to hand-eye laterality profiles. In some works, we also found the term
562
“dominance” instead of “laterality” to refer to the preference for one side of the body over the
563
other. We haven’t found enough evidence to use either of the two general terms. However, we
564
have chosen for our review the most widely used form in the available studies for referring
565
specifically to the type of dominance or laterality: uncrossed profile (UC-HELP) when the
566
dominant eye and hand are on the same side of the body and crossed profile (C-HELP) when the
567
dominance of the hand and the eye are on opposite sides. 568 558
length to the eyes), and using the examiner's nose or a camera lens at three meters of distance as
559
a target, and also considering its repetition for detecting possible central dominance cases. 558
length to the eyes), and using the examiner's nose or a camera lens at three meters of distance as
559
a target, and also considering its repetition for detecting possible central dominance cases. 560
From our review, we have noticed an important terminological dispersion between the studies
561
when referring to hand-eye laterality profiles. In some works, we also found the term
562
“dominance” instead of “laterality” to refer to the preference for one side of the body over the
563
other. We haven’t found enough evidence to use either of the two general terms. However, we
564
have chosen for our review the most widely used form in the available studies for referring
565
specifically to the type of dominance or laterality: uncrossed profile (UC-HELP) when the
566
dominant eye and hand are on the same side of the body and crossed profile (C-HELP) when the
567
dominance of the hand and the eye are on opposite sides. Manuscript to be reviewed 568
569
Limitations and future lines of research
570
Concerning laterality profile distribution, one of the biggest limitations that we find is that the
571
distribution of C-HELPs and UC-HELPs in the normal population (non-athletes) is not yet clear,
572
and therefore we cannot compare sports values with a standard value. While the hand-eye
573
laterality meta-analysis of Bourassa et al. (1996) compiled a 34.8% prevalence of C-HELPs,
574
MacManus et al. (1999) reported a range between 24% and 27% for C-HELP prevalence, and
575
other studies reported a range between 10% and 30% crossed (Robinson, Jacobsen & Heintz,
576
1997). Further investigation is needed to clarify and determine more objective and recent data
577
about hand-eye laterality profiles in the general population. 578
It is clear that the study of laterality profiles is not as relevant in sports with “symmetrical”
579
laterality, such as swimming, cycling or athletics (footraces), as it is in sports which require
580
asymmetrical actions for throwing, hitting or shooting. In that sense, it would be necessary to
581
corroborate the results and hypotheses about the effects of laterality profiles on performance in
582
“asymmetrical” sports such as soccer, tennis, basketball, or hockey, and in target sports such as
583
archery or shooting. 584
The methods or measurements used to establish the favorable C-HELP distribution in some
585
studies are unknown and not published, as in the studies of Dorochenko (2013) or Bache &
586
Orellana (2014), while in other studies they are improper and subjective, as in the work of
587
Ziagkas, Mavvidis & Georgios (2018). This could lead to hasty conclusions in sports like tennis,
588
where more data is needed. In addition, to clarify these possible relationships between
589
performance and hand-eye laterality, studies on specific performance indicators would be
590
convenient, in addition to the standardization of the methods for assessing laterality. 591
Another goal we had set ourselves in this review was to relate the hand-eye laterality profile with
592
psychological traits of the athletes. Some recognized experts from different disciplines point to a
593
relationship between the different hand-eye laterality profiles and certain behavioral models,
594
associating the dominance of the eye with the corresponding cerebral hemisphere (Dorochenko,
595
2009). 569
Limitations and future lines of research Although this relationship and its applications seem to be very widespread in some
596
specific areas such as professional tennis training, we did not find any study in our review that
597
related laterality with psychological aspects of athletes. Research is needed on this possible
598
association in the field of sports, through the application of behavioral and cognitive style
599
questionnaires in conjunction with the application of consensual laterality tests, such as the Sport
600
Orientation Questionnaire (SOQ) from Gill and Deeter (1988), the Sport Competition Anxiety
601
Test (SCAT) developed by Martens (1977), the Coaching Behavior Assessment System (CBAS) 570
Concerning laterality profile distribution, one of the biggest limitations that we find is that the
571
distribution of C-HELPs and UC-HELPs in the normal population (non-athletes) is not yet clear,
572
and therefore we cannot compare sports values with a standard value. While the hand-eye
573
laterality meta-analysis of Bourassa et al. (1996) compiled a 34.8% prevalence of C-HELPs,
574
MacManus et al. (1999) reported a range between 24% and 27% for C-HELP prevalence, and
575
other studies reported a range between 10% and 30% crossed (Robinson, Jacobsen & Heintz,
576
1997). Further investigation is needed to clarify and determine more objective and recent data
577
about hand-eye laterality profiles in the general population. 584
The methods or measurements used to establish the favorable C-HELP distribution in some
585
studies are unknown and not published, as in the studies of Dorochenko (2013) or Bache &
586
Orellana (2014), while in other studies they are improper and subjective, as in the work of
587
Ziagkas, Mavvidis & Georgios (2018). This could lead to hasty conclusions in sports like tennis,
588
where more data is needed. In addition, to clarify these possible relationships between
589
performance and hand-eye laterality, studies on specific performance indicators would be
590
convenient, in addition to the standardization of the methods for assessing laterality. 591
Another goal we had set ourselves in this review was to relate the hand-eye laterality profile with
592
psychological traits of the athletes. Some recognized experts from different disciplines point to a
593
relationship between the different hand-eye laterality profiles and certain behavioral models,
594
associating the dominance of the eye with the corresponding cerebral hemisphere (Dorochenko,
595
2009). 606
Conclusions DOI:
637
https://doi.org/10.2466/pms.1976.43.3f.1031
638
Azemar G & Ripoll J (1987) Neurosciences du Sport Paris: INSEP; p 228–42 607
The study of the relationship between hand-eye laterality and sports performance is an
608
underdeveloped field of knowledge, although it is notable that more than half of the publications
609
found in this review are from the last decade. Our review provides information that could help
610
shape future research in this area. Certain sports have different prevalences of hand-eye laterality
611
profiles than the normal population. In sports such as golf, tennis, and team sports (soccer,
612
volleyball, handball, basketball, hockey, softball, and water polo) the percentage of C-HELP is
613
higher in amateur and high-level athletes than in the normal population. In target sports (archery
614
and shooting) the UC-HELP seems to confer an advantage, given the significant concentration of
615
this profile in the highest performing populations, and some studies directly confirm these effects
616
on biathlon shooting. In basketball, cricket and golf, the literature under review reported
617
biomechanical differences between the two profiles in the execution of some techniques. It is
618
worth highlighting the need for further scientific research on the distribution of hand-eye
619
laterality profiles in asymmetrical sports like tennis, golf, basketball, or soccer, in order to study
620
the mechanisms that produce direct effects on performance. The results shown in this review
621
must be taken with caution as many of them refer to indirect effects. Manuscript to be reviewed 602
by Smith, Smoll and Hunt (1977), the Revised Competitive Anxiety Inventory-2 (CSAI-2R)
603
from Cox, Martens y Russell (2003), or the Profile of Mood States (POMS) from McNair, Lorr
604
and Droppleman (1971). 602
by Smith, Smoll and Hunt (1977), the Revised Competitive Anxiety Inventory-2 (CSAI-2R)
603
from Cox, Martens y Russell (2003), or the Profile of Mood States (POMS) from McNair, Lorr
604
and Droppleman (1971). 635
Abigail, E.R, Johnson, E.G. (1976). Ear and Hand Dominance and Their Relationship with
636
Reading Retardation. Perceptual and Motor Skills, 43:1031–1036. DOI:
637
https://doi.org/10.2466/pms.1976.43.3f.1031
638
Azemar, G & Ripoll, J. (1987). Neurosciences du Sport. Paris: INSEP; p. 228–42.
639
Azemar, G. (2003). De l’oeil à la main. In CNRS Éditions (Ed.), L’homme asymétrique (pp. 221-
640
280) Paris: CNRS Editions
641
Azémar, G., Stein, J.-F., & Ripoll, H. (2008). Effects of ocular dominance on eye-hand
642
coordination in sporting duels . Science and Sports, 23(6), 263–277.
643
https://doi.org/10.1016/j.scispo.2008.06.004 569
Limitations and future lines of research Although this relationship and its applications seem to be very widespread in some
596
specific areas such as professional tennis training, we did not find any study in our review that
597
related laterality with psychological aspects of athletes. Research is needed on this possible
598
association in the field of sports, through the application of behavioral and cognitive style
599
questionnaires in conjunction with the application of consensual laterality tests, such as the Sport
600
Orientation Questionnaire (SOQ) from Gill and Deeter (1988), the Sport Competition Anxiety
601
Test (SCAT) developed by Martens (1977), the Coaching Behavior Assessment System (CBAS) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) 606
Conclusions 607
The study of the relationship between hand-eye laterality and sports performance is an
608
underdeveloped field of knowledge, although it is notable that more than half of the publications
609
found in this review are from the last decade. Our review provides information that could help
610
shape future research in this area. Certain sports have different prevalences of hand-eye laterality
611
profiles than the normal population. In sports such as golf, tennis, and team sports (soccer,
612
volleyball, handball, basketball, hockey, softball, and water polo) the percentage of C-HELP is
613
higher in amateur and high-level athletes than in the normal population. In target sports (archery
614
and shooting) the UC-HELP seems to confer an advantage, given the significant concentration of
615
this profile in the highest performing populations, and some studies directly confirm these effects
616
on biathlon shooting. In basketball, cricket and golf, the literature under review reported
617
biomechanical differences between the two profiles in the execution of some techniques. It is
618
worth highlighting the need for further scientific research on the distribution of hand-eye
619
laterality profiles in asymmetrical sports like tennis, golf, basketball, or soccer, in order to study
620
the mechanisms that produce direct effects on performance. The results shown in this review
621
must be taken with caution as many of them refer to indirect effects. 622
We did not find any study in our review that related hand-eye laterality with psychological
623
aspects of athletes. The incorporation of cognitive and behavioral indicators would provide very
624
valuable information about the relationship between hand-eye laterality profiles and
625
psychological or tactical sports patterns. In short, the advancement of knowledge about hand-eye
626
laterality could also contribute to more effective athlete development plans and could
627
complement talent detection. 628
Finally, to ameliorate the terminological dispersion that we found in our review, we propose the
629
term hand-eye laterality profile as a general topic and crossed profiles (C-HELP) and uncrossed
630
profiles (UC-HELP) as the specific patterns. We also propose a combination of direct
631
observation and the Edinburgh Handedness Inventory in handedness, and the application of the
632
sighting test for eyedness as a protocol for hand-eye laterality measurement in sports. 633
634
References
635
Abigail, E.R, Johnson, E.G. (1976). Ear and Hand Dominance and Their Relationship with
636
Reading Retardation. Perceptual and Motor Skills, 43:1031–1036. 606
Conclusions DOI:
637
https://doi.org/10.2466/pms.1976.43.3f.1031 607
The study of the relationship between hand-eye laterality and sports performance is an
608
underdeveloped field of knowledge, although it is notable that more than half of the publications
609
found in this review are from the last decade. Our review provides information that could help
610
shape future research in this area. Certain sports have different prevalences of hand-eye laterality
611
profiles than the normal population. In sports such as golf, tennis, and team sports (soccer,
612
volleyball, handball, basketball, hockey, softball, and water polo) the percentage of C-HELP is
613
higher in amateur and high-level athletes than in the normal population. In target sports (archery
614
and shooting) the UC-HELP seems to confer an advantage, given the significant concentration of
615
this profile in the highest performing populations, and some studies directly confirm these effects
616
on biathlon shooting. In basketball, cricket and golf, the literature under review reported
617
biomechanical differences between the two profiles in the execution of some techniques. It is
618
worth highlighting the need for further scientific research on the distribution of hand-eye
619
laterality profiles in asymmetrical sports like tennis, golf, basketball, or soccer, in order to study
620
the mechanisms that produce direct effects on performance. The results shown in this review
621
must be taken with caution as many of them refer to indirect effects. 622
We did not find any study in our review that related hand-eye laterality with psychological
623
aspects of athletes. The incorporation of cognitive and behavioral indicators would provide very
624
valuable information about the relationship between hand-eye laterality profiles and
625
psychological or tactical sports patterns. In short, the advancement of knowledge about hand-eye
626
laterality could also contribute to more effective athlete development plans and could
627
complement talent detection. 628
Finally, to ameliorate the terminological dispersion that we found in our review, we propose the
629
term hand-eye laterality profile as a general topic and crossed profiles (C-HELP) and uncrossed
630
profiles (UC-HELP) as the specific patterns. We also propose a combination of direct
631
observation and the Edinburgh Handedness Inventory in handedness, and the application of the
632
sighting test for eyedness as a protocol for hand-eye laterality measurement in sports. 633
634
References
635
Abigail, E.R, Johnson, E.G. (1976). Ear and Hand Dominance and Their Relationship with
636
Reading Retardation. Perceptual and Motor Skills, 43:1031–1036. 634
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280) Paris: CNRS Editions 641
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coordination in sporting duels . Science and Sports, 23(6), 263–277. 643
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809
contralateral eye-hand dominance in tennis serve accuracy of amateur tennis players. 810
Journal of Physical Education and Sport, 17(2), 867–870. DOI:
811
https://doi.org/10.7752/jpes.2017.02132 812
Zouhal, H., Abderrahman, A. B., Dupont, G., Truptin, P., Le Bris, R., Le Postec, E., … Bideau,
813
B. (2018). Laterality influences agility performance in elite soccer players. Frontiers in
814
Physiology, 9(807). https://doi.org/10.3389/fphys.2018.00807 816 Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Figure 1 Figure 1.PRISMA flow chart of the process of identifying and selecting studies PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Figure 2. Percentage of reviewed studies by publication date Figure 2. Percentage of reviewed studies by publication date PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Figure 3 Figure 3. Distribution of crossed profiles by sport modality and skill level. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Figure 3. Distribution of crossed profiles by sport modality and skill level. Note: HPA: high-performance athletes; RA: regular athletes; BA: beginner athletes; NA: non-
athletes; *: weighted percentage. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed Figure 4 Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Figure 4 Figure 4. Effects of laterality profiles reported by number of selected studies. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Table 1(on next page) Table 1. General characteristics of the reviewed studies Table 1. General characteristics of the reviewed studies Note: HPA: high-performance athletes; AA: amateur athletes; BA: beginner athletes; NA: non-
athletes; PR: prevalence; CS: cross-sectional; QE: quasi-experimental; --: not reported. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed Table 1. General characteristics of the reviewed studies. Study
Country
Sport
n (men)
Age Standard
Deviation
Research
design
Dalton, Guillon &
Naroo (2015)
United States
Golf
HPA: 10 (--)
RA: 7 (--)
BA: 14 (--)
--
CS
Laborde et al. (2009)
France
Archery
BA: 82 (48)
RA: 1323 (--)
BA: 19.3 1.7
RA: --
CS
Lopez-Diaz et al. (2015)
Spain
Basketball
RA: 34 (24)
12.94 0.35
QE
Mann, Runswick &
Allen (2016)
England
Cricket
HPA: 43 (43)
BA: 93 (93)
HPA: 29.6 5.6
BA: 24.1 7.2
CS
Nosek, Hurdálková, &
Cihlář (2018)
Czech
Republic
Biathlon
RA: 37 (--)
16.4 ± 1.24
CS
Pointer (2008)
United
Kingdom
Motorsports
RA: 60 (54)
19.9 ± 9.6
CS
Portal & Romano
(1998)
United States
Baseball
RA: 23 (--)
NA: 100 (--)
--
CS
Quevedo et al. (2014)
Spain
Multiple sports
RA: 536 (315)
17.4 ± 3.7
CS
Razeghi et al. (2012)
Iran
Darts
BA: 20 (20)
21.43 ± 1.33
QE
Shick (1971)
United States
Basketball
RA: 32 (0)
--
CS
Shick (1977)
United States
Basketball
RA: 86 (0)
--
CS
Sugiyama & Lee
(2005)
Japan
Golf
RA: 47 (37)
20.2 0.8
CS
Ziagkas, Mavvidis &
Georgios (2018). Greece
Tennis
HPA:50 (50)
--
PR
Zouhal et al. (2018)
France
Soccer
HPA: 72 (72)
RA: 9 (9)
HPA:18.2 2.2
RA: 19.6 2.1
CS
Note: HPA: high-performance athletes; RA: regular athletes; BA: beginner athletes; NA: non-athletes; PR: prevalence; CS:
cross-sectional; QE: quasi-experimental; --: not reported. 1
Table 1. General characteristics of the reviewed studies. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Table 2(on next page) Table 2(on next page) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Table 2(on next page) Table 2. Main results on the relationship between hand-eye laterality and sports
performance and skill level. Note: C-HELP: hand-eye laterality crossed profile; UC-HELP: hand-eye laterality uncrossed
profile; HPA: high-performance athletes; RA: regular athletes; BA: beginner athletes; NA: non-
athletes; --: not assessed. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed 1
Table 2. Main results on the relationship between hand-eye laterality and sports performance and skill level. Stud
S
S
HELP
terminology
C-HELP%, UC-
HELP%
Handedness
assessment
Eye preference
assessment
HELP and sports performance/skill
level relationship
Favourable
Direct Effects
Favourable
Indirect Effects
Dalton,
Guillon &
Naroo (2015)
Golf
Crossed,
Uncrossed
dominance
HPA (50, 50)
RA (80, 20)
BA (14.4, 76.6)
Author self-
report
questionnaire
Pointing Test
The distribution of C-HELP and UC-
HELP was statistically different
between the different skill groups
--
C-HELP
Laborde et al. (2009)
Archery
Crossed,
Uncrossed
laterality
BA (34.1, 65.9)
RA (17.7, 82.2)
Edinburgh
Inventory
Pointing Test
An analysis of variance indicated that
beginners with an uncrossed pattern
scored significantly more points than
those with a crossed pattern
UC-HELP
--
Lopez-Diaz
et al. (2015)
Basketball
Crossed,
Homogeneous
laterality
RA (27.8, 72.2)
Harris Test
Sighting Test
O
of C-HELP at
young high-level basketball players. Technical effect found: the shoot
mechanics should be adapted on UC-
HELP players
Biomechanical effects
Mann,
Runswick &
Allen (2016)
Cricket
Do not refer to
this relation
HPA (26, 74)
BA (19, 71)
Edinburgh
Inventory
Pointing Test
Technical effects found: placing the
dominant hand in the top of the bat
(reverse stance) offer a very significant
advantage. Placing the dominant eye in
front of the stance did not affect the
performance
--
--
Nosek,
Hurdálková
& Cihlář
(2018)
Biathlon
Crossed,
Identical
laterality
Not reported
T-116 test
T-116 test
UC-HELP shooters were more accurate
UC-HELP
Pointer
(2008)
Motorsports
Crossed,
Uncrossed
laterality
RA (31.7, 68.3)
NA (30, 70)
Author self-
report
questionnaire
Hole-in-the-card
Test
No relation found
--
No effects found
Portal &
Romano
(1998)
Baseball
Crossed,
Uncrossed
laterality
RA (35, 39)
NA (18, 65)
Direct
preference
observation
Pointing Test
There are twice C-HELP in the group
of baseball players than in normal
controls. --
C-HELP
Quevedo et
al. Manuscript to be reviewed Table 2(on next page) (2014)
Multi-Sport
Crosslateral,
Homolateral
dominance
HPA (39.9, 61.1)
Interview
Pointing Test
and Sighting
Test
There are more UC-HELP shooters and
C-HELP in golf and team sports than in
normal population
--
Golf and team
sports: C-HELP
Shooting: UC-
HELP
Razeghi et al. (2012)
Darts
Crosslateral,
Unitaleral
dominance
Not reported
Edinburgh
Inventory
Hole-in-the-card
and Pointing
Test
No significant differences between C-
HELP and UC-HELP in skill with darts
--
No effects found 1
Table 2. Main results on the relationship between hand-eye laterality and sports performance and skill level. 1
Table 2. Main results on the relationship between hand-eye laterality and spor PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed 2
Note: C-HELP: hand-eye laterality crossed profile; UC-HELP: hand-eye laterality uncrossed profile; HPA: high-performance athletes; RA: regular athletes; BA: beginner athletes;
3
NA: non-athletes; --: not assessed. 4
Shick (1971)
Basketball
Contralateral,
Unilateral
dominance
Not reported
Direct
preference
observation
Hole-in-the-card
test
UC-HELP registered more lateral
errors towards de side of nondominant
hand
C-HELP
--
Shick (1977)
Basketball
Contralateral,
Unilateral
dominance
RA (32.7, 67.2)
Direct
preference
observation
Hole-in-the-card
test
No relation found on lateral errors in
free- throw shooting for college women
and HEL
--
No effects found
Sugiyama &
Lee (2005)
Golf
Crossed dextral,
Pure dextral
Not reported
Hand
Dominance
Questionnaire
Pointing Test
. No effects found
Ziagkas,
Mavvidis &
Georgios
(2018)
Tennis
Contralateral,
Ipsilateral
dominance
HPA (42, 58)
Direct
preference
observation
Direct
preference
observation
through pictures
There are more C-HELP in the 50 best
world tennis players than in normal
populations
--
C-HELP
Zouhal et al. (2018)
Soccer
Crossed, Non
crossed
laterality
HPA (53, 47)
RA (33, 67)
Individual
laterality in
sports. Azemar
(2003)
Individual
laterality in
sports. Azemar
(2003)
There are more C-HELP in the soccer
elite players than in the amateur group. C-HELP PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Table 3(on next page) Table 3(on next page) Table 3. Handedness and eyedness assessment methods. Note: Number of reviewed studies applying the instrument; most cited referring the
instrument; --: not reported PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1
Table 3. Handedness and eyedness assessment methods. 2
3
Note: 1 Number of reviewed studies applying the instrument; 2 most cited referring the instrument; --: not reported. 4
Assessment
Type
Instrument
Administration (items)
Author
Reliability Studies1
Sport
Edimburgh
Inventory
Self-reported
(20 items)
Olfield (1971)
Yes
3
Archery;
Cricket; Darts
Hand Dominance
Questionnaire
Self-reported
(13 items)
Chapman &
Chapman (1987)
Yes
1
Golf
Test
Harris Test
Performance task
(11 items)
Harris (1947)
Yes
1
Basketball
Self-reported
(1 item)
Daltlon, Guillon &
Naroo (2015)
--
1
Golf
Self-reported
(--)
Pointer (2008)
--
1
Motorsport
Autor
questionnaire
Self-reported
(3 items)
Quevedo et al. (2014)
--
1
Multisports
Handedness
Direct
observation
Observation of hand
preference on the task of
basketball
Shick (1971, 1977)
2
Basketball
Pointing/Porta
Test
Performance task
Porta (1593)2:
Yes
6
Archery;
Baseball;
Darts; Golf
Hole-in-the-card-
test
Performance task
Crider, (1944);
Coren & Kaplan
(1973); Rice et al. (2008)
Yes
4
Basketball;
Darts;
Motorsport
Eyedness
Direct
observation
Sighting/Miles
Test
Performance task
Zazzo (1960)2
--
2
Basketball;
Multisports
T-116 Test
Performance task (12
items)
Matějček (2007)
--
1
Biathlon
Test
Individual
laterality in sports
Self-reported
(11 items)
Azemar (2003)
--
1
Soccer
Handedness
and eyedness
Direct
observation
Web photographies
Ziagkas, Mavvidis
& Georgios (2018)
1
Tennis 1
Table 3. Handedness and eyedness assessment methods. 3
Note: 1 Number of reviewed studies applying the instrument; 2 most cited referring the instrument; --: not reported. Manuscript to be reviewed Table 4(on next page) Table 4(on next page) Table 4(on next page) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Table 4(on next page) Table 4. Variations of the Pointing Test (Porta Test) depending on the target (type and
distance), the pointing technique and the identification method of the dominant eye. Note: --: not reported. Note: --: not reported. PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) Manuscript to be reviewed Manuscript to be reviewed 1
Table 4. V
of the Pointing Test (Porta Test) depending on the target (type and dista
2
the pointing technique and the identification method of the dominant eye. 3
Study
Sport
Target
Target distance
Pointing
technique
Assessment variations
Dalton,
Guillon &
Naroo
(2015)
Golf
Chart
(Michel
Guillon
vision clinic)
Scalable at any
distance
Index finger
on both arms
alternately
Evaluators cover both eyes
alternately, and subject indicate
where the finger and target still
aligned (that is the dominant eye)
Laborde
et al. (2009)
Archery
Any object
>2 meters
Index finger
on one arm
Subject close one eye at a time. The
eye aligned with the object and
the finger is dominant sighting eye
Mann,
Runswick
& Allen
(2016)
Criquet
Camera
3 meters
Thumb finger
on both arms
alternately in
specific
batting
stance
Photograph
Portal &
Romano
(1998)
Baseball
--
--
--
--
Razeghi
(2012)
Darts
Any object
--
One arm
index finger
Subjects close the eyes alternately
or draw the finger back to the head
Sugiyama
& Lee
(2005)
Golf
Examiner
nose
--
Index or
thumb finger
on both arms
alternately
The eye with which the finger was
aligned was noted
45
Note: --: not reported. 6 1
Table 4. V
of the Pointing Test (Porta Test) depending on the target (type and distance),
2
the pointing technique and the identification method of the dominant eye. 3 PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022) PeerJ reviewing PDF | (2022:05:73773:1:0:NEW 4 Oct 2022)
|
https://openalex.org/W3164647363
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https://www.researchsquare.com/article/rs-109728/v1.pdf?c=1605826245000
|
English
| null |
Vascular endothelial growth factor-A (VEGFA) gene polymorphisms may serve as genetic marker for Coronary Heart Disease (CHD)
|
Research Square (Research Square)
| 2,020
|
cc-by
| 5,945
|
Central South University Xiangya School of Medicine Affiliated Haikou Hospital
Shijuan Lu
(
lushijuan_361@163.com
) Central South University Xiangya School of Medicine Affiliated Haikou Hospital
Shijuan Lu
(
lushijuan_361@163.com
) Shijuan Lu
(
lushijuan_361@163.com
)
Central South University Xiangya School of Medicine Affiliated Haikou Hospital
Yilei Zhou
Central South University Xiangya School of Medicine Affiliated Haikou Hospital
Dehong Lin
Central South University Xiangya School of Medicine Affiliated Haikou Hospital
Zibin Chen
Central South University Xiangya School of Medicine Affiliated Haikou Hospital
Zanrui Zhong
Central South University Xiangya School of Medicine Affiliated Haikou Hospital
Jianghua Zhong
Central South University Xiangya School of Medicine Affiliated Haikou Hospital Central South University Xiangya School of Medicine Affiliated Haikou Hospital
Yilei Zhou Research Keywords: VEGFA, coronary heart disease (CHD), susceptibility, polymorphism, case - control study
Posted Date: November 19th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-109728/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Keywords: VEGFA, coronary heart disease (CHD), susceptibility, polymorphism, case - control study License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/11 Page 1/11 2.1. Study participants Using a case-control study, 501 CHD patients and 496 unrelated healthy controls in a large cohort of Han Chinese population were recruited from Central
South University Xiangya School of Medicine Affiliated Haikou Hospital to investigate whether the VEGFA variants have influence on CHD. All CHD patients
were diagnosed including classic ischemic symptoms, plus one or more electrocardiographic (ECG) changes (ST-segment depression or elevation of ≥
0.5 mm, T-wave inversion of ≥ 3 mm in ≥ 3 leads, or left bundle branch block), in addition to increased cardiac markers such as creatinine kinase-MB and
troponin T (9). Patients with other diseases, such as complex hematologic, autoimmune, congenital cardiac structural or functional abnormalities, or tumors
were excluded. At the same time, the 496 healthy controls were randomly selected from the same hospital in the same period and were diagnosed without
heart disease or other diseases. All subjects were unrelated individuals and at least three generations of Han ancestors. 2 2 D t
ll
ti 1. Introduction Coronary artery disease (CAD) continues to be one of the most common cardiovascular disease with high morbidity and mortality (1), which represents a
public health challenge in both industrialized and developing countries (2). Coronary heart disease (CHD) is the most common and severe manifestation of
CAD (3). CHD, the myocardial functional or organic lesion, is caused by coronary artery stenosis or occlusion, or shortage of blood and oxygen supply (4). According to the data from the American Heart Association in 2009, cardiovascular disease has become the leading cause of death in the world (5). As China
gradually enters an aging society and the number of the elderly gradually increases, CHD is the second leading cause of death after malignant tumors (6). Numerous studies of its etiology and pathogenesis indicated that many factors have been proved to be associated with the occurrence of CHD, such as age,
gender, diabetes, diet, and genetic factors (7, 8). Despite the specific factors influencing susceptibility of CHD is still unknown, recent molecular
epidemiological studies have pointed to the potential and significant role of gene variants associated CHD, such as CCDC92 (9), AdipoQ (10). The vascular
permeability factor (VEGF) gene, consisting of VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, VEGFF and placental growth factor, is located on chromosome 6p21.3
and could express different isoforms of proteins (11). Usually, VEGFA is referred as VAGF. The human VEGFA gene is very polymorphic. Doxing Liu et al (12)
showed that 3 SNPs (rs699947, rs3025039 and rs1570360) of VEGFA were remarkably associated with the susceptibility to CHD in a Han Chinese population. In addition, a systematic review and meta-analysis showed that VEGFA rs699947 polymorphism was not associated with CHD (13). However, there are few
studies which are able to clarify the potential relationship between VEGFA genetic polymorphisms and the susceptibility to CHD in a Han Chinese population. Therefore, a case-control study was carried out to evaluate the influence of VEGFA polymorphisms at allele, genotype, and haplotype interface with
development of CHD among Han Chinese population. Abstract Purpose: Coronary heart disease (CHD) is a common cardiovascular disease resulting from interaction of multiple environmental and genetic factors. This
study aimed to confirm whether single nucleotide polymorphisms (SNPs) in VEGFA gene were associated with CHD in the Han Chinese population. Materials and Methods: Blood samples were collected from 501 CHD patients and 496 healthy individuals. Genotyping of five SNPs within VEGFA was
performed using Agena MassARRAY platform. Odd ratios (ORs) and 95% confidence intervals (95%CIs) were calculated to evaluate the association between
SNPs and CHD risk. Results: The genotype “C/T” of rs3025021 in VEGFA was found to be associated with CHD susceptibility (OR = 1.35, 95% CI = 1.02- 1.80, p = 0.038) in the
overall. Rs833068 was observed to be associated with the reduced risk of CHD at age > 61 years and ≤ 61 years, respectively. And three loci (rs833068,
rs3025021 and rs6905288) were related to the CHD risk in males. In addition, rs3025021 was associated with increased risk of CHD in patients with
hypertension or diabetes. Conversely, three loci (rs833068, rs3025030 and rs6905288) were related to the CHD risk in patients without hypertension. The
rs833068 was associated with reduced risk of CHD in patients without diabetes. Finally, we found a strong LD between rs833068 and rs833070. Conclusion: Gene polymorphisms in VEGFA were notably correlated with altered CHD risk in the Han Chinese population. Large sample size and well -
designed studies are needed to further clarify the potential mechanisms underlying the CHD. 2.2 Data collection The protocol of the present study was approved by the clinical investigative ethical committee of Central South University Xiangya School of Medicine
Affiliated Haikou Hospital, and all procedures were performed in compliance with medical research involving humans of the World Medical Association
Declaration of Helsinki. Informed consent forms were obtained from all participants after a full explanation. Blood samples from each individual were
harvested respectively at the time of initial diagnosis. Subsequently, about 5 ml venous blood samples were collected from each participant into tubes
containing ethylenediamine tetraacetic acid (EDTA) for anticoagulation. A Whole Blood Genomic DNA Extraction Kit (Tiangen Biotech, Beijing, China) was
used to extract genomic DNA from peripheral blood samples according to the manufacturer’s instructions. And the purity and concentration of the DNA
samples were evaluated with the NanoDrop 2000C (Thermo Scientifc, Waltham, Massachusetts, USA) (14). The isolated DNA samples were stored at − 80 °C
until analysis. 2.3 SNP selection and genotyping 2.3 SNP selection and genotyping Page 2/11 Page 2/11 Five candidate SNPs (rs833068, rs833070, rs3025021, rs3025030, rs6905288) in the VEGFA gene were selected with the minor allele frequency (MAF) > 0.05 in
Han Chinese from the 1000 Genomes Project (http://www.1000genomes.org/). The primers for amplification and extension reactions were designed with
Agena MassARRAY Assay Design 3.0 Software (Table 1) (15). The SNP genotyping was performed with Agena MassARRAY RS1000 according to the
manufacturer’s instruction. The data management and analysis were carried using Agena Typer 4.0 software (15, 16). Table 1
PCR primer for this study. SNP-ID
Foward primer (5'-3') for PCR
Reverse primer (5'-3') for PCR
UEP_DIR
UEP SEQ
rs833068
ACGTTGGATGGGGAGAGTGGACATTTAGTG
ACGTTGGATGGAGATCCCATTAGGCTGAG
R
GCTCACACAGGAAGGGT
rs833070
ACGTTGGATGGCTCAGCCTAATGGGATCTC
ACGTTGGATGAGTTCACAGCACCCGAACAT
R
cattaACAGCACCCGAACATAGTCAA
rs3025021
ACGTTGGATGCACAGAGGCCTCCTTGCAG
ACGTTGGATGGGTGTGATGGGAGGCTAAG
R
CACAGCCTCCGACCC
rs3025030
ACGTTGGATGTGGCTGTTCGTTTAGGATGG
ACGTTGGATGGACTGGTGGAGGATTAAAGG
R
GGAGGATTAAAGGTATCTAGTATT
rs6905288
ACGTTGGATGTGAGGAATAATAACCACCCC
ACGTTGGATGAAGTAGAGAGAAGCCATCCC
F
GCCATCCCTGCCCCA
SNP, Single-nucleotide polymorphism; UEP SEQ, Unextended mini-sequencing primer; DIR: direction. 2.4 Statistical analysis Statistical analyses were analyzed using SPSS 19.0 (SPSS, Chicago, IL, USA) and Microsoft Excel. The two-sided Chi-square tests and independent sample
Student’s t-test were applied to assess the differences in the distribution of gender and age between cases and controls, respectively. The genotype
frequencies of the control group were tested for the Hardy-Weinberg equilibrium (HWE) using Chi-square test. Chi-squared test was used to calculate the allele
and genotype frequencies of each SNP between cases and controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the
association between VEGFA gene polymorphisms and the CHD risk using logistic regression analysis with or without adjustment for age and gender (17). The
wild type allele was used as a reference. The four genetic models analyses were applied using PLINK software (Version 1.07) to evaluate the associations
between SNPs and the CHD risk (18). Then, we conducted stratification analysis on age, gender, CHD-associated diseases. Finally, we performed linkage
disequilibrium (LD) and haplotype analysis using the Haploview software package (version 4.2). All p values of statistical tests in this study were two-sided,
and p < 0.05 indicated statistical significance. 3.1 Participant characteristics. We recruited 501 CHD patients (mean age 61.32 ± 11.70 years old) consisting of 320 males and 181 females and 496 unrelated healthy individuals (mean age
60.69 ± 6.42 years old) containing of 318 healthy males and 178 females in this study. There were no statistically significant differences on the distribution of
gender and age between the cases and controls (p = 0.885, p = 0.289, respectively). In the case group, there were 296 patients with hypertension and 101
patients with diabetes, while there were 205 patients without hypertension and 400 patients without diabetes, respectively. The basic characteristics of all
subjects were shown in Table 2. Page 3/11 Page 3/11 Table 2
Characteristics of the cases and controls. Variables
Cases (n = 501)
Controls (n = 496)
p
Gender
0.885a
Male
320(64%)
318(64%)
Female
181(36%)
178(36%)
Age
61.32 ± 11.70
60.69 ± 6.42
0.289b
> 61
250(50%)
233(47%)
≤ 61
251(50%)
263(53%)
Hypertension
yes
296(60%)
no
205(40%)
Diabetes
yes
101(20%)
no
400(80%)
ap value was calculated from two-sided Chi-squared tests; bp value was calculated from Student’s t test. p < 0.05 indicates statistical significance. 3.2. Association between VEGFA variations and CHD risk. In our study, the basic information about the selected SNPs in VEGFA gene was listed in Table 3. The genotype distribution of all candidate SNPs in control
group were in accordance with HWE (p value > 0.05). We had not found that any SNPs were significantly associated with the risk of CHD in the allele model (all
p > 0.05). Four genetic models, including the codominant model, the dominant model, the recessive model, and the Log-additive model were carried out to
analyze the relationship between SNPs genotypes and CHD risk. The results were showed in Table 4. The significantly positive association was found
between the “C/T” of rs3025021 in VEGFA and CHD susceptibility in the codominant model (OR = 1.35, 95% CI = 1.02–1.80, p = 0.038) after adjustment for
gender and age, which showed that the rs3025021 was a risk factor in the development of CHD. However, we observed no significant correlation between other
SNPs and CHD risk. Table 3
Basic information of the 5 SNPs in this study. SNP-ID
Chr. Position
Allele
MAF
pa-HWE
Allele model
(A/B)
Cases
Controls
OR(95%CI)
pb
rs833068
6
43774790
A/G
0.411
0.440
0.412
0.89(0.74–1.06)
0.185
rs833070
6
43774889
C/T
0.238
0.229
0.252
1.05(0.85–1.30)
0.631
rs3025021
6
43781426
C/T
0.168
0.145
0.204
1.19(0.93–1.51)
0.167
rs3025030
6
43782850
C/G
0.178
0.156
0.393
1.17(0.92–1.48)
0.200
rs6905288
6
43791136
A/G
0.262
0.268
0.210
0.97(0.8–1.19)
0.774
SNP, single-nucleotide polymorphism; MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium; OR, odds ratio; CI, confidence interval. p < 0.05 indicates statistical significance. Page 4/11 Page 4/11 Page 4/11 SNPs associated with the CHD with adjustments for gender and age. 3.2. Association between VEGFA variations and CHD risk. SNP
Model
Genotype
Case
Control
Before adjusted
After adjusted
OR(95%CI)
p-value
OR(95%CI)
p-value
rs833068
Co-dominant
G/G
175(35.00%)
150(30.31%)
1[Ref]
1[Ref]
A/G
239(47.80%)
254(51.31%)
0.81(0.61–1.07)
0.133
0.80(0.61–1.07)
0.128
A/A
86(17.20%)
91(18.38%)
0.81(0.56–1.17)
0.260
0.81(0.56–1.17)
0.254
Dominant
G/G
175(35.00%)
150(30.31%)
1[Ref]
1[Ref]
A/G-A/A
325(65.00%)
345(69.69)
0.81(0.62–1.05)
0.114
0.80(0.62–1.05)
0.110
Recessive
G/G-A/G
414(82.80%)
404(81.62%)
1[Ref]
1[Ref]
A/A
86(17.20%)
91(18.38%)
0.92(0.67–1.28)
0.625
0.92(0.67–1.28)
0.623
Log-additive
-
-
-
0.89(0.74–1.06)
0.182
0.88(0.74–1.06)
0.177
rs833070
Co-dominant
T/T
285(57.34%)
289(58.38%)
1[Ref]
1[Ref]
C/T
187(37.63%)
185(37.38%)
1.03(0.79–1.33)
0.853
1.02(0.79–1.33)
0.881
C/C
25(5.03%)
21(4.24%)
1.21(0.66–2.21)
0.540
1.20(0.66–2.19)
0.558
Dominant
T/T
285(57.34%)
289(58.38%)
1[Ref]
1[Ref]
C/T-C/C
212(42.66%)
206(41.62%)
1.04(0.81–1.34)
0.740
1.04(0.81–1.34)
0.770
Recessive
T/T-C/T
472(94.97)
474(95.76%)
1[Ref]
1[Ref]
C/C
25(5.03%)
21(4.24%)
1.20(0.66–2.17)
0.556
1.19(0.66–2.15)
0.569
Log-additive
-
-
-
1.06(0.85–1.31)
0.623
1.05(0.85–1.3)
0.651
rs3025021
Co-dominant
T/T
344(68.66%)
366(73.79%)
1[Ref]
1[Ref]
C/T
146(29.14%)
116(23.39%)
1.34(1.01–1.78)
0.044*
1.35(1.02–1.80)
0.038*
C/C
11(2.20%)
14(2.82%)
0.84(0.37–1.87)
0.662
0.85(0.38–1.89)
0.687
Dominant
T/T
344(68.66%)
366(73.79%)
1[Ref]
1[Ref]
C/T-C/C
157(31.34%)
130(26.21%)
1.29(0.98–1.69)
0.074
1.30(0.99–1.71)
0.063
Recessive
T/T-C/T
490(97.80%)
482(97.18%)
1[Ref]
1[Ref]
C/C
11(2.20%)
14(2.82%)
0.77(0.35–1.72)
0.528
0.78(0.35–1.74)
0.544
Log-additive
-
-
-
1.19(0.93–1.51)
0.168
1.20(0.94–1.53)
0.147
rs3025030
Co-dominant
G/G
339(67.66%)
350(70.56%)
1[Ref]
1[Ref]
C/G
146(29.14%)
137(27.62%)
1.10(0.83–1.45)
0.499
1.10(0.83–1.45)
0.507
C/C
16(3.19%)
9(1.81%)
1.84(0.80–4.21)
0.152
1.81(0.79–4.15)
0.164
Dominant
G/G
339(67.66%)
350(70.56%)
1[Ref]
1[Ref]
C/G-C/C
162(32.34%)
146(29.43%)
1.15(0.88–1.50)
0.322
1.14(0.87–1.50)
0.333
Recessive
G/G-A/G
485(96.81%)
487(98.19%)
1[Ref]
1[Ref]
C/C
16(3.19%)
9(1.81%)
1.79(0.78–4.08)
0.169
1.76(0.77–4.02)
0.182
Log-additive
-
-
-
1.17(0.92–1.49)
0.196
1.17(0.92–1.48)
0.208
rs6905288
Co-dominant
G/G
280(55.89%)
260(52.42%)
1[Ref]
1[Ref]
A/G
179(35.73%)
206(41.53%)
0.81(0.62–1.05)
0.108
0.81(0.62–1.05)
0.105
A/A
42(8.38%)
30(6.05%)
1.30(0.79–2.14)
0.302
1.30(0.79–2.15)
0.296
Dominant
G/G
280(55.89%)
260(52.42%)
1[Ref]
1[Ref]
A/G-A/A
221(44.11%)
236(47.58%)
0.87(0.68–1.12)
0.272
0.87(0.68–1.12)
0.268
Recessive
G/G-A/G
459(91.62%)
466(93.95%)
1[Ref]
1[Ref]
A/A
42(8.38%)
30(6.05%)
1.42(0.87–2.31)
0.156
1.43(0.88–2.32)
0.152
SNP: single-nucleotide polymorphism; EC: Esophageal cancer; OR, odds ratio; CI, confidence interval. p < 0.05 indicates statistical significance. Page 5/11 SNP
Model
Genotype
Case
Control
Before adjusted
After adjusted
OR(95%CI)
p-value
OR(95%CI)
p-value
Log-additive
-
-
-
0.97(0.80–1.18)
0.775
0.97(0.80–1.18)
0.775
SNP: single-nucleotide polymorphism; EC: Esophageal cancer; OR, odds ratio; CI, confidence interval. p < 0.05 indicates statistical significance. 3.3 Stratification analysis by age and gender. According to gender and age parameters, stratified analysis regarding the effects of SNPs on CHD risk was summarized in Table 5. 3.2. Association between VEGFA variations and CHD risk. The stratification analysis
by age adjusted for age and gender showed that rs833068 in VEGFA was observed to be associated with the reduced risk of CHD at age > 61 years in the
codominant model (OR = 0.64, 95% CI = 0.42–0.98, p = 0.042 for the “A/G” genotype). Meanwhile, the rs833068 was also associated with the reduced risk of
CHD at age ≤ 61 years in the recessive model (OR = 0.58, 95% CI = 0.34–0.98, p = 0.043). In addition, the stratification analysis by gender adjusted for age
found that three loci (rs833068, rs3025021 and rs6905288) in VEGFA were related to the CHD risk in males. The rs833068 was associated with reduced risk of
CHD in the allele model (adjusted, OR = 0.75, 95% CI = 0.01–0.75, p = 0.010), the codominant model (adjusted, OR = 0.58, 95% CI = 0.37–0.92, p = 0.020 for
“A/A” genotype; OR = 0.65, 95% CI = 0.45–0.92, p = 0.016 for “A/G” genotype), the dominant model (OR = 0.63, 95% CI = 0.45–0.88, p = 0.006), the log-additive
model (OR = 0.74, 95% CI = 0.60–0.93, p = 0.010). The rs3025021 was associated with increased risk of CHD in the codominant model (adjusted, OR = 1.49,
95% CI = 1.04–2.13, p = 0.029 for “C/T” genotype), the dominant model (OR = 1.43, 95% CI = 1.01–2.02, p = 0.041). The rs6905288 was associated with
increased risk of CHD in the recessive model (OR = 2.03, 95% CI = 1.08–3.78, p = 0.027). However, we found no any genotypes of the selected SNPs were
associated with CHD in females in any genetic model. SNP
Model
Genotype
Case
Control
Before adjusted
After adjusted
OR(95%CI)
p-value
OR(95%CI)
p-value
Log-additive
-
-
-
0.97(0.80–1.18)
0.775
0.97(0.80–1.18)
0.775
SNP: single-nucleotide polymorphism; EC: Esophageal cancer; OR, odds ratio; CI, confidence interval. p < 0.05 indicates statistical significance. 3.3 Stratification analysis by age and gender. 3.3 Stratification analysis by age and gender. According to gender and age parameters, stratified analysis regarding the effects of SNPs on CHD risk was summarized in Table 5. The stratification analysis
by age adjusted for age and gender showed that rs833068 in VEGFA was observed to be associated with the reduced risk of CHD at age > 61 years in the
codominant model (OR = 0.64, 95% CI = 0.42–0.98, p = 0.042 for the “A/G” genotype). Meanwhile, the rs833068 was also associated with the reduced risk of
CHD at age ≤ 61 years in the recessive model (OR = 0.58, 95% CI = 0.34–0.98, p = 0.043). In addition, the stratification analysis by gender adjusted for age
found that three loci (rs833068, rs3025021 and rs6905288) in VEGFA were related to the CHD risk in males. The rs833068 was associated with reduced risk of
CHD in the allele model (adjusted, OR = 0.75, 95% CI = 0.01–0.75, p = 0.010), the codominant model (adjusted, OR = 0.58, 95% CI = 0.37–0.92, p = 0.020 for
“A/A” genotype; OR = 0.65, 95% CI = 0.45–0.92, p = 0.016 for “A/G” genotype), the dominant model (OR = 0.63, 95% CI = 0.45–0.88, p = 0.006), the log-additive
model (OR = 0.74, 95% CI = 0.60–0.93, p = 0.010). The rs3025021 was associated with increased risk of CHD in the codominant model (adjusted, OR = 1.49,
95% CI = 1.04–2.13, p = 0.029 for “C/T” genotype), the dominant model (OR = 1.43, 95% CI = 1.01–2.02, p = 0.041). The rs6905288 was associated with
increased risk of CHD in the recessive model (OR = 2.03, 95% CI = 1.08–3.78, p = 0.027). However, we found no any genotypes of the selected SNPs were
associated with CHD in females in any genetic model. Page 6/11 Page 6/11 Table 5
Stratified analysis on association between selected SNPs and CHD risk. 3.3 Stratification analysis by age and gender. SNP ID
p†, OR (95% CI)
Allele
Homozygote
Heterozygote
Dominant
Recessive
Additive
Age
> 61
rs833068
0.219,0.86(0.67–
1.10)
0.875,0.96(0.56–
1.64)
0.042*,0.64(0.42–
0.98)
0.105,0.72(0.48–
1.07)
0.343,1.26(0.78–
2.02)
0.586,0.93(0.71–
1.21)
≤ 61
rs833068
0.551,0.92(0.72–
1.20)
0.150,0.65(0.36–
1.17)
0.375,1.22(0.79–
1.88)
0.851,1.04(0.69–
1.57)
0.043*,0.58(0.34–
0.98)
0.335,0.87(0.66–
1.15)
Gender
Male
rs833068
0.010*,0.75(0.01–
0.75)
0.020*,0.58(0.37–
0.92)
0.016*,0.65(0.45–
0.92)
0.006*,0.63(0.45–
0.88)
0.172,0.76(0.50–
1.13)
0.010*,0.74(0.60–
0.93)
rs3025021
0.087,1.30(0.09–
1.30)
0.966,0.98(0.37–
2.58)
0.029*,1.49(1.04–
2.13)
0.041*,1.43(1.01–
2.02)
0.796,0.88(0.34–
2.31)
0.089,1.3(0.96–
1.76)
rs6905288
0.449,1.10(0.45–
1.10)
0.05,1.89(1.00-
3.59)
0.332,0.85(0.61–
1.18)
0.817,0.96(0.71–
1.32)
0.027*,2.03(1.08–
3.78)
0.453,1.1(0.86–
1.41)
Female
rs833068
0.213,1.21(0.90–
1.62)
0.227,1.48(0.79–
2.77)
0.506,1.17(0.73–
1.87)
0.344,1.24(0.79–
1.94)
0.309,1.34(0.76–
2.35)
0.228,1.21(0.89–
1.64)
rs3025021
0.993,1.00(0.67–
1.51)
0.622,0.69(0.16–
2.99)
0.63,1.13(0.70–
1.81)
0.734,1.08(0.68–
1.72)
0.591,0.67(0.16–
2.88)
0.885,1.03(0.68–
1.56)
rs6905288
0.137,0.78(0.56–
1.08)
0.341,0.66(0.29–
1.54)
0.161,0.73(0.47–
1.13)
0.125,0.72(0.47–
1.10)
0.508,0.76(0.33–
1.72)
0.135,0.77(0.55–
1.08)
Hypertension
yes
rs3025021
0.088,1.27(0.96–
1.67)
0.824,1.11(0.45–
2.69)
0.029*,1.44(1.04-
2.00)
0.035*,1.41(1.02–
1.93)
0.999,1.00(0.41–
2.42)
0.067,1.29(0.98–
1.70)
no
rs833068
0.046,0.79(0.62-
1.00)
0.037*,0.58(0.35–
0.97)
0.212,0.79(0.55–
1.14)
0.084,0.74(0.52–
1.04)
0.087,0.67(0.42–
1.06)
0.034*,0.77(0.60–
0.98)
rs3025030
0.047,1.35(1.00-
1.82)
0.041*,2.7(1.04–
6.99)
0.206,1.26(0.88–
1.81)
0.090,1.35(0.95–
1.91)
0.055,2.52(0.98–
6.47)
0.037*,1.38(1.02–
1.87)
rs6905288
0.930,0.99(0.76–
1.28)
0.102,1.64(0.91–
2.97)
0.051,0.7(0.49-
1.00)
0.241,0.82(0.59–
1.14)
0.031*,1.89(1.06–
3.37)
0.983,1.00(0.77–
1.29)
Diabetes
yes
rs3025021
0.025*,1.55(1.05–
2.27)
0.928,0.93(0.20–
4.25)
0.002*,2.06(1.30–
3.27)
0.004*,1.94(1.24–
3.05)
0.704,0.75(0.17–
3.37)
0.016,1.60(1.09–
2.37)
no
rs833068
0.130,0.86(0.72–
1.04)
0.246,0.80(0.54–
1.17)
0.035*,0.73(0.54–
0.98)
0.039*,0.75(0.56–
0.99)
0.821,0.96(0.68–
1.36)
0.130,0.86(0.72–
1.04)
SNP: single-nucleotide polymorphism; OR: odds ratio; CI: confidence interval; CHD: coronary heart disease. Table 5
b t Table 5
b 3.4 Stratification analysis with/without hypertension or diabetes. 3.4 Stratification analysis with/without hypertension or diabetes. We next analyzed the relationship between selected SNPs and CHD-associated diseases, which include hypertension and diabetes. In CHD patients with
hypertension, we found that rs3025021 was associated with increased risk of CHD in the codominant model (adjusted, OR = 1.44, 95% CI = 1.04–2.00, p =
0.029 for “C/T” genotype), the dominant model (OR = 1.41, 95% CI = 1.012–1.93, p = 0.035). Conversely, in the CHD patients without hypertension, three loci
(rs833068, rs3025030 and rs6905288) in VEGFA were related to the CHD risk. The rs833068 was associated with reduced risk of CHD in the codominant
model (adjusted, OR = 0.58, 95% CI = 0.35–0.97, p = 0.037 for “A/A” genotype), the log-additive model (OR = 0.77, 95% CI = 0.60–0.98, p = 0.034). 4. Discussion In this case-control study, allele, genotype and haplotype frequencies of five SNPs in the VEGFA gene between CHD patients and healthy controls were
compared and stratification analyses were conducted. The genotype“C/T”of rs3025021 in VEGFA was found to be associated with CHD susceptibility in the
codominant model (adjusted, OR = 1.35, 95% CI = 1.02–1.80, p = 0.038) in the overall. The stratification analysis by age showed that rs833068 in VEGFA were
observed to be associated with the reduced risk of CHD at age > 61 years and age ≤ 61 years, respectively. And the stratification analysis by gender found that
three loci (rs833068, rs3025021 and rs6905288) in VEGFA were related to the CHD risk in males. In addition, we found that rs3025021 was associated with
increased risk of CHD in patients with hypertension. Conversely, three loci (rs833068, rs3025030 and rs6905288) in VEGFA were related to the CHD risk in
patients without hypertension. Furthermore, rs3025021 was associated with increased risk of CHD in patients with diabetes. The rs833068 was associated
with reduced risk of CHD in patients without diabetes. Finally, we found a strong LD between rs833068 and rs833070. To our knowledge, this is the first study
that evaluate and show an association of VEGFA genetic variants with risk of developing CHD in Han Chinese population. Coronary heart disease (CHD) is a common and complicated cardiovascular disease in the worldwide, and caused by narrowing of the coronary arteries and a
lack of blood supply (12). However, the etiological factors for CHD are not fully understood. Like lung cancer (19) and glioma (20), it's clear that genetic
factors, such as single nucleotide polymorphism (SNPs) may also play a pivotal role in determining susceptibility of CHD (21). VEGF can not only promote the
vascular recanalization and establishment of collateral circulation, but also enhance the dependent vasodilatation of endothelial cells which are closely
related to coronary heart disease (22). Several studies have shown that appropriate timing and dose of VEGF is essential to avoid cardiovascular defects
during heart development (23, 24). VEGFA, as a member of VEGF, has been proved to promote the differentiation, proliferation and migration of microvascular
endothelial cells by binding to their receptors, thus improving the formation and development (24). In addition, the study has shown that VEGFA may play an
important role in the process of epithelial-mesenchymal transformation (EMT) and regulate the formation of endocardial cushion (22). 3.3 Stratification analysis by age and gender. The
rs3025030 was associated with increased risk of CHD in the codominant model (adjusted, OR = 2.70, 95% CI = 1.04–6.99, p = 0.041 for “C/C” genotype), the
log-additive model (OR = 1.38, 95% CI = 1.02–1.87, p = 0.037). The rs6905288 was associated with increased risk of CHD in the recessive model (adjusted, OR
= 1.89, 95% CI = 1.06–3.37, p = 0.031). In CHD patients with diabetes, we found that rs3025021 was associated with increased risk of CHD in the allele model (adjusted, OR = 1.55, 95% CI = 1.05–
2.27, p = 0.025), the codominant model (adjusted, OR = 2.06, 95% CI = 1.30–3.27, p = 0.002 for “C/T” genotype), the dominant model (OR = 1.94, 95% CI = 1.24–
3.05, p = 0.004). In CHD patients without diabetes, the rs833068 was associated with reduced risk of CHD in the codominant model (adjusted, OR = 0.73, 95%
CI = 0.54–0.98, p = 0.035 for “A/G” genotype), the dominant model (OR = 0.75, 95% CI = 0.56–0.99, p = 0.039). p
g
yp )
3.5 Haplotype association g
y
)
3.5 Haplotype association )
3.5 Haplotype association Page 7/11 Page 7/11 Finally, the LD block and haplotype analyses of the selected SNPs in all subjects were further studied. And we found a strong LD between rs833068 and
rs833070 with D’ = 1.00 (Fig. 1). The results of the association between haplotypes and the CHD risk were shown in Table 6. However, we found no haplotypes
were associated with the risk of CHD even if after logistic regression analysis adjustment for age and gender. Table 6
Haplotype analysis results in this study. Haplotype
Frequency
Before adjusted
After adjusted
Case
Control
OR(95%CI)
p-value
OR(95%CI)
pa-value
rs833068|rs833070
GT
0.239
0.230
1.06(0.85–1.31)
0.622
1.05(0.85–1.30)
0.651
AC
0.412
0.439
0.89(0.75–1.07)
0.221
0.89(0.75–1.07)
0.215
GC
0.651
0.669
0.92(0.77–1.11)
0.407
0.92(0.76–1.11)
0.378
SNP: single-nucleotide polymorphism; OR: odds ratio; CI: confidence interval. Pa: Adjusted by gender and age. 4. Discussion Recently, several studies have explored the association between the SNPs in VEGFA and the susceptibility to CHD (12, 25, 26). Han et al (25) showed that
rs3025039 in VEGFA gene was remarkably associated with CHD risk in Han Chinese population. On the contrary, Griffin et al (26) conducted a meta-analysis
which demonstrated VEGFA polymorphisms may not be associated with CHD. Furthermore, Dong et al (12) found three SNPs (rs699947, rs3025039, and
rs1570360) were remarkably correlated with the susceptibility to CHD. To further clarify the relationship between SNPs within VEGFA and CHD,we genotyped
five SNPs (rs833068, rs833070, rs3025021, rs3025030, rs6905288) in this study and discovered that all the five SNPs were significantly with the risk of CHD. Inevitably, several limitations should be addressed with regard to the case-control study. First, our sample was limited to the Han Chinese population, thus the
investigation results might not be applicable to other Chinese populations or additional ethnic groups. Larger and more diversity sample is needed to identify
the role of VEGFA genetic polymorphisms in CHD risk. Besides, it could not be ignored that the uniqueness of samples might exaggerate the correlation
between the selected 5 SNPs and CHD risk, or neglect the role of some other vital polymorphisms in VEGFA in susceptibility to CHD. Therefore, large
prospective cohort studies or combined meta-analyses should be designed to address these limitations. 5. Conclusion In summary, the present study indicated that gene polymorphisms in VEGFA gene were notably correlated with altered CHD risk in the Han Chinese population. Thus, the VEGFA mutations might be used to clinical medicine as genetic marker. Competing interests None References 1. Pfisterer ME, Zellweger MJ, Gersh BJ. Management of stable coronary artery disease. Lancet (London, England). 2010;375(9716):763-72. doi:
10.1016/s0140-6736(10)60168-7. 2. Ross R. Atherosclerosis--an inflammatory disease. The New England journal of medicine. 1999;340(2):115-26. doi: 10.1056/nejm199901143400207. 2. Ross R. Atherosclerosis--an inflammatory disease. The New England journal of medicine. 1999;340(2):115-26. 3. Heusch G, Libby P, Gersh B, Yellon D, Bohm M, Lopaschuk G, et al. Cardiovascular remodelling in coronary artery disease and heart failure. Lancet
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different arterial oxygen pressures. Basic research in cardiology. 1990;85(5):467-80. 4. Walfridsson H, Lund N. Tissue oxygen pressure in normal myocardium and across the border zone during coronary artery occlusion in the pig. Effects of
different arterial oxygen pressures. Basic research in cardiology. 1990;85(5):467-80. 5. Lloyd-Jones D, Adams R, Carnethon M, De Simone G, Ferguson TB, Flegal K, et al. Heart disease and stroke statistics--2009 update: a report from the
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American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2009;119(3):480-6. doi:
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Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medicine. 2007;27(8):689-91. 6. Chen BJ, Pan ZQ, Su XX. [Study on changes of TCM syndrome in patients with coronary heart disease before and after intervention treatment]. Zhongguo
Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medicine. 2007;27(8):689-91. 6. Chen BJ, Pan ZQ, Su XX. [Study on changes of TCM syndrome in patients with coronary heart disease before and after intervention treatment]. Zhonggu
Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medicine. 2007;27(8):689-91. 7. Pogosova N, Kotseva K, De Bacquer D, von Kanel R, De Smedt D, Bruthans J, et al. References Psychosocial risk factors in relation to other cardiovascular risk factors
in coronary heart disease: Results from the EUROASPIRE IV survey. A registry from the European Society of Cardiology. European journal of preventive
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journal of cardiology. 2014;172(1):e220-4. doi: 10.1016/j.ijcard.2013.12.156. 13. Chen HL, Liu K. Vascular endothelial growth factor polymorphisms and coronary artery disease: a systemic review and meta-analysis. International
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population. World journal of gastroenterology. 2015;21(22):6898-904. doi: 10.3748/wjg.v21.i22.6898. 14. Geng TT, Xun XJ, Li S, Feng T, Wang LP, Jin TB, et al. Association of colorectal cancer susceptibility variants with esophageal cancer in a Chinese
population. World journal of gastroenterology. 2015;21(22):6898-904. doi: 10.3748/wjg.v21.i22.6898. 15. Gabriel S, Ziaugra L, Tabbaa D. SNP genotyping using the Sequenom MassARRAY iPLEX platform. Current protocols in human genetics. 2009;Chapter
2:Unit 2.12. doi: 10.1002/0471142905.hg0212s60. 16. Thomas RK, Baker AC, Debiasi RM, Winckler W, Laframboise T, Lin WM, et al. High-throughput oncogene m
genetics. 2007;39(3):347-51. doi: 10.1038/ng1975. 16. Thomas RK, Baker AC, Debiasi RM, Winckler W, Laframboise T, Lin WM, et al. High-throughput oncogene mutation profiling in human cancer. Nature
genetics. 2007;39(3):347-51. doi: 10.1038/ng1975. s notes. The odds ratio. BMJ (Clinical research ed). 2000;320(7247):1468. doi: 10.1136/bmj.320.7247.1468. 17. Bland JM, Altman DG. Statistics notes. Acknowledgement We thank all of the participants for their involvement in this study. We are very grateful to the clinicians and other hospital staff for providing blood samples
and data collection for this study. Page 8/11 Page 8/11 Authors' contributions Conceived and designed the experiments: Shijuan Lu. Performed the experiments: Kang Huang, Yilei Zhou. Analyzed the data: Dehong Lin, Zibin Chen. Contributed reagents/materials/analysis tools: Zanrui Zhong. Wrote the paper: Jianghua Zhong. Revised：Shijuan Lu. F
di References The odds ratio. BMJ (Clinical research ed). 2000;320(7247):1468. doi: 10.1136/bmj.320.7247.1468. 7. Bland JM, Altman DG. Statistics notes. The odds ratio. BMJ (Clinical research ed). 2000;320(7247):1468. doi: 1 18. Clarke GM, Anderson CA, Pettersson FH, Cardon LR, Morris AP, Zondervan KT. Basic statistical analysis in genetic case-control studies. Nature protocols
2011;6(2):121-33. doi: 10.1038/nprot.2010.182. 19. Gao L, Thakur A, Liang Y, Zhang S, Wang T, Chen T, et al. Polymorphisms in the TERT gene are associated with lung cancer risk in the Chinese Han
population. European journal of cancer prevention : the official journal of the European Cancer Prevention Organisation (ECP). 2014;23(6):497-501. doi:
10.1097/cej.0000000000000086. 19. Gao L, Thakur A, Liang Y, Zhang S, Wang T, Chen T, et al. Polymorphisms in the TERT gene are associated with lung cancer risk in the Chinese Han
population. European journal of cancer prevention : the official journal of the European Cancer Prevention Organisation (ECP). 2014;23(6):497-501. doi
10.1097/cej.0000000000000086. 20. Li G, Jin TB, Wei XB, He SM, Liang HJ, Yang HX, et al. Selected polymorphisms of GSTP1 and TERT were associated with glioma risk in Han Chinese. Cancer epidemiology. 2012;36(6):525-7. doi: 10.1016/j.canep.2012.06.008. 20. Li G, Jin TB, Wei XB, He SM, Liang HJ, Yang HX, et al. Selected polymorphisms of GSTP1 and TERT were associated with glioma risk in Han Chinese. Cancer epidemiology. 2012;36(6):525-7. doi: 10.1016/j.canep.2012.06.008. 20. Li G, Jin TB, Wei XB, He SM, Liang HJ, Yang HX, et al. Selected polymorphisms of GSTP1 and TERT were as
Cancer epidemiology. 2012;36(6):525-7. doi: 10.1016/j.canep.2012.06.008. 21. Song C, Chang Z, Magnusson PK, Ingelsson E, Pedersen NL. Genetic factors may play a prominent role in the development of coronary heart disease
dependent on important environmental factors. Journal of internal medicine. 2014;275(6):631-9. doi: 10.1111/joim.12177. 21. Song C, Chang Z, Magnusson PK, Ingelsson E, Pedersen NL. Genetic factors may play a prominent role in the development of coronary heart disease
dependent on important environmental factors. Journal of internal medicine. 2014;275(6):631-9. doi: 10.1111/joim.12177. Funding This study was supported by Hainan Natural Science Foundation Project (No. 20168320.00). Competing interests Page 9/11 Page 9/11 Page 9/11 Page 9/11 22. Wang E, Wang Z, Liu S, Gu H, Gong D, Hua K, et al. Polymorphisms of VEGF, TGFbeta1, TGFbetaR2 and conotruncal heart defects in a Chinese population. Molecular biology reports. 2014;41(3):1763-70. doi: 10.1007/s11033-014-3025-9. 22. Wang E, Wang Z, Liu S, Gu H, Gong D, Hua K, et al. Polymorphisms of VEGF, TGFbeta1, TGFbetaR2 and conotruncal heart defects in a Chinese populatio
Molecular biology reports. 2014;41(3):1763-70. doi: 10.1007/s11033-014-3025-9. 23. Miquerol L, Langille BL, Nagy A. Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development (Cambridge, England). 2000;127(18):3941-6. 24. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, et al. Abnormal blood vessel development and lethality in embryos lacking a single
VEGF allele. Nature. 1996;380(6573):435-9. doi: 10.1038/380435a0. 25. Han X, Liu L, Niu J, Yang J, Zhang Z, Zhang Z. Association between VEGF polymorphisms (936c/t, -460t/c and -634g/c) with haplotypes and coronary
heart disease susceptibility. International journal of clinical and experimental pathology. 2015;8(1):922-7. 26. Griffin HR, Hall DH, Topf A, Eden J, Stuart AG, Parsons J, et al. Genetic variation in VEGF does not contribute significantly to the risk of congenital
cardiovascular malformation. PloS one. 2009;4(3):e4978. doi: 10.1371/journal.pone.0004978. 22. Wang E, Wang Z, Liu S, Gu H, Gong D, Hua K, et al. Polymorphisms of VEGF, TGFbeta1, TGFbetaR2 and conotruncal heart defects in a Chinese population.
Molecular biology reports. 2014;41(3):1763-70. doi: 10.1007/s11033-014-3025-9. 23. Miquerol L, Langille BL, Nagy A. Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression.
Development (Cambridge, England). 2000;127(18):3941-6. Figures Figures
Figure 1
The LD block and haplotype analyses of the selected SNPs in all subjects were further studied. And we found a strong LD between rs833068 and rs833070
with D’ = 1.00 (Figure 1). Figures Figure 1 The LD block and haplotype analyses of the selected SNPs in all subjects were further studied. And we found a strong LD between rs833068 and rs833070
with D’ = 1.00 (Figure 1). The LD block and haplotype analyses of the selected SNPs in all subjects were further studied. And we found a strong LD between rs833068 and rs833070
with D’ = 1.00 (Figure 1). Page 10/11 Figure 1
The LD block and haplotype analyses of the selected SNPs in all subjects were further studied. And we found a strong LD between rs833068 and rs833070
with D’ = 1.00 (Figure 1). Figure 1 The LD block and haplotype analyses of the selected SNPs in all subjects were further studied. And we found a strong LD between rs833068 and rs833070
with D’ = 1.00 (Figure 1). The LD block and haplotype analyses of the selected SNPs in all subjects were further studied. And we found a strong LD between rs833068 and rs833070
with D’ = 1.00 (Figure 1). Page 11/11
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The physiotherapy workforce in the Brazilian Unified Health Care System
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Abstract Background: Maintaining sufficient health care workforce is a global priority to achieve universal health coverage. Therefore this study addresses the availability of physiotherapists in Brazil. Objective: To describe secular trends of the physiotherapy workforce-to-population ratio in the Unified Health Sys-
tem, considering public and private sector and care level (primary, secondary, tertiary) in Brazil and its regions. Method: Descriptive exploratory quantitative study based on secondary sources. All data related to the distribution
of physiotherapists between August 2007 and September 2016 regarding facilities types, location and public and pri-
vate sectors was obtained from the Brazilian National Registry of Health Care Facilities. Data related to the population
of Brazil was extracted from Brazilian Institute of Geography and Statistics. The physiotherapy workforce-to-population
ratio was calculated by the number of physiotherapists per 1000 population (public and private sector and care level)
by ANOVA test. The distribution trends are represented on maps. Annual growth rates were estimated with Prais–Win-
sten linear regression models, with a significance level of 0.05, autocorrelation was checked by the Durbin–Watson
test. Results: The physiotherapists ratio in Brazil was 0.22/1000 population in 2007 and 0.41 in 2016, showing growth
of 86%, with an increasing trend of 0.5% on an annual average. The public sector had the biggest physiotherapy
workforce in the country in 2007 and 2016. The primary health care had the smallest physiotherapy workforce-to-
population ratio (2007: p > 0.001 and 2016: p = 0.003), even though it had the largest growth trend in annual average
(0.9% p > 0.001), followed by public and private tertiary health care sectors (0.8% p > 0.001). The workforce in second-
ary health care was bigger in the private sector than in the public sector (0.6% p > 0.001 vs. 0.2% p = 0.004). Overall, all
regions had greater growth of physiotherapy workforce-to-population ratio in public primary and tertiary health care
sectors, and private secondary health care sector, mainly the Southeast, South and Central-West regions. Conclusion: Although the physiotherapy workforce in Brazil is relatively small, there was a trend towards growth
with differences among care levels, and public and private sectors. The physiotherapy workforce-to-population ratio
is bigger in the private secondary health care sector, followed by public tertiary, secondary and primary health care
sectors. Sub-national regions show similar trends to the national estimates, with minor variations by region. Keywords: Physiotherapists, Health workforce/statistics & numerical data, Time factors Rodés et al. Hum Resour Health (2021) 19:101
https://doi.org/10.1186/s12960-021-00642-8 Rodés et al. Hum Resour Health (2021) 19:101
https://doi.org/10.1186/s12960-021-00642-8 Open Access The physiotherapy workforce in the Brazilian
Unified Health Care System Carolina Hart Rodés1, João Vitor Lovato Daré1, Bruna Carolina de Araujo1, Leonardo Graciani2,
Silvia Maria Amado João1, Ana Claudia Camargo Gonçalves Germani3 and Ana Carolina Basso Schmitt1* © The Author(s) 2021. 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/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background In order to identify actions that will promote advances
in global health, the World Health Organization (WHO)
periodically updates epidemiological data related to the
healthcare workforce of partner countries. At the Third *Correspondence: carolinaschmitt@usp.br
1 Departamento de Fisioterapia, Fonoaudiologia e Terapia Ocupacional da
Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil
Full list of author information is available at the end of the article *Correspondence: carolinaschmitt@usp.br
1 Departamento de Fisioterapia, Fonoaudiologia e Terapia Ocupacional da
Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil
Full list of author information is available at the end of the article Methods Despite the recommendation regarding universal
health systems, considering health workers’ availability,
accessibility, acceptability and quality to assure effective
coverage [3], WHO has identified only five specific occu-
pations (doctors, nurses, midwives, dentists and phar-
macists) as indicators of the Sustainable Development
Goals’ Target related to health workforce. That partly
explains why most published literature about the health
workforce ratio and its distribution only consider doctors
[4–6] or doctors, nurses and midwives [7, 8]. A descriptive exploratory quantitative study based on
secondary sources were obtained from the Brazilian
National Registry of Health Care Facilities (Cadastro
Nacional de Estabelecimentos de Saúde—CNES), the
main national information system on health establish-
ments, maintained and made publicly available by the
Brazilian National Ministry of Health [20], available on
the DATASUS website. Because this study used second-
ary data, it was exempt from the need for approval by the
local Research Ethics Committee [21–23]. In contrast, it is known that “one in every three people
in the world would benefit from rehabilitation at some
point during the course of their illness”, and most of them
due to musculoskeletal disorders [9]. Despite the critical
demand for rehabilitation workforce due to the grow-
ing burden of disability related to chronic conditions [9],
there are no recommendations from the WHO regard-
ing the ideal physiotherapy workforce-to-population
ratio, nor regarding the criteria for activities and services
related to health promotion, protection, recovery [3] and
palliative care. The discussion about physiotherapy den-
sity has been included in the rehabilitation health work-
force [10–12], and also in studies involving specifically
the physiotherapy workforce-to-population ratio in dif-
ferent countries [13–16].i [
]
All data of the distribution of physiotherapists were
collected monthly between August 2007 (when the Bra-
zilian Classification of Occupations was updated in the
DATASUS, equivalent to the physiotherapist at ISCO
[24] code 2264) and September 2016 regarding facilities
types, Brazilian regions and public and private sectors. Primary, secondary, and tertiary facilities were defined
as follows: Primary Health Care (PHC) facilities—fit-
ness centres, family health support clinics, general clin-
ics (with various designations in Portuguese), clinics for
indigenous peoples, mixed-use facilities, and mobile clin-
ics; Secondary Health Care (SHC) facilities—transfusion
medicine/hematology centres, psychosocial care centres,
maternity centres, specialized outpatient clinics, physi-
cian offices, pharmacies, “polyclinics”, orthopaedic work-
shops, home care services, residential care clinics, and
centres for diagnostic/therapeutic support; and Tertiary
Health Care (THC) facilities—specialized hospitals, day
hospitals, and general hospitals. © The Author(s) 2021. 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/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Rodés et al. Hum Resour Health (2021) 19:101 Page 2 of 11 Page 2 of 11 Global Forum on Human Resources for Health, an analy-
sis of the WHO Global Health Observatory Data Reposi-
tory containing information from 36 countries showed
that maintaining a sufficient health care workforce is a
global priority and that the effectiveness of that work-
force should be determined by calculating the healthcare
workforce-to-population ratio [1]. At the Fourth Global
Forum on Human Resources for Health, four years later,
besides acknowledging the urge to increase the recruit-
ment and development of the health workforce, especially
in developing countries, it was once more emphasized
the fundamental importance of an optimally organized
and distributed health workforce [2]. analysis of the physiotherapy workforce for each region
of the country. The investigation of the nationwide dis-
tribution of physiotherapists in Brazil is necessary to fur-
ther examine the relationship between supply and health
needs and to support the attainment of the Universal
Health Coverage. Thus, the objective of this study was to
describe secular trends of the physiotherapists workforce-
to-population ratio among the public and private health
care sectors and across care levels (primary, secondary,
tertiary) of the Unified Health System (Sistema Único de
Saúde—SUS), by the five Brazilian geographical regions
(North, Northeast, Central-west, Southeast, and South). Methods Statistical procedures were per-
formed in the Stata program, version 13.0 (StataCorp LP,
College Station, TX, USA). established by the WHO in 2007 [25] (for example: 4782
physiotherapists of public PHC sector in August 2017 in
Brazil/191741381 Brazilian population in August 2017
* 1.000), public and private health care facilities being
evaluated separately. Data related to the population of
Brazil for the 2007–2016 period were obtained from
the Brazilian Institute of Geography and Statistics [26],
which provides population estimates by performing geo-
metric progression. The difference in means of the physi-
otherapy workforce-to-population ratio between the care
level and between public and private sectors in Brazil was
used one-way ANOVA and Bonferroni corrections were
applied to the many levels, with a significance level of
0.05. The trends of the physiotherapy workforce-to-pop-
ulation ratio were visualized on maps generated through
geoprocessing. Prais–Winsten linear regression models
were used in order to estimate trends in the annual aver-
age of the physiotherapy workforce-to-population ratio,
with a significance level of 0.05. The autocorrelation in
the time series of the annual coefficients was checked by
the Durbin–Watson test. Statistical procedures were per-
formed in the Stata program, version 13.0 (StataCorp LP,
College Station, TX, USA). physiotherapists; 191,741,381 population) and 0.41 per
1000 population in September 2016 (89,352 physiothera-
pists; 208,846,074 population), representing growth of
86%, with an increasing trend of 0.5% on an annual aver-
age (CI 95% 0.5–0.7%). There were clear increases in the
physiotherapy workforce in all care levels of the public
sector and in the private SHC sector. In 2016, the physi-
otherapy workforce-to-population ratio was higher for
the private SHC level, followed by the public THC sector. There were regional differences, showing higher ratios
mainly in the Southeast, South and Central-West regions
(Fig. 1). After the workforce in public SHC dropped in
2009–2010, the public THC sector workforce surpassed
the public SHC level (Fig. 2).h The public sector had the biggest physiotherapy work-
force in the country in 2007 (65.1% for the three care
levels p = 0.027) and 2016 (64.4% for the three care lev-
els p = 0.0209). In 2016, the physiotherapy workforce-
to-population ratio was bigger in the private SHC sector
(0.1282 physiotherapist/1000 population), followed by
the public THC sector (0.1145 physiotherapist/1000
population), the public SHC sector (0.0896 physiothera-
pist/1000 population) and the public PHC sector (0.0630
physiotherapist/1000 population). Methods Among the care lev-
els, PHC (p > 0.0001 for both years) and THC (p > 0.0001
for 2007 and p = 0.0420 for 2016) levels had the smallest
physiotherapy workforce-to-population ratio than SHC. In the private sector, workforce availability is highest at Methods The Brazilian regions are
North, Northeast, Southeast, South and Central-west. The public sector is subsidized by the State and the pay-
ment in the private sector is exclusively an individual’s
responsibility. Everyone in Brazil can use the public sec-
tor, while only people who can afford to pay for health
care use the private sector.fi In Brazil, the Unified Health System (Sistema Único
de Saúde—SUS) comprises both public and private sec-
tors, and has been progressively evolving since 1988
(over 30 years), delivering universal and comprehensive
health care to the Brazilian population. Different kinds of
health professionals, including physiotherapists, have to
work together in an integrated network to offer different
services considering the three care levels: primary, sec-
ondary, and tertiary [17]. In all three care levels, physi-
otherapists skills have an important role in rehabilitation,
while also embracing health promotion and injury pre-
vention, contributing to a more comprehensive care [18].i We calculated coefficients for the number of physi-
otherapists per 1000 population in Brazil (nationwide
and by region) to obtain the physiotherapy workforce-
to-population ratio, in accordance with the guidelines Costa et al. [19] identified the distribution of physio-
therapists among the various types of healthcare facilities
in Brazil, but there is no data concerning the time series Rodés et al. Hum Resour Health (2021) 19:101 Page 3 of 11 Page 3 of 11 established by the WHO in 2007 [25] (for example: 4782
physiotherapists of public PHC sector in August 2017 in
Brazil/191741381 Brazilian population in August 2017
* 1.000), public and private health care facilities being
evaluated separately. Data related to the population of
Brazil for the 2007–2016 period were obtained from
the Brazilian Institute of Geography and Statistics [26],
which provides population estimates by performing geo-
metric progression. The difference in means of the physi-
otherapy workforce-to-population ratio between the care
level and between public and private sectors in Brazil was
used one-way ANOVA and Bonferroni corrections were
applied to the many levels, with a significance level of
0.05. The trends of the physiotherapy workforce-to-pop-
ulation ratio were visualized on maps generated through
geoprocessing. Prais–Winsten linear regression models
were used in order to estimate trends in the annual aver-
age of the physiotherapy workforce-to-population ratio,
with a significance level of 0.05. The autocorrelation in
the time series of the annual coefficients was checked by
the Durbin–Watson test. Discussion the SHC level, whereas in the public sector it is highest at
the THC level. The workforce is larger in the public sec-
tor, p = 0.0274 for 2007 and p = 0.0209 for 2016 (Table 1). Complementing Table 1, sub-national regions show
similar trends to the national estimates, with a difference
for the North and Northeast regions. In 2007, North and
Northeast region had the highest ratio in the public SHC
sector (0.0309 and 0.0581 physiotherapist/1000 popula-
tion, respectively) and the Southeast, South and Central-
west regions in the private SHC sector (0.845, 0.1153 and
0.0802 physiotherapist/1000 population, respectively). In
2016, the private SHC sector in Brazil and its Southeast,
South and Central-west regions had the potentialized
rates (0.1592, 01,967 and 0.1236 physiotherapist/1000
population, respectively) as well as the public THC sector
in North and Northeast (0.0842 and 0.1126 physiothera-
pist/1000 population, respectively). the SHC level, whereas in the public sector it is highest at
the THC level. The workforce is larger in the public sec-
tor, p = 0.0274 for 2007 and p = 0.0209 for 2016 (Table 1). Complementing Table 1, sub-national regions show
similar trends to the national estimates, with a difference
for the North and Northeast regions. In 2007, North and
Northeast region had the highest ratio in the public SHC
sector (0.0309 and 0.0581 physiotherapist/1000 popula-
tion, respectively) and the Southeast, South and Central-
west regions in the private SHC sector (0.845, 0.1153 and
0.0802 physiotherapist/1000 population, respectively). In
2016, the private SHC sector in Brazil and its Southeast,
South and Central-west regions had the potentialized
rates (0.1592, 01,967 and 0.1236 physiotherapist/1000
population, respectively) as well as the public THC sector
in North and Northeast (0.0842 and 0.1126 physiothera-
pist/1000 population, respectively). As the workforce-to-population ratio approach is a criti-
cal component of monitoring and strengthening the per-
formance of national health systems [18], this study was
relevant to determine the availability of physiotherapists
to the population of Brazil. Regarding the physiotherapy workforce in Brazil and in
other countries, the present study shows that the physi-
otherapy workforce-to-population ratio in Brazil (0.41
for 1000 people) is low in comparison with that estimated
for other countries [13–16, 27]. According to Jesus et al. [13] Portugal had a ratio of 0.78 per 1000 people in 2014
and the United States a ratio of 0.65 per 1000 people. Resultsh The physiotherapy workforce-to-population ratio in
Brazil (public and private PHC, SHC and THC sectors)
was 0.22 per 1000 population in August 2007 (42,164 Fig. 1 Physiotherapy workforce-to-population ratio, according to care level in public and private sectors, in the five geographic regions of Brazil (N:
North, NE: Northeast, CE: Central-west, SE: Southeast, S: South). 2007 and 2016 ig. 1 Physiotherapy workforce-to-population ratio, according to care level in public and private sectors, in the five geograph
orth, NE: Northeast, CE: Central-west, SE: Southeast, S: South). 2007 and 2016 Rodés et al. Hum Resour Health (2021) 19:101 Page 4 of 11 Fig. 2 Physiotherapy workforce-to-population ratio, by sector and care levels, in Brazil. 2007–2016. PHC: primary health care; SHC: secondary health
care; THC: tertiary health care Fig. 2 Physiotherapy workforce-to-population ratio, by sector and care levels, in Brazil. 2007–2016. PHC: primary health care; SHC: secondary health
care; THC: tertiary health care Discussion On the other hand, considering this study, Brazil has a
greater workforce-to-population ratio when compared to
Singapore (0.15) and Bangladesh (0.01). Also, Landry [14,
15] estimated the Canadian physiotherapy workforce-
to-population ratio to be 0.5 in 2000. All these data may
indicate that the physiotherapy workforce in Brazil is still
insufficient to cover the population health needs. For the trends of the annual average of the physiothera-
pists workforce-to-population ratio (Table 2), in Brazil
from 2007 to 2016, the largest growth of the physiother-
apy workforce-to-population ratio annual growth was in
the public PHC sector (0.9% p > 0.001), followed by the
public and private THC sectors (0.8% p > 0.001) and the
SHC level, bigger in the private sector when compared
to the public sector (0.6% p > 0.001 vs. 0.2% p = 0.004). There were some regional trend differences in the annual
average of physiotherapy workforce-to-population ratio
to private PHC and THC sectors in North, Northeast,
South and Central-West, but in general all five regions
had greater growth of physiotherapy workforce-to-popu-
lation ratio in the public PHC and THC sectors, followed
by the private SHC sector. fi
However, our study used a method and a database that
focused on physiotherapists that were available to pro-
vide health care in various facilities, while all compared
studies used the number of registered professionals. Registered professionals may or may not be actually and
directly providing health care, since they may be involved
in management positions, research, education, or even
unemployed. Our study analysed the availability of the
physiotherapy workforce in various facilities, and also
its distribution in the care levels and public and private
sectors, bringing different perspectives to workforce data
analysis and research. Rodés et al. Hum Resour Health (2021) 19:101 Page 5 of 11 Table 1 Physiotherapy workforce-to-population ratio, by sector and care level, in Brazil and regions. Discussion 2007 and 2016
Region
Year
Sector
Care level
Total
PHC
SHC
THC
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
Brazil
2007
Public
0.0249
11.5
0.0673
31.1
0.0488
22.5
0.1411
65.1
p = 0.0274a
Private
0.0005
0.2
0.0669
30.9
0.0084
3.9
0.0757
34.9
Total
0.0254
11.7
0.1342
61.9
0.0572
26.4
0.2168
100
p < 0.0001b
p < 0.0001c
p = 0.0660d
2016
Public
0.063
15.2
0.0896
21.6
0.1145
27.6
0.2671
64.4
p = 0.0209a
Private
0.0006
0.1
0.1282
30.9
0.0187
4.5
0.1474
35.6
Total
0.0636
15.3
0.2178
52.5
0.1331
32.1
0.4145
100
p < 0.0001b
p = 0.0420c
p = 0.1230d
North
2007
Public
0.0139
14.3
0.0309
32
0.0314
32.5
0.0762
78.8
p < 0.0001a
Private
0
0
0.0181
18.7
0.0025
2.5
0.0205
21.2
Total
0.0139
14.3
0.049
50.7
0.0339
35
0.0967
100
p = 0.0030b
p = 0.3690d
p = 0.1470d
2016
Public
0.0452
19
0.0527
22.2
0.0842
35.5
0.1821
76.7
p < 0.0001a
Private
0.0003
0.1
0.0516
21.7
0.0034
1.4
0.0554
23.3
Total
0.0455
19.2
0.1043
43.9
0.0877
36.9
0.2375
100
p = 0.0580a
p = 1.0000c
p = 0.2630d North Page 6 of 11 Rodés et al. Hum Resour Health (2021) 19:101 Table 1 (continued)
Region
Year
Sector
Care level
Total
PHC
SHC
THC
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
Northeast
2007
Public
0.0163
10.1
0.0581
36.2
0.0454
28.3
0.1198
74.6
p = 0.0003a
Private
0.0002
0.1
0.0363
22.6
0.0044
2.7
0.0408
25.4
Total
0.0164
10.2
0.0944
58.8
0.0498
31
0.1606
100
p < 0.0001b
p = 0.0060c
p = 0.0630d
2016
Public
0.0788
22.2
0.0789
22.2
0,1126
31.7
0.2703
76.1
p < 0.0001a
Private
0.0008
0.2
0.0773
21.8
0,0065
1.8
0.0847
23.8
Total
0.0796
22.4
0.1562
44
0,1191
33.6
0.355
100
p = 0.1280a
p = 0.9170c
p = 0.8650d
Southeast
2007
Public
0.0326
12
0.079
29
0.0612
22.5
0.1728
63.5
p = 0.0394a
Private
0.0006
0.2
0.0845
31
0.0145
5.3
0.0996
36.5
Total
0.0332
12.2
0.1635
60
0.0756
27.8
0.2724
100
p < 0.0001b
p < 0.0001c
p = 0.0370d
2016
Public
0.0593
12.3
0.0933
19.4
0.1314
27.3
0.2839
59.1
p = 0.1564a
Private
0.0005
0.1
0.1592
33.2
0.0366
7.6
0.1963
40.9
Total
0.0598
12.4
0.2525
52.6
0.1679
35
0.4803
100
p < 0.0001b
p = 0.0600c
p = 0.0150d Page 7 of 11 Rodés et al. Discussion Hum Resour Health (2021) 19:101 Table 1 (continued)
Region
Year
Sector
Care level
Total
PHC
SHC
THC
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
Physiotherapist/1.000
population
%
South
2007
Public
0.0343
11.8
0.0971
33.4
0.0389
13.4
0.1703
58.5
p = 0.3463a
Private
0.0011
0.4
0.1153
39.6
0.0042
1.5
0.1207
41.5
Total
0.0354
12.2
0.2124
73
0.0431
14.8
0.2909
100
p < 0.0001b
p < 0.0001c
p = 1.0000d
2016
Public
0.0635
12.7
0.1351
27.1
0.0948
19.1
0.2933
58.9
p = 0.2607a
Private
0.0005
0.1
0.1967
39.5
0.0072
1.5
0.2044
41.1
Total
0.0639
12.8
0.3318
66.6
0.1021
20.5
0.4978
100
p < 0.0001b
p < 0.0001c
p = 0.8020d
Central-west
2007
Public
0.0213
9.3
0.0539
23.6
0.0575
25.2
0.1327
58.1
p = 0.2379a
Private
0.0077
3.4
0.0802
35.1
0.0079
3.4
0.0957
41.9
Total
0.029
12.7
0.1341
58.7
0.0653
28.6
0.2285
100
p < 0.0001b
p = 0.0010c
p = 0.0830d
2016
Public
0.057
14,4
0.0801
20.2
0.119
30.1
0,256
64,7
p = 0.0315a
Private
0.0101
2,5
0.1236
31.2
0.0062
1.6
0.1398
35,3
Total
0.067
16.9
0.2036
51.4
0.1252
31.6
0.3958
100
p = 0.0010a
p = 0.0860c
p = 0.3350d
PHC, primary health care; SHC, secondary health care; THC, tertiary health care
a Anova between public and private
b Anova and Bonferroni correction between PHC and SHC level
c Anova and Bonferroni correction between SHC and THC level
d Anova and Bonferroni correction between PHC and THC level
Values of p showing significant differences are represented in bold Rodés et al. Discussion Hum Resour Health (2021) 19:101 Page 8 of 11 Table 2 Trends of the annual average of the physiotherapists workforce-to-population ratio
Brazil and regions, 2007–2016
PHC, primary health care; SHC, secondary health care; THC, tertiary health care
a Growth from 2007 to 2016
b Significant difference in relation to the care level
c Reduction from 2007 to 2016
Region
Sector
Trends of Physiotherapy Workforce %change annual average (95% CI) 2007–2016
PHC
SHC
THC
Brazil
Public
0.9 (0.7;1.1)ab
p < 0.001
0.2 (0.1;0.4)a
p = 0.004
0.8 (0.6;0.9)ab
p < 0.001
Private
0.2 (− 0.2;0.6)
p = 0.362
0.6 (0.5;0.7)ab
p < 0.001
0.8 (0.5;1.0)ab
p < 0.001
North
Public
1.0 (0.9;1.2)ab
p < 0.001
0.4 (0.2;0.6)a
p < 0.001
0.8 (0.7;0.9)ab
p < 0.001
Private
1.4 (0.6;2.1)a
p < 0.001
0.9 (0.7;1.2)a
p < 0.001
0.5 (0.2;0.9)a
p = 0.007
Northeast
Public
1.5 (1.1;1.8)ab
p < 0.000
0.3 (0.1;0.4)a
p < 0.000
0.8 (0.7;0.9)ab
p < 0.000
Private
1.5 (0.4;2.8)a
p = 0.007
0.7 (0.5;0.8)a
p < 0.001
0.3 (0.2;0.5)a
p < 0.001
Southeast
Public
0.6 (0.4;0.7)ab
p < 0.001
0.1 (−0.01;0.2)
p = 0.117
0.7 (0.5;0.9)ab
p = 0.014
Private
−0.2 (−0.4;−0.04)
p < 0.001
0.6 (0.4;0.8)a
p < 0.001
0.9 (0.6;1.2)a
p < 0.001
South
Public
0.6 (0.5;0.7)ab
p < 0.000
0.3 (0.1;0.4)a
p < 0.000
0.8 (0.7;0.9)ab
p < 0.000
Private
−0.8 (−1.6;−0.1)c
p = 0.019
0.5 (0.4;0.6)a
p < 0.001
0.4 (0.2;0.6)a
p < 0.001
Central-west
Public
0.9 (0.7;1.1)ab
p < 0.001
0.3 (0.2;0.4)a
p < 0.001
0.7 (0.6;0.7)ab
p < 0.001
Private
0.3 (0.1;0.5)a
p = 0.004
0.4 (0.2;0.6)a
p < 0.0001
−0.08 (−0.2;0.04)
p < 0.221 Table 2 Trends of the annual average of the physiotherapists workforce-to-population ratio Brazil and regions, 2007–2016
PHC, primary health care; SHC, secondary health care; THC, tertiary health care
a Growth from 2007 to 2016
b Significant difference in relation to the care level
c Reduction from 2007 to 2016 Brazil and regions, 2007–2016
PHC, primary health care; SHC, secondary health care; THC, tertiary health care
a Growth from 2007 to 2016
b Significant difference in relation to the care level
c Reduction from 2007 to 2016 An already known strategy used by many countries
to increase the size of the workforce, is to increase the
number of students. Discussion In Israel, the National Ministry of
Health sought to control the size of the physician work-
force increasing the number of medical students by 52%,
resulting in a 32.5% increase in the size of the workforce
[5]. But each country and region has an unique context,
resulting in significant variability and requiring tailored
solutions [13].h study the markers of an effective workforce, to manage
the demands related to access, and to create and imple-
ment public policies aimed at improving their health care
systems. We could also notice a plausible relation between the
distribution of the physiotherapy workforce in Brazil and
the impact of public policies. We found that the major-
ity of physiotherapists in Brazil worked in the SHC sec-
tor, probably because the SHC sector was the “cradle” of
physiotherapy in Brazil [19]. That is why many studies value the benchmark as an
important tool to improve health care workforce analy-
sis [4, 17, 27]. Brazil lags behind in the treatment of
these data and the development of criteria to assess the
demand for physiotherapy in Brazil as a whole and in its
various regions, as well as the size of the physiotherapy
workforce accordingly. Since an effective workforce is
one of the global priorities highlighted by the WHO [1,
2], Brazil could find inspiration in other countries such
as Australia and Israel [4, 5], as well as cities such as Han-
nover, Germany, and provinces as Saskatchewan, Canada
[6, 16, 27]. Those places have all convened committees to The trends related to growth of the physiotherapy
workforce-to-population ratio in the SHC level were
comparable between public and private sectors until
2009. In 2009 there was a decrease in the rate of physio-
therapists in the public sector, whereas it kept growing in
the private sector. This drop in the public SHC level could
be explained by a partial targeting of professionals to the
public PHC level, due to the creation and financing of
the Family Health Support Center in 2008, that encour-
aged the position of the physiotherapist at the PHC level,
expanding the physiotherapy workforce nationwide [28]. Rodés et al. Hum Resour Health (2021) 19:101 Page 9 of 11 Rodés et al. Hum Resour Health (2021) 19:101 Page 9 of 11 Another public policy that clearly affected the physio-
therapy workforce distribution was a resolution launched
in 2010 by the Brazilian Health Regulatory Agency
(Anvisa) [29]. Discussion The resolution made it mandatory for
every intensive care unit to have at least one physiothera-
pist team coordinator and at least one physiotherapist for
every 10 beds [29], therefore increasing the physiother-
apy workforce at the THC level. Three years after the
enactment of the law, that workforce had grown by 0.8%
in the public and private THC sectors.i only 27.9% of the Brazilian population has access to some
type of private (i.e., only people who can afford to pay for
health care) medical or dental insurance [30]. Overall, the imbalance found in the workforce distribu-
tion among the three levels of public and private health
care and among the geographical regions may be a bar-
rier to meeting the health needs and promoting access to
health care services, especially for the poorest population
[31]. Mcintyre et al. [32] discussed that access is more
than simply having an opportunity to use the health care
system. The end users of health care services should be
well informed and must be encouraged to seek care and
recognize health care as a right, as well as to understand
that each individual plays an active role in requesting
services and managing their own health care. However,
promoting such empowerment is the responsibility of the
healthcare system (to devise ways to overcome barriers)
and of health professionals themselves (to be sources of
information). With the objective of improving the Unified Health
System, the structured implementation of the afore-
mentioned public policies seems to be resulting in the
inclusion of physiotherapy in various facilities and care
levels, proving to be an important resource for modu-
lating the healthcare workforce [4]. That is why, in order
to adequately improve the access and distribution of the
physiotherapy workforce in the five Brazilian regions and
across care levels, it is indispensable to further investi-
gate health needs with a local and community-centred
approach. We hope that the data in this article will stimulate fur-
ther studies on the relationship between the need for
physiotherapy care and available physiotherapy work-
force and their geographic distribution. It may also con-
tribute to research regarding the rehabilitation workforce
with the composition of other health professionals. Discussion While Brazilian public policies stipulate fixed and gen-
eralized incentives, without distinguishing the particu-
lar context, gaps and demands of the care levels and/or
regions [5], the Israeli National Ministry of Health used
the strategy of offering financial incentives according to
their own scales, to bridge the gap between supply and
demand [10]. Also, some countries like Israel and Aus-
tralia have committees focused on understanding how
to stimulate the health workforce towards the specializa-
tions and regions of greatest need [4, 5]. Author details
1 1 Departamento de Fisioterapia, Fonoaudiologia e Terapia Ocupacional da
Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil. 2 Departamento de Geografia da Faculdade de Filosofia, Letras e Ciências
Humanas, Universidade de São Paulo, São Paulo, SP, Brazil. 3 Departamento de
Medicina Preventiva da Faculdade de Medicina, Universidade de São Paulo,
São Paulo, SP, Brazil. There is a trend towards growth in the physiotherapy
workforce in Brazil with differences between health care
levels, and among public and private sectors. However,
the physiotherapy workforce-to-population ratio appears
to be still insufficient to meet the health needs of the
population. The physiotherapy workforce-to-population
ratio in Brazil is bigger in the private SCH sector, fol-
lowed by the public THC, SHC and PHC sectors. The
Brazilian regions followed similar patterns, with minor
regional differences, mainly showing greater availability
of the workforce in the Southeast, South and Central-
West regions. Also, the physiotherapy workforce seems
to have a close relationship with public policies related to
human resources for health, therefore underscoring the
importance of planning and regulation to meet the health
needs of the population. Received: 12 February 2020 Accepted: 10 August 2021 Received: 12 February 2020 Accepted: 10 August 2021 Limitations of this studyh Hum Resour Health (2021) 19:101 Page 10 of 11 Page 10 of 11 Funding
Thi
d 10. Jesus TS, Landry MD, Dussault G, Fronteira I. Human resources for health
(and rehabilitation): six Rehab-Workforce Challenges for the century. Human Resour Health. 2017;15:8. This study did not receive any type of funding from public, private, or non-
profit institutions. 11. Rodes CH, Kurebayashi R, Kondo VE, Luft VD, Góes AB, Schmitt ACB. The
access and rehabilitation working in Primary Health Care. Fisioter Pesqui. 2017;24:74–82. References
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J Phys Rehab Med. 2017;53(Suppl 1):1–6. All of the undersigned authors participated actively in the study. JVLD contrib-
uted with the analysis and interpretation of data and was a major contributor
in writing the manuscript. CHR contributed with the acquisition, analysis and
interpretation of data, and revision of the manuscript. BCA contributed with
the analysis and interpretation of data. LG contributed with the geospatial
analysis of data. SMAJ contributed with the writing and revision of the manu-
script. ACCGG contributed with the writing and revision of the manuscript. ACBS contributed with the conception of the work, the acquisition, analysis
and interpretation of data, and with the writing of the manuscript. All authors
have read and approved the manuscript in its present form and have agreed
to its submission to the Human Resources for Health. All authors read and
approved the final manuscript. 7. Crisp N, Chen L. Global supply of health professionals. N Engl J Med. 2014;370:950–7. 7. Crisp N, Chen L. Global supply of health professionals. N Engl J Med. 2014;370:950–7. 8. Pozo-Martin F, Nove A, Lopes SC, Campbell J, Buchan J, Dussault G, Kunju-
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Study 2019. Lancet. 2021. https://doi.org/10.1016/S0140-6736(20)
32340-0. Abbreviations
CNES N i
l R CNES: National Registry of Health Care Facilities (Cadastro Nacional de Estabel-
ecimentos de Saúde); PHC: Primary Health Care; SHC: Secondary Health Care;
THC: Tertiary Health Care; WHO: World Health Organization. 4. Joyce CM. The medical workforce in 2025: what’s in the numbers? Med J
Aust. 2013;199:S6. Consent for publication
Not applicable. 16. Shah TI, Milosavljevic S, Trask C, et al. Mapping physiotherapy use in
canada in relation to physiotherapist distribution. Physiotherapy Canada
Physiotherapie Canada. 2019;71(3):213–9. https://doi.org/10.3138/
ptc-2018-0023. 16. Shah TI, Milosavljevic S, Trask C, et al. Mapping physiotherapy use in
canada in relation to physiotherapist distribution. Physiotherapy Canada
Physiotherapie Canada. 2019;71(3):213–9. https://doi.org/10.3138/
ptc-2018-0023. Limitations of this studyh The number of active physiotherapists might have been
overestimated, because the CNES does not consider
overlaps related to professionals with dual practice [17]. Since the CNES provides their data regarding the num-
ber of professionals registered in each facility and not
about the amount of their working hours, an important
limitation to this study methodology is the inability to
present and calculate the professionals’ availability by
full time equivalents. Other studies should consider and
explore other methods of data collection that allow the
adjustment of the professionals’ availability and working
hours in each facility by full time equivalents. In Brazil we found a higher physiotherapy workforce
mainly in the Southeast, South and Central-West regions,
which can indicate bigger gaps in access to physiotherapy
in other regions, which require further investigation. In a study conducted in Canada, Shah et al. [16] found
that the physical and social barriers to physiotherapy
healthcare access are greater in the smaller cities, where
incomes are also lower. While the majority of physi-
otherapists work in urban areas, they found large gaps
in access to physiotherapy in rural and remote areas. In
order to improve the access to physiotherapy with greater
equity, a similar analysis could contribute to planning a
more appropriate physiotherapy assistance in the health
system. Also, this study focused specifically on the physi-
otherapy workforce. Other studies comparing the work-
force-to-population ratio, and its distribution and trends
among other health professionals and health profession-
als overall would contribute for better understanding
where the physiotherapy workforce stands in Brazil. Nonetheless, there are some important issues to point
out regarding the access to physiotherapy and effective
coverage in the Brazilian context. Despite the concen-
tration of physiotherapists being the highest at the SHC
level, especially within the private sector (ratio of 0.13),
that does not mean that the population has greater access
to private SHC services of physiotherapy. According to
the data from the 2013 Brazilian National Health Survey, It is important to state the exploratory nature of the
study. The adequate physiotherapy workforce-to-pop-
ulation ratio in order to assure effective health coverage
is still unknown, therefore more investigation is needed
to better understand and guarantee the access to physi-
otherapy, sufficiently meeting regional focused health
needs for each level of care. Rodés et al. Hum Resour Health (2021) 19:101 Rodés et al. Declarations 15. Landry MD, Hack LM, Coulson E, et al. Workforce projections 2010–2020:
annual supply and demand forecasting models for physical therapists
across the United States. Phys Ther. 2016;96(1):71–80. https://doi.org/10.
2522/ptj.20150010. Ethics approval and consent to participate
Not applicable. Ethics approval and consent to participate
Not applicable. Consent for publication
Not applicable. Acknowledgements
h
k h
h Acknowledgements
We thank the researcher Larissa Costa, who inspired this study. Competing interests Competing interests
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. Rodés et al. Hum Resour Health (2021) 19:101 Rodés et al. Hum Resour Health (2021) 19:101 Page 11 of 11 17. World Health Organization. Establishing and monitoring benchmarks for
human resources for health: the workforce density approach. Spotlight
on health workforce statistics. 2008. https://www.who.int/hrh/statistics/
Spotlight_6_Nov2008_benchmarking.pdf?ua=1. Accessed 10 Feb 2020. 27. McFadden B, McGrath KJ, Lowe T, Thiessen C, Irinici S, Shah T, Milosavlje-
vic S, Bath B. Examining the supply of and demand for physiotherapy in
Saskatchewan: the relationship between where physiotherapists work
and population health need. Physiother Can. 2016;68(Suppl 4):335–45. p
p y
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and population health need. Physiother Can. 2016;68(Suppl 4):335– 18. Paim J, Travassos C, Almeida C, Bahia L, Macinko J. The Brazilian health
system: history, advances, and challenges. Lancet. 2011. https://doi.org/
10.1016/S0140-6736(11)60054-8. 28. Brasil. Ministério da Saúde. Portaria nº 154, de 24 de janeiro de 2008. Cria
os Núcleos de Apoio à Saúde da Família–NASF. Diário Oficial da República
Federativa do Brasil. Brasília-DF, 2008. 19. Costa LR, Costa JLR, Oishi J, Driusso P. Distributions of physical therapist
working on public and private establishment in different levels of com-
plexity of health care in Brazil. Rev Bras Fisioter. 2012;16(Suppl 5):422–30. 29. Conselho Nacional de Fisioterapia e Terapia Ocupacional – Crefito 5. Cartilha de Políticas Públicas: Gestão 2010–14. Porto Alegre: Crefito. 2014. http://www.crefito5.org.br/wp-content/uploads/2014/06/cartilha_polit
icas_publicas.pdf. Accessed 6 Aug 2017. p
y
pp
20. Datasus. Brasil, Ministério da Saúde. http://datasus.saude.gov.br/sistemas-
e-aplicativos/cadastros-nacionais/cnes. Accessed 11 Oct 2016. 30. IBGE. Brasil: Instituto Brasileiro de Geografia e Estatística - Diretoria de
Pesquisas, Coordenação de Trabalho e Rendimento, Pesquisa Nacional
de Saúde 2013. Cobertura de plano de saúde. https://ww2.ibge.gov.br/
home/estatistica/populacao/pns/2013_vol2/default_ods.shtm. Accessed
15 Jun 2018. 21. Brasil. Ministério da Saúde. Conselho Nacional de Saúde. Resolução nº
196, de 1996. Aprova as diretrizes e normas regulamentadoras de pesqui-
sas envolvendo seres humanos. Conselho Nacional de Saúde. Bioética. 1996; (Supl 4–2):15–25. 22. Brasil. Lei no 12.527, de 18 de novembro de 2011. Regula o acesso a
informações previsto no inciso XXXIII do art. 5o, no inciso II do § 3o do
art. 37 e no § 2o do art. Competing interests 216 da Constituição Federal; altera a Lei no 8.112,
de 11 de dezembro de 1990; revoga a Lei no 11.111, de 5 de maio de
2005, e dispositivos da Lei no 8.159, de 8 de janeiro de 1991; e dá outras
providências. Diário Oficial da União, Brasília. 2011. 31. Jacobs B, Ir P, Bigdeli M, Annear PL, Van Damme W. Addressing access
barriers to health services: an analytical framework for selecting appropri-
ate interventions in low-income Asian countries. Health Policy Plan. 2012;27(Suppl 4):288–300. 32. Mcintyre D, Thiede M, Birch S. Access as a policy-relevant concept
in low- and middle-income countries. Health Econ Policy and Law. 2009;4:179–93. 32. Mcintyre D, Thiede M, Birch S. Access as a policy-relevant concept
in low- and middle-income countries. Health Econ Policy and Law. 2009;4:179–93. 23. Brasil. Ministério da Saúde. Conselho Nacional de Saúde. Resolução nº
510, de 2016. Dispõe sobre as normas aplicáveis a pesquisas em Ciências
Humanas e Sociais. Diário Oficial da União, Brasília, DF. 2016. Publisher’s Note 24. International Standard Classification of Occupations: ISCO-08/Interna-
tional Labour Office, - Geneva: ILO, 2012. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. 25. World Health Organization. The World Health Report 2006 - working
together for health. 2007. https://www.who.int/whr/2006/whr06_en.pdf?
ua=1. Accessed 10 Feb 2020. 26. IBGE. Brasil: Instituto Brasileiro de Geografia e Estatística. https://www.
ibge.gov.br/estatisticas-novoportal/sociais/populacao/9103-estimativas-
de-populacao.html?&t=downloads. Accessed 11 Jan 2017. •
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https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_5_from_Nutlin-3a_Efficacy_in_Sarcoma_Predicted_by_Transcriptomic_and_Epigenetic_Profiling/22402145/1/files/39847937.pdf
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Supplementary Figure 5 from Nutlin-3a Efficacy in Sarcoma Predicted by Transcriptomic and Epigenetic Profiling
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A
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p=0.042
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100
% GADD45A CpG methylaon
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Supplementary Fig 5: Methyla!on of GADD45A and sarcoma pathology
(A) GADD45A CpG methylaon was determined using Sequenom MassARRAY on bilsulphite converted DNA
from 24 untreated sarcoma cases. Hypermethylaon was defined as methylaon exceeding the highest level
observed in germline samples (>12%). Horizontal bars indicate the mean of data. Open symbols denote mutant
TP53 sarcomas. WD: Well-differentiated, DD: De-differenated. (B) Correlaon between sarcoma TP53 status
and GADD45A CpG methylaon levels. Asterisk denotes stascal significance (p<0.05). (C) Global DNA
methylaon was quanfied using 5-mC DNA ELISA assay as per manufacturer’s instrucons (Zymo Research,
Irvine, CA). 100ng of denatured sarcoma genomic DNA was assayed per reacon. Assay was read at 405nm
20mins a!er the addion of HRP developer. Global tumour methylaon levels ranged from 2.6-12.1% (mean ±
STDEV from duplicate reacons shown). (D) Lack of correlaon between global methylaon (%5-mC) and
GADD45A CpG methylaon.
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https://www.biorxiv.org/content/biorxiv/early/2022/11/04/2022.11.03.515031.full.pdf
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English
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Generalized peakgroup scoring boosts identification rates and accuracy in mass spectrometry based discovery proteomics
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bioRxiv (Cold Spring Harbor Laboratory)
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Abstract The statistical validation of peptide and protein identifications in mass spectrometry proteomics
is a critical step in the analytical workflow. This is particularly important in discovery experiments
to ensure only confident identifications are accumulated for downstream analysis and biomarker con-
sideration. However, the inherent nature of discovery proteomics experiments leads to scenarios
where the search space will inflate substantially due to the increased number of potential proteins
that are being queried in each sample. In these cases, issues will begin to arise when the machine
learning algorithms that are trained on an experiment specific basis cannot accurately distinguish
between correct and incorrect identifications and will struggle to accurately control the false discov-
ery rate. Here, we propose an alternative validation algorithm trained on a curated external data
set of 2.8 million extracted peakgroups that leverages advanced machine learning techniques to cre-
ate a generalizable peakgroup scoring (GPS) method for data independent acquisition (DIA) mass
spectrometry. By breaking the reliance on the experimental data at hand and instead training on a
curated external dataset, GPS can confidently control the false discovery rate while increasing the
number of identifications and providing more accurate quantification in different search space sce-
narios. To first test the performance of GPS in a standard experimental environment and to provide
a benchmark against other methods, a novel spike-in data set with known varying concentrations
was analyzed. When compared to existing methods GPS increased the nunmber of identifications
by 5-18% and was able to provide more accurate quantification by increasing the number of ratio
validated identifications by 24-74%. To evaluate GPS in a larger search space, a novel data set of 141
blood plasma samples from patients developing acute kidney injury after sepsis was searched with a
human tissue spectral library (10000+ proteins). Using GPS, we were able to provide a 207-377% in-
crease in the number of candidate differentially abundant proteins compared to the existing methods
while maintaining competitive numbers of global identifications. Finally, using an optimized human
tissue library and workflow we were able to identify 1205 proteins from the 141 plasma samples and
increase the number of candidate differentially abundant proteins by 70.87%. With the addition of
machine learning aided differential expression, we were able to identify potential new biomarkers
for stratifying subphenotypes of acute kidney injury in sepsis. Abstract These findings suggest that by using
a generalized model such as GPS in tandem with a massive scale spectral library it is possible to
expand the boundaries of discovery experiments in DIA proteomics. GPS is open source and freely
available on github at (https://github.com/InfectionMedicineProteomics/gscore). November 3, 2022 November 3, 2022 Generalized peakgroup scoring boosts identification rates and
accuracy in mass spectrometry based discovery proteomics Aaron M. Scott1, Christofer Karlsson1, Tirthankar Mohanty1, Suvi T. Vaara2, Adam
Linder1, Johan Malmstr¨om1, and Lars Malmstr¨om1 Aaron M. Scott1, Christofer Karlsson1, Tirthankar Mohanty1, Suvi T. Vaara2, Adam
Linder1, Johan Malmstr¨om1, and Lars Malmstr¨om1 ron M. Scott1, Christofer Karlsson1, Tirthankar Mohanty1, Suvi T. Vaara2, Adam
Linder1, Johan Malmstr¨om1, and Lars Malmstr¨om1 1Division of Infection Medicine, Department of Clinical Sciences, Lund University,
Lund, Sweden
2Division of Anaesthesia and Intensive Care Medicine Department of Surgery, Intensive
Care Units, Helsinki University Central Hospital, Box 340, 00029 HUS, Helsinki,
Finland 1Division of Infection Medicine, Department of Clinical Sciences, Lund University,
Lund, Sweden
2Division of Anaesthesia and Intensive Care Medicine Department of Surgery, Intensive
Care Units, Helsinki University Central Hospital, Box 340, 00029 HUS, Helsinki,
Finland .
CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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;
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bioRxiv preprint Generalized peakgroup scoring boosts identification rates and
accuracy in mass spectrometry based discovery proteomics
Aaron M. Scott1, Christofer Karlsson1, Tirthankar Mohanty1, Suvi T. Vaara2, Adam
Linder1, Johan Malmstr¨om1, and Lars Malmstr¨om1
1Division of Infection Medicine, Department of Clinical Sciences, Lund University,
Lund, Sweden
2Division of Anaesthesia and Intensive Care Medicine Department of Surgery, Intensive
Care Units, Helsinki University Central Hospital, Box 340, 00029 HUS, Helsinki,
Finland .
CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for this
this version posted November 4, 2022.
;
https://doi.org/10.1101/2022.11.03.515031
doi:
bioRxiv preprint 1
Introduction However, if the library filtering is
done too strictly potential true peptides are eliminated unnecessarily from the library and the benefits
that could be gleaned from using these massive libraries are reduced, decreasing the potential depth of
a discovery analysis. If the filtering is done too liberally, where too many false peptides are left in the
library, the same issue arises where validation algorithms have trouble distinguishing true signal in the
large search space. In both cases, this can result in an unreliable estimation of the FDR, so care must
be taken when filtering large spectral libraries for analysis. Alternative to the filtering and sub-setting of spectral libraries to analyze data with large spectral
libraries, one case that has not been investigated with the same vigor is the effect of the choice of statis-
tical validation algorithms on their ability to navigate this large search space and control the FDR in a
stable manner. The algorithm of choice in the validation of DIA mass spectrometry data is the mProphet
algorithm [49], which is similar to the percolator algorithm commonly used in DDA proteomics [58]. This
mProphet algorithm, implemented in the python package PyProphet [50] for the use of validating DIA
data extracted with the OpenSwath software [52], uses a semi-supervised method to combine all calcu-
lated sub-scores into a final classification score used to control the FDR. This method works by selecting
positive training targets above a particular q-value cutoffin an iterative fashion once they are identified
and validated by the algorithm in the previous iteration. The initial targets are identified using a selected
sub-score and a broad q-value cutoff(typically 0.15 or 15% FDR) and the method progresses until no
more new identifications are found to pass below a selected FDR threshold (1%). In most cases, where
the library very closely matches with the data contained in the sample of interest, this method works
exceedingly well. However, when searching a sample with a repository scale library, where the majority
of precursors in the library are likely not contained in the sample, a situation arises where the normally
”true” target labels are noisy. This means that the peakgroups labeled as a ”target” in the spectral
library and assumed to be in the sample are not actually contained in the sample. 1
Introduction One disadvantage of data independent acquisition (DIA) proteomics is that a spectral library based on
previously identified peptides in a sample is required to interpret the complex signal and quantify pep-
tides. In the past, this has made the practice of discovery proteomics in DIA difficult, as samples would
first need to be run with data dependent acquisition (DDA) and extensive offline fractionation to reach
the depth needed to see the benefits of the more sensitive DIA methods. However, recent computational 1 . CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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bioRxiv preprint . CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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bioRxiv preprint advances in the development of fragment spectra prediction [19, 65, 59], and the creation of repository
scale spectral libraries for selected organisms and sample types [51, 66, 39, 47, 6, 31] have allowed for
the exploration of DIA as a means for discovery proteomics. Although these large libraries can help
facilitate the identification of novel markers in a DIA experiment, they present significant computational
difficulties, particularly when attempting to control the false discovery rate (FDR) using the target-decoy
approach [14]. The increased search space of massive libraries can cause a decrease in sensitivity and
statistical power as more false positives are introduced into the library, leading to less precursors being
correctly identified when they are in fact in the sample [42, 18, 17]. In these cases, validation algorithms
struggle to distinguish the true signal from the false, resulting in low numbers of validated peakgroups
and imprecise FDR control. Attempts have been made to filter down these massive libraries to man-
ageable sample-type specific libraries in a data-dependent fashion to a more manageable search space
that statistical validation algorithms can readily deal with [26, 18]. 2.1
Generalizable model training To enable the generalized scoring of peakgroups, we first aimed to train a machine learning model that can
detect true peakgroups in a sample with high precision. 129 yeast samples were run with varying gradient
lengths (30, 45, 60, 90, 120 minutes) and analyzed with OpenSWATH [52] to generate 1893804 target
peakgroups and 1857563 decoy peakgroups using the ms2-level sub-scores calculated by OpenSWATH
as features. We split the dataset so 80% remained for training and 20% was reserved for evaluating the
performance the machine learning models. Because many of the target labels in the dataset are noisy (ie. they originate from false targets) we developed an algorithm to filter out noisy labels from the dataset
that we will refer to as the denoising algorithm. The denoising algorithm trains an ensemble of weak
learners using bagging [7] that vote on each peakgroup from a particular mass spectrometric sample. If every single classifier in the ensemble voted that the peakgroup was a target, then the peakgroup is
kept as a target in the training set, otherwise it is removed. The overall workflow for this method is
depicted in Figure 1B. This results in a filtered training set with 1278834 true targets, marking a 15.53%
decrease in the number of targets used for training, along with 1486043 decoy peakgroups. In Figure 1A
the UMAP [38] projection shows that the cluster of decoy datapoints (labeled 0.0) is contaminated with
target labels, making the cluster appear almost purple. After denoising, the effects can be visualized in
Figure 1C which depicts the purity of the projected components for the target and decoy data points
in the training set. To evaluate the performance of the denoising algorithm in generating a classifier to
identify only true peakgroups, we trained 2 classifiers, 1 based on the unfiltered training data, and the
2nd of the filtered training set, and analyzed a held-out subset of the unfiltered training data that we will
refer to as the testset. Overall on this testset we observed an 11.66% increase in the precision using the
filtered model, from 89.19% to 99.58%, and a 96.13% decrease in the false discovery rate (FDR), from
10.81% to 0.42%. In D-E of Figure 1 the confusion matrices display the overall classification numbers
that were used to calculate these metrics, and the overall score distributions for each method. 1
Introduction ;
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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bioRxiv preprint . CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for this
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bioRxiv preprint setting by analyzing blood plasma samples from patients with septic acute kidney injury using the repos-
itory scale Pan Human Library (PHL) [51] and evaluate the possibility of using this type of framework
for the discovery of potential disease markers. GPS is a freely available as an open source Python package
and command line tool on github (https://github.com/InfectionMedicineProteomics/gscore). setting by analyzing blood plasma samples from patients with septic acute kidney injury using the repos-
itory scale Pan Human Library (PHL) [51] and evaluate the possibility of using this type of framework
for the discovery of potential disease markers. GPS is a freely available as an open source Python package
and command line tool on github (https://github.com/InfectionMedicineProteomics/gscore). 2.1
Generalizable model training The true
and false labels widened substantially and the portion of targets deemed false more accurately model the
decoy distribution in the filtered model as seen in D-E of Figure 1. 1
Introduction This is a particularly
tricky area of machine learning research, and research has been done to develop methods that identify
noisy labels and stabilize the training of models in the presence of noisy labels [43, 11]. PyProphet, and
additionally the tool Percolator [58] designed for validating identifications in data dependent acquisi-
tion, attempt to mitigate this issue with the iterative semi-supervised learning approach, but when the
number of true positives in the sample is so low, it is not guaranteed that each iteration can actually
select true positives for training. If some true targets are identified, it would be an exceedingly small
amount compared to the negative decoys in the data, creating a overwhelming class imbalance, which
can destabilize the training of machine learning algorithms if not dealt with in an appropriate manner [2]. Instead of dealing with these types of noisy label and class imbalance problems on the fly during sam-
ple specific model training, we propose to create a stable, generalizable machine learning model that can
be used to combine sub-scores from DIA extraction software by training on a curated data set of known
true and false positive peakgroups. This approach has been demonstrated to work using static models
with percolator in the context of DDA proteomics [58] but the static models are trained on the sample
types that they are used to evaluate, so it is unclear if they would generalize to diverse datasets of unre-
lated sample types. We hypothesize that a good peakgroup is a good peakgroup, no matter the sample
type, and that statistical validation models can be trained on an unrelated external data set if the data
is curated properly. To that end, we have trained a generalizable scoring model and implemented a suite
of algorithms to provide the stable validation of extracted peakgroups through the spectral library size-
agnostic Generalizable Peakgroup Scoring (GPS) framework. Here, we compare the GPS to two existing
validation tools, PyProphet[50] and Percolator[58] and evaluate the performance on 3 different novel
datasets using different spectral libraries and search spaces. We demonstrate the use of GPS in a practical 2 . CC-BY 4.0 International license
perpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for this
this version posted November 4, 2022. 2.2
Method Benchmark Comparison To establish the validity of data produced using GPS with the filtered model described above, we first
performed an analysis on samples with known ratios of 4X more yeast peptides in one sample group
spiked-in into a constant mouse kidney proteome background. To verify that our method was validating
correct peakgroups, we monitored the expected ratios to make sure that the quantification is accurate,
while still maintaining high levels of identifications. We used OpenSwath [52] to perform signal extraction
and then GPS, PyProphet [50], and Percolator [58] to validate the extracted peakgroups and compare
between the different tools. To ensure that we were directly comparing the ability of only each machine
learning tool to validate peakgroups, the scoring functions of each tool were used and then the scored
samples were compiled and exported into global quantification matrices in the exact same way. At a 1%
FDR, we observed 15.69% increase in precursors, an 18.20% percent increase in peptides, and a 14.79%
increase in proteins identified by GPS compared to PyProphet and a 12.68% increase in precursors, a
14.40% increase in peptides, and a 4.7% increase in proteins identified by GPS compared to Percolator
(Figure 2). To measure the accuracy of each validation method at identifying precursors correctly, we
measured the number of true ratio validated identifications in a ±0.2 log2 fold-change (log2FC) window
around the known yeast and mouse log2 fold concentrations as done previously [41, 26]. Compared
to PyProphet, we observed a 24.30% increase in the number of ratio validated yeast identifications
and a 28.83% increase in the number of mouse ratio validated identifications (Figure 2). Compared to
Percolator, we observed a 68.52% increase in the number of ratio validated yeast identifications and a
74.06% increase in the number of mouse ratio validated identifications (Figure 2). The log2 fold change
distributions and the ratio validation windows of interest for each method can be seen in A, C, and E of
Figure 2. To measure the quantification accuracy beyond an arbitrary window of ±0.2, we measured the 3 . CC-BY 4.0 International license
perpetuity. It is made available under a
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Method Benchmark Comparison It is made available under a
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bioRxiv preprint Figure 1: a) The UMAP projection of the unfiltered training labels. 1.0 is a target label and 0.0 is a
decoy label. As visible by the projection, the cluster on the left corresponding to the decoy labels, is
also fully populated with target labels. This means that during training, noisy target labels that are
indistinguishable from decoy labels will be used as positive training instances when they should not be
b) The overall workflow for denoising the noisy target labels in the training set. The training data is first
split into k number of folds, for each held-out fold, the remaining training data is randomly sampled n-
times with replacement to train n-classifiers that are then used to vote on the held-out data to determine
if an instance is a true target or not. The probability threshold to consider a vote positive can be adjusted
for stringency. c) The UMAP projection of the filtered training labels with a 0.75 probability vote cutoff
after denoising. Each individual cluster is very pure, with only a few overlapping data points in the
middle, meaning that the target labels used for training are much more confident that they are a true
positive instance. d) The confusion matrices and score distributions for the classifier trained using the
noisy unfiltered labels. e) The confusion matrices and score distributions for the classifier trained using
the filtered labels. Figure 1: a) The UMAP projection of the unfiltered training labels. 1.0 is a target label and 0.0 is a
decoy label. As visible by the projection, the cluster on the left corresponding to the decoy labels, is
also fully populated with target labels. This means that during training, noisy target labels that are
indistinguishable from decoy labels will be used as positive training instances when they should not be
b) The overall workflow for denoising the noisy target labels in the training set. 2.4
Acute kidney injury (AKI) analysis in patient blood plasma To provide a biological context to the improvements provided by GPS, we analyzed 141 previously
unpublished blood plasma samples from a subcohort of sepsis patients with acute kidney injury from
the FINNAKI study [48, 20]. These 141 samples are comprised of 2 established subphenotypes, based
on severity of the illness, that were developed from a combination of multiple clinical markers [61]. We
interrogated this data using the repository scale Pan Human Library (PHL) (10838 proteins) [51] and
an optimized PHL where we corrected the retention time and appended spectra from identifications
obtained by searching the DIA data directly with MSFragger-DIA (v3.5) [30] [57] (10952 proteins). This
analysis puts into context the benefits that GPS provides when querying a large search space and the
benefit of using extensive curated repository spectral libraries in discovery DIA approaches and how they
can be optimized. 2.2
Method Benchmark Comparison It is made available under a
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bioRxiv preprint average precision of ratio validations at increasing thresholds from the known concentrations of yeast and
mouse proteins (Figure 2F). The average precision is calculated as the number of yeast identifications
in the yeast window plus the number of mouse identifications in the mouse window divided by the total
number of identifications found both windows at each given threshold. As seen in Figure 2F, GPS starts
out with the highest precision closest to the known ratios and then drops as the windows widen while
maintaining comparable precision to PyProphet until around the ±0.4 window when it is slightly lower
than PyProphet (0.9744 PyProphet compared to 0.9746 GPS at the final cutoff). Percolator displays a
lower precision at all windows compared to GPS and PyProphet, although it is worth noting that the
precision of each method here is very high with 0.97 being the minimum precision found among all three
methods. 2.3
Percentage of Incorrect Target (PIT) Analysis To further establish confidence in the GPS method and to investigate the necessity of down-weighting
decoys with the PIT during q-value calculation, we analyzed samples comprised of mouse kidney tissue
and queried it with the same spectral library from the mouse-yeast spike-in analysis described above. The PIT (or pi0) correction has been used previously to allow for more identifications to be identified
at the same FDR threshold due to the downweighting of decoys [27]. This method is implemented
in both Percolator and PyProphet in order to increase the number of identifications overall, but it
is possible that the inclusion of these identifications can lead to less accurate quantification and the
inclusion of incorrect identifications in the resulting quantitative matrices. To test this, we used the
denoising algorithm developed for model training above and applied it to the classification of putative
peakgroups in a sample. The denoising voting ensemble proves accurate in identifying which portion
of the target distribution should be classified as the false target distribution, and the overlay of these
predicted distributions can be seeing in Figure 3A. To measure the true FDR, we used these false target
classifications to perform a PIT correction during q-value calculation and then measured the number of
yeast identifications that passed at the equivalent estimated FDR (Figure 3B). At a 1.0% FDR, GPS
obtained the lowest Yeast FDR (1.94%), while the Yeast FDR calculated using GPS with the estimated
PIT correction (2.15%) was 10.97% higher. The PyProphet (2.38%) and Percolator (2.13%) Yeast FDRs
were 22.40% and 9.97% higher respectively. We also compared the true positive rate (TPR), measured
using mouse identifications, and the false positive rate (FPR), measured using yeast identifications, at
increasing FDR thresholds (Figure3C-D). At all thresholds, Percolator displays the lowest FPR, but also
identifies significantly less mouse identifications at the same cutoffs (GPS with PIT estimation increases
the number of identifications at a 1.0% FDR by 71.01% compared to Percolator). At a 1.0% FDR GPS
with PIT estimation identifies the most correct mouse identifications, while maintaining a comparable
FPR and Yeast FDR. 2.2
Method Benchmark Comparison The training data is first
split into k number of folds, for each held-out fold, the remaining training data is randomly sampled n-
times with replacement to train n-classifiers that are then used to vote on the held-out data to determine
if an instance is a true target or not. The probability threshold to consider a vote positive can be adjusted
for stringency. c) The UMAP projection of the filtered training labels with a 0.75 probability vote cutoff
after denoising. Each individual cluster is very pure, with only a few overlapping data points in the
middle, meaning that the target labels used for training are much more confident that they are a true
positive instance. d) The confusion matrices and score distributions for the classifier trained using the
noisy unfiltered labels. e) The confusion matrices and score distributions for the classifier trained using
the filtered labels. 4 Figure 1: a) The UMAP projection of the unfiltered training labels. 1.0 is a target label and 0.0 is a
decoy label. As visible by the projection, the cluster on the left corresponding to the decoy labels, is
also fully populated with target labels. This means that during training, noisy target labels that are
indistinguishable from decoy labels will be used as positive training instances when they should not be
b) The overall workflow for denoising the noisy target labels in the training set. The training data is first
split into k number of folds, for each held-out fold, the remaining training data is randomly sampled n-
times with replacement to train n-classifiers that are then used to vote on the held-out data to determine
if an instance is a true target or not. The probability threshold to consider a vote positive can be adjusted
for stringency. c) The UMAP projection of the filtered training labels with a 0.75 probability vote cutoff
after denoising. Each individual cluster is very pure, with only a few overlapping data points in the
middle, meaning that the target labels used for training are much more confident that they are a true
positive instance. d) The confusion matrices and score distributions for the classifier trained using the
noisy unfiltered labels. e) The confusion matrices and score distributions for the classifier trained using
the filtered labels. 4 4 . CC-BY 4.0 International license
perpetuity. 2.4.1
Large Search Space Comparison When repository scale spectral libraries are used to query samples, classic peakgroup scoring methods
can struggle to correctly validate a large portion of the proteins that truly exist in the sample due to
the increased complexity of the data extracted. We analyzed the 141 blood plasma samples with the
PHL and then used GPS, PyProphet, and Percolator to score the extracted signal, calculate q-values
at the precursor, global peptide, and global protein levels, and aggregate the data into a quantitative
matrix. The precursors validated by the 3 tools were rolled up into their proteins using a Python
implementation of the relative quantification iq algorithm [46]. The resulting volcano plots in Figure 5 . CC-BY 4.0 International license
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bioRxiv preprint Figure 2: a) Distributions of the fold changes for GPS of the precursors with at least 2 measurements
per group. The 0.2 log2FC windows around the expected mouse and yeast ratios are highlighted. b)
Global precursor, peptide, and protein counts for each tool in the benchmark at a 1.0% global protein
and peptide FDR. c) Distributions of the fold changes for PyProphet of the precursors with at least
2 measurements per group. The 0.2 log2FC windows around the expected mouse and yeast ratios are
highlighted. d) Barplot showing the percent increase of ratio validated identifications for PyProphet and
Percolator for the expected yeast ratio and the expected mouse ratio. This measures the accuracy and
number of identifications in the indicated areas from a, c, and e in the figure. e) Distributions of the
fold changes for Percolator of the precursors with at least 2 measurements per group. The 0.2 log2FC
windows around the expected mouse and yeast ratios are highlighted. 2.4.1
Large Search Space Comparison f) Lineplot that visualizes the
precision of measurements of each tool at increasing log2FC threshold windows around the expected Figure 2: a) Distributions of the fold changes for GPS of the precursors with at least 2 measurements
per group. The 0.2 log2FC windows around the expected mouse and yeast ratios are highlighted. b)
Global precursor, peptide, and protein counts for each tool in the benchmark at a 1.0% global protein
and peptide FDR. c) Distributions of the fold changes for PyProphet of the precursors with at least
2 measurements per group. The 0.2 log2FC windows around the expected mouse and yeast ratios are
highlighted. d) Barplot showing the percent increase of ratio validated identifications for PyProphet and
Percolator for the expected yeast ratio and the expected mouse ratio. This measures the accuracy and
number of identifications in the indicated areas from a, c, and e in the figure. e) Distributions of the
fold changes for Percolator of the precursors with at least 2 measurements per group. The 0.2 log2FC
windows around the expected mouse and yeast ratios are highlighted. f) Lineplot that visualizes the
precision of measurements of each tool at increasing log2FC threshold windows around the expected
mouse and yeast ratios. Precision is measured by the proportion of correct identifications divided by all
identifications within the tolerance windows. 6 Figure 3: a) Density plots of the target, decoy, and false target distributions from a particular mouse
kidney sample. The predicted false target distribution is determined using the denoising algorithm to
correct q-value calculation during PIT estimation. The overlay of the decoy and false target distributions
show the accuracy of the denoising method in predicting targets and false targets. b) The number of
yeast identifications that pass at particular FDR thresholds for each measured method. Since no yeast
identifications should be found in the sample, a completely linear relationship (y=x) would indicate
perfect entrapment FDR control c) The true positive rate of mouse proteins identified with increasing
q-value cutoffs for all measured methods. This acts as a proxy for the number of identifications at
particular q-value cutoffs. d) The false positive rate as a measurement of the yeast peptides that pass
at certain FDR thresholds compared to all yeast peptides in the library. Figure 3: a) Density plots of the target, decoy, and false target distributions from a particular mouse
kidney sample. .
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bioRxiv preprint 2.4.1
Large Search Space Comparison The predicted false target distribution is determined using the denoising algorithm to
correct q-value calculation during PIT estimation. The overlay of the decoy and false target distributions
show the accuracy of the denoising method in predicting targets and false targets. b) The number of
yeast identifications that pass at particular FDR thresholds for each measured method. Since no yeast
identifications should be found in the sample, a completely linear relationship (y=x) would indicate
perfect entrapment FDR control c) The true positive rate of mouse proteins identified with increasing
q-value cutoffs for all measured methods. This acts as a proxy for the number of identifications at
particular q-value cutoffs. d) The false positive rate as a measurement of the yeast peptides that pass
at certain FDR thresholds compared to all yeast peptides in the library. 7 . CC-BY 4.0 International license
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bioRxiv preprint 4A-C visualize the differentially abundant proteins between the 2 subphenotypes of AKI (81 samples
were from subphenotype 2 and 60 samples were from subphenotype 1) above an absolute value of 1
log2FC and an adjusted p-value of 0.1. At these cutoffs GPS provides a 377.78% increase in the number
of differentially expressed proteins compared to PyProphet, and a 207.14% increase in differentially
expressed proteins compared to Percolator (Figure 4F). Percolator identified the most proteins at a
global 1.0% FDR at the protein level with 937, compared to 768 using GPS, and 164 with PyProphet
(Figure 4D). 2.4.1
Large Search Space Comparison The quantitative matrices produced by Percolator and GPS contain 87.91% and 78.29%
missing values (MV) respectively while PyProphet produced substantially less MVs overall (35.74%),
although the number of proteins at the global level was also much lower (Figure 4). Overall GPS was
able to identify 768 proteins globally, 91 differentially abundant proteins (adjusted p-value ¡ 0.1), and
381 potentially differentially abundant proteins (proteins found in at least 2 samples per group). The
large increases in the number of differentially abundant proteins in Figure 4F that pass the specified
1.0 log2FC and 0.1 adjusted p-value cutoffs can be attributed to the fact that GPS provides a more
complete data matrix in the sense that a greater number of proteins have multiple measurements per
subphenotype, so more proteins can be accurately compared (a 38.55% increase compared to Percolator
and a 154.0% increase compared to PyProphet). Figure 4: a) Volcano plot visualizing the distribution of differential abundance values (log2FC) for GPS
on the AKI data and their -log10(adjusted p-value). The cutoffs are set at a 0.1 adjusted p-value and
1 log2FC. b) Volcano plot visualizing the distribution of differential abundance values (log2FC) for
Percolator on the AKI data and their -log10(adjusted p-value). The cutoffs are set at a 0.1 adjusted p-
value and 1 log2FC. c) Volcano plot visualizing the distribution of differential abundance values (log2FC)
for PyProphet on the AKI data and their -log10(adjusted p-value). The cutoffs are set at a 0.1 adjusted
p-value and 1 log2FC. d) Barplot of the overall global protein counts that pass a 1.0% FDR cutofffor
the compared methods. e) Barplot of the percent of missing values in the dataset for proteins that
pass a 1.0% FDR cutofffor the compared methods. f) Barplot of the number of differentially abundant
proteins that pass a 1.0% FDR cutofffor the compared methods. Figure 4: a) Volcano plot visualizing the distribution of differential abundance values (log2FC) for GPS
on the AKI data and their -log10(adjusted p-value). The cutoffs are set at a 0.1 adjusted p-value and
1 log2FC. b) Volcano plot visualizing the distribution of differential abundance values (log2FC) for
Percolator on the AKI data and their -log10(adjusted p-value). The cutoffs are set at a 0.1 adjusted p-
value and 1 log2FC. c) Volcano plot visualizing the distribution of differential abundance values (log2FC)
for PyProphet on the AKI data and their -log10(adjusted p-value). 2.4.1
Large Search Space Comparison The cutoffs are set at a 0.1 adjusted
p-value and 1 log2FC. d) Barplot of the overall global protein counts that pass a 1.0% FDR cutofffor
the compared methods. e) Barplot of the percent of missing values in the dataset for proteins that
pass a 1.0% FDR cutofffor the compared methods. f) Barplot of the number of differentially abundant
proteins that pass a 1.0% FDR cutofffor the compared methods. 3
Discussion With the implementation of our new methods, we show an increase in the precision of quantification,
while also increasing the number of identifications when compared to the established methods. We were
also able to demonstrate how these established methods can suffer when the spectral library search space
is too large and does not match the sample, while GPS is able to score data in this scenario in a stable
manner. These combined improvements allow for the deep and in-depth analysis of plasma proteome
samples using repository scale spectral libraries to boost the power of discovery DIA experiments. 2.4.2
Discovery DIA in plasma proteomics To provide biological context, and to show the benefit of using full-tissue libraries in discovery DIA
proteomics, we reanalyzed the 141 blood plasma samples using the optimized PHL with an iterative
retention time alignment workflow described in the methods. Differential expression was performed
between the 2 subphenotypes to identify potential proteins that could act as differentiating markers in
stratifying AKI subtypes in sepsis. Using this optimized PHL and workflow we were able to detect 1205
proteins overall (a 56.90% increase compared to the standard PHL), 170 differentially abundant proteins
(adjusted p-value ¡ 0.1), and 651 potentially differentially abundant proteins (proteins found in at least 2 8 . CC-BY 4.0 International license
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bioRxiv preprint samples per group). In Figure 5A we can see the overall distribution of differentially abundant proteins
in the dataset. 2.4.3
Machine learning aided differential expression Utilizing the boosted number of potential marker proteins detected with the optimized PHL, we wanted to
investigate the potential to train a machine learning model to differentiate between the 2 subphenotypes of
AKI mentioned above, interpret the importance of each protein in the model, and use this interpretation
to guide the selection of interesting proteins for further study. Using 10-fold cross validation we trained
a Random Forest classifier which resulted in a mean accuracy of 0.84 (0.12 standard deviation). To
quantitatively understand the importance of each protein used in the model as a feature we leveraged
the explainable machine intelligence algorithms in the SHAP python package[35]. From the trained
model, we calculated the SHAP values for each protein and have visualized the top 20 proteins with the
greatest mean importance (Figure 5B). Using these top 20 proteins we performed an average hierarchical
clustering with correlation similarity using a maximum of 2 groups and saw that the subphenotypes
clustered together with 76% accuracy (0.76 Rand score) and we could easily visualize a clear separation
of the 2 groups (Figure 5C). For the purposes of the visualization, we capped the normalized protein
abundances at -1.0 and 1.0. Additionally, we annotated the volcano plot in Figure 5A to showcase that
the top 20 most important proteins in differentiating between the 2 subtypes using the trained random
forest model would not pass the arbitrary canonical cutoffs used to identify interesting differentially
expressed proteins. In Figure 5e we plot these top 20 selected proteins to visualize their abundances
across the 2 subphenotypes. 3.1
Benchmark comparison with existing methods It is made available under a
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bioRxiv preprint Figure 5: a-c) Volcano plots for the differential abundant proteins analyzed with the optimized PHL
library. Red corresponds to proteins upregulated in subphenotype 2 and blue corresponds to proteins
upregulated in subphenotype 1. The labeled proteins correspond to the top 20 most important proteins
found that differentiate between subphenotypes with a Random Forest model. It is worth noting that
many of the most important proteins do not fall outside of the arbitrary cutoffs commonly used to identify
proteins of interest in biomarker studies. b) Violin plots of the SHAP scores for the top 20 proteins
indicated in Figure5A. A red color indicates a higher abundance for a protein and a blue color indicates
a lower abundance for a protein. SHAP scores greater than 0.0 on the x-axis indicate features that
drive classification towards subphenotype 2 and scores less than 0.0 indicate protein values that drive
classification towards subphenotype 1. c) Heatmap and cluster analysis of the top 20 proteins identified
using SHAP scores from the Random Forest model. There is a clear grouping of the subphenotypes
(0 76 accuracy) and 2 main clusters of proteins the first indicating proteins of lower abundance in Figure 5: a-c) Volcano plots for the differential abundant proteins analyzed with the optimized PHL
library. Red corresponds to proteins upregulated in subphenotype 2 and blue corresponds to proteins
upregulated in subphenotype 1. The labeled proteins correspond to the top 20 most important proteins
found that differentiate between subphenotypes with a Random Forest model. It is worth noting that
many of the most important proteins do not fall outside of the arbitrary cutoffs commonly used to identify
proteins of interest in biomarker studies. b) Violin plots of the SHAP scores for the top 20 proteins
indicated in Figure5A. 3.1
Benchmark comparison with existing methods PyProphet and Percolator both use a semi-supervised learning technique to train on a particular data
set by identifying new true targets every iteration and re-training until the number of peakgroups that
pass a q-value threshold no longer increase. This type of method can provide great results on individual
experiments, particularly by increasing the number of identifications, as it maximizes the local number of
targets that are validated at the end, but could potentially introduce false positives that are elucidated
only when analyzing the precision of the quantitative accuracy on known spiked-in proteins. It has also
been observed that these semi-supervised methods can struggle when the number of samples analyzed
is relatively low (low-n) [17] or when the spectral library used contains substantially more proteins than
it is possible to find in the sample (Figure 4). To minimize these issues, we looked to leverage one of
the core strengths of machine learning algorithms by training a generalizable model on a diverse dataset
that can accurately separate true targets from false targets independent of the search space size. Since
it is difficult to directly quantify the performance of new computational methods based on individual
metrics, we created a species mixture data set of known ratios of yeast peptides spiked in to a constant
mouse kidney proteome background. This allows us to evaluate the efficiency of different peakgroup
validation methods and compare them directly based on the number of identifications and the accuracy
of quantification based on how close the produced quantitative values are to their expected ratios. We
demonstrate here that the optimized GPS model outperforms existing methods based on the number of
global identifications because the performance of GPS is not hindered by the specificity of the samples
being analyzed since we trained the generalizable model on millions of extracted peakgroups from an
external dataset. We also demonstrate a boost in accuracy and precision of quantification due to the
automated curation of the training data to ensure only true peakgroups are included when building the
model without over-fitting on experiment specific data. These performance improvements suggest that
generalized scoring models can be leveraged in diverse experiment types to provide boost identification 9 . CC-BY 4.0 International license
perpetuity. 3.3
Boosted performance in library-sample size mismatches In theory, it is particularly beneficial to search blood plasma samples with massive scale complex libraries
to delve deeper into the proteome as it could be possible to pick up and identify potential disease markers
missed in standard analysis, especially in the case of low abundant proteins. In practice, however, classic
peakgroup validation algorithms struggle when the true signal extracted with the library is much smaller
than the library itself, or when the library size is significantly larger than the amount of proteins contained
in the sample. For example, when a blood plasma sample is searched with a repository scale library
containing 500,000 precursors there may only be 15,000 identifiable precursors in the sample, so a massive
imbalance in search space is created. As seen in Figure 4, the existing semi-supervised algorithms can
struggle in these search space imbalances and lead to significantly less candidate differentially expressed
proteins being identified. One potential reason for this is that the library search space imbalance creates
a situation where the true target labels are extremely noisy, ie. only 3% of the target peakgroups
extracted by the library are true peakgroups. Semi-supervised methods attempt to get around this in
an iterative fashion using an algorithm where new targets are selected based on q-value cutoffs [50, 58]. When the true target to decoy ratio is so small, it is not guaranteed that a correct set of true targets
are picked up for training during the semi-supervised iteration, creating a massive imbalance in the ratio
of true training targets to false training targets. There are a few different ways to try and mitigate
training set imbalances, such as down-sampling the majority class, up-sampling the minority class [2],
which Percolator does, or by using the synthetic minority oversampling technique (SMOTE) where
synthetic training instances are created based on the distributions of features found in the minority
class [15]. It is also possible to provide the class ratios to certain machine learning algorithms to ensure
that over-represented classes do not dominate the training loops. Unfortunately, these methods are not
implemented in all common peakgroup validation algorithms, so even though some may work effectively
in situations where the library size is very similar to the sample composition, they can still struggle in
situations as the library grows larger and larger compared to the sample. 3.2
PIT estimation PIT estimation has become an established method to boost the number of identifications that pass
through at a given FDR cutoffin mass spectrometry proteomics. However, predicting this percentage
of false targets is computationally difficult, as it is unknown which targets are in fact false, or where
to split the target distribution to estimate pi0. Existing methods use certain heuristics to estimate
pi0 by calculating the difference between the decoy counts and target counts at certain score cutoffs
[27], or provide naive counts based on the number of targets below a 1.0% FDR, but this can lead to
inaccurate results depending on the shapes of the score distributions. To accurately estimate pi0, we
leveraged the ensemble denoising algorithm used to filter the training data for the high precision GPS
model. By measuring the number of targets below a 100% vote percentage we can accurately model the
false target distribution and use it to down-weight the decoy counts when calculating q-values. This can
considerably increase the number of identifications that pass at certain thresholds, especially in large
search space scenarios (ie. respository scale spectral library searches). It is possible to underestimate
or overestimate pi0 by changing the probability thresholds of the denoising classifier, and these can be
leveraged in different situations depending on the goal of the particular experiment. The implications
of false target prediction goes beyond the downweighting of decoys during q-value calculation, it also
opens up the possibility of decoy free scoring methods that utilize the predicted false target distributions
directly to calculate q-values. This could massively cut down the search space, particularly in discovery
DIA settings using repository scale or deep learning predicted libraries. 3.1
Benchmark comparison with existing methods A red color indicates a higher abundance for a protein and a blue color indicates
a lower abundance for a protein. SHAP scores greater than 0.0 on the x-axis indicate features that
drive classification towards subphenotype 2 and scores less than 0.0 indicate protein values that drive
classification towards subphenotype 1. c) Heatmap and cluster analysis of the top 20 proteins identified
using SHAP scores from the Random Forest model. There is a clear grouping of the subphenotypes
(0.76 accuracy), and 2 main clusters of proteins, the first indicating proteins of lower abundance in
subphenotype 2 and the second of higher abundance in subphenotype 2. d) Box plots of the abundance
of the top 20 selected proteins compared between subphenotypes. It is interesting to see that many of the
proteins do not have clear separation of the means between the groups, but are still distinctly important
in classifying between subphenotypes. 10 . CC-BY 4.0 International license
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bioRxiv preprint numbers and quantitative accuracy without the need to train new models for each experiment. Ad-
ditionally, there is no need to optimize hyperparameters to squeeze the best performance out of GPS,
as the generalized model will score peakgroups accurately in a stable manner no matter the conditions
of the data. Existing methods can be optimized in different conditions, but a meticulous optimization
of hyperparameters is required. This non-trivial time consuming parameter optimization task can be
avoided completely by implementing peakgroup scoring with generalizable models such as with GPS. 3.4
Analysis of AKI samples in human plasma Taking the algorithmic benefits of GPS into consideration, we decided to apply the new method to the
computationally difficult data set of AKI plasma samples described above to provide some biological
context. Using our 1205 globally identified proteins, and instead of selecting candidate proteins for fur-
ther analysis based on arbitrary q-value and log2 fold change cut offs, we trained a machine learning
model to differentiate between the 2 subphenotypes of AKI using the candidate differential proteins in
the dataset and then calculated the importance of each protein to the model during classification us-
ing SHAP values [35]. The high accuracy and separating power of this panel of 20 proteins indicates
that they would provide a good starting point for investigation as potential clinical markers. In fact,
many of these proteins have already been identified and studied as potential clinical sepsis markers or
markers for infection and inflammation [21, 44, 60, 16, 45, 36, 37, 67, 64, 10, 62, 23, 9, 55, 1, 25]. The
majority of the top 20 proteins were up-regulated in the more severe subphenotype 2. Some examples
which were higher in subphenotype 2 are the neutrophil-enriched NGAL (Neutrophil-gelatinase associ-
ated lipocalin), a previously described marker of severe AKI [60]; CD14 (Cluster of differentiation 14), a
marker of monocyte infiltration; serum acute phase protein LBP (Lipopolysaccharide binding protein);
neutrophil-specific DEF3 (Neutrophil defensin 3); and mitochondria specific MDHM (Malate dehydro-
genase, mitochondrial). Proteins that were downregulated in subphenotype were the liver-specific acute
phase protein ITIH-1, -2 (Inter-alpha-trypsin inhibitor heavy chain-1, -2)[56] and the albumin-like AFAM
(Afamin) whose levels are known to be lowered with increase in sepsis severity [33]. This panel could
further be expanded to any protein that has a significant weight in classifying the severity of AKI based
on a combination of SHAP values and differential expression analysis in an effort to identify novel disease
biomarkers. These findings suggest that it is possible to identify potential sepsis markers in plasma samples and
accurately quantify them using repository scale spectral libraries and statistical validation with GPS. These added benefits could significantly aid in the stratification of sepsis subphenotypes by allowing
for a deeper exploratory investigation of the plasma proteome on a systematic basis and the informed
data-driven selection of potential biomarkers for further validation. 3.4
Analysis of AKI samples in human plasma This approach would also generalize
to other biological conditions or diseases easily, providing a systematic method towards discovery DIA
for a wide range of experiment types. 4
Conclusion We have proposed GPS as a method for the statistical validation of DIA mass spectrometry data, and
provided evidence that generalized scoring models can outperform dynamically trained models especially
in a large search space environment. GPS provides stable validation that leads to more accurate down-
stream quantification and provides evidence that sophisticated generalized scoring models can be used
in tandem with massive scale spectral libraries to support the development of discovery proteomics in
DIA mass spectrometry. the box. the box. 3.3
Boosted performance in library-sample size mismatches In the case of GPS, instead of
choosing to implement imbalanced learning methods to validate the data with sample specific classifiers
we chose to build a static classifier that leverages modern machine learning technologies and generalizes
to diverse sample and experiment types. In these cases, it is also extremely beneficial that there is no
need to optimize the parameters of GPS to get these results, it can realize these improvements out of 11 . CC-BY 4.0 International license
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bioRxiv preprint the box. 5.1.4
AKI data AKI plasma samples used in the study belong to the FINNAKI study [48], a prospective, observational,
multicenter study evaluating development of AKI in ICU patients with sepsis and septic shock. AKI
was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria based on
changes in serum creatinine[29]. 51% of the patients developed AKI within the first 5 days in the ICU,
with 30% diagnosed <12 hours from admission. Approximately 100 patients each developed stage 1, 2,
and 3 AKI. 91 patients received RRT and the 90-day mortality for AKI patients was 33.7%. 141 samples
were chosen for up to 5 time points from 23 acute kidney injury patients. The patients were from 2
distinct subphenotypes that were previously defined using a panel of clinical markers and latent class
analysis [61]. 5.1.3
Mouse kidney data 31 mouse kidney samples were selected from a previous study [40] to provide a base for the entrapment
FDR analysis. The samples are from the same study as the mouse kidney material used to prepare the
spike-in data, and follow the same ethical considerations and approvals (ethical permit number 03681-
2019), as well as sample preparation methods described above. 5.1.2
Spike-in data We generated a spike-in dataset of known concentrations of Yeast tryptic digest peptides (Promega)
spiked in to a constant mouse kidney background. The mouse kidney material was obtained from the a
previous project [40]. All animal use and procedures were approved by the local Malm¨o/Lund Institu-
tional Animal Care and Use Committee, ethical permit number 03681-2019. C57BL/6 mice (Janvier, Le
Genest-Saint-Isle, France) were sacrificed, and kidneys were isolated into a tube containing DPBS and
silica beads (1 mm diameter, Techtum). The kidneys were then homogenized using MagNAlyser (Roche)
and stored at -80°C. Homogenates were then thawed and centrifuged at 10,000g for 10’ at 4°C. The
supernatant containing the soluble proteins was collected and protein content was estimated using BCA
(Pierce). 25ug of protein was taken for reduction, alkylation, digestion and C18 clean up, as described
below. A serial dilution series of yeast peptides (1X, 2X, 4X, 8X, 16X, 32X) was performed, and 10
technical replicates of each concentration were sampled for a total of 60 samples. Internal retention time
peptides were also added in to each sample. Each of the 60 samples were analyzed on the an Orbitrap
HF-X using both DDA and DIA. 5.1
Datasets Dataset
MS Acquisition
N Samples
Technical Replicates
Usage
HFX-SPD
DIA
129
All
Generalizable model training
Mouse-Yeast
DDA
60
10
Spectral library generation
Mouse-Yeast
DIA
60
10
GPS Benchmark
Mouse Kidney
DIA
31
None
Entrapment FDR Analysis
AKI
DIA
141
None
Large Search Space Comparison
Optimized Library Analysis
ML Differential Expression 12 . CC-BY 4.0 International license
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GPS model training data: HFX-SPD In order to provide a chromatographically diverse set of training data, we used a data set comprised of
129 different samples of 500ng Yeast tryptic digest (Promega) with varying gradient lengths (30, 45, 60,
90, 120 minutes) and acquired with DIA. This data set will be referred to as the HFX-SPD data set. 5.3.2
Pan Human Library The Pan Human Library [51] was downloaded in its original form and then converted to a PQP spectral
library using the OpenSwathAssayGenerator tool available from OpenMS [54]. 5.2
Mass spectrometry sample preparation and data acquisition
5.2.1
AKI sample preparation and analysis The cartridges were washed with 0.1% TFA before the peptides were eluted with
80% ACN/0.1% TFA. The eluted peptides were dried in a SpeedVac (Concentrator plus Eppendorf) and
resuspended in 25 µL of 2% ACN/0.1% TFA. The peptide concentration was measured using the Pierce
Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific, Rockford, IL, USA). The samples,
10 µL, were diluted with 10 µL 2% ACN/0.1% TFA and spiked with synthetic iRT peptides (JPT Peptide
Technologies, GmbH, Berlin, Germany) before liquid chromatography-mass spectrometry (LC-MS/MS)
analysis. flow rate of 5 µL/min. The cartridges were washed with 0.1% TFA before the peptides were eluted with
80% ACN/0.1% TFA. The eluted peptides were dried in a SpeedVac (Concentrator plus Eppendorf) and
resuspended in 25 µL of 2% ACN/0.1% TFA. The peptide concentration was measured using the Pierce
Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific, Rockford, IL, USA). The samples,
10 µL, were diluted with 10 µL 2% ACN/0.1% TFA and spiked with synthetic iRT peptides (JPT Peptide
Technologies, GmbH, Berlin, Germany) before liquid chromatography-mass spectrometry (LC-MS/MS)
analysis. 5.3.1
Experiment specific spike-in library An experiment specific library for the spike-in data was built by analyzing the samples acquired using
DDA using FragPipe (v18.0). First the samples were searched using MSFragger (v3.5) [30] with default
parameters using a fasta file of Swiss-prot reviewed saccharomyces cerevisiae and mus musculus proteomes
concatenated with reverse sequence decoy proteins. Peptide spectrum matches (PSMs) were validated
using Percolator[58]. The Philosopher toolkit (v4.4.0) was used to perform protein level FDR control with
ProteinProphet, generate downstream reports, and filter the resulting identifications[34]. The python
package easypqp was then used to convert and format the library for use by OpenSwath and the OpenMS
(2.6.0) tool chain for formatting PQP files was used to optimize the assays in the library [54]. 10 spiked-in
retention time peptides (iRT) were used for alignment and retention time correction for each sample. 5.2.2
All other sample preparations and analysis All protein samples were denatured with 8M urea and reduced with 5 mM Tris(2-carboxyethyl)phosphine
hydrochloride, pH 7.0 for 45 min at 37 °C, and alkylated with 25 mM iodoacetamide (Sigma) for 30 min
followed by dilution with 100 mM ammonium bicarbonate to a final urea concentration below 1.5 M. Proteins were digested by incubation with trypsin (1/100, w/w, Sequencing Grade Modified Trypsin,
Porcine; Promega) for at least 9 h at 37 °C. Digestion was stopped using 5% trifluoracetic acid (Sigma)
to pH 2 to 3. The peptides were cleaned up by C18 reversed-phase spin columns as per the manufac-
turer’s instructions (Silica C18 300 ˚A Columns; Harvard Apparatus). Solvents were removed using a
vacuum concentrator (Genevac, miVac) and were resuspended in 50 µl HPLC-water (Fisher Chemical)
with 2% acetonitrile and 0.2% formic acid (Sigma). Peptide analyses were performed on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific)
connected to an EASY-nLC 1200 ultra-HPLC system (Thermo Fisher Scientific). Peptides were trapped
on precolumn (PepMap100 C18 3 µl; 75 µl Ö 2 cm; Thermo Fisher Scientific) and separated on an EASY-
Spray column (ES903, column temperature 45 °C; Thermo Fisher Scientific). Equilibrations of columns
and sample loading were performed per manufacturer’s guidelines. Mobile phases of solvent A (0.1%
formic acid), and solvent B (0.1% formic acid, 80% acetonitrile) was used to run a linear gradient from
5% to 38% over various gradient length times at a flow rate of 350 nl/min. The 44 variable windows
DIA acquisition method is described by Bruderer et al [[8]]. MS raw data was stored and managed
by openBIS (20.10.0) [[3]] and converted to centrioded indexed mzML files with ThermoRawFileParser
(1.3.1) [[24]]. 5.2
Mass spectrometry sample preparation and data acquisition
5.2.1
AKI sample preparation and analysis 141 samples were chosen for up to 5 time points from 23 acute kidney injury patients. All sample
preparation steps, including desalting and protein digestion, used the Agilent AssayMAP Bravo Platform
(Agilent Technologies, Inc.) per manufacture’s protocol. Each plasma sample was diluted 1:10 (100-mM
ammonium bicarbonate (AmBic); Sigma-Aldrich Co, St Louis, MO, USA), and 10 l of each diluted plasma
sample were transferred to a 96-well plate (Greiner G650201) where 40 µL of 4 M urea (Sigma-Aldrich)
in 100 mM AmBic was manually added with a pipette for a final volume of 50 µL. The proteins were
reduced with 10 µL of 60 mM dithiothreitol (DTT, final concentration of 10 mM, Sigma-Aldrich) for one
hour at 37 °C followed by alkylation with 20 µL of 80 mM iodoacetamide (IAA, final concentration of 20
mM, Sigma-Aldrich) for 30 min in a dark at room temperature. The plasma samples were digested with
2 µg Lys-C (FUJIFILM Wako Chemicals U.S.A. Corporation) for five hours at room temperature and
further digested with 2µg trypsin (Sequencing Grade Modified, Promega, Madison, WI, USA) overnight
at room temperature [5]. The digestion was stopped by pipetting 20 µL of 10% trifluoroacetic acid (TFA,
Sigma-Aldrich) and the digested peptides were desalted on Bravo platform. To prime and equilibrate
the AssayMAP C18 cartridges (Agilent, PN: 5190-6532), 90% acetonitrile (ACN, Sigma-Aldrich) with
0.1% TFA and 0.1% TFA were used, respectively. The samples were loaded into the cartridges at the 13 . CC-BY 4.0 International license
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bioRxiv preprint flow rate of 5 µL/min. 5.3.3
Optimized Pan Human Library To augment the PHL with additional identifications and correct the retention time to the experiment
at hand, we first searched the 141 AKI plasma samples using MSFragger-DIA (v3.5) [30, 57]. Using
the resulting set of identifications, shared precursors between the PHL and direct search were selected
for retention time alignment. LOWESS was first used to smooth the correlation between the direct
search results and the PHL and then an interpolated univariate spline function was fit on top of this to
adjust the retention time in the direct search to the scale of the PHL. The shared proteins between the
2 libraries were replaced in the PHL with proteins, and associated precursors, from the direct search, 14 . CC-BY 4.0 International license
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bioRxiv preprint and the proteins not contained in the PHL were appended to the library. 10 spiked-in retention time
peptides (iRT) were used for alignment and retention time correction for each sample. and the proteins not contained in the PHL were appended to the library. 10 spiked-in retention time
peptides (iRT) were used for alignment and retention time correction for each sample. 5.5.1
Implementation GPS is a Python library and command line utility for the generalized statistical validation of peakgroups. The source can be found here (https://github.com/InfectionMedicineProteomics/gscore). GPS
leverages the package numpy [22] for efficient processing of numerical data, scikit-learn, sklearn and
xgboost for implementing machine learning algorithms, and numba [32] for its just-in-time (JIT) com-
pilation that compiles Python to machine code for optimization in performance critical areas of the
library. 5.5.2
Denoising algorithm The denoising algorithm used to filter the HFX-SPD training set and predict the false target distribution
for PIT correction is based on the concept of bagging from machine learning[7]. The data to be analyzed
is first split into k number of folds (default is 10, and what is used throughout the study). Each fold
is scored by training an ensemble of n logistic regression classifiers (default is 10, and what is used
throughout the study) using stochastic gradient descent [53]) on data that is randomly sampled with
replacement from the data left out of the selected k-fold. The ensemble of classifiers is then used to score
the k-fold data, providing an average target probability for each peakgroup if the fold, and voting on
each peakgroup to determine the vote percentage. A vote is considered a positive vote if the predicted
probability for the individual classifier in the ensemble exceeds a threshold. 5.4
Refined OpenSwath analysis We adapted the previously published DIAnRT workflow [13] to optimize signal extraction at the sample
level before combining the analysis to control for the global FDR. To do this, a first iteration is performed
where sub-optimal retention time peptides are provided to align the experimental chromatograms to
the spectral library. The first pass of signal extraction is scored and validated, and then the highest
scoring peakgroups from a specified number of bins are selected and written out to a sample specific
retention time library. This sample specific retention time library is used to align and correct the
retention time to the spectral library with more stringent parameters in a second pass. Again, the
second pass analysis is scored and validated and the highest scoring peakgroups for a specified number
of bins are extracted and used as sample specific retention time libraries for a final pass. The final
pass uses these sample specific retention time libraries to additionally correct mz and rentention time
in an even more stringent manner. These final validated peakgroups are then used to infer peptide
and proteins in a global manner to control for FDR and are finally combined in an output matrix
that can be analyzed using the DPKS package mentioned above. Software to perform the sample specific
retention time library extraction can be found in combination with the GPS python package and complete
snakemake workflows and corresponding command line options for the different tools used can be found
at (https://github.com/InfectionMedicineProteomics/gscore). 5.5.3
Filtering training data In an attempt to remove the noisy labels from the training dataset, the denoising algorithm described
above was used to calculate a vote percentage for each peakgroup. If the calculated vote percentage was
100% then the peakgroup was kept as a true target. The probability to accept a positive vote was set at
0.75 to more strictly filter out potential false positives at the risk of losing some true identifications in
the dataset. The negative training set, the decoy peakgroups, remained unfiltered. 5.6
Downstream statistical analysis All down stream analysis was performed using the Data Processing Kitchen Sink (DPKS) Python package
for general purpose data processing of mass spectrometry proteomics data. (https://github.com/
InfectionMedicineProteomics/DPKS) For all datasets, a retention time-mean sliding window normalization method was used based on the
implementation in the NormalyzerDE R package [63]. Proteins were quantified for the AKI analysis
using an implementation of the iQ relative quantification algorithm [46] (also known as MaxLFQ[12]). Differential expression was preformed using linear models, at the precursor level for the spike-in analysis
and protein level for the AKI analysis. Multiple testing correction was performed using the Benjamini-
Hochberg method [4]. All of these methods, including other options, are available in the DPKS package. 5.5.6
Global FDR control Global FDR control is implemented in a similar manner to PyProphet[50], where all scored samples
in an experiment are aggregated and the highest scoring precursor is selected to represent either the
peptide or the protein at the desired level. Once the highest scoring precursors are selected, q-values are
estimated using the method described above and PIT corrected using the global PIT correction method. The resulting scoring models are exported as serialized Python objects that can then easily be used from
the command line by GPS to export an annotated quantitative matrix. 5.5.5
Peakgroup scoring and q-value calculation The algorithm used to score each peakgroup in GPS is very straightforward. The peakgroups and their
associated sub-scores are read in and parsed into a data structure that maps each scored peakgroup
to the potential precursor. All of the peakgroups are scored using the filtered model trained above. Each peakgroup is additionally ran through the denoising algorithm to provide the needed scores to
estimate the PIT. After scoring, the top ranked peakgroup for each precursor is selected to represent
that precursor and the PIT is estimated. Q-values are calculated using an implementation of the qvality
algorithm[28], where an interpolated spline is fit to the distributions of the target and decoy scores. A
q-value for a particular peakgroup is calculated by first integrating the area under the curve of the decoy
distribution from that particular score to the max and multiplying it by the PIT to get the decoy counts
at a particular score threshold. The target counts are then obtained by integrating the area under the
curve of the target distribution from that particular score to the max. Finally, the decoy counts are
divided by the target counts plus the decoy counts to calculate a q-value, which can be used to filter
for a given FDR. The highest scoring peakgroup for each precursor and the corresponding scores and
q-value are written out to a file for downstream processing. 5.5.7
Quantitative matrix export GPS can aggregate all scored samples, and the global peptide and protein models, into a quantitative
matrix for downstream analysis. Each sample is read in to a data structure that filters the samples in the
precursor based on their individual q-values. Once all samples have been parsed, they are annotated with
their global peptide and protein level q-values using the score distribution objects that were previously
built. The resulting annotated quantitative matrix is then written out for downstream analysis by the
tool of your choosing. 5.5.4
PIT Estimation To estimate the PIT, the denoising algorithm was used to calculate the vote percentage for each peak-
group in the dataset, where only the top scoring peakgroup was kept per precursor. For Figure3 0.75
was the probability threshold for a positive vote, and all peakgroups below a 100% vote percentage are
considered false targets. The PIT is calculated by then dividing the number of false targets by the
number of decoys. 15 . CC-BY 4.0 International license
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bioRxiv preprint For global PIT calculations on the peptide and protein level, the PIT is estimated by counting the
number of peptides or proteins below a 1% FDR cutoffand then dividing that by the number of decoys. For global PIT calculations on the peptide and protein level, the PIT is estimated by counting the
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https://openalex.org/W4382795727
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https://esj.eastasouth-institute.com/index.php/eslhr/article/download/94/74
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English
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Protecting Civil and Political Rights: Comparative Analysis of International Human Rights Mechanisms
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The Easta Journal Law and Human Rights
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cc-by-sa
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ABSTRACT This research conducts a comparative analysis of international human
rights mechanisms to examine their effectiveness in protecting civil and
political rights. The research aims to assess the strengths, limitations
and contributions of various international mechanisms in protecting
these fundamental rights. Part of the study process included a
thorough analysis of international human rights treaties, agreements,
reports, and case law from regional and international human rights
organizations. The focus of the examination is on contrasting the
mandates, responsibilities, and management styles of important
international human rights institutions, including the Inter-American
Commission on Human Rights, the European Court of Human Rights,
and the UN Human Rights Council. These international organizations
use a variety of tactics and resources to safeguard civil and political
rights, which are made evident in its conclusions. The report lists
common problems that these systems encounter, such as poor
enforcement capacities, disparities in membership and participation,
and the potential impact of political interests on decision-making
processes. It also looks at how these processes have helped advance
civil and political rights all throughout the world. The article discusses
significant cases and rulings that have helped establish international
and state standards for human rights legislation. This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author:
Name: Ade Risna Sari
Institution: Tanjungpura University
Email: a.risna.sari@fisip.untan.ac.id Protecting Civil and Political Rights: Comparative Analysis of
International Human Rights Mechanisms
Ade Risna Sari1, Amtai Alaslan2
1 Tanjungpura University
2 Lelemuku Saumlaki University Ade Risna Sari1, Amtai Alaslan2
1 Tanjungpura University
2 Lelemuku Saumlaki University The Easta Journal Law and Human Rights
Vol. 1, No. 03, June, pp. 176 - 184
ISSN: 2985-7112, DOI: 10.58812/eslhr.v1.i03 The Easta Journal Law and Human Rights
Vol. 1, No. 03, June, pp. 176 - 184
ISSN: 2985-7112, DOI: 10.58812/eslhr.v1.i03 This is an open access article under the CC BY-SA license. Journal homepage: https://esj.eastasouth-institute.com/index.php/eslhr 1. INTRODUCTION ILO
cooperation,
The
Commission
for
Economic, Social, and Cultural Rights and
European Court of Human Rights assess and
interpret cases [1]. Labor rights problems
often use ILO principles and legal philosophy. They are a "significant addition" to the
European Covenant on Human Rights and
International Covenant on Economic, Social,
and Cultural Rights [1]. Another such is the
UN human rights treaty organizations, which International
human
rights
mechanisms
are
organizations
and
procedures designed to advance and defend
human rights on a global scale. Treaties,
conventions, monitoring organizations, and
courts are only a few examples of these
systems' many manifestations [1]. ILO is a
worldwide human rights mechanism. With Journal homepage: https://esj.eastasouth-institute.com/index.php/eslhr The Easta Journal Law and Human Rights (ESLHR) 177 Indonesian law, human rights reform has
stalled. Another article [8] covers Indonesia's
criminal
justice
system
and
legislative
framework for law enforcement, with a focus
on protecting human rights principles.. keep tabs on how states parties are enforcing
the agreements. One of the most important
global tools for protecting human rights is
reporting on UN human rights treaties [2]. Recent research have linked government
reporting to human rights monitoring groups
to greater human rights [3]. Another article [9] studies the efforts
made
by
Palestinian
human
rights
organizations to accomplish their objectives in
order to analyze the part these organizations
play in defending civil and political rights. According to the study, human rights
organizations support the defense of many
different rights, such as the right to life, bodily
integrity, freedom of expression, and a fair
trial. Sustainability
and
international
human
rights
may
overlap. The
UN
framework for action emphasizes human
rights and sustainable development, and the
SDGs promote human rights in numerous
ways
[4]. International
human
rights
processes promote and protect human rights
worldwide. These methods monitor and
enforce human rights standards to guarantee
states respect, safeguard, and fulfill them. These articles may shed light on
human rights reform and judicial reform in
Indonesia and the role of human rights groups
in preserving civil and political rights abroad. International human rights treaties
protect and enhance human rights. Key
international human rights accords [5]. The
1948
UN
General
Assembly
Universal
Declaration
of Human
Rights
(UDHR)
establishes universal freedoms and rights. The
1966 UN General Assembly established the
International Covenant on Civil and Political
Rights. ICCPR protects life, expression, and
religion. 1. INTRODUCTION The maintenance of civil and political
rights is a critical component of promoting
human rights, democracy, and justice around
the world. These liberties include several
critical ones, such as the right to life, liberty,
and personal security, freedom of expression
and association, and the right to participate in
political processes. Despite the fact that
nations
are
ultimately
responsible
for
preserving these rights, international human
rights mechanisms are critical for monitoring
compliance
and
holding
governments
accountable. The UN General Assembly adopted
the International Covenant on Economic,
Social, and Cultural Rights (ICESCR) in 1966,
guaranteeing labor, education, and healthcare
rights [6]. CEDAW passed in 1979. This
enforceable
agreement
reduces
gender
imbalance and discrimination against women. This study compares international
human rights systems' civil and political
rights
protection. This
study
evaluates
regional human rights tribunals, treaty
bodies, and special procedures to understand
their strengths and limitations and their
potential to defend these rights. The 1989 UN General Assembly
Convention on the Rights of the Child (CRC)
defines children's rights. These rights include
freedom from abuse, medical care, and
education. In 2006, the UN General Assembly
approved the CRPD. Justice, employment,
and education are covered under this treaty. It
promotes human rights and is recognized by
many states. 2.1 International Human Rights Mechanisms The phrase "international human
rights
mechanisms"
refers
to
the
institutional structures and procedures
that have been built at the international
level in order to monitor and safeguard
human rights. The regional human rights
tribunals,
treaty
organizations,
and
special processes are all examples of these
types of systems. This
study
investigates
the
justifications for state adherence to
international human rights law. The
study
demonstrates
that
domestic
compliance
mechanisms
driven
by
constituents are in charge of compliance. Strong public support for compliance
drives these systems, which increases
elected officials' willingness to comply
[10]. This
study
presents
a
novel
hypothesis of the politicization of the
enforcement of human rights laws. The
study
demonstrates
that
nations
frequently attack their rivals on delicate
matters that threaten the authority and
viability of the target regime, while
conversing with friends about more
neutral subjects. The study demonstrates
that governments penalize human rights
infractions differently depending on their
perceived "sensitivity" towards the target
state by using data from the UN Universal
Periodic Review, a complex human rights
system [11]. The chances for a regional
East Asian human rights framework are
evaluated in this study. The theoretical
foundation
for
developing
regional
human rights regimes is examined, as is
the effect of globalization on human
rights, as well as an evaluation of other
regional human rights regimes and NGO
contributions
to
them. The
report
evaluates the past attempts to create
regional human rights structures in Asia,
the current state of human rights in East
Asia, and the status of human rights in
East Asian NGOs [12]. This
study
investigates
whether
national
legislatures
can
effectively
establish the legitimacy of international
human rights treaties. According to the
research,
those
who
exercise
the
parliamentary
veto
by
blocking
legislation raise the price of formalistic
repressive tactics. The use of extralegal
tactics by executives may become more
expensive as a result, reducing repression. This study demonstrates that the number
of legislative veto players has a favorable
impact on human rights treaties [14]. 2.2 Regional Human Rights Courts Regional human rights tribunals
protect civil and political rights in certain
regions. Examples are the AfCHPR,
IACtHR, and ECtHR. These courts allow
protests against governments that violate
civil and political rights. They also
interpret
regional
human
rights
legislation, set precedent, and rule. 2. LITERATURE REVIEW International
human
rights
organizations safeguard civil and political
rights
using
several
theoretical
underpinnings. Human rights are based on
the belief in each person's dignity and some
basic
freedoms. Cultural
relativism
emphasizes cultural diversity and local
context in defining and enforcing human One article [7] evaluates Indonesia's
progress on justice reform and human rights
since the fall of the Suharto administration in
1998, claiming that despite initial efforts to
incorporate transitional justice systems and
international human rights standards into Vol. 1, No. 03, June 2023: pp. 176 - 184 The Easta Journal Law and Human Rights (ESLHR) 178 The rhetoric of international human
rights in contrast to the reality of women's
rights In order to assess whether or if
international
human
rights
law
on
women's rights is effective in promoting
women on a global scale, the topic of this
article is investigated. The argument
made in this article is that despite the
rapid expansion of formal international
human rights law, there is still a
significant gap between the aspirational
wording of this legislation and its
insufficient ability to be put into practice
and enforced. This disparity between
rhetoric and reality demonstrates that
gender equality and rights cannot be
improved by the use of universalist legal
frameworks [13]. rights, while universalism emphasizes their
inherent and universal nature. 2.6 Gaps in the Existing Literature Experts
compared
international
human rights frameworks' civil and
political rights protection. Global human
rights protection requires international
human
rights
mechanisms. Treaties,
agreements, and international courts
promote human rights. A research
contrasted the Inter-American Court of
Human Rights, the European Court, and
the UN Human Rights Committee. Origins, goal, legal framework, and
efficiency were examined [15]. While there is a large body of
literature on international human rights
mechanisms, there are still gaps in
understanding
their
effectiveness
in
protecting civil and political rights. Some
areas that require further exploration
include the interaction and cooperation
between
different
mechanisms,
the
impact of regional court decisions on state
compliance, the effectiveness of remedies
provided by treaty bodies, and the
influence of political factors on the
functioning of these mechanisms. Another
study
looked
at
the
criminality of human trafficking as well as
the strategies for preventing, suppressing,
and combating it. The study found
legislative flaws in the systems used to
combat human trafficking, particularly
against people with impairments [16]. By
addressing
these
gaps,
this
research aims to contribute to the existing
literature and deepen our understanding
of the strengths and weaknesses of
international human rights mechanisms
in protecting civil and political rights. A research examined Poland and
Russia's COVID-19 pandemic human
rights. The
authors
contrasted
EU
legislation to those of non-EU nations,
which commonly deploy undemocratic
means of control [17]. 2.5 Comparative Studies on International
Human Rights Mechanisms 2.5 Comparative Studies on International
Human Rights Mechanisms
Experts
compared
international
human rights frameworks' civil and
political rights protection. Global human
rights protection requires international
human
rights
mechanisms. Treaties,
agreements, and international courts
promote human rights. A research
contrasted the Inter-American Court of
Human Rights, the European Court, and
the UN Human Rights Committee. Origins, goal, legal framework, and
efficiency were examined [15]. 2.4 Special Procedures 2.4 Special Procedures
The
UNHRC
selects
special
rapporteurs,
independent
experts,
working groups, and commissions of
inquiry to address thematic or country-
specific human rights issues. These
processes
conduct
investigations,
generate reports, and provide advice to
the state. They are critical in addressing
serious human rights challenges, raising
awareness, and advocating the defense of
civil and political rights. These works examine international
human rights systems such tribunals,
human
trafficking
criminalization,
human rights deterioration, and human
rights-based development. 2.3
Treaty Bodies The International Covenant on Civil
and Political Rights (ICCPR) and the
Convention against Torture (CAT) create
treaty bodies. These organizations are
made up of unbiased experts who
monitor how states adhere to their
respective treaty duties. They review
national reports, interact favorably with Vol. 1, No. 03, June 2023: pp. 176 - 184 179 The Easta Journal Law and Human Rights (ESLHR) nations, make recommendations, and
provide trustworthy interpretations of
treaty clauses. Finally,
a
study
contrasted
the
employment
of
human
rights-based
approaches (HRBA) in development by
international
non-governmental
organizations (INGOs) and government
agencies. The study looked into the cases
of ActionAid and Sida and discovered
that both have HRBAs in place but are
working to completely integrate them. In
practice, the biggest distinction is their
interaction with the government and the
poor [19]. 4. RESULTS AND DISCUSSION 4.1 The European Court of Human Rights
(ECtHR) 4.1 The European Court of Human Rights
(ECtHR) 3.3 Data Collection Methods The main data sources for this
research are academic literature, reports,
case studies, and official documents
related to international human rights
mechanisms. A comprehensive literature
review will be conducted to gather
existing knowledge and insights on the
subject. In addition, document analysis
will involve an examination of relevant
legal
instruments,
court
decisions,
reports, and recommendations issued by
regional human rights courts, treaty
bodies, and special procedures. The main data sources for this
research are academic literature, reports,
case studies, and official documents
related to international human rights
mechanisms. A comprehensive literature
review will be conducted to gather
existing knowledge and insights on the
subject. In addition, document analysis
will involve an examination of relevant
legal
instruments,
court
decisions,
reports, and recommendations issued by
regional human rights courts, treaty
bodies, and special procedures. 3.1 Comparative Analysis Framework This research compares international
human rights frameworks' ability to
protect civil and political rights. Regional
human rights tribunals, treaty bodies, and
special procedures are examined in this
study to assess their strengths and
limitations
and
shed light
on
the
challenges
and
opportunities
for
preserving these rights. Thematic analysis will be used to
identify
and
analyze
key
themes
emerging from the data. These themes
will be linked to evaluation criteria to
assess the strengths and weaknesses of
each mechanism. The findings will be
synthesized
and
presented
comprehensively to provide a deeper
understanding
of
the
role
and
effectiveness of the mechanisms. 3.2 Evaluation Criteria
Several civil and political rights-
related
factors
will
be
evaluated. Accessibility, independence, efficiency,
enforcement mechanisms, due process,
impact on civil and political rights, and
responsiveness to societal changes and
developing human rights concerns are
examples. These standards will evaluate
regional human rights courts, treaty
bodies, and special procedures. 4.1 The European Court of Human Rights
(ECtHR) The
European
Regional
Human
Rights Court (ECtHR) was formed under
the European Convention on Human
Rights
(ECHR). Council
of Europe
members elect court judges. The ECtHR
interprets and implements the ECHR and
ensures Member States comply. Regional human rights court historic
cases will be reviewed. These examples
will be chosen for their relevance to civil
and political rights and their potential to
reveal these systems' strengths and
limitations. ECtHR case law covers many civil
and
political
legal
concerns. The
European Court of Human Rights has
ruled on freedom of expression, fair trials,
torture, and minority rights. Case law
interprets her ECHR in an authoritative 3.5 Case Selection To conduct the comparative analysis,
a purposive sampling method will be
used to select relevant cases from regional
human rights courts. The selection will be
based on the importance of the cases in
addressing civil and political rights issues
and their potential to shed light on the
functioning of these mechanisms. A
variety of cases will be selected from
different
regions
to
ensure
a
comprehensive analysis. 3. METHOD This study uses a qualitative research
approach to conduct a comparative analysis
of international human rights mechanisms in
protecting
civil
and
political
rights. Qualitative
research
allows
in-depth
exploration of complex phenomena and
provides a rich understanding of the strengths
and weaknesses of these mechanisms. The
research design incorporates a comprehensive Another research examines Ukraine's
compliance of international human rights
abatement laws. The paper examines the
nature,
causes,
procedures,
idiosyncrasies, and issues of human rights
derogations [18]. Vol. 1, No. 03, June 2023: pp. 176 - 184 The Easta Journal Law and Human Rights (ESLHR) 180 literature review, document analysis, and case
law review as primary data sources [20]. analysis. This will involve coding and
categorizing the data to identify recurring
themes,
patterns,
and
trends. Comparative analysis techniques will be
used
to
examine
similarities
and
differences among international human
rights mechanisms in terms of their
effectiveness in protecting civil and
political rights. 3.1 Comparative Analysis Framework 4.2 Inter-American Court of Human Rights
(IACHR) 4.2 Inter-American Court of Human Rights
(IACHR) The American Convention on Human
Rights created the Inter-American Court
of Human Rights (IACtHR). It enforces
and interprets American human rights
laws. Judges
are
elected
by
OAS
members. The AfCHPR has promoted and
protected African civil and political
rights. Its rulings have changed laws and
policies, improving human rights across
the continent. The court's decisions have
allowed civil society organizations and
individuals to seek justice for human
rights breaches. Human rights in the Americas have
been shaped by IACtHR rulings. The
IACtHR
has
addressed
enforced
disappearances, extrajudicial murders,
indigenous rights, and free speech. The
court's case law interprets the American
Convention and helps member nations
protect civil and political rights. However, the effectiveness of the
AfCHPR faces various challenges. The
court's
limited
awareness
and
accessibility
among
the
African
population, as well as limited resources
and
jurisdictional
constraints,
may
hamper its impact. The court's reliance on
state referrals and individual petitions
may also limit its capacity to effectively
address human rights issues. The IACtHR has promoted and
protected
civil
and
political
rights
throughout the Americas. Its verdicts
have led to substantial legal reforms, truth
commissions, and reparations for human
rights breaches. The court's rulings have
also affected member states' legal systems
and policies. 3.4 Data Analysis Methods Data collected through the literature
review, document analysis, and case law
review will undergo qualitative data Vol. 1, No. 03, June 2023: pp. 176 - 184 The Easta Journal Law and Human Rights (ESLHR) 181 way, serving as a blueprint for member
states' national legal systems. way, serving as a blueprint for member
states' national legal systems. However,
the
IACtHR
faces
challenges in terms of its effectiveness. Some member states are reluctant to fully
comply with the tribunal's judgments,
leading to delays in implementing the
judgments and ensuring redress for
victims. The limited resources of the court
and the geographical diversity of the
region pose additional challenges to its
functioning. In Europe, the ECtHR has played a
crucial role in defending civil and political
rights. In order to ensure compliance with
the ECHR, its judgements have prompted
considerable legal and policy reforms in
member nations. The judgements of the
court have also helped to shape regional
norms and standards for human rights. 4.3 African Court on Human and Peoples'
Rights (AfCHPR) 4.3 African Court on Human and Peoples'
Rights (AfCHPR) The ECtHR does, however, face a
number of obstacles to its functioning. Concerns have been made about timely
access to justice due to the backlog of
cases and the length of the legal processes. ECtHR
rulings
have
not
all
been
implemented equally by member states,
and some have had trouble properly
adhering
to
the
court's
decisions. Additionally, because of its reliance on
individual petitions, the ECtHR may not
be able to adequately address systemic
human rights problems. The ECtHR does, however, face a
number of obstacles to its functioning. Concerns have been made about timely
access to justice due to the backlog of
cases and the length of the legal processes. ECtHR
rulings
have
not
all
been
implemented equally by member states,
and some have had trouble properly
adhering
to
the
court's
decisions. Additionally, because of its reliance on
individual petitions, the ECtHR may not
be able to adequately address systemic
human rights problems. Africa's regional human rights court
is AfCHPR. The Court hears African
human rights cases under the African
Charter on Human Rights and Peoples'
Rights. The tribunal has African Union-
elected judges. Africa's civil and political rights are
covered by the AfCHPR's expanding case
law. The court has ruled on freedom of
expression, fair trials, women's rights,
and indigenous rights. The court's case
law guides member states' human rights
enforcement. Vol. 1, No. 03, June 2023: pp. 176 - 184 4.4 Comparative Assessment These regional human rights courts
perform similarly and differently. All
three courts interpret regional human
rights instruments, produce case law, and Vol. 1, No. 03, June 2023: pp. 176 - 184 The Easta Journal Law and Human Rights (ESLHR) 182 182 and improvements. The HRC receives
individual complaints alleging ICCPR
violations. and improvements. The HRC receives
individual complaints alleging ICCPR
violations. enforce human rights norms. They have
affected
domestic
legal
systems,
redressed human rights violations, and
reformed laws. 4.6 Impact and Challenges However,
the
tribunals
face
challenges, including a backlog of cases,
delays
in
implementing
judgments,
uneven compliance from member states,
and limited resources and jurisdiction. To
overcome these challenges, continued
efforts are needed to improve the
efficiency, accessibility, and effectiveness
of regional human rights courts. The HRC's recommendations and
interpretations
of
the
ICCPR
have
influenced domestic legal systems and
policies in states parties. The committee's
jurisprudence has contributed to the
development of international human
rights law, particularly in areas such as
freedom of expression, the right to a fair
trial, and protection against torture. This comparative analysis provides
insight into the strengths and weaknesses
of these mechanisms, which can inform
discussions on potential reforms and
improvements. By
considering
the
experiences and lessons learned from
regional
human
rights
courts,
policymakers,
human
rights
organizations, and civil society can work
to strengthen the protection of civil and
political rights at both the regional and
national levels. However, the HRC faces challenges
in terms of its effectiveness. Backlogs of
state reports, limited resources, and
varying levels of state compliance can
affect the committee's ability to effectively
monitor and address civil and political
rights
violations. The
HRC's
recommendations are non-binding, and
enforcement mechanisms depend on the
political will of states parties. In addition to the ICCPR, other treaty
bodies also oversee the implementation of
human rights treaties relevant to civil and
political rights. These include: Treaty bodies are committees of
experts
established
under
specific
international human rights treaties. They
play an important role in monitoring state
compliance
with
treaty
obligations
relating to civil and political rights. Treaty
bodies are composed of independent
experts who review state reports, engage
in
dialog
with
states,
issue
recommendations,
and
provide
authoritative interpretations of treaty
provisions. The Convention against Torture and
Other Cruel, Inhuman and Degrading
Treatment or Punishment (CAT). The
Committee against Torture monitors state
compliance with CAT. It reviews country
reports, engages in dialog with states, and
investigates allegations of torture or ill-
treatment. The
Committee
issues
recommendations
and
may
conduct
investigations in cases of systematic or
gross violations. 5. CONCLUSION The comparative analysis shows that
regional human rights courts and treaty
bodies have different but complementary
roles in protecting civil and political rights. Regional
courts
provide
adjudicative
functions, issuing binding decisions and
orders, while treaty bodies offer monitoring
and
reporting
functions,
providing
recommendations and guidance to states
parties. 4.5 International Covenant on Civil and
Political Rights (ICCPR) 1. Convention on the Elimination of
All
Forms
of
Racial
Discrimination (CERD) The ICCPR is a key international
human rights treaty. The HRC monitors
its
implementation. Independent
specialists analyze state reports and
discuss state parties with the HRC. The
Committee
on
the
Elimination of Racial Discrimination
monitors
state
compliance
with
CERD. It reviews state reports and
issues recommendations to combat
racial discrimination. It also considers The HRC requires ICCPR states to
report on their implementation efforts. The HRC reviews these reports and
makes conclusions on progress, concerns, Vol. 1, No. 03, June 2023: pp. 176 - 184 183 The Easta Journal Law and Human Rights (ESLHR) individual
complaints
alleging
violations of the convention. Efforts to strengthen treaty bodies
include
improving
reporting
mechanisms,
addressing
resource
limitations, and encouraging states to
implement recommendations. Regular
reviews
of
the
functioning
and
effectiveness of treaty bodies can identify
areas for improvement and promote the
protection of civil and political rights at
the international and national levels. 2. Convention on the Rights of the
Child (CRC) 2. Convention on the Rights of the
Child (CRC) The Committee on the Rights of
the Child monitors state compliance
with the CRC. It reviews country
reports, issues recommendations on
child rights issues, and considers
individual complaints relating to
child rights violations. Overall,
treaty
bodies
play
an
important
role
in
advancing
and
protecting civil and political rights, and
their work is critical to advancing human
rights. 3. Convention on the Elimination of
All
Forms
of Discrimination
against Women (CEDAW) The
Committee
on
the
Elimination
of
Discrimination
Against Women (CEDAW) oversees
state conformity with the Convention
on the Elimination of All Forms of
Discrimination Against Women. It
examines
nation
reports,
makes
recommendations to improve gender
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M. Kim, “Public and elite opinion on international human rights law: Completing the causal chain of the
domestic compliance mechanism,” J. Hum. Rights, vol. 18, no. 4, pp. 419–438, Aug. 2019, doi:
10.1080/14754835.2019.1629891. [11]
R. TERMAN and J. BYUN, “Punishment and Politicization in the International Human Rights Regime,”
Am. Polit. Sci. Rev., vol. 116, no. 2, pp. 385–402, 2022, doi: DOI: 10.1017/S0003055421001167. [12]
H. Hashimoto, The Prospects for a Regional Human Rights Mechanism in East Asia (RLE Modern East and
South East Asia). 2015. doi: 10.4324/9781315697796. [13]
D. L. DeLaet, “Lost in legation: the gap between rhetoric and reality in international human rights law
governing women’s rights,” Glob. Discourse, vol. 8, no. 3, pp. 387–404, Jul. 2018, doi:
10.1080/23269995.2018.1520006. The Easta Journal Law and Human Rights (ESLHR) The Easta Journal Law and Human Rights (ESLHR) compliance, and ensuring accountability for
human rights violations at both the regional
and international levels. Overall, the combination of regional
human rights courts and treaty bodies
provides a comprehensive framework for
protecting civil and political rights, promoting 4.7 Comparative Assessment Both mechanisms have strengths and
weaknesses. Regional
courts
have
the
advantage of binding decisions and the
potential to have a direct impact on domestic
law and policy. However, they may face
challenges relating to access, resources and
varying levels of state compliance. On the
other hand, treaty bodies have a wider reach
through their periodic reviews and the ability
to consider individual complaints. However,
their recommendations are non-binding,
dependent
on
state
cooperation
and
implementation. Treaty bodies play an important role
in monitoring and promoting civil and
political rights globally. Each treaty body
has its own mandate, reporting processes,
and review mechanisms. Although they
operate under different treaties, they
share the common goal of protecting civil
and political rights and ensuring state
compliance. A comparative analysis of the treaty
bodies reveals strengths and challenges. Their
recommendations
and
interpretations
contribute
to
the
development of international human
rights law, influence domestic legal
systems, and empower civil society
organizations and individuals. However,
challenges such as a backlog of reports,
limited resources, and varying levels of
state
compliance
impact
their
effectiveness. To improve the effectiveness of these
mechanisms, efforts should be focused on
addressing challenges such as case backlogs,
processing delays, resource limitations, and
uneven
state
compliance. Strengthening
cooperation between regional courts and
treaty bodies can also facilitate harmonization
and consistent interpretation of human rights
standards. Vol. 1, No. 03, June 2023: pp. 176 - 184 184 REFERENCE [14]
Y. Lupu, “Legislative Veto Players and the Effects of International Human Rights Agreements,” Am. J. Pol. Sci., vol. 59, no. 3, pp. 578–594, Jun. 2015, [Online]. Available: http://www.jstor.org/stable/24583084 [15]
R. David, “Comparative Study of Three International Human Rights Systems and Their Enforcement
Mechanisms,” SSRN Electron. J., 2012, doi: 10.2139/ssrn.1566495. 6]
L. A. Abu Dalu, “The crime of human trafficking against persons with disabilities. A comparative study
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M. Pietras-Eichberger, “Human rights restrictions during the COVID-19 pandemic in Poland and Russia. Legal and comparative aspects,” Gubernaculum Adm., vol. 2(24), pp. 323–340, Jan. 2021, doi:
10.16926/gea.2021.02.35. g
[18]
V. Denysov and L. Falalieieva, “Derogation in the Field of Human Rights: International Legal
Instruments and Practice of Ukraine,” Int. Comp. Law Rev., vol. 20, no. 2, pp. 7–37, 2020, doi:
doi:10.2478/iclr-2020-0016. [19]
L. Tolander, “A Human Rights-Based Approach to Development-from Policy to Practice A Comparative
Study between ActionAid and Sida,” 2013. [20]
J. W. Creswell, “Research Desain: Pendekatan Kualitatif, Kualitatif, Dan Mixed (Edisi Keti).” Yogyakarta,
2013. Vol. 1, No. 03, June 2023: pp. 176 - 184
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A Study of Industry-university-institute Cooperative Education in Colleges and Universities against the Background of Emerging Engineering Education
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Emerging Engineering Education
Universities
against
the
Background
of
Cooperative
Education
in
Colleges
and
A
Study
of
Industry-university-institute 5NingboTech University, Ningbo 315100, China
4NingboTech University, Ningbo 315100, China
3NingboTech University, Ningbo 315100, China
2Zhejiang Post And Telecommunication College, Shaoxing, China
1NingboTech University, Ningbo 315100, China Abstract. With emerging engineering education becoming a new strategic
direction of the higher engineering education reform in China, it is an
important issue faced by colleges and universities to comprehensively
improve their abilities of training talent, conducting scientific research and
serving the society. Promoting industry-university-institute cooperation is a
key measure for colleges and universities to keep up with the pace of
higher education and socio-economic development. Colleges and
universities need to improve the industry-university-institute cooperative
talent training mechanism, and establish an industry-university-institute
cooperative education system based on public technology service platforms,
to promote the combination of technology and production through
cooperative education based on their current situation of research, push
forward the supply side reform of higher education, and provide human
resources, technical support and industrial services for social development
against the background of emerging engineering education. While
enhancing their levels of scientific research and education, colleges and
universities can promote social progress and help enterprises create
economic benefits, to achieve win-win cooperation with all relevant parties
in the society. Keywords:
Industry-University-Institute
cooperative,
Emerging
Engineering Education, Education system Keywords:
Industry-University-Institute
cooperative,
Emerging
Engineering Education, Education system Keywords:
Industry-University-Institute
cooperative,
Emerging
Engineering Education, Education system * Corresponding author: fyz@zptc.cn SHS Web of Conferences 96, 03001 (2021)
IAFSM 2020 SHS Web of Conferences 96, 03001 (2021)
IAFSM 2020 https://doi.org/10.1051/shsconf/20219603001 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction Based on the background of emerging engineering education, and in order
to adapt to the development of the times, actively integrate into the driving force of social
innovation and industrial transformation and upgrading, further promote the structural
reform on the supply side of education, and strengthen the cultivation of students' basic
theory, innovation spirit and practical ability, universities need to carry out industry
university research cooperation with enterprises. It has become the consensus of the whole
society to accelerate the construction and development of emerging engineering education,
cultivate talents in urgent need of the new economy, and cultivate talents who will lead the
development of technology and industry in the future [2]. At the same time, the
construction of emerging engineering education brings opportunities and new challenges to
the upgrading of the cooperation of production, learning and research in local universities. The industry-university-institute cooperation education is the only way for the development
of high-level universities and first-class universities. Because of the continuous innovation
of the practice of industry-university-research cooperation education, universities strive to
set up a platform for industry university research cooperation education, scientific research
and technical service, which provides strong talent support and scientific and technological
services for the local economic development and industrial technological progress [3]. Fig. 1. Industry-university-institute cooperation. 2 Improve the industry-university-institute cooperative talent
training mechanism
The guidelines for the construction of emerging engineering education clearly put forward
that it is necessary to ask for the construction of industries, update the traditional disciplines,
and promote the cross combination of existing engineering disciplines; it is necessary to ask
for the content of technological development and reform, and update the knowledge system
of engineering talents. In order to meet the needs of social development and solve the
problem of disconnection between talent training, economy and science and technology, it Fig. 1. Industry-university-institute cooperation. Fig. 1. Industry-university-institute cooperation. 1 Introduction In order to cultivate high-quality composite talents with innovation ability, engineering
practice ability and international competitiveness that meet the needs of emerging industries
and economies, the government has proposed an important measure for the upgrading and
economic transformation of the Emerging Engineering Education construction. The system © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). SHS Web of Conferences 96, 03001 (2021)
IAFSM 2020 https://doi.org/10.1051/shsconf/20219603001 of industry-university-institute cooperation is a complex structure system with the
participation of enterprises, universities, scientific research institutes and social service
institutions [1]. Based on the background of emerging engineering education, and in order
to adapt to the development of the times, actively integrate into the driving force of social
innovation and industrial transformation and upgrading, further promote the structural
reform on the supply side of education, and strengthen the cultivation of students' basic
theory, innovation spirit and practical ability, universities need to carry out industry
university research cooperation with enterprises. It has become the consensus of the whole
society to accelerate the construction and development of emerging engineering education,
cultivate talents in urgent need of the new economy, and cultivate talents who will lead the
development of technology and industry in the future [2]. At the same time, the
construction of emerging engineering education brings opportunities and new challenges to
the upgrading of the cooperation of production, learning and research in local universities. The industry-university-institute cooperation education is the only way for the development
of high-level universities and first-class universities. Because of the continuous innovation
of the practice of industry-university-research cooperation education, universities strive to
set up a platform for industry university research cooperation education, scientific research
and technical service, which provides strong talent support and scientific and technological
services for the local economic development and industrial technological progress [3]. of industry-university-institute cooperation is a complex structure system with the
participation of enterprises, universities, scientific research institutes and social service
institutions [1]. 2 Improve the industry-university-institute cooperative talent
training mechanism The guidelines for the construction of emerging engineering education clearly put forward
that it is necessary to ask for the construction of industries, update the traditional disciplines,
and promote the cross combination of existing engineering disciplines; it is necessary to ask
for the content of technological development and reform, and update the knowledge system
of engineering talents. In order to meet the needs of social development and solve the
problem of disconnection between talent training, economy and science and technology, it 2 2 SHS Web of Conferences 96, 03001 (2021)
IAFSM 2020 https://doi.org/10.1051/shsconf/20219603001 is urgent to innovate and reform the talent training mode in universities. The cooperative
education mode pays attention to the relationship among production, research and learning,
which not only increases the cooperation among the three, but also optimizes the function
of talent training in universities. is urgent to innovate and reform the talent training mode in universities. The cooperative
education mode pays attention to the relationship among production, research and learning,
which not only increases the cooperation among the three, but also optimizes the function
of talent training in universities. The defect of talent training mode is a great resistance to the development of production,
research and learning[4]. Although the education reform has been highly valued by the
society, the current part of colleges and universities still follow the pattern of the traditional
teaching mode, which is embodied in the emphasis on knowledge teaching and the neglect
of the practice link. The students cultivated are difficult to have the ability to solve complex
problems [5,6]. The main purpose of professional learning is to complete classroom
assignments or experiments, which leads to students' lack of ability to think and explore. The domestic industry-university-institute cooperative model started relatively late, and
colleges and universities can rely on industry-university-institute cooperative to strengthen
students' corresponding practical and innovative capabilities. Although the industry-
university-institute cooperative model has gradually risen to the national level, it is still not
clear how to develop talents based on the actual situation of universities. Colleges and
universities should take social needs and professional characteristics as the guidance to set
up talent training objectives, so as to improve the fit between talent training objectives and
social needs. In this way, we can take full advantages of collaborative innovation of
production, learning and research, and construct the training mode of excellent talents. 2 Improve the industry-university-institute cooperative talent
training mechanism The
university should pay attention to the cooperative training of students' basic abilities and
professional skills, so as to promote the development of talents towards the application-
oriented direction. 2.1 Course teaching Colleges and universities should not only attach importance to professional knowledge and
basic knowledge, but also consider the necessary skills needed by society and employers. Teaching should be carried out with the ability of scientific and technological innovation as
the core, which needs to reflect the epochal character of teaching. The society needs
colleges and universities to develop an open, advanced and practical curriculum system of
collaborative education. In addition, universities should also improve the advanced nature
and comprehensiveness of the courses from the elective courses, effectively expand their
horizons and cultivate their independent thinking and work ability. The construction of
theoretical curriculum system highlighting the cultivation of innovation ability, while
making full use of the technical force of front-line production units, introducing their new
technology and new equipment in the classroom, to ensure that the content of the
curriculum keeps pace with the times. 2.2 Scientific research training Teachers should bring advanced research methods and analysis methods into the curriculum
system, promote the integration of analysis tools and applications, and ensure that students
can choose matching coping styles when facing different problems. Students should be
encouraged to actively participate in the project discussion and contact the advanced
research platform as much as possible, so as to master the advanced scientific research
means and improve their scientific research literacy. Establish a new system of senior with
junior, let senior students bring junior students into the scientific research work together,
junior students preliminarily master the scientific research analysis methods and test
methods in the learning process, senior students improve their scientific research ability and
stimulate scientific research innovation in the teaching process. 3 3 https://doi.org/10.1051/shsconf/20219603001 SHS Web of Conferences 96, 03001 (2021)
IAFSM 2020 SHS Web of Conferences 96, 03001 (2021) 2.3 Innovative practice The construction of the practical curriculum system that emphasizes the cultivation of
practical ability. The biggest difference between emerging engineering education mode and
traditional teaching mode is that it pays more attention to the cultivation of students'
practical ability [7-8]. The cooperative education mode of industry-university-institute
should be used to guide students to participate in project development and production
practice for a long time, so as to ensure that they are familiar with the corresponding
production process and application needs. Then relying on the guidance of the tutors inside
and outside the school, let students clear the direction of the subject, and improve their
innovation practice ability. Universities should strengthen the integration of production and
learning, make full use of the practice platform of enterprises and institutions, let students
learn the required knowledge in the real entrepreneurship and innovation work, make
students feel the "real scene" of the work, effectively improve students' lack of
understanding of society and occupation, and minimize the employment adaptation period
of students[9]. 4 Conclusion The construction of emerging engineering education is still making progress, when colleges
and universities should ascertain the status quo of emerging engineering education
construction and industry-university-institute cooperation, figure out problems in existing
industry-university-institute cooperation among all parties, and present new paths for this
cooperation, thereby promoting the reform of the industry-university-institute cooperative
education model. Through close cooperation among universities, enterprises and
governments, we can improve the teaching system, improve the quality of personnel
training, and promote the transformation of scientific research results into productivity. Pay
attention to the cultivation of individual ability of students, in order to achieve the purpose
of individualized development and comprehensive development of students. Eventually,
achieve win-win cooperation with all relevant parties in the society. Acknowledgement This research was financially supported by the Zhejiang Province Higher Education "13th
Five-year" Teaching Reform Research Project (jg20180442). 3 Establish
an
industry-university-institute
cooperative
education system based on public technology service platforms University Science Park is an important medium of university technology transfer. Local
colleges and universities need to constantly improve the corresponding school enterprise
cooperation mechanism in combination with the characteristics of colleges and enterprises,
and promote the construction of industry university research cooperation. For school
enterprise cooperation, it is necessary to select a matching cooperation mode according to
the enterprise situation. Both sides need to clarify the core areas and cooperation
mechanism of cooperation, and regularly organize exchange meetings. Colleges and
universities can set up off campus practice bases with the help of school enterprise
cooperation mode, so that students can go deep into the actual production links of
enterprises, clarify the application needs of enterprises, so as to formulate corresponding
research directions, and actively promote the transformation of scientific research results
into productivity. Universities should build and utilize the technology service platform of
academic research cooperation to continuously improve their social service ability. The school uses the technical strength of enterprises and large-scale instrument test
equipment, according to the needs of the core technology research in the real society, and
under the cooperation of the government, schools and enterprises, establishes a public
technical service platform with complete functions. In the science and technology park,
vigorously carry out technical consultation, technical services, technological development
and technology transfer. Which will promote the trade, promotion and diffusion of
technological achievements and intellectual property rights, and promote technology
transfer and transformation of achievements. Based on the public technology service
platform, students can participate in relevant work as much as possible, so that students in
school can understand the needs of enterprise work, social science and technology
development and enterprise engineering. The each major should also establish a dual tutor
guidance mechanism, which can employ excellent enterprise talents as external tutors, and
establish a matching training program combining with students' cognition and thinking
mode. The internal and external tutors should jointly undertake the teaching task. 4 https://doi.org/10.1051/shsconf/20219603001 SHS Web of Conferences 96, 03001 (2021)
IAFSM 2020 SHS Web of Conferences 96, 03001 (2021) Fig. 2. Functions of technical service platform. Fig. 2. Functions of technical service platform. References 1. M Zhu, C Chang, L Yu, Research on construction of harmony evaluation index system
of industry-university-research cooperation system, Journal of Shenyang University of
Technology. 12, 67-72, (2019) 1. M Zhu, C Chang, L Yu, Research on construction of harmony evaluation index system
of industry-university-research cooperation system, Journal of Shenyang University of
Technology. 12, 67-72, (2019) 1. M Zhu, C Chang, L Yu, Research on construction of harmony evaluation index system
of industry-university-research cooperation system, Journal of Shenyang University of
Technology. 12, 67-72, (2019) 2. S Gao, Implement the emerging engineering education F-plan and cultivate
engineering leaders, Research in Higher Education of Engineering. 04, 19-25, (2019) 3. W Liu, M Chen, X Zheng, Research and Practice of the industry-university-institute
cooperative in the New Era, Chinese University Technology Transfer. 12, 80-82, (2019) 4. M Gao, Q Wang, C Liu, Teaching reform and exploration of PLC technology and
application course of applied undergraduate colleges under the background of 4. M Gao, Q Wang, C Liu, Teaching reform and exploration of PLC technology and
application course of applied undergraduate colleges under the background of 5 https://doi.org/10.1051/shsconf/20219603001 SHS Web of Conferences 96, 03001 (2021)
IAFSM 2020 emerging engineering education, Journal of Langfang Normal University. 18, 124-128,
(2018) emerging engineering education, Journal of Langfang Normal University. 18, 124-128,
(2018) emerging engineering education, Journal of Langfang Normal University. 18, 124-128,
(2018) 5. X Chen, W Luo, S Luo, Research on the upgrading of the emerging engineering
education under the background of emerging engineering education, Education
Modernization. 06, 7-8, (2019) 6. J Tan, L Hua, Improvement and upgrading of emerging engineering education in local
universities under the background of emerging engineering education, Industrial
Innovation. 10, 238-244, (2019) 7. C Xiu, B Ma, Z Xu, Reform and Practice of Production-University Integration and
Cooperative Education Model for Surveying and Mapping Engineering Specialty
under the Background of New Engineering, Education Teaching Forum. 49, 85-88
(2019) 8. J Luo, X Li, Y Xi, X Li. Exploration and practice of innovation and entrepreneurship
education reform, Beijing Education: Higher Education Edition, 10, 37-39, (2015). 9. Z Wang, Y Shan, S Wang, Constructing a New System of " Five in One " Innovation
and Entrepreneurship Education - - Exploration and Practice of Innovation and
Entrepreneurship Education Reform in Inner Mongolia University, teaching of
university of china, 06, 29-32, (2017) 6 6
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First inverse kinematics measurement of key resonances in the 22Ne(p,γ)23Na reaction at stellar temperatures
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First inverse kinematics measurement of key resonances in the
22Ne(p, γ )23Na reaction at stellar temperatures A. Lennarz a,∗, M. Williams a,b, A.M. Laird b,1, U. Battino c,1, A.A. Chen d, D. Connolly a,2,
B. Davids a, N. Esker a,3, R. Garg b,4, M. Gay e, U. Greife f, U. Hager g, D. Hutcheon a, J. José h,
M. Lovely f, S. Lyons g,i, A. Psaltis d,1, J.E. Riley b, A. Tattersall c, C. Ruiz a a TRIUMF, 4004 Wesbrook Mall, Vancouver, BC, V6T 2A3, Canada a TRIUMF, 4004 Wesbrook Mall, Vancouver, BC, V6T 2A3, Canada
b Department of Physics, University of York, Heslington, York, YO10 5DD, UK
c University of Edinburgh, School of Physics and Astrophysics, Edinburgh EH9 3FD, UK
d Department of Physics and Astronomy, McMaster University, Hamilton, ON, L8S 4L8, Canada
e Columbia University, 116th St & Broadway, New York, NY 10027, USA
f Colorado School of Mines, Golden, CO, USA
g National Superconducting Cyclotron Laboratory, Michigan State University, East Lansing, MI 4882
h Departament de Física, Universitat Politècnica de Catalunya & Institut d’Estudis Espacials de Cata
Nexus-201 C Gran Capità 2-4 E-08034 Barcelona Spain a TRIUMF, 4004 Wesbrook Mall, Vancouver, BC, V6T 2A3, Canada
b Department of Physics, University of York, Heslington, York, YO10 5DD, UK
c University of Edinburgh, School of Physics and Astrophysics, Edinburgh EH9 3FD, UK
d Department of Physics and Astronomy, McMaster University, Hamilton, ON, L8S 4L8, Canada
e Columbia University, 116th St & Broadway, New York, NY 10027, USA
f Colorado School of Mines, Golden, CO, USA f
g National Superconducting Cyclotron Laboratory, Michigan State University, East Lansing, MI 48824, USA
h g National Superconducting Cyclotron Laboratory, Michigan State University, East Lansing, MI 48824, USA
h Departament de Física, Universitat Politècnica de Catalunya & Institut d’Estudis Espacials de Catalunya (
N
201 C G
C
ità 2 4 E 08034 B
l
S
i g National Superconducting Cyclotron Laboratory, Michigan State University, East Lansing, MI 48824, USA
h Departament de Física, Universitat Politècnica de Catalunya & Institut d’Estudis Espacials de Catalunya (IEEC), C. Eduard Mari
Nexus-201, C. Gran Capità, 2-4, E-08034, Barcelona, Spain h Departament de Física, Universitat Politècnica de Catalunya & Institut d’Estudis Espacials de Catalunya (IEEC), C. Eduard Maristany 10, E-080
Nexus-201, C. Gran Capità, 2-4, E-08034, Barcelona, Spain p
p
i The Joint Institute for Nuclear Astrophysics–Center for the Evolution of the Elements, Michigan State University, East Lansing, MI 48824, USA a b s t r a c t Article history:
Received 27 February 2020
Received in revised form 25 May 2020
Accepted 3 June 2020
Available online 5 June 2020
Editor: W. Haxton
Keywords:
Inverse kinematics measurements
Radiative capture reactions
Stellar nucleosynthesis Article history:
Received 27 February 2020
Received in revised form 25 May 2020
Accepted 3 June 2020
Available online 5 June 2020
Editor: W. Haxton
Keywords:
Inverse kinematics measurements
Radiative capture reactions
Stellar nucleosynthesis Article history:
Received 27 February 2020
Received in revised form 25 May 2020
Accepted 3 June 2020
Available online 5 June 2020
Editor: W. Haxton In this Letter we report on the first inverse kinematics measurement of key resonances in the
22Ne(p, γ )23Na reaction which forms part of the NeNa cycle, and is relevant for 23Na synthesis in
asymptotic giant branch (AGB) stars. An anti-correlation in O and Na abundances is seen across all
well-studied globular clusters (GC), however, reaction-rate uncertainties limit the precision as to which
stellar evolution models can reproduce the observed isotopic abundance patterns. Given the importance
of GC observations in testing stellar evolution models and their dependence on NeNa reaction rates, it
is critical that the nuclear physics uncertainties on the origin of 23Na be addressed. We present results
of direct strengths measurements of four key resonances in 22Ne(p, γ )23Na at Ec.m. = 149 keV, 181 keV,
248 keV and 458 keV. The strength of the important Ec.m. = 458 keV reference resonance has been
determined independently of other resonance strengths for the first time with an associated strength
of ωγ = 0.439(22) eV and with higher precision than previously reported. Our result deviates from the
two most recently published results obtained from normal kinematics measurements performed by the
LENA and LUNA collaborations but is in agreement with earlier measurements. The impact of our rate
on the Na-pocket formation in AGB stars and its relation to the O-Na anti-correlation was assessed via
network calculations. Further, the effect on isotopic abundances in CO and ONe novae ejecta with respect
to pre-solar grains was investigated. © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. Contents lists available at ScienceDirect Contents lists available at ScienceDirect E-mail address: lennarz@triumf.ca (A. Lennarz).
1 * Corresponding author. shed by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by Physics Letters B 807 (2020) 135539 Physics Letters B 807 (2020) 135539 * Corresponding author.
E-mail address: lennarz@triumf.ca (A. Lennarz).
1 The NuGrid collaboration, http://www.nugridstars .org.
2 Present address: Los Alamos National Laboratory, Los Alamos, New Mexico
87545, USA.
3 Present address: San José State University, 1 Washington Square, Duncan Hall
518 San José, CA 95192-0101.
4 Present address: University of Edinburgh, School of Physics and Astrophysics,
Edinburgh EH9 3FD, UK. 1 The NuGrid collaboration, http://www.nugridstars .org. 2. Experimental details The recoil-
detection system consisted of a double-sided silicon strip detector
(DSSSD) [21,22]. (
) [
]
A high intensity (∼2 × 1012 ions/sec) isotopically pure 22Ne4+
beam was delivered to the hydrogen-filled gas target. 23Na recoils
were transmitted through the separator and detected in the DSSSD. To contain the entire yield profile of the resonances within the
target, an average gas pressure of 5 Torr was used (∼3.9×1018 hy-
drogen atoms/cm2). The maximum charge state was selected by
transmitting the beam through the magnetic dipoles. Equilibrium
charge-state distributions for 23Na ions in hydrogen were mea-
sured at recoil energies to eliminate systematic uncertainties asso-
ciated with semi-empirical calculations. Two silicon surface barrier
detectors positioned at 30◦and 57◦relative to the beam axis in-
side the target detected elastically scattered protons for a relative
measure of the beam intensity. The elastic scattering rate was nor-
malized to automated hourly Faraday Cup readings. The energy
loss across the target was determined by measuring the incom-
ing and outgoing beam energy via the magnetic field of the first
magnetic dipole, which centered the beam on-axis. The incoming
beam-energy spread was ∼0.1% FWHM [23]. Stopping powers were
calculated based on the energy loss, the gas density derived from
continuously recorded pressure and temperature, and the effective
target length [21]. This reduces uncertainties induced by the com-
monly used software packages SRIM [24] and LISE [25]. The beam
heating effect on the measured yield under the experimental con-
ditions, i.e., beam powers, beam energies and gas pressures given
in this work has been found to be negligible with a dissipated
power of ∼0.12 Watts across the target and an average heating
of ∼0.18 to ∼0.24 K. For further details on the effect of intense
ion beams on gas target densities we refer to Ref. [26]. Resonance
energies were determined via the position sensitive BGO array by
relating the centroid of the distribution (γ yield vs target position)
to the incoming and outgoing beam energy [23]. [
]
In recent years the 22Ne(p, γ )23Na reaction has been targeted
intensively at three facilities, all employing normal kinematics
techniques [11–14]. The low-energy regime was investigated by
the LUNA and LENA collaborations, since the rate is dominated
by narrow low-energy resonances. With the exception of the low-
energy resonance strength measurements by LUNA [13,14] with
Ec.m. 2. Experimental details variability in observed abundances, some ubiquitous trends be-
come apparent, such as the anti-correlation in oxygen and sodium
abundances [3]. Currently stellar models are unable to reproduce
many of the abundance patterns present in GC stars along the
red-giant branch (RGB), but absent in their field star counter-
parts [2,4,5]. AGB stars undergoing Hot Bottom Burning (HBB) are
currently the most favored astrophysical sites to explain the O-
Na anti-correlation [6,7]. HBB occurs during the quiescent phase
between two thermal pulses (TP) when part of the H-shell is in-
cluded in the envelope convection and the H-shell has enhanced
access to fuel which is convectively mixed into its outer layers. In
TP-AGB stars, sodium is primarily synthesized by proton-capture
on 22Ne in the outer-most layer of the core-envelope transition
zone, resulting in the formation of a so-called 23Na pocket [8,9]. This pocket forms when 22Ne and 12C abundances are compara-
ble, and the 22Ne(p, γ )23Na and 12C(p, γ )13N reactions compete. In low-mass AGB stars, at solar metallicity, models predict the 23Na
pocket to be the main sodium source, and the overproduction of
sodium to result from the ingestion of the 23Na pocket during
the thermal dredge up [8]. The 22Ne(p, γ )23Na reaction further af-
fects the 20Ne/22Ne, 21Ne/22Ne and 20Ne/21Ne abundance ratios of
pre-solar grains found in meteorites. These grains are important
signatures of nucleosynthesis in different stellar environments and
mixing in stellar ejecta before the formation of our solar system. The 22Ne(p, γ )23Na reaction is also influential in nova nucleosyn-
thesis as has been identified by a sensitivity study by Iliadis et
al., showing that nuclear uncertainties associated with this reac-
tion rate can significantly influence the final abundances of 22Ne
and 23Na [10]. 22
23 The measurement was performed using the DRAGON (Detec-
tor of Recoil and Gammas Of Nuclear reactions) recoil separa-
tor [21] at the ISAC beam facility at TRIUMF, Vancouver, Canada. DRAGON is designed to conduct studies of radiative capture reac-
tions in inverse kinematics and consists of: (1) a windowless, dif-
ferentially pumped, recirculated gas target surrounded by a high-
efficiency γ -detector array consisting of 30 BGO detectors; (2) a
high-suppression electromagnetic mass separator with two stages
of charge and mass selection; (3) a variable heavy ion detection
system in combination with two micro-channel plate (MCP) based
timing detectors for time-of-flight (TOF) measurements. 1. Introduction Globular clusters (GCs) are dense aggregates of predominantly
old stars found in the galactic halo and have long fascinated as-
tronomers for the unique insight they provide into the processes
driving galaxy formation and chemical evolution. In particular,
GCs are ideal test sites for answering open questions about the
interplay between primordial and evolutionary chemical enrich-
ment [1]. These objects have therefore warranted significant ob-
servational efforts and, through recent studies a complex picture
of GCs abundance patterns has emerged, with strong evidence
supporting multiple epochs of star formation [2]. Despite clear 0370-2693/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativeco
SCOAP3. A. Lennarz et al. / Physics Letters B 807 (2020) 135539 2 2. Experimental details ≤248 keV, all previously reported strengths were either
measured relative to reference resonances at Ec.m. = 458 keV or
1222 keV or depended on these resonances to determine target
stoichiometries. The 458 keV resonance strength directly influ-
ences the strengths of the low-energy resonances reported by the
LENA collaboration [12], and was used as reference for target sto-
ichiometries in 22Ne+α [15] and normal kinematics studies of the
22Ne(p, γ )23Na reaction [11]. Moreover, this resonance is particu-
larly relevant for reaction-rate compilations conducted by Sallaska,
Iliadis et al. [16], for which all other measured strengths were nor-
malized to the 458 keV strength value of ωγ = 0.524(51) eV [17]. The latter was determined relative to the Ep = 405.5(3) keV (ωγ =
(8.63(52)×10−3) eV [18]) resonance strength in 27Al(p, γ )28Si, and
depends on the background contribution of the Ep = 326 keV and
447 keV resonances in the same reaction. We note that there
is a more recent result for the 405.5(3) keV strength of ωγ =
1.04(5)×10−2 eV [19]. Using this value for re-normalization would
reduce the 458 keV strength reported by Longland et al. to ωγ =
0.435(42) eV. Further, the strengths of the resonances affecting the
background in that measurement have also been normalized to the
405.5(3) keV strength. Though the 458 keV resonance has been
investigated numerous times [17,11,20], our measurement reveals
that the situation for its strength is still not resolved. In fact, the
strength of this resonance has never been measured independently
of other resonances. However, this work puts forward a direct,
reference-independent measurement which is largely independent
of knowledge of the relevant branching ratios (BRs). This letter
further presents the results of the direct strengths measurements
of the astrophysically important resonances in 22Ne(p, γ )23Na at
Ec.m. = 149 keV, 181 keV, 248 keV. Table 1 m
m + M ,
(1) (1) with the recoil yield, Y , the stopping power in the laboratory sys-
tem, ϵ, the center-of-mass de-Broglie wavelength, λ2
c.m. as well as
the proton (m) and 22Ne (M) masses. Our result for the 458 keV
strength of ωγcoinc = 0.441(50) eV (ωγsingles = 0.439(22) eV) is
lower and not in agreement within errors with the two lat-
est results [11,20]. However, it agrees with three previous val-
ues [17,31,32]. The result from Meyer et al. [32] was normalized to
the 612 keV resonance strength, and the Endt et al. [31] value is
based on Ref. [32], however, normalized the 1.222 MeV resonance
strength from Ref. [33]. The sensitivity of former studies to refer-
ence resonances underlines the necessity of reference-independent
measurements as well as more precise measurements of reference-
resonance strengths. with the recoil yield, Y , the stopping power in the laboratory sys-
tem, ϵ, the center-of-mass de-Broglie wavelength, λ2
c.m. as well as
the proton (m) and 22Ne (M) masses. Our result for the 458 keV
strength of ωγcoinc = 0.441(50) eV (ωγsingles = 0.439(22) eV) is
lower and not in agreement within errors with the two lat-
est results [11,20]. However, it agrees with three previous val-
ues [17,31,32]. The result from Meyer et al. [32] was normalized to
the 612 keV resonance strength, and the Endt et al. [31] value is
based on Ref. [32], however, normalized the 1.222 MeV resonance
strength from Ref. [33]. The sensitivity of former studies to refer-
ence resonances underlines the necessity of reference-independent
measurements as well as more precise measurements of reference-
resonance strengths. 4. Results For the 149 keV resonance we report a strength of ωγ (149) =
(1.67 ± 0.28 (sys) +0.39
−0.28 (stat))×10−7 eV, which is lower but
in agreement with all previous values. Our 181 keV strength of
ωγ (181) = (2.17+0.32
−0.31 (sys) +0.2
−0.17 (stat))×10−6 eV is in good agree-
ment with the LUNA HPGe result [14] and lower but also in agree-
ment with the TUNL result. Further, our result is 20% lower than
the LUNA BGO measurement [13] (compare Table 1), though the
two values are still consistent within 1 σ . Regarding the 248 keV
resonance we report a strength of ωγ (248) = 8.5(1.4)×10−6 eV. The dominant contributions to the systematic uncertainty result
from uncertainties on coincidence efficiency (10%), stopping power
(4.3 - 5.9%), charge-state fraction (1.8%(181 keV) - 2.4%(149 keV)),
MCP efficiency (5%) and beam normalization (1.1 - 4.9%). To determine the 149 keV, 181 keV and 248 keV resonance
strengths, conservative recoil gates for DSSSD and BGO energy
were placed on the separator TOF vs MCP TOF spectrum or sep-
arator TOF spectrum (Fig. 3). The 248 keV yield measurement does
not have an associated separator vs MCP TOF spectrum since the
MCP detection efficiency was too low to give enough statistics; this
issue was resolved for the lower energy measurements. In view of the significant deviation of the DRAGON ωγ (458 keV)
result from the value used to normalize the strengths of the low-
energy resonances in the TUNL measurement [12], we carefully
reviewed the latter. In fact, re-normalizing the TUNL 149 keV
strength to our ωγ (458 keV) result, brings it into better agreement
with DRAGON, and a re-normalized 181 keV strength is compatible
with the DRAGON and LUNA HPGe results. For the analysis of the 149 keV and 181 keV yield mea-
surements the branching ratios for the Ex = 8943(3) keV and
8972(3) keV levels given in Ref. [12] were used for the GEANT3
simulation. The BRs from Ref. [12] were chosen over those re-
ported in Ref. [34] as the analysis in Ref. [12] did not require
additional background subtraction or coincidence-summing correc-
tions, and accounted for escape peaks and Compton continuum. To 3. Analysis A difference of 1.1 % and 3.7% in
simulated efficiency was found for the 149 keV and 181 keV res-
onances, respectively, which has been taken into account in the
uncertainty budget. Table 1
Overview of resonance strengths. (S) marks results from a singles analysis. Ec.m.[keV]
ωγ [eV]
Lit. This work
458.0(3) [35]
0.583(43) [20]
0.441(50)
0.439(22) (S)
0.594(38) [11]
248.3(6) [36]
8.2(7)×10−6 [14]
8.5(1.4)×10−6
9.7(7)×10−6 [13]
181.2(7) [36]
2.2(2)×10−6 [14]
2.7(2)×10−6 [13]
2.17+0.37
−0.35×10−6
2.32(32)×10−6 [12]
149.4(7) [36]
1.8(2)×10−7 [14]
2.2(2)×10−7 [13]
1.67+0.48
−0.40×10−7
2.03(40)×10−7 [12]
Ref. [12] re-normalized to this work
181.2(7) [36]
1.75(29)×10−6
149.4(7) [36]
1.53(33)×10−7 Table 1 Table 1
Overview of resonance strengths. (S) marks results from a singles analysis. Fig. 1. Singles (black histogram) and coincidence (blue histogram) DSSSD energy
spectra of the 458 keV yield measurement. In red, the triple Gaussian fit of the
singles spectrum is shown. The black dashed line denotes the unreacted beam com-
ponent (not present in coincidence measurement) of the fit and the red dashed
vertical lines indicate the recoil gate. model. High statistics and a clear separation of unreacted beam
and recoils also allowed for a singles analysis of the 458 keV reso-
nance to eliminate uncertainties introduced by the dependence of
the coincidence analysis on BRs and BGO efficiency. Using the fit
parameters of the coincidence spectrum as guide for the singles
analysis, a triple Gaussian function was applied to the DSSSD en-
ergy spectrum, and the integral of the main recoil peak and satel-
lite peak comprises the number of recoil events. Fig. 2 presents the
458 keV resonance-strength values based on coincidence and sin-
gles analysis, which are mutually consistent, relative to previous
measurements. investigate how the choice of BRs propagates to the BGO detection
efficiencies and resonance strengths, respectively, simulations were
performed for both sets of BRs. A difference of 1.1 % and 3.7% in
simulated efficiency was found for the 149 keV and 181 keV res-
onances, respectively, which has been taken into account in the
uncertainty budget. investigate how the choice of BRs propagates to the BGO detection
efficiencies and resonance strengths, respectively, simulations were
performed for both sets of BRs. A difference of 1.1 % and 3.7% in
simulated efficiency was found for the 149 keV and 181 keV res-
onances, respectively, which has been taken into account in the
uncertainty budget. investigate how the choice of BRs propagates to the BGO detection
efficiencies and resonance strengths, respectively, simulations were
performed for both sets of BRs. A difference of 1.1 % and 3.7% in
simulated efficiency was found for the 149 keV and 181 keV res-
onances, respectively, which has been taken into account in the
uncertainty budget. The resonance strengths were calculated using the standard for-
mula for thick target yield in inverse kinematics [30], ωγ = 2ϵY
λ2
c.m. m
m + M , ωγ = 2ϵY
λ2
c.m. 3. Analysis For improved background suppression, the resonance strengths
were extracted in a coincidence analysis, where the GEANT3 [27]
simulation used to determine the BGO detection efficiency relies
on literature BRs. For the 458 keV measurement the DSSSD en-
ergy spectrum was fitted with a double Gaussian to set appro-
priate energy cuts for the “golden” recoil gate at ±3.5σ relative
to the peak centroids, and to account for the satellite peak at
the low energy side of the main recoil peak (Fig. 1). The satel-
lite peak results from the additional energy loss of ions passing
the ∼3% aluminum DSSSD grid [28]. Accounting for satellite peak
and inter-strip events results in a DSSSD efficiency of (96.15 ±
0.1stat. ± 0.43sys.)% [29]. The established DSSSD and BGO energy
gates were then placed on the separator TOF, i.e., the time be-
tween γ - and recoil event detection, spectrum to extract the num-
ber of recoils. The background was estimated by sampling the
time-random background and calculating an average expectation
over the width of the signal region using a poissonian background A. Lennarz et al. / Physics Letters B 807 (2020) 135539 3 3 Fig. 1. Singles (black histogram) and coincidence (blue histogram) DSSSD energy
spectra of the 458 keV yield measurement. In red, the triple Gaussian fit of the
singles spectrum is shown. The black dashed line denotes the unreacted beam com-
ponent (not present in coincidence measurement) of the fit and the red dashed
vertical lines indicate the recoil gate. Fig. 1. Singles (black histogram) and coincidence (blue histogram) DSSSD energy Fig. 2. Previous 458 keV strength values (black circles) in relation to the DRAGON
results (red squares) obtained from singles and coincidence analysis. Fig. 2. Previous 458 keV strength values (black circles) in relation to the DRAGON
results (red squares) obtained from singles and coincidence analysis. Table 1
Overview of resonance strengths. (S) marks results from a singles analysis. Ec.m.[keV]
ωγ [eV]
Lit. This work
458.0(3) [35]
0.583(43) [20]
0.441(50)
0.439(22) (S)
0.594(38) [11]
248.3(6) [36]
8.2(7)×10−6 [14]
8.5(1.4)×10−6
9.7(7)×10−6 [13]
181.2(7) [36]
2.2(2)×10−6 [14]
2.7(2)×10−6 [13]
2.17+0.37
−0.35×10−6
2.32(32)×10−6 [12]
149.4(7) [36]
1.8(2)×10−7 [14]
2.2(2)×10−7 [13]
1.67+0.48
−0.40×10−7
2.03(40)×10−7 [12]
Ref. [12] re-normalized to this work
181.2(7) [36]
1.75(29)×10−6
149.4(7) [36]
1.53(33)×10−7
investigate how the choice of BRs propagates to the BGO detection
efficiencies and resonance strengths, respectively, simulations were
performed for both sets of BRs. 5. Astrophysical impact A 5 M⊙model
with metallicity z = 0.006 was utilized to study the impact of
our rate on HBB in TP-AGB stars, using the STARLIB2013 rate
as reference. We observe a close mapping of [Na/Fe] as a func-
tion of [s/Fe] for the two rates, confirming the robustness of
the STARLIB2013 rate. This contradicts the factor of ∼3 en-
hancement in 23Na production for 5 M⊙AGB stars stated by
Slemer et al. [43] based on the LUNA rate, which includes the
tentative Ec.m. = 68 keV and 100 keV resonances. Even though Sle-
mer et al. use a code that couples mixing and burning during HBB,
and adopt a similar list of isotopes as NuGrid, neutron captures
are not included. Thus, the important 23Na destruction channel
23Na(n, γ )24Na stated in Ref. [44] remains unconsidered. Further,
the effect of the 22Ne(p, γ )23Na rate on the sodium abundance
was studied. When closing the (p,γ ) channel, the abundance drops
to almost zero, confirming the 22Ne(p, γ )23Na reaction as main
sodium production channel in massive AGB stars. Further, the ef-
fect on the 23Na-pocket formation in low-mass AGB stars (2M⊙,
z = 0.001 and z = 0.006) using the DRAGON rate relative to the
STARLIB2013 rate was investigated by evaluating the abundance
profile of 23Na when the sodium pocket is fully formed (Fig. 5). Switching off the 22Ne(p, γ )23Na reaction results in a significant
sodium abundance reduction. However, in contrast to the 5 M⊙
model, the sodium abundance stays relatively high due to the sec-
ond production channel 22Ne(n,γ )23Ne(β−)23Na, which is active DRAGON reaction rate evaluation, the analysis results from higher
energy resonances at 610 keV, 632 keV and 1222 keV as well as
the direct-capture contribution as detailed in Ref. [37] were in-
cluded in addition to the here discussed resonance strengths. The
dramatic enhancement of the LUNA rate upper limit is mainly due
to the inclusion of the Ec.m. = 68 keV resonance, which has been
excluded in the median rate and for which only an upper limit
has been reported [13]. Our rate maps closely with the TUNL rate,
with a slight reduction due to our reduced 149 keV and 181 keV
strengths. 5. Astrophysical impact Abundances of 23Na, 24Mg
or higher mass isotopes remain unaffected. Instead, the observed
difference in 22Ne abundances may be relevant for studies of pre-
solar grains. For further details on the impact of the rate from this
work on isotopic abundances compared to the STARLIB2013 rate
the reader is referred to Ref. [37]. ity of Mg synthesis to the peak temperature [41]. Due to the larger
rate, the mass flow is pushed up to Mg synthesis temperatures. As a result of this correlation these ratios become relevant in the
identification of pre-solar grains, as they function as probe for the
peak temperature reached in the outburst, and the underlying WD
mass. In a sensitivity study [10], the final abundances of 24,25Mg
for 1.0 M⊙CO novae varied by up to a factor of 5, when varying
the 22Ne(p, γ )23Na rate (STARLIB2013) within its uncertainties,
whereas the DRAGON rate, which as stated above closely maps
with the TUNL rate, strongly limits the reaction rate uncertainty
in the temperature range of interest (Tpeak = 170 MK). Varying the
new rate within its limits only changes the Mg isotope mass frac-
tions by up to 7% in the 1.15 M⊙CO nova model. For ONe novae,
the cycling back to 20Ne is irrelevant for both mass models, as
20Ne is sufficiently available. This is reflected in the same 20,21Ne
final yield, independent of the model. Abundances of 23Na, 24Mg
or higher mass isotopes remain unaffected. Instead, the observed
difference in 22Ne abundances may be relevant for studies of pre-
solar grains. For further details on the impact of the rate from this
work on isotopic abundances compared to the STARLIB2013 rate
the reader is referred to Ref. [37]. Fig. 4. The 22Ne(p, γ )23Na reaction rate normalized to the STARLIB2013 rate [16]. Shaded areas bound the 1 −σ upper and lower limits of each calculated rate. The
DRAGON rate was calculated using the same RateMC code [38] used for the TUNL
rate. [
]
The NuGrid multi-zone post-processing code MPPNP [42] was
used to implement our rate in nucleosynthesis network calcula-
tions, and to model the [Na/Fe] abundance ratio on the AGB star
surface at the end of the evolution of stable isotopes for var-
ious masses and metallicities (compare Fig. 5). 5. Astrophysical impact Fig. 4 displays an overlay of the rates determined from this
work and those of LUNA and TUNL measurements. For the 4
A. Lennarz et al. / Physics Letters B 807 (2020) 135539
Fig. 3. Separator TOF spectrum for the 248 keV resonance, and separator vs MCP TOF spectra for the 181 keV and 149 keV yield measurements. The red dashed lines
represent the recoil timing gates. Each spectrum is gated on the recoil peak in the DSSSD energy spectrum and a minimum BGO energy threshold of Eγ > 2.2, 2.0, and 2.5
MeV, respectively. A. Lennarz et al. / Physics Letters B 807 (2020) 135539 4 Fig. 3. Separator TOF spectrum for the 248 keV resonance, and separator vs MCP TOF spectra for the 181 keV and 149 keV yield measurements. The red dashed lines
represent the recoil timing gates. Each spectrum is gated on the recoil peak in the DSSSD energy spectrum and a minimum BGO energy threshold of Eγ > 2.2, 2.0, and 2.5
MeV, respectively. Fig. 4. The 22Ne(p, γ )23Na reaction rate normalized to the STARLIB2013 rate [16]. Shaded areas bound the 1 −σ upper and lower limits of each calculated rate. The
DRAGON rate was calculated using the same RateMC code [38] used for the TUNL
rate. ity of Mg synthesis to the peak temperature [41]. Due to the larger
rate, the mass flow is pushed up to Mg synthesis temperatures. As a result of this correlation these ratios become relevant in the
identification of pre-solar grains, as they function as probe for the
peak temperature reached in the outburst, and the underlying WD
mass. In a sensitivity study [10], the final abundances of 24,25Mg
for 1.0 M⊙CO novae varied by up to a factor of 5, when varying
the 22Ne(p, γ )23Na rate (STARLIB2013) within its uncertainties,
whereas the DRAGON rate, which as stated above closely maps
with the TUNL rate, strongly limits the reaction rate uncertainty
in the temperature range of interest (Tpeak = 170 MK). Varying the
new rate within its limits only changes the Mg isotope mass frac-
tions by up to 7% in the 1.15 M⊙CO nova model. For ONe novae,
the cycling back to 20Ne is irrelevant for both mass models, as
20Ne is sufficiently available. This is reflected in the same 20,21Ne
final yield, independent of the model. 6. Summary [13] F. Ferraro, M.P. Takács, D. Piatti, F. Cavanna, R. Depalo, M. Aliotta, D. Bemmerer,
A. Best, A. Boeltzig, C. Broggini, et al., Phys. Rev. Lett. 121 (2018) 172701. In summary, key resonances in the 22Ne(p, γ )23Na reaction
have been investigated in inverse kinematics for the first time us-
ing the DRAGON recoil separator. The strength of the important
reference resonance at 458 keV has been determined more pre-
cisely via a direct measurement, and does not agree within errors
with the two most recent normal kinematics results. Our result
affects resonance strengths that have been determined relative to
the strength of this resonance, as well as neon-target stoichiome-
tries determined based on its strength. A new reaction rate was
calculated based on the DRAGON measurement, which confirms
the accuracy of the current 23Na production results in AGB stars in
relation to the behavior of the 22Ne(p, γ )23Na reaction and under-
lines the importance of this reaction for the sodium production in
AGB stars. Further work is needed to reassess the sensitivity of Mg
isotopic ratios in CO novae to rate variations in the Ne-Al region
to use said ratios as a probe of the underlying WD peak tempera-
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burning [44]. during radiative 13C burning as well as during convective 22Ne
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U. Greife, A. Hussein, D.A. Hutcheon, D. Ottewell, et al., Nucl. Instrum. Methods
Phys. Res., Sect. B 266 (2008) 4167. 5. Astrophysical impact José acknowledges
support from the Spanish MINECO grant AYA2017-86274-P, the EU
FEDER funds and the AGAUR/Generalitat de Catalunya grant SGR-
661/2017. Authors from the Colorado School of Mines acknowl-
edge funding via the U.S. Department of Energy grant DE-FG02-
93ER40789. The authors also thank R. Longland for his support in
calculating the thermonuclear reaction rate presented in this work. 5. Astrophysical impact g
The effect of the DRAGON rate compared to the Iliadis 2010
rate [38] on the sodium and neon abundances in neon-oxygen
(ONe) novae with underlying white-dwarf (WD) masses of 1.15 M⊙
and 1.25 M⊙, as well as carbon-oxygen (CO) novae (1.15 M⊙
and 1.00 M⊙) was investigated using hydro-dynamical nova mod-
els [39,40]. Changes of more than 10% in the isotopic abundances
within the Ne-Al region (20,21,22Ne, 22,23Na, 25,26Mg, 26,27Al) in
1.15 M⊙CO novae, and a factor of 2 enhancement in 23Na abun-
dance are observed for both CO nova mass models. For ONe novae,
a factor of 2 reduction of the 22Ne content is observed for both WD
mass models. Further, the 24Mg abundance is enhanced by ∼15%
in the 1.25 M⊙model, whereas only slight differences are seen
for the remaining isotopes considered in both models. Regarding
CO novae, our rate increases the differences in the 25Mg/26Mg and
26Mg/25Mg ratios between the 1.0 and 1.15 M⊙models. Using the
DRAGON rate in the 1.15 M⊙model increases the 25Mg/24Mg ra-
tio by 24% and decreases the 26Mg/25Mg ratio by 13% compared
to the STARLIB2013 rate. This can be explained by the sensitiv- A. Lennarz et al. / Physics Letters B 807 (2020) 135539 5 5 Fig. 5. Predicted surface [Na/Fe] abundance ratio as a function of s process element
abundances [s/Fe] for a 5M⊙(top) at z = 0.006 and a 2M⊙(bottom) AGB star model
at different metallicities (z = 0.001 and z = 0.006) using the rate from this work
relative to the STARLIB rate. “ChETEC” COST Action (CA16117), supported by COST 116 (Euro-
pean Cooperation in Science and Technology). MW, AML, JR were
supported by the UK Science and Technology Facilities Council
(STFC) ST/P003885/1. UB acknowledges support from the European
Research Council ERC-2015-STG Nr. 677497. J. José acknowledges
support from the Spanish MINECO grant AYA2017-86274-P, the EU
FEDER funds and the AGAUR/Generalitat de Catalunya grant SGR-
661/2017. Authors from the Colorado School of Mines acknowl-
edge funding via the U.S. Department of Energy grant DE-FG02-
93ER40789. The authors also thank R. Longland for his support in
calculating the thermonuclear reaction rate presented in this work. “ChETEC” COST Action (CA16117), supported by COST 116 (Euro-
pean Cooperation in Science and Technology). MW, AML, JR were
supported by the UK Science and Technology Facilities Council
(STFC) ST/P003885/1. UB acknowledges support from the European
Research Council ERC-2015-STG Nr. 677497. J. Acknowledgements [26] J. Görres, K.U. Kettner, H. Krawinkel, C. Rolfs, Nucl. Instrum. Methods 177
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EE-84-1 1987. The authors thank the ISAC operations and technical staff at
TRIUMF. TRIUMF’s core operations are supported via a contribu-
tion from the federal government through the National Research
Council Canada, and the Government of British Columbia pro-
vides building capital funds. DRAGON is supported by funds from
the National Sciences and Engineering Research Council of Canada
SAPPJ-2019-00039. The authors acknowledge support from the [28] Micron Semiconductor Ltd, micronsemiconductor.co .uk, 2017. [29] C. Wrede, A. Hussein, J.G. Rogers, J.M. D’Auria, Nucl. Instrum. Methods Res.,
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4817–4837. [37] M. Williams, A. Lennarz, A.M. Laird, U. Battino, J. José, D. Acknowledgements Connolly, C. Ruiz, A. Chen, B. Davids, N. Esker, et al., submitted to Phys. Rev. C (2020). [44] S. Cristallo, R. Gallino, O. Straniero, L. Piersanti, I. Domínguez, Mem. Soc. Astron. Ital. 774 (2006) 77. [38] C. Iliadis, R. Longland, A.E. Champagne, A. Coc, Nucl. Phys. A 841 (2010)
251–322. [39] J. José, M. Hernanz, Astrophys. J. 494 (1998) 680. [40] J. José, Stellar Explosions: Hydrodynamics and Nucleosynthesis, CRC Press, Boca
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https://www.emerald.com/insight/content/doi/10.1108/IJPH-08-2013-0041/full/pdf?title=alcohol-related-risk-and-harm-amongst-young-offenders-aged-11-17
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Alcohol-related risk and harm amongst young offenders aged 11-17
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Abstract Research limitations/implications – This research was confined to one geographical area of England,
however, the results show that even in this area of high drinking by young people the levels of AUDs amongst
young people in the CJS are very high. Social implications – There are major social implications to this research. It is imperative for changes to be
made to the care pathways in place in the UK for young people coming through the CJS with alcohol-related
issues. Social implications – There are major social implications to this research. It is imperative for changes to be
made to the care pathways in place in the UK for young people coming through the CJS with alcohol-related
issues. Originality/value – This paper adds to the evidence base by using well-validated tools to measure alcohol
use amongst young people in the CJS in the UK. Originality/value – This paper adds to the evidence base by using well-validated tools to measure alcohol
use amongst young people in the CJS in the UK. Received 23 August 2013
Revised 24 October 2014
Accepted 18 December 2014 Keywords Criminal justice system, Prison, Alcohol, Substance abuse, Young offenders,
Youth offending service Paper type Research paper © Dorothy Newbury-Birch,
Katherine Jackson,
Tony Hodgson, Eilish Gilvarry,
Paul Cassidy, Simon Coulton,
Vicky Ryan, Graeme B. Wilson,
Ruth McGovern, Eileen Kaner. Published by Emerald Group
Publishing Limited. This article is
published under the Creative
Commons Attribution (CC BY 3.0)
licence. Anyone may reproduce,
distribute, translate and create
derivative works of this article
(for both commercial & non-
commercial purposes), subject to
full attribution to the original
publication and authors. The full
terms of this licence may be seen
at http://creativecommons.org/
licences/by/3.0/legalcode Alcohol-related risk and harm amongst
young offenders aged 11-17 Dorothy Newbury-Birch, Katherine Jackson, Tony Hodgson, Eilish Gilvarry, Paul Cassidy,
Simon Coulton, Vicky Ryan, Graeme B. Wilson, Ruth McGovern and Eileen Kaner The authors affiliations can be
found at the end of this article. The authors affiliations can be
found at the end of this article. Abstract Purpose – The purpose of this paper is to examine the prevalence of alcohol use disorders (AUDs) amongst
young people in the criminal justice system (CJS) in the North East of England and to compare the ability of
the Alcohol Use Disorders Identification Test (AUDIT) to the Youth Justice Board ASSET tool in identifying
alcohol-related need in Youth Offending Team (YOT) clients. Design/methodology/approach – A validated screening tool (AUDIT) was used to identify alcohol-related
health risk or harm. Findings from AUDIT were compared with those of the standard criminogenic risk
screening tool used in CJS (ASSET). An anonymous cross-sectional questionnaire was administered during
a one-month period in 2008. The questionnaires were completed by 11-17-year-old offenders who were in
contact with three YOTs, one Youth Offending Institution and one Secure Training Estate. Design/methodology/approach – A validated screening tool (AUDIT) was used to identify alcohol-related
health risk or harm. Findings from AUDIT were compared with those of the standard criminogenic risk
screening tool used in CJS (ASSET). An anonymous cross-sectional questionnaire was administered during
a one-month period in 2008. The questionnaires were completed by 11-17-year-old offenders who were in
contact with three YOTs, one Youth Offending Institution and one Secure Training Estate. Findings – In total, 429 questionnaires were completed out of a possible 639 (67 per cent). The majority
(81 per cent) of the young offenders were identified as experiencing alcohol-related health risk or harm and
77 per cent scored within a possibly alcohol-dependent range. In total, 77 (30 per cent) of young people
completing both assessments were identified as having an AUD by AUDIT but not identified as needing
alcohol-related treatment using ASSET. Findings – In total, 429 questionnaires were completed out of a possible 639 (67 per cent). The majority
(81 per cent) of the young offenders were identified as experiencing alcohol-related health risk or harm and
77 per cent scored within a possibly alcohol-dependent range. In total, 77 (30 per cent) of young people
completing both assessments were identified as having an AUD by AUDIT but not identified as needing
alcohol-related treatment using ASSET. Research limitations/implications – This research was confined to one geographical area of England,
however, the results show that even in this area of high drinking by young people the levels of AUDs amongst
young people in the CJS are very high. Introduction Adolescents in England are amongst the heaviest drinkers in Europe (Hibbell et al., 2012). The
percentage of adolescents who have ever had an alcoholic drink in England increases with age
from 12 per cent of those aged 11 to 74 per cent of those aged 15, and the prevalence of
drinking in the last week rises from 1 per cent of those aged 11 to 25 per cent of those aged 15
(Fuller et al., 2013). Although the proportion of adolescents in England aged between 11 and 15
years who report that they have ever drunk alcohol decreased from 54 to 43 per cent between
2007 and 2012, the mean amount in those that drank increased from 10.4 standard drink units
per week in 2011 to 12.5 units per week in 2012 (Fuller et al., 2013). This clearly shows that
drinking increases over adolescence, but that this is not immutable, with changes in trends over
time and with age. The North East of England has been shown to have the highest rates of j INTERNATIONAL JOURNAL OF PRISONER HEALTH VOL. 11 NO. 2 2015, pp. 75-86, Emerald Group Publishing Limited, ISSN 1744-9200 VOL. 11 NO. 2 2015, pp. 75-86, Emerald Group Publishing Limited, ISSN 1744-9200 j INTERNATIONAL JOURNAL OF PRISONER HEALTH j P DOI 10.1108/IJPH-08-2013-0041 alcohol misuse by adolescents in England, with 51 per cent of 11-15-year-olds reporting having
ever drunk alcohol (Fuller et al., 2013). This compares to 48 per cent in the South East,
46 per cent in the North West and 31 per cent in London (Fuller et al., 2013). Further, the mean
alcohol consumption in the previous week for adolescents in England is highest in the North East
and North West (15.7 units per week) compared to the South East (11.0) and London (9.4 units)
(Fuller et al., 2013). alcohol misuse by adolescents in England, with 51 per cent of 11-15-year-olds reporting having
ever drunk alcohol (Fuller et al., 2013). This compares to 48 per cent in the South East,
46 per cent in the North West and 31 per cent in London (Fuller et al., 2013). Further, the mean
alcohol consumption in the previous week for adolescents in England is highest in the North East
and North West (15.7 units per week) compared to the South East (11.0) and London (9.4 units)
(Fuller et al., 2013). Introduction The impact of alcohol on the development and behaviour of adolescents has been well
researched in early (Zucker et al., 2009), middle (Windle et al., 2009) and late adolescence
(Brown et al., 2009). It is now well known that adolescents are much more vulnerable than
adults to the adverse effects of alcohol, due to a range of physical and psycho-social factors
which often interact (Newbury-Birch et al., 2009a; Marshall, 2014). These adverse effects include:
physiological factors resulting from a typically lower body mass and less efficient metabolism
of alcohol (Windle et al., 2009; Zucker et al., 2009); neurological factors due to changes that
occur in the developing adolescent brain after alcohol exposure (Windle et al., 2009; Squeglia
et al., 2012); cognitive factors due to psychoactive effects of alcohol which impair judgement
and increase the likelihood of accidents and trauma (Rodham et al., 2006); and social factors
which arise from a typically high-intensity drinking pattern (often called “binge drinking”) which
leads to intoxication and risk-taking behaviour (MacArthur et al., 2012). The latter are
compounded by the fact that adolescents have less experience of dealing with the effects of
alcohol than adults (Murgraff et al., 1999), and they have fewer financial resources to help buffer
the social and environmental risks that result from drinking alcohol (Brown et al., 2009). The Chief Medical Officer for England has provided recommendations on alcohol consumption in
young people (Donaldson, 2009) based on an evidence review of the risks and harms of alcohol
to young people (Newbury-Birch et al., 2009a). The recommendations state that children should
abstain from alcohol before the age of 15 and those aged 15-17 are advised not to drink, but if
they do drink it should be no more three to four units and two to three units per week in males and
females, respectively, on no more than one day per week (Donaldson, 2009) which equates to
adult daily drinking recommendations. A particular group of adolescents at risk due to their drinking is young offenders (Kennedy, 2013). Evidence shows that drinking amongst adolescents under the age of 18 years, especially
frequent drinking, is associated with criminal and disorderly behaviour (Fuller et al., 2013). Introduction Alcohol
consumption amongst adolescents aged ten to 17 years is estimated to be responsible for
80,640 violent offences per year (Budd et al., 2005) and to cost in excess of £5 million per year for
criminal activity to the Criminal Justice System (CJS) (Bellis et al., 2007). Moreover, adolescents
who drink are more likely than non-drinkers to be both perpetrators and victims of violence
(Newburn and Shiner, 2001). These data have culminated in a joint health and criminal justice
policy focusing on identifying and tackling youth drinking and social disorder in the UK (HM
Government, 2009a, b). In the UK, higher rates of alcohol misuse have been found at various stages in the CJS for adults,
compared to the 20-30 per cent observed in primary care populations (Funk et al., 2005; Coulton
et al., 2012), with around two-thirds of men and women having established problematic alcohol
use. This encompasses those arrested at police stations (Brown et al., 2010), within probation
settings (Newbury-Birch et al., 2009b; Orr et al., 2015) and those in prison (Graham et al., 2012;
Fazel et al., 2006; Newbury-Birch et al., 2009b). High rates are also found among adolescents
within the CJS with a study of 16-20-year-olds in the prison system in England and Wales
reporting that 62 per cent of males and 13 per cent of females on remand and 70 per cent of
males and 51 per cent of females of those sentenced experienced an Alcohol Use Disorder (AUD)
using a cut-off of eight on the ten question Alcohol Use Disorders Identification Test (AUDIT)
(Lader et al., 2000). Identifying alcohol-related risk and harm in adolescents The AUDIT is a screening tool that was developed to help practitioners identify people who
drink excessively. The AUDIT consists of ten questions about alcohol consumption, alcohol
dependence and alcohol-related problems. Shortened versions have also been validated for use. j INTERNATIONAL JOURNAL OF PRISONER HEALTH j VOL. 11 NO. 2 2015 In adult populations, the AUDIT ten question tool is regarded to be the “gold standard” screening
tool for identifying alcohol-related risk or harm (Saunders et al., 1993). At a cut-off point of eight
(out of a possible total score of 40), it has a sensitivity and specificity of identifying alcohol-related
risk or harm of 92 and 94 per cent, respectively (Saunders et al., 1993). A further positive feature
of AUDIT is that it can distinguish between different types of alcohol-related risk: hazardous
drinking (scores 8-15) which identifies an amount or pattern of drinking that increases the risk of
physical or psychological problems; harmful drinking (scores 16-19) which is defined by
the presence of physical or psychological symptoms; and probable alcohol dependence
(scores 20+) which is a cluster of physiological, behavioural and cognitive phenomena
conforming to the “alcohol dependence syndrome” (Babor et al., 1989). Occurrence of any of
these three drinking profiles is called an AUD (Saunders et al., 1993). A systematic review commissioned by the English National Institute for Health and Clinical
Excellence examined the validity of alcohol screening in a wide range of settings for both
adolescents (age ten to 17 years) and adults (aged over 18 years). This review found that
questionnaires performed better than blood markers or breath alcohol concentration for adults
and adolescents (Jackson et al., 2009). AUDIT has been found to have greater sensitivity and
specificity for identifying likely AUDs than other youth-focused alcohol screening questionnaires
(Cook et al., 2005). Thus it is currently regarded as the best alcohol screening tool available for
adolescents (Cook et al., 2005). In adolescent populations, AUDIT sensitivities have ranged
from 54 to 87 per cent, specificities from 65 to 97 per cent. Optimal scores for identifying likely
AUDs using the ten question AUDIT range from two to ten (Clark and Moss, 2010). Across a
14-18-year-old age range, likely hazardous or harmful drinking has been identified at an AUDIT
score of 2+ and probable dependent drinking at an AUDIT score of 3+ with sensitivity and
specificity values of 83 and 93 per cent, respectively (Knight et al., 2003). Identifying alcohol-related risk and harm in adolescents These are the
adolescent scores used for the present study. The ASSET tool ASSET is a standardised assessment tool, developed within the CJS in England and Wales,
which aims to identify the underlying causes of a young person’s offending behaviour and to plan
appropriate interventions (Youth Justice Board, 2006b). It is often used on multiple occasions
to help measure changes in young offenders’ health and social needs and the risk of reoffending
over time. ASSET has been used with all young offenders in England and Wales since 2000
and it examines 12 dynamic risk factors: living arrangements; family and personal relationships;
education, training and employment; neighbourhood; lifestyle; substance use; physical health;
emotional health; perception of self and others; thinking and behaviour; attitudes to offending;
and motivation to change. The extent to which each section is associated with the likelihood of
further offending is rated on a 0-4 scale (Youth Justice Board, 2006b). The current research recorded the substance misuse section of ASSET (section 6) which looks at
alcohol misuse as well as other drug misuse and includes questions on whether the young
person has ever used, or recently used a variety of substances including alcohol. It also looks at
the age of first misuse and whether the substance is linked to the young person’s offending
(Youth Justice Board, 2006a, b). A score of two or more indicates referral to colleagues who
specialise in assessment and intervention to ameliorate substance use. This work is often
conducted within the secure estate (National Treatment Agency for Substance Misuse, 2012)
or Youth Offending Team (YOT) but can occur in liaison with local adolescent substance misuse
services (Youth Justice Board, 2006a). There is however a lack of data on alcohol-related risk or harm in younger adolescents
(aged 11-14). Moreover, there is relatively little research conducted in the CJS with young
drinkers. The purpose of this present study was to identify alcohol-related risk or harm in the full
age range of adolescents in contact with the CJS and to include both community-based and
institutional settings. The study sought to compare the risk profile obtained via AUDIT, using both
the adult and the suggested adolescent cut-offs, with the standard criminogenic risk assessment
tool used in the CJS with young offenders (ASSET). The purpose of this comparison is to
determine the sensitivity of identifying likely AUDs using the ASSET. The study will also compare
the prevalence and severity of AUD by offence type. VOL. 11 NO. Measures AUDIT. In this present study both the adult cut-off ranges of 8-15 (hazardous drinking) 16-19
(harmful drinking) and 20+ (probable dependence) (Saunders et al., 1993) and the adolescent
cut-off ranges of 2+ (hazardous/harmful drinking) and 3+ (probable dependence) previously used
by Knight et al. (2003) were used. ASSET. The ASSET score currently recorded by the YOT/secure estate was used within this
study. A score of 2 or more (which indicates referral for specialist intervention) was used as the
cut-off for identification of alcohol-related risk. Study questionnaire A one page questionnaire was developed which consisted of: the ten items of AUDIT;
demographic data including age (at time of completing the questionnaire) and gender. Boxes
were included for the staff member to record the young person’s ethnicity; offence and sentence
(no categories were given for this). A visual aid of alcoholic drinks and related units was included. The adolescents ASSET score relating to substance misuse (ASSET section 6) was added by the
staff member onto the questionnaire. Sample The study was based in community-based YOTs and secure establishment institutions in the
North East of England. All 11 YOTs and all three secure establishments were approached to
be involved in the study. Three YOTs and the two secure establishments (one Secure Training
Estate and one Youth Offending Institution) agreed to be involved. The reason for
non-participation in the study was workload. Approval for the study was obtained from the
local Youth Justice Board (YJB) and the prison service. The methods in this study are identical to
those used in our previous study of alcohol prevalence rates in adult offenders in the North East of
England (Newbury-Birch et al., 2009b). A convenience sample of staff from YOTs and secure estates recruited young offenders into
the study during a one-month period in 2008. Staff administered questionnaire that included no
identifiable information from the young person. Cross-sectional designs entail the collection of data of a large sample and at a single point in time
in order to collect a body of data in connection with multiple variables which can be examined
to detect patterns of association (Bryman, 2004). It was agreed with participating staff that
collecting data over a one-month period would encourage maximum participation in
questionnaire administration without being too burdensome in the longer-term. For the secure
establishments this was March 2008 and for the YOTs September 2008. Due to staffing
constraints at the sites it was not possible to carry out the research in the same time period. Questionnaires were administered by criminal justice staff that had been trained by the research
team. This training included how to gather informed consent from young people and how to
administer and score the AUDIT tool as well as gather the information required to complete the
questionnaire. Criminal justice staff were involved to enable explanation of the questions when
needed by the young people. The ASSET tool 2 2015 j INTERNATIONAL JOURNAL OF PRISONER HEALTH Procedures All staff in the participating sites that came into contact with the young offenders were asked to
complete a questionnaire with every consenting young offender they engaged with during the
study period. Staff asked the questions and completed the questionnaire with the respondents’
answers. Files were marked to ensure that adolescents were only asked once to participate in
the study. However there is a small possibility that a young offender asked in the YOT could have
been asked in the secure estate also. The inclusion criterion was all adolescents in the age range
who had not already completed the questionnaire (as marked on their file). In accordance with
ethical guidelines (Shaw et al., 2011), the adolescents were informed by the staff at the sites that 78 j INTERNATIONAL JOURNAL OF PRISONER HEALTH j VOL. 11 NO. 2 2015 their participation was voluntary and would not have any effect on their usual care. By returning
the anonymous questionnaire filled in, participants were told that they were deemed to have given
consent to participate in the study. Completed questionnaires were stored securely in each of the
sites and were collected regularly by members of the research team. Sample In total, 429 questionnaires were completed with young offenders by the YOTs and secure
establishments during the study time period. In total, 18 of these questionnaires were excluded
(12 who were aged 18 or over; six with no age given). Of the 411 that were included in the
analysis, 227 (55 per cent) were from the YOT and 184 (45 per cent) from secure
accommodation. The response rate was 67 per cent (97 per cent secure accommodation,
n ¼ 189; 53 per cent YOTs, n ¼ 240/450). Demographics In total, 85 per cent of participants were male (n ¼ 349) and 15 per cent female (n ¼ 60). Two
questionnaires did not have gender recorded. This is a higher proportion of males: females than
the proportion of young male offenders recorded for the North East in the CJS in the region in
2008-2009 (75 per cent male) (Youth Justice Board, 2010). The majority of the females were from
the YOT setting (93 per cent). In total, 97 per cent who gave their ethnicity described themselves
as white or white British which is comparable with the general population in the North East of
England at the same time point (Office for National Statistics, 2011). The age range of participants was 11-17 years with 6 per cent (n ¼ 26) aged between 11 and 13;
25 per cent (n ¼ 100) aged 14 or 15 and 69 per cent (283) aged 16-17 (two did not give age). Analysis plan The data were subsequently entered into SPSS. Analysis was undertaken using the statistical
software package PASW 18 (SPSS Inc.). Offences were categorised as either violent (assaults,
robbery, rape, possession of offensive weapons or causing death) or non-violent (affray, arson,
burglary, criminal damage, drugs, motor offences, theft, trespass, perverting the course of justice
and public order) as has been used in previous criminal justice studies (Sherman et al., 2007). The
statistical analysis was primarily descriptive and focused on the prevalence of alcohol misuse;
AUDIT scores less than 2 for low-risk drinkers, 2 for hazardous or harmful drinkers and 3 or more
for probably dependent, and their relationship to offence type and ASSET score for substance
misuse (a score of 2 or more). Where appropriate, comparisons were made across different
demographic groups of adolescents. χ2 statistics were used to assess categorical data. We
explored the sensitivity, the number of true positive cases identified, and specificity, the number
true negative cases identified, of ASSET score of 2 or more vs AUDIT score of 2 or more as an
indicator of AUDs and AUDIT score of 2 or more vs. Binge drinking derived from question three of
the AUDIT which relates to binge drinking (How often do you have six or more standard drinks on
one occasion) was used (Saunders et al., 1993). AUDIT results In total, 25 of the 411 (6 per cent) respondents did not complete the AUDIT fully, therefore 386
(male 329; 85 per cent; females 55; 14 per cent) individuals with an AUDIT score and age
recorded were included in the analysis. The mean age of all included was 15.9 SD 1.2; median 16
(range 11-17). In total, 16 per cent reported that they had not drunk in the last year (50 per cent n ¼ 12/24 of
those aged between 11 and 13; 24 per cent n ¼ 23/95 of those aged 14-15 and 9 per cent
n ¼ 25/267 of those aged 16-17). Those with a likely AUD of the highest severity (probably
dependent) were high in all age groups (33 per cent n ¼ 8/24 of those aged between 11 and 13;
62 per cent n ¼ 59/95 of those aged 14-15; 86 per cent n ¼ 229/267 of those aged between
16 and 17 (Table I). VOL. 11 NO. 2 2015 j INTERNATIONAL JOURNAL OF PRISONER HEALTH j PAGE Table I AUDIT scores by age (using young people’s cut-offs of 2+ for hazardous harmful
and 3+ for possible dependence)
Abstainers
Low risk
Hazardous/harmful
Probably dependent
Total AUD
Age
n
%
n
%
n
%
n
%
n
%
11-13 (n ¼ 24)
12
50.0
1
4.2
3
12.5
8
33.3
11
45.8
14-15 (n ¼ 95)
23
24.2
9
9.5
4
4.2
59
62.1
63
66.3
16-17 (n ¼ 267)
25
9.4
4
1.5
9
3.4
229
85.8
238
89.1
Total (n ¼ 386)
60
15.5
14
3.6
16
4.1
296
76.7
312
80.8
Notes: Not recorded: age ¼ 3 Table I AUDIT scores by age (using young people’s cut-offs of 2+ for hazardous harmful
and 3+ for possible dependence) Table I The mean AUDIT score was 13.3 (SD 10.6; median 12; CI 3-21). When the non-drinkers
were removed from the sample the mean AUDIT score was 15.8 (SD 9.7; median 14;
CI 8-23). Adolescent AUDIT cut-offs. Using adolescent cut-offs, 81 per cent of participants scored 2+ on
the AUDIT which identifies likelihood of an AUD. In total, 77 per cent scored 3+ on the AUDIT
which identifies a likely AUD of greater severity (probable alcohol dependence). Adult AUDIT cut-offs. Offence type In total, 309 (75 per cent) questionnaires had data recorded relating to both offence
type and AUDIT score. Significantly more offences were classed as non-violent offences
(n ¼ 170, 55 per cent) than violent offences (n ¼ 139, 45 per cent; χ2 5.825; df ¼ 1; p ¼ 0.01)
(Table III). In total, 84 per cent of the adolescents convicted of a violent offence scored positive for an AUD
(score of 2+) compared to 83 per cent of those convicted of a non-violent offence. This difference
was not statistically significant (χ2o0.0001; df ¼ 1; p ¼ 0.9866). However, significantly more individuals convicted of a violent offence (38 per cent) scored as
probable dependent (3+) compared to those with a non-violent offence (24 per cent) (χ2 6.094;
df ¼ 1; p ¼ 0.0136). Furthermore a significant difference was observed in mean AUDIT score
between violent and non-violent offenders; 14.92 vs 12.57 (p ¼ 0.03). All of those convicted of an
assault against a police officer, 78 per cent of those convicted of all other assaults; 81 per cent of
those convicted of robberies; 92 per cent of those convicted of use of an offensive weapon;
50 per cent of those convicted of rapes and 78 per cent of those convicted of violence-related
public order offences scored in the probable dependent range of an AUD for adolescents (3+)
using the AUDIT. AUDIT results Using adult cut-offs 64 per cent of participants scored 8+ on the AUDIT
which identifies likelihood of AUD (22 per cent hazardous; 12 per cent harmful; 30 per cent
probable alcohol dependence). Further to this, the percentage of adolescents who were likely to have an AUD increased from 46
per cent of those aged 11-13 to 66 per cent of those aged 14-15 to 89 per cent of those aged 16
or above (χ2 43.78; df ¼ 2; p ¼ o0.001). The full profile of responses (number and percentage) to
the ten AUDIT questions is displayed in Table II. ASSET scores Of the 429 questionnaires completed, 259 (60 per cent) had both an AUDIT and an ASSET score
for substance misuse available. In total, 24 per cent scored zero on ASSET (n ¼ 63); 23 per cent
had a score of one (n ¼ 62); 24 per cent had a score of two (n ¼ 65); 21 per cent had a score of
three (n ¼ 56) and 8 per cent had a score of four (n ¼ 20). Overall 30 per cent (n ¼ 77) of the
adolescents identified by AUDIT as at risk of an AUD were not identified as being at risk due to
their substance misuse using ASSET (score 2+) alone. The ASSET has relatively low sensitivity
(0.626) and higher specificity (0.793) (Table IV) at identifying AUDIT positives compared to PAGE 80 j INTERNATIONAL JOURNAL OF PRISONER HEALTH j VOL. 11 NO. 2 2015 q
1. How often do you have a drink containing alcohol? Never
(n ¼ 64; 17%)
Monthly or less
(n ¼ 77; 20%)
2-4 times a month
(n ¼ 90; 23%)
2-3 times a week
(n ¼ 82; 21%)
W4 times a week
(n ¼ 73; 19%)
2. How many standard drinks containing alcohol do you have on a typical day when you are
drinking? 1 or 2
(n ¼ 87; 23%)
3 or 4
(n ¼ 37; 10%)
5 or 6
(n ¼ 57; 15%)
7-9
(n ¼ 76; 20%)
10 or more
(n ¼ 129; 33%)
Never
Less than monthly
Monthly
Weekly
Daily or almost daily
3. How often do you have 6 or more standard drinks on one occasion? (n ¼ 98; 25%)
(n ¼ 57; 15%)
(n ¼ 57; 15%)
(n ¼ 112; 29%)
(n ¼ 62; 16%)
4. How often during the last year have you found that you were not able to stop drinking
once you had started? (n ¼ 257; 67%)
(n ¼ 41; 11%)
(n ¼ 20; 5%)
(n ¼ 35; 9%)
(n ¼ 33; 9%)
5. How often during the last year have you failed to do what was expected of you because of
your drinking? (n ¼ 235; 61%)
(n ¼ 51; 14%)
(n ¼ 43; 11%)
(n ¼ 39; 10%)
(n ¼ 18; 5%)
6. How often during the last year have you needed an alcoholic drink in the morning to get
yourself going after a heavy drinking session? ASSET scores This indicates that
this question alone has advantages over the ASSET as a screening tool for identifying likely AUDs
in this group of individuals. question 3 of the AUDIT (How often do you have six or more standard drinks on one occasion),
which shows high levels of sensitivity (0.917) and specificity (0.987) (Table V). This indicates that
this question alone has advantages over the ASSET as a screening tool for identifying likely AUDs
in this group of individuals. ASSET scores Discussion
This cross-sectional study found high rates of drinking amongst adolescents attending youth
justice settings in the North East of England and a high prevalence of likely AUDs as indicated
i
th AUDIT I t t l 81
t
f th
ff
d
i thi
t d
id
tifi d
lik l
Table III Offence types of included sample
YOT
PRISON
n
%
n
%
Violent offences
3
5.0
2
2.5
Assault PC
1
1.7
3
3.8
Death (murder or manslaughter)
25
41.7
6
7.6
Assault (no category given)
7
11.7
5
6.3
Offensive weapon
3
5.0
6
7.6
Public order (including violence)
0
0.0
4
5.1
Rape
3
5.0
24
30.4
Robbery
6
10.0
19
24.1
S18 or S20 assault
10
16.7
5
6.3
S47 assault
2
3.3
5
6.3
Total
60
100.1
79
100.0
Non-violent offences
Burglary
17
19.1
28
34.6
Public order (no violence)
13
14.6
7
8.6
Arson
2
2.2
2
2.5
Criminal damage
14
15.7
6
7.4
Drugs offences
4
4.5
3
3.7
Drunk and disorderly
3
3.4
1
1.2
Motoring offences
7
7.9
3
3.7
Theft
28
31.5
27
33.3
Trespass
1
1.1
1
1.2
Perverting the course of justice
0
0.0
3
3.7
Total
89
100.0
81
99.9
Table IV Sensitivity and specificity of ASSET score of 2 or more in identifying those with
AUD, AUDIT score 2 or more
Value (95% CI)
Sensitivity
0.626 (0.558; 0.689)
Specificity
0.793 (0.665; 0.880) Table III Offence types of included sample
YOT
PRISON
n
%
n
%
Violent offences
3
5.0
2
2.5
Assault PC
1
1.7
3
3.8
Death (murder or manslaughter)
25
41.7
6
7.6
Assault (no category given)
7
11.7
5
6.3
Offensive weapon
3
5.0
6
7.6
Public order (including violence)
0
0.0
4
5.1
Rape
3
5.0
24
30.4
Robbery
6
10.0
19
24.1
S18 or S20 assault
10
16.7
5
6.3
S47 assault
2
3.3
5
6.3
Total
60
100.1
79
100.0
Non-violent offences
Burglary
17
19.1
28
34.6
Public order (no violence)
13
14.6
7
8.6
Arson
2
2.2
2
2.5
Criminal damage
14
15.7
6
7.4
Drugs offences
4
4.5
3
3.7
Drunk and disorderly
3
3.4
1
1.2
Motoring offences
7
7.9
3
3.7
Theft
28
31.5
27
33.3
Trespass
1
1.1
1
1.2
Perverting the course of justice
0
0.0
3
3.7
Total
89
100.0
81
99.9 Table III Table IV Sensitivity and specificity of ASSET score of 2 or more in identifying those with
AUD, AUDIT score 2 or more
Value (95% CI)
Sensitivity
0.626 (0.558; 0.689)
Specificity
0.793 (0.665; 0.880) Sensitivity
Specificity question 3 of the AUDIT (How often do you have six or more standard drinks on one occasion),
which shows high levels of sensitivity (0.917) and specificity (0.987) (Table V). ASSET scores (n ¼ 310; 80%)
(n ¼ 35; 9%)
(n ¼ 9; 2%)
(n ¼ 13; 3%)
(n ¼ 19; 5%)
7. How often during the last year have you had a feeling of guilt or remorse after drinking? (n ¼ 261; 68%)
(n ¼ 65; 17%)
(n ¼ 28; 7%)
(n ¼ 25; 6%)
(n ¼ 7; 2%)
8. How often during the last year have you been unable to remember what happened the
night before because you had been drinking? (n ¼ 164; 42%)
(n ¼ 82; 21%)
(n ¼ 56; 15%)
(n ¼ 64; 17%)
(n ¼ 20; 5%)
Never
Yes, but not in the
last year
Yes, in the
last year
9. Have you or somebody else been injured as a result of your drinking? (n ¼ 214; 55%)
(n ¼ 37; 10%)
(n ¼ 135; 35%)
10. Has a relative or friend or a doctor or health worker been concerned about your drinking
or suggested you cut down? (n ¼ 233; 60%)
(n ¼ 15; 4%)
(n ¼ 138; 36%)
Note: n
386 VOL. 11 NO. 2 2015 j INTERNATIONAL JOURNAL OF PRISONER HEALTH j PAGE 81 question 3 of the AUDIT (How often do you have six or more standard drinks on one occasion),
which shows high levels of sensitivity (0.917) and specificity (0.987) (Table V). This indicates that
this question alone has advantages over the ASSET as a screening tool for identifying likely AUDs
in this group of individuals. Discussion The ASSET is
completed by workers who are not specialists in identifying alcohol and substance misuse and
the primary intention of ASSET screening is to identify factors linked to offending behaviour (either
disclosed by the young person or known to the YOT officer from previously recorded information). Thus it is possible that both the limited skill set of youth justice staff in identifying alcohol-related
risk or harm and the criminogenic focus of ASSET may be limiting factors for the full recognition of
alcohol-related problems in this setting as has been found elsewhere in the CJS (Newbury-Birch
et al., 2009b). Nevertheless, this study shows that even adding a single question from AUDIT
(question 3) to the ASSET might significantly increase the ability of youth justice professionals to
identify alcohol-related risk or harm in young offenders. However, the broader issue of whether
young offenders are willing to disclose details of a behaviour that might be linked to offending to
youth justice staff needs to be explored in more depth. There were a number of limitations to the data presented in this paper. The initial response from
the YOTs was low with only three out of a possible 11 YOTs agreeing to participate in the study,
impacting upon the representativeness of the sample of young people recruited. It is possible that
a small number of young offenders were asked at both a YOT and within the secure estate. Moreover, the research was only carried out in one geographical area of England and therefore
the results may not be indicative of the whole country. In addition, the administration of
questionnaires by youth justice staff may have influenced young offenders reporting of their
drinking behaviour. However, any suggestion that the young offenders may have been inclined to
under-report so as not to prejudice their future management in the CJS seems to be contradicted
by the high rates of drinking and likely AUDs reported. Furthermore, the ASSET substance
section includes alcohol and drugs so it is not possible to extrapolate data relating only to alcohol,
however, this is the current method of identifying young offenders with alcohol issues in England
and Wales. Lastly, the high response rates achieved during routine youth justice practice,
including 97 per cent of all adolescents in the secure estate, suggests that a realistic reflection
of young offender behaviour was achieved. Discussion This cross-sectional study found high rates of drinking amongst adolescents attending youth
justice settings in the North East of England and a high prevalence of likely AUDs as indicated
using the AUDIT. In total, 81 per cent of the young offenders in this study were identified as likely
having an AUD using a cut-off of 2+ on AUDIT and the likely AUD rate was also higher than that e V Sensitivity and specific of AUDIT score of 2 or more identifying any binge
consumption Sensitivity
Specificity 82 j INTERNATIONAL JOURNAL OF PRISONER HEALTH j VOL. 11 NO. 2 2015 VOL. 11 NO. 2 2015 reported for adults in the CJS in the North East of England (63 per cent) (Newbury-Birch et al.,
2009b). Moreover, 77 per cent of young offenders were identified as
probably dependent
(cut-off of 3+); a pattern of use associated with high levels of health and social risk. This study also
showed that the percentage of adolescents in youth justice settings who were likely to have an
AUD increased by age from aged 11 to 17 as in the general population, however, at much higher
rates at all ages. The general population rate for likely AUD in adults in England and Wales is 26 per cent
(Drummond et al., 2004). Indeed, the more conservative analysis (using adult cut-offs of 8+) of
alcohol-related risk or harm would have indicated that 22 per cent of adolescents were likely to
be drinking hazardously and 42 per cent likely to be drinking in the harmful or probable
alcohol-dependent range. These compare to general adult population rates of 23 per cent in the
likely hazardous or harmful range and 4 per cent in the probable dependent range (Drummond
et al., 2004). Furthermore, although numbers were small, the results showed that nearly half of
those aged 11-13 were categorised as likely having an AUD with a third of this age group being
classified as probably dependent to alcohol. This information is vital for staff training to deal with
severe issues with such a young age group. Significantly more adolescents convicted of a violent offence (compared to a non-violent offence)
scored within the probable alcohol-dependent range. The present study further showed that
30 per cent of all adolescents attending the YOT identified by AUDIT as likely to have an AUD
were not identified as having an alcohol-related need using the ASSET tool. Discussion Workload was the reason given by staff in the YOTs
for why some questionnaires were not completed. A key issue identified in this study related to the accurate measurement of alcohol-related risk and
harm in young offenders. Although the AUDIT has been identified as being the best tool to use
with adolescents (Cook et al., 2005), there needs to be further investigation of the validity and
reliability of the tool with younger age groups, particularly in a UK context (Reinert and Allen,
2007). In particular, it is important to investigate whether all the questions on AUDIT have the
same meaning (and signify the same categories of risk, harm and dependence) for adolescents
as they do for adults. In particular, the proportion of young offenders who showed probable signs
of alcohol dependence (75 per cent) in our sample was extremely high. Since adolescents have VOL. 11 NO. 2 2015 j INTERNATIONAL JOURNAL OF PRISONER HEALTH INTERNATIONAL JOURNAL OF PRISONER HEALTH PAGE 83 different levels of understanding compared to adults, and since young offenders often have lower
levels of educational attainment than non-offenders, it is possible that questions relating to
physical or psychological dependence may have been misinterpreted. Furthermore, behaviour
that is clearly indicative of alcohol dependence in adults (such as drinking alcohol in the morning
to prevent withdrawal effects) may not have the same meaning for adolescents. Nevertheless,
regardless of whether drinking in the morning occurs to prevent alcohol withdrawal or for an
entirely different reason still indicates a high degree of alcohol-related risk for adolescents and
possibly for recipients of alcohol-related crime and disorder. A further concern was the high levels of likely AUDs at all ages; with rates rising from 46 per cent in
11-13-year-olds to 89 per cent in 16-17-year-olds. This present study suggests that alcohol-
related risk or harm identified by the AUDIT tool in around 30 per cent of young offenders is not
correctly identified using the current systems and procedures in youth offending organisations. This
has significant implications for the YJB since the ASSET tool may be failing to identify significant
numbers of young offenders with alcohol-related problems using a tool that measures both alcohol
and drug use. In addition to compromising the well-being of young offenders, this is an important
workload issue. Problematic drinking behaviour is highly likely to increase the risk of future offending
behaviour. Discussion More research needs to be carried out in the youth justice system in order to identify the
best alcohol screening and assessment tool for use with different age groups of young offenders
who drink. Professionals in the youth justice system need more training in alcohol-related issues so
that they are more clearly aware of the link between alcohol and crime and better able to identify risk
of AUDs. Furthermore, there is a need for alcohol treatment services which are specifically geared
towards adolescents. All young people deserve access to the best possible care in order to give
them the best chance in life and a valuable opportunity is being missed for the many young
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Vol. 32 No. 1, pp. 16-29. Authors bibliography Dr Dorothy Newbury-Birch is Lecturer in Public Health Research at the Institute of Health and
Society, Newcastle University, Newcastle upon Tyne, UK. Katherine Jackson is based at the
Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK. Dr Dorothy Newbury-Birch is Lecturer in Public Health Research at the Institute of Health and
Society, Newcastle University, Newcastle upon Tyne, UK. Katherine Jackson is based at the
Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK. Tony Hodgson is based at the North East England Team, Youth Justice Board, Leeds, UK. Tony Hodgson is based at the North East England Team, Youth Justice Board, Leeds, UK. Professor Eilish Gilvarry is at the Northern Regional Drug and Alcohol Services, Northumbria,
Tyne and Wear NHS Trust, Newcastle upon Tyne, UK. Professor Eilish Gilvarry is at the Northern Regional Drug and Alcohol Services, Northumbria,
Tyne and Wear NHS Trust, Newcastle upon Tyne, UK. Dr Paul Cassidy is at the Teams Family Practice, Gateshead, UK. Dr Paul Cassidy is at the Teams Family Practice, Gateshead, UK. Professor Simon Coulton is at the Centre for Health Services Studies, University of Kent,
Canterbury, UK. Professor Simon Coulton is at the Centre for Health Services Studies, University of Kent,
Canterbury, UK. Professor Simon Coulton is at the Centre for Health Services Studies, University of Kent,
Canterbury, UK. Vicky Ryan, Dr Graeme B. Wilson, Dr Ruth McGovern and Professor Eileen Kaner are based at
the Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK. Vicky Ryan, Dr Graeme B. Wilson, Dr Ruth McGovern and Professor Eileen Kaner are based at
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Or contact us for further details: permissions@emeraldinsight.com AGE 86 j INTERNATIONAL JOURNAL OF PRISONER HEALTH j VOL. 11 NO. 2 2015 TERNATIONAL JOURNAL OF PRISONER HEALTH j VOL. 11 NO. 2 2015 VOL. 11 NO. 2 2015
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Assessing the competence of midwives to provide care during labor, childbirth and the immediate postpartum period – A cross sectional study in Tigray region, Ethiopia
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RESEARCH ARTICLE Methods A cross-sectional study design was employed to collect data through direct observation of
the performance of 144 midwives selected from 57 health facilities. Data were collected
from January to February 2015 by 12 experienced midwives who were trained on basic
emergency obstetric care and had previous experience with data collection. Using a stan-
dardized competence checklist, adapted from International confederation of midwives, data
collectors interviewed and directly observed the performance of midwives from admission of
laboring mothers to six hours after delivery. Multivariable linear regression was used to iden-
tify predicators associated with overall clinical competence of midwives. Editor: Cheryl A. Moyer, University of Michigan
Medical School, UNITED STATES
Received: March 27, 2017
Accepted: October 12, 2018
Published: October 31, 2018 Copyright: © 2018 Goshu 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. Result The mean competence score of midwives was found to be 51%. In multivariable linear
regression, male midwifery professionals (p = 0.022), availability of up to date job aids in
work place (p = 0.04) and being recognized for improved performance (p = 0.005) were sig-
nificantly associated with competence of midwives in the provision of care during labor,
childbirth and immediate postpartum period. Data Availability Statement: We don’t have any
legal restriction or ethical issue to share the data
for the journal. Therefore we have included the data
set using SPSS version in the supporting
information file. OPEN ACCESS The availability of a skilled birth attendant is widely recognized as a critical factor in reducing
maternal and newborn mortality. Competence of maternal healthcare providers directly
affects quality of care and health outcomes. This study assessed competence of midwives
and associated factors in provision of care during labor, and the immediate postpartum
period at public health facilities in Tigray, Ethiopia. Citation: Goshu M, Godefay H, Bihonegn F, Ayalew
F, Haileselassie D, Kebede A, et al. (2018)
Assessing the competence of midwives to provide
care during labor, childbirth and the immediate
postpartum period – A cross sectional study in
Tigray region, Ethiopia. PLoS ONE 13(10):
e0206414. https://doi.org/10.1371/journal. pone.0206414 Assessing the competence of midwives to
provide care during labor, childbirth and the
immediate postpartum period – A cross
sectional study in Tigray region, Ethiopia Miruts GoshuID1*, Hagos Godefay2, Fantaw Bihonegn1, Firew Ayalew3,
Daniel Haileselassie1, Abebe Kebede1, Girma Temam3, Gebreamlak Gidey4 Miruts GoshuID1*, Hagos Godefay2, Fantaw Bihonegn1, Firew Ayalew3,
Daniel Haileselassie1, Abebe Kebede1, Girma Temam3, Gebreamlak Gidey4 1 Jhpiego, Mekelle, Ethiopia, 2 Tigray Regional Health Bureau, Mekelle, Ethiopia, 3 Jhpiego, Addis Ababa,
Ethiopia, 4 Department of Midwifery, College of Health Sciences, Aksum University, Aksum, Ethiopia a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * Miruts.Goshu@jhpiego.org * Miruts.Goshu@jhpiego.org An assessment of competence of midwives in public health facilities for better performance were predicted competence. This requires attention and investment
from Tigray regional health bureau and health development partners working on maternal
and child health. Competence based in-service training, on-the-job mentoring, availing up to
dated standard job aids, recognition of high performing midwives are recommended to
improve the quality of maternity care in public health facilities of the region. Moreover, affir-
mative actions including on-the-job training and supervision are needed to improve the com-
petence of female midwives. of Ethiopia and the United States under the
Cooperative Agreement AID-663-A-12-00008. The
authors thank USAID for funding the study. of Ethiopia and the United States under the
Cooperative Agreement AID-663-A-12-00008. The
authors thank USAID for funding the study. Competing interests: The authors have declared
that no competing interests exist. Introduction The shortage of human resources for health in sub-Saharan African countries severely limits
the ability of countries to meet national and global maternal and neonatal health targets and
goals. [1, 2] The health worker shortages are further exacerbated by a lack of appropriate
knowledge and skills among the existing workforce. [3, 4] Ethiopia has a maternal mortality
ratio (MMR) of 412 deaths per 100,000 live births and a neonatal mortality rate of 29 deaths
per 1000 live births. [5] Maternal and Child health is at the center of the five year Health Trans-
formational Plan of the Ethiopian government, [6] and its Human Resources for Health strat-
egy has set a target to train 8,635 midwives by the year 2015. [7] The country has also set a
target to reduce its MMR to 199 per 100,000 live births by the year 2020. [6] A study conducted
in the Tigray region of the country estimated that the MMR for the region was 266 deaths per
100,000 live births [8], and skilled birth attendance coverage was reported to be 59%. [5] One
of the factors contributing to the high level of maternal and perinatal mortality and morbidity
is poor quality of care. [9] Without ensuring the availability of adequate numbers of competent
providers at health facilities, other strategies designed to reduce maternal, neonatal mortality
and morbidity cannot be effective. [10] In Ethiopia in general and in the Tigray region in par-
ticular, there is lack of evidence on the competence of midwives. This study explored the fol-
lowing research questions: 1) How competent are midwives in providing care during labor,
childbirth and the immediate postpartum period in public health facilities? and 2) What are
the factors associated with competence of midwives in provision of care during labor, and the
immediate postpartum period at public health facilities? Conclusion Funding: The study was made possible through
the Strengthening Human Resources for Health
Project, which is a five year (2012–2017) bilateral
cooperative agreement between the Governments Competence of midwives was found to be low to provide safe and quality maternity care in
the region. Male gender, availability of complete job aids and receiving recognition/awards PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 1 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 N = total number of midwives working in health facilities of Tigray region (N = 662). N = total number of midwives working in health facilities of Tigray region (N = 662). Since ratio of n/N is > 0.5 in the region, the sample required adjustment using the formula; Since ratio of n/N is > 0.5 in the region, the sample required adjustment using the formula;
n/ (1+n/N) and 10% nonresponse rate. Hence, the final adjusted sample size for inclusion
in the study was 144 midwives. Data from the Tigray regional health bureau suggested that an average of ten midwives was
employed at each public hospital, and three midwives at each heath center. Considering
resources and the total number of midwives available, a sample of four midwives per public
hospital and 2 midwives per health center was determined to be optimal for observation and
interview. Accordingly, four midwives were randomly selected in each hospital and two mid-
wives randomly selected in each sampled health center for observation. In order to get hetero-
geneous information, we divided the total estimated sample of 144 midwives into hospitals
and health centers using a proportional allocation technique. This resulted in a sample of 52
midwives in 13 hospitals and 92 midwives in 46 randomly selected health centers in the study. Thirteen out of the 14 existing hospitals were included; one hospital was excluded because of
geographic inaccessibly. The 46 sample health centers were randomly selected from a list of
total 214 health centers. Two health centers from the sample 46 health centers were omitted
from the study because there were no laboring mothers during the data collection period. Assessors invited randomly selected midwives to observe their performance when they were
providing labor and delivery services. Assessors did not observe midwives who were not
selected randomly even if she or he provided labor and delivery care. Additional midwives
were observed from hospitals to replace midwives working in health centers because of the
lack laboring mothers during data collection time at these health centers. Z1-α = 95% confidence interval with critical value of 1.96 Z1-α = 95% confidence interval with critical value of 1.96 SD = Standard deviation with value 15.3 Deff = Design effect value of 1.2 d = assumed margin of error with value 2.59 (or 5% of relative error from the mean score or
51.8), PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 Study design and sample size The study employed a cross-sectional design using direct observation of midwives while per-
forming labor and delivery, and postpartum care in public health facilities in Tigray region. There were a total of 228 health facilities (14 hospitals and 214 health centers) and 662 mid-
wives in the region during the study period. An optimum sample size was calculated to provide
regionally representative information using the following sample size calculation assumptions:
95% level of confidence, a design effect of 1.2, proxy reference values of 51.8% mean compe-
tence score of midwives, with standard deviation value of 15.3 [11], 5% of relative error from
percentage mean competence score (equivalent to 2.58 margin of error) and adding 10% non-
response rate. Based on these statistical parameters, an estimated sample of 144 midwives was
calculated as outlined below: Minimum sample size required ðnÞ ¼ Z21 aðSD2ÞDeff
d2
¼ 1:962X15:32X1:2
2:592
¼ 161 Where: Minimum sample size required ðnÞ ¼ Z21 aðSD2ÞDeff
d2
¼ 1:962X15:32X1:2
2:592
¼ 161 Where: 2 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 An assessment of competence of midwives in public health facilities Z1-α = 95% confidence interval with critical value of 1.96
SD = Standard deviation with value 15.3
Deff = Design effect value of 1.2
d = assumed margin of error with value 2.59 (or 5% of relative error from the mean score or
51.8), Data collection Twelve basic emergency obstetric care (BEmONC) trained expert midwives who had previ-
ously been selected by the regional health bureau to assess midwifery competence of graduat-
ing students in the region for another study were recruited to conduct the data collection. Before deployment, data collectors were trained for three days on how to obtain consent,
observation approaches, interviewing skills, and maintaining data confidentiality. During the
training they pre-tested the tools in four health centers and demonstrated their performance
in conducting the competence assessment of midwives using role play. observation approaches, interviewing skills, and maintaining data confidentiality. During the
training they pre-tested the tools in four health centers and demonstrated their performance
in conducting the competence assessment of midwives using role play. Data were collected by six teams with two data collectors and one supervisor in each team. Each team of data collectors was immediately deployed to assigned health facilities after the
training. Data were collected from January to February 2015. The data collection team spent a
maximum of two days at each health center, and a maximum of 4 days at each hospital. Four
co-investigators and two faculty members from Mekelle University supervised the field data
collection process, verifying that data collectors were recording data appropriately. The study
team first met the person in charge of each health facility and explained the purpose of the
study, presented letters of approval from the Regional Health Bureau and answered questions
before beginning any data collection. Study team members then met all eligible participants at
each facility and explained the study, and obtained verbal consent. Observations were documented using a standardized checklist on the various domains of
competencies. Midwives were directly observed while providing care during labor, delivery
and in the first 6 hours after birth. One labor and delivery procedure was observed per midwife
provided that the woman went through all stages of labor and delivery. If observation was
incomplete (that is the provider did not complete labor and delivery observation), data collec-
tors observed the performance of the midwife during her/his care of the next laboring woman. After the observation, midwives were interviewed using a structured questionnaire to cap-
ture socio-demographic characteristics and availability of an enabling environment for mater-
nity care. The interview took place at a time and place that was convenient for the midwives
and lasted approximately 15 minutes. An assessment of competence of midwives in public health facilities availability of relevant trainings, practice-based learnings, supervisory support, availability of
standard and up-to dated job aids, recognition/awards, infection prevention equipment and
supplies, medical equipment (such as sphygmomanometer, thermometer, delivery kits, episi-
otomy sets, vacuum, bag and mask for newborn resuscitation, medical supplies (like glove,
sutures), emergency medications and equipment (such as utero-tonics, magnesium sulphate,
IV solutions, needle, syringe), records and forms (Partograph, delivery log or register), a toilet
in the delivery area, and water in delivery area and new born care unit. The response options
for the availability of trainings, practice based learnings opportunities; supportive supervision,
job aids, equipment, supplies and infrastructure were “yes” or “no”. Instruments The contents of the midwifery competence assessment checklist were adapted from the essen-
tial competencies for basic midwifery practice defined by the International Confederation of
Midwives (ICM). [12] Twelve competence areas were included in the study to observe mid-
wives while attending laboring mothers. Each competence domain had a description explain-
ing what the competence criterion includes, which guided assessors to give appropriate scores. The competencies included 1) performing rapid initial evaluation at first contact; 2) introduc-
tion & history taking; 3) perform physical examination; 4) use of partograph to monitor labor
progress; 5) assist the woman to have a safe and clean birth; 6) providing immediate postpar-
tum care; 7) clinical judgment/ decision-making in providing care; 8) responding to problems/
irregularities if any; 9) infection prevention; 10) communication; 11) organization, efficiency
and team work; and 12) humanistic qualities/ professionalism. For each competence item,
assessors provided a rating ranging from 1 to 9 indicating unsatisfactory (1 to 3) when the pro-
vider did not perform the task or attempted to perform the task but was below expectation;
satisfactory (4 to 6) when the provider performed the task correctly & met expectations/mini-
mum standards, and superior (7 to 9) when the performance was above expectation and the
provider could be considered expert or a master, able to teach others. Midwives’ socio-demo-
graphic characteristics were included in the questionnaire, as were questions related to the PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 3 / 12 Midwives response about their working environment Majority of the midwives reported that there was regular supportive supervision and on-the-
job learning which included case presentations, seminars and bedside rounds. Only 38.2% of
the midwives reported that they had adequate equipment and supplies, 42.4% had a toilet in
the delivery room, 51.2% had water in the delivery room and 28.5% had received awards for
good performance (Table 2). Data management and analysis Epi-data version 2.0.2.13 was used for data entry and SPSS version 23 was used for further sta-
tistical analysis. Descriptive analysis performed to calculate frequencies and mean competence
percentages. Linear regression analyses was used to explore relationship of providers’ mean
competence score and predictors, including: background characteristics (gender, age, educa-
tion level, education program attended, facility type and experience), technical update training
in the last two years, equipment and supplies, availability of standard and up-to date job aids,
availability of forms and records, toilet, water, newborn care unit, regular supportive supervi-
sion, regular seminar/case presentation and award/recognition. Predictors in the bivariate PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 4 / 12 An assessment of competence of midwives in public health facilities linear regression models were selected as candidate predictors for Multivariable linear regres-
sion using p-value of 0.25. Before fitting the final model, regression model assumptions
including multicollinearity, normality, homoscedasticity and potential outliers were checked,
Based on these criterion, thirteen predictors were included in the final multivariable regression
analysis. A p-value of less than 0.05 was used for making statistical significance decision. Socio demographic characteristics of midwives The response rate of participants was 100%. Majority (70.8%) of study participants were
female, and had a diploma/level IV education (73.6%). The mean age of the participants was
33.9 ± 9.5 years, and 41.7% had five or more service years. Most (56.9%) were working at a
health center (Table 1). Ethical consideration The study protocol was approved by the Johns Hopkins School of Public Health Institutional
Review Board (IRB#6118). A letter of support was sent from the Tigray regional health bureau
to each participating health facility. Informed oral consent was obtained from all participants
(midwives and laboring mothers or their kinship) after the aim of the study was explained–the
consent form was translated to the local language, Tigrinya. The steps taken to preserve confi-
dentiality of information includes: de-identification of 07X aster, able to teach others bureau
to assess competence of midwives before graduation of students in the region. Personal infor-
mation, completed assessment study tools were kept in locked cabinet and electronic datasets
were protected with a password. PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 An assessment of competence of midwives in public health facilities Table 1. Characteristics of midwives, Tigray region, Ethiopia (N = 144). Variable
Number
%
Mean
SD
Range (Min, Max)
Facility type
Health Center
82
56.9
Hospital
62
43.1
Gender
Male
42
29.2
Female
102
70.8
Education level
BSc. Degree/Bachelor/
38
26.4
Diploma/level IV
106
73.6
Service year
less than one year
29
20.1
1 to<5year
55
38.2
> = 5years
60
41.7
Education program
Generic
67
46.5
Upgrade
77
53.5
Average deliveries per midwife per day
2.5
1.3
1, 5
Age in years
33.9
9.5
33 (20, 53)
SD: Standard Deviation
https://doi.org/10.1371/journal.pone.0206414.t001 Table 1. Characteristics of midwives, Tigray region, Ethiopia (N = 144). Overall and specific mean percentage competencies of midwives The overall mean competence score reflecting average competence across 11 areas was 51%. One competence area (responding to problems/irregularities) was excluded because very few
midwives were observed providing this service. The mean score was comparable for both facil-
ity type and education level, however, being male was positively associated with having a
higher competency score. The midwives scored highest in organization, efficiency and team
work (58%) followed by humanistic qualities/professionalism (57%). Their competence was
lowest in conducting rapid initial evaluation at first contact (41%) followed by use of parto-
graph to monitor labor progress (43%). Being male was associated with achievement of higher
scores in specific competencies including performing rapid initial evaluation at first contact of
women, introduction and history taking, perform physical examination, use of partograph to
monitor labor progression, assist the women during labor to have safe and clean delivery, pro-
vide immediate postpartum care and clinical judgment/decision making in providing care
(Table 3) 5 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 Factors associated with overall mean competence of midwives The bivariate linear regression model found that the mean competence score of midwives was
significantly associated with male gender, experience, availability of complete job aids, avail-
ability of water, conducting regular on-the-job learning and recognition for good perfor-
mance. Three variables: male gender (p = 0.022), availability of complete job aids (p = 0.04)
and recognition for good performance (p = 0.005) remained significant in a multivariable lin-
ear regression (Table 4). PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 Discussion Ensuring the equitable provision of quality maternal health services is one of the essential strat-
egies to reduce maternal and neonatal morbidity and mortality in Ethiopia. [5] Our study Table 2. Midwives’ positive responses on characteristics of their work environment, Tigray region, Ethiopia. Variables
Number (Yes)
Percent (%)
Availability of complete job aids
55
38.2
Availability of medical equipment and supplies
75
52.1
Availability of forms and records
71
49.3
Availability of a toilet in the delivery room
61
42.4
Availability of water in the delivery room
74
51.4
Availability of a new born care unit
78
54.2
Technical update training offered within the previous two years
98
68.1
Availability of regular supportive supervision
123
85.4
Availability of on-the-job learning opportunity
(case presentations, seminars and rounds)
118
81.9
Received recognition/awards for good performance
41
28.5
https://doi.org/10.1371/journal.pone.0206414.t002 able 2. Midwives’ positive responses on characteristics of their work environment, Tigray region, Ethiopia. 6 / 12 Type of competence
Percentage Mean
competence
Gender
Type of facility
Level of education
Male
Female P-value Hospital
Health
Center
P-value Bachelor
Diploma P-value
Perform Rapid initial Evaluation at first
contact
41.0
48.9
37.7
0.001
42.1
40.1
0.513
46.4
39.0
0.028
Introduction and history taking
45.0
54.4
41.1
0.000
46.1
44.2
0.523
51.8
42.6
0.007
Perform physical examination
52.0
56.7
49.4
0.019
50.3
52.4
0.474
54.1
50.6
0.271
Use partograph to monitor labor
progression
43.0
51.6
40.6
0.005
44.1
43.4
0.861
50.8
41.3
0.019
Assist the women to have a safe and
clean birth
55.0
59.8
53.2
0.022
52.9
56.9
0.125
55.6
55.0
0.860
Provide immediate postpartum care
50.0
55.8
47.4
0.001
50.0
49.9
0.952
50.6
49.7
0.724
Clinical judgment in providing care
53.0
60.3
50.0
0.000
51.7
54.1
0.335
55.2
52.2
0.299
Responding to problems if any (n = 28)
54.0
60.3
51.9
0.324
51.0
58.6
0.317
55.6
53.2
0.771
Infection prevention
51.0
52.1
50.4
0.571
50.7
51.1
0.893
51.2
50.9
0.914
Communication
52.0
60.6
55.9
0.100
56.1
58.1
0.417
57.3
57.2
0.979
Organization, efficiency and teamwork
58.0
61.3
57.0
0.068
57.3
58.9
0.473
58.2
58.3
0.970
Humanistic qualities/ professionalism
57.0
56.9
57.2
0.917
54.7
58.9
0.115
55.0
57.9
0.345
Overall mean competence
51.0
56.4
49.0
0.001
50.6
51.6
0.607
53.6
50.3
0.164
Independent sample t-test
https://doi.org/10.1371/journal.pone.0206414.t003
Table 4. Bivariate and multivariate linear regression analysis of factors associated with competence score of Midwives. Tigray region Ethiopia. Variable
Bivariate linear regression
Multivariate linear regression
Coefficient (95%CI)
P-Value
Coefficient (95%CI)
P-value
Male gender (reference: Female)
7.10(2.90, 11.3)
0.001
5.45 (0.79, 10.1)
0.022
Age (in years)
-0.20 (-0.40, 0.01)
0.062
-0.201 (-0.57, 0.17)
0.28
Education level (reference : Diploma)
2.80 (-1.63, 7.27)
0.212
2.31 (-2.57, 7.18)
0.35
Experience in months
-0.03 (-0.05, -0.004)
0.021
-0.01 (-0.04, 0.012)
0.31
Education program (reference : upgrade)
2.55 (-1.39, 6.48)
0.20
-4.22 (-10.96, 2.52)
0.22
Availability of complete job aid (reference : No)
6.21 (2.28, 10.13)
0.002
4.38 (0.198, 8.57)
0.04
Availability of forms and records (reference : No)
0.67 (-3.27, 4.62)
0.74
1.77 (-2.32, 5.87)
0.39
Availability of toilet in delivery room (reference : No)
2.76 (-1.20, 6.73)
0.17
-1.40 (-5.70, 2.91)
0.52 found that the overall mean competence score of midwives working in Tigray public health
facilities was low, which is consistent with other studies [11, 13–17] conducted in low resource
settings. An assessment of competence of midwives in public health facilities Table 3. Overall and specific mean percentage competence scores of study participants by gender, type of facility and level of education. Tigray region, Ethiopia. Type of competence
Percentage Mean
competence
Gender
Type of facility
Level of education
Male
Female P-value Hospital
Health
Center
P-value Bachelor
Diploma P-value
Perform Rapid initial Evaluation at first
contact
41.0
48.9
37.7
0.001
42.1
40.1
0.513
46.4
39.0
0.028
Introduction and history taking
45.0
54.4
41.1
0.000
46.1
44.2
0.523
51.8
42.6
0.007
Perform physical examination
52.0
56.7
49.4
0.019
50.3
52.4
0.474
54.1
50.6
0.271
Use partograph to monitor labor
progression
43.0
51.6
40.6
0.005
44.1
43.4
0.861
50.8
41.3
0.019
Assist the women to have a safe and
clean birth
55.0
59.8
53.2
0.022
52.9
56.9
0.125
55.6
55.0
0.860
Provide immediate postpartum care
50.0
55.8
47.4
0.001
50.0
49.9
0.952
50.6
49.7
0.724
Clinical judgment in providing care
53.0
60.3
50.0
0.000
51.7
54.1
0.335
55.2
52.2
0.299
Responding to problems if any (n = 28)
54.0
60.3
51.9
0.324
51.0
58.6
0.317
55.6
53.2
0.771
Infection prevention
51.0
52.1
50.4
0.571
50.7
51.1
0.893
51.2
50.9
0.914
Communication
52.0
60.6
55.9
0.100
56.1
58.1
0.417
57.3
57.2
0.979
Organization, efficiency and teamwork
58.0
61.3
57.0
0.068
57.3
58.9
0.473
58.2
58.3
0.970
Humanistic qualities/ professionalism
57.0
56.9
57.2
0.917
54.7
58.9
0.115
55.0
57.9
0.345
Overall mean competence
51.0
56.4
49.0
0.001
50.6
51.6
0.607
53.6
50.3
0.164
Independent sample t-test found that the overall mean competence score of midwives working in Tigray public health
facilities was low, which is consistent with other studies [11, 13–17] conducted in low resource
settings. This may be a reflection of approaches that on shorter-term solutions that aim to
increase the numbers of providers [18] rather than a focus on ensuring that they have the
required competencies. [3]. This low competence score of midwives in our study could be
attributed to the students’ perceived poor quality of health care providers’ pre service educa-
tion in Ethiopia [11,19] including gaps in availability of quality and adequacy of teachers,
Table 3. Overall and specific mean percentage competence scores of study participants by gender, type of facility and level of education. Tigray region, Ethiopia. An assessment of competence of midwives in public health facilities educational resources and the practical sites. In a study conducted in Addis Ababa, insufficient
in-service and pre-service trainings were also identified as barriers to providing quality basic
emergency obstetric and neonatal care. [20] The poor competence of midwives demonstrated
in our study underscores the urgent need to refocus on both pre service and in-service training
of midwives. Major international consortiums and initiatives, including the United Nations
Global Strategy for Women’s and Children’s Health [21] and the United States of America
government Global Health Initiative [22] have identified health workforce capacity building as
a key element in strengthening health systems and for achieving improved health outcomes,
particularly for women and children. Strategies common to programs for enhancing health
workforce capacity include workforce training, improved supervision, and the use of monetary
and non-monetary incentives. [23–27]. Female midwives scored lower than their male counterpart in certain areas. These results
are consistent with previous studies conducted in Ethiopia looking at graduating midwifery
[11] and anesthesia students.[19] These persistent findings of lower competence of female
health care providers requires further investigation to identify challenges faced by female
health care providers in gaining and maintaining the required skills and competencies. We found that the availability of job aids was significantly associated with improved compe-
tence of midwives. Our findings were consistent with studies conducted in Benin and Uganda
in which interventions focused on job aids were linked with positive outcomes on providers’
practices. [28–29] Moreover, recent evidence shows that job aids can serve health care provid-
ers as an acceptable, low-cost alternative to improve their performance when combined with
minimal training and supervision. [30–34] In line with this finding, it is important for the
Tigray regional health bureau and its stakeholders to avail relevant job aids in all health facili-
ties to guide midwives’ performance. The study also found that recognition for good performance is significantly associated with
higher competence of midwives. This finding is consistent with a Ugandan study [29] which
found that recognition of health care providers was associated with improved performance in
counselling. Therefore, performance appraisal linked with recognition needs to be strength-
ened as a means to improve performance of midwives in provision of maternity care in the
region. One limitation of the study is that the Hawthorne effect–the performance of the midwives
may have been altered because they were aware of being observed. Another limitation of the
study could be lack of national benchmarks to define the competence level of midwives. How-
ever, we believe that the findings are generalizable to the Tigray region, and provide useful
insights for policy makers and other stakeholders. PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 This may be a reflection of approaches that on shorter-term solutions that aim to
increase the numbers of providers [18] rather than a focus on ensuring that they have the
required competencies. [3]. This low competence score of midwives in our study could be
attributed to the students’ perceived poor quality of health care providers’ pre service educa-
tion in Ethiopia [11,19] including gaps in availability of quality and adequacy of teachers, Table 4. Bivariate and multivariate linear regression analysis of factors associated with competence score of Midwives. Tigray region Ethiopia. Variable
Bivariate linear regression
Multivariate linear regression
Coefficient (95%CI)
P-Value
Coefficient (95%CI)
P-value
Male gender (reference: Female)
7.10(2.90, 11.3)
0.001
5.45 (0.79, 10.1)
0.022
Age (in years)
-0.20 (-0.40, 0.01)
0.062
-0.201 (-0.57, 0.17)
0.28
Education level (reference : Diploma)
2.80 (-1.63, 7.27)
0.212
2.31 (-2.57, 7.18)
0.35
Experience in months
-0.03 (-0.05, -0.004)
0.021
-0.01 (-0.04, 0.012)
0.31
Education program (reference : upgrade)
2.55 (-1.39, 6.48)
0.20
-4.22 (-10.96, 2.52)
0.22
Availability of complete job aid (reference : No)
6.21 (2.28, 10.13)
0.002
4.38 (0.198, 8.57)
0.04
Availability of forms and records (reference : No)
0.67 (-3.27, 4.62)
0.74
1.77 (-2.32, 5.87)
0.39
Availability of toilet in delivery room (reference : No)
2.76 (-1.20, 6.73)
0.17
-1.40 (-5.70, 2.91)
0.52
Availability of Water in delivery room (reference : No)
4.32 (0.44, 8.20)
0.03
2.77 (-1.21, 6.75)
0.17
Availability of new born unit (reference : No)
3.79 (-0.12, 7.70)
0.057
0.48 (-3.65, 4.60)
0.82
Availability of regular supportive supervision (reference : No)
4.71 (-0.82, 10.25)
0.09
3.26 (-2.18, 8.71)
0.24
Availability of on job learning (case presentation, seminars and rounds) (reference : No)
6.35 (1.33, 11.37)
0.013
3.10 (-1.85, 8.06)
0.22
Received recognition/awards for better performance (reference : No)
6.31 (2.07,10.55)
0.004
5.75 (1.76, 9.75)
0.005
Variables significantly associated with mean competence of midwives (P-Value <0.05) in bivariate and multivariate analysis. https://doi.org/10.1371/journal.pone.0206414.t004 n analysis of factors associated with competence score of Midwives. Tigray region Ethiopia. PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 7 / 12 Acknowledgments We are grateful for the midwives, health facility leaders, and Tigray regional health bureau
experts who participated in the study, as well as expert midwife assessors who participated in
data collection. We extend our gratitude to Tegbar Yigzaw and Sharon Kibwana for their sup-
port during study design. Conclusion The competence of midwives working in public health facilities in Tigray was found to be low,
thus suggesting potential concerns regarding the quality of maternal and child health services
in the region. Male midwives, availability of complete job aids and receiving recognition for
good performance predicted competence. Urgent attention and more investment from the
Ethiopian Ministry of health, the Tigray regional health bureau and health development part-
ners working in improving maternal care is required to attain the 2015–2020 health sector
transformation plan [5] and sustainable development Goal (SDG) target of reducing MMR to
less than 70/100,000 [35]. Competence based in-service training, on-the-job mentoring, avail-
ing up to dated standard job aids, and recognition of high performing midwives is recom-
mended. Moreover, affirmative actions including on-the-job training and supervision are
needed to improve the competence of female midwives. 8 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0206414
October 31, 2018 An assessment of competence of midwives in public health facilities Supporting information
S1 File. Recruitment & oral consent script #1 (Tigrigna language translation). (PDF)
S2 File. Recruitment & oral consent script #2 (Tigrigna language translation). (PDF)
S3 File. Recruitment & oral consent script #1 to be read aloud to midwives. (PDF)
S4 File. Recruitment & oral consent script #2 to be read aloud to client (women in labor
and delivery). (PDF)
S5 File. Performance assessment of midwives in provision of care during labor, childbirth,
and immediate postpartum period in Tigray and Amhara regions, Ethiopia. (PDF)
S1 Dataset. SPSS data Competence Assessement of Midwives in Tigray health facilities. (SAV) S4 File. Recruitment & oral consent script #2 to be read aloud to client (women in labor
and delivery).
(PDF) S5 File. Performance assessment of midwives in provision of care during labor, childbirth,
and immediate postpartum period in Tigray and Amhara regions, Ethiopia. (PDF) S5 File. Performance assessment of midwives in provision of care during labor, childbirth,
and immediate postpartum period in Tigray and Amhara regions, Ethiopia. (PDF) S1 Dataset. SPSS data Competence Assessement of Midwives in Tigray health facilities. (SAV) S1 Dataset. SPSS data Competence Assessement of Midwives in Tigray health facilities. (SAV) References 1. Dreesch N, Dolea C, Roberto Dal Poz M, Goubarev A, Adams O, Aregawi M, et al. An approach to esti-
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newborn care services in Addis Ababa, Ethiopia; a cross sectional study. Author Contributions Conceptualization: Miruts Goshu, Hagos Godefay, Firew Ayalew, Daniel Haileselassie, Abebe
Kebede. Conceptualization: Miruts Goshu, Hagos Godefay, Firew Ayalew, Daniel Haileselassie, Abebe
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selassie, Abebe Kebede, Girma Temam, Gebreamlak Gidey. Formal analysis: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew, Daniel Hai-
leselassie, Abebe Kebede, Girma Temam, Gebreamlak Gidey. Funding acquisition: Miruts Goshu, Firew Ayalew. Investigation: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew, Daniel Haile-
selassie, Abebe Kebede, Girma Temam, Gebreamlak Gidey. Methodology: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew, Daniel Haile-
selassie, Abebe Kebede, Girma Temam, Gebreamlak Gidey. Project administration: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew,
Daniel Haileselassie, Abebe Kebede, Girma Temam. Resources: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew, Daniel Hailese-
lassie, Abebe Kebede, Girma Temam, Gebreamlak Gidey. Software: Fantaw Bihonegn, Firew Ayalew, Girma Temam. Software: Fantaw Bihonegn, Firew Ayalew, Girma Temam. Supervision: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Daniel Haileselassie, Abebe
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October 31, 2018 An assessment of competence of midwives in public health facilities Validation: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew, Daniel Hailese-
lassie, Abebe Kebede, Girma Temam, Gebreamlak Gidey. Visualization: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew, Daniel Haile-
selassie, Abebe Kebede, Girma Temam, Gebreamlak Gidey. Writing – original draft: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew,
Daniel Haileselassie, Abebe Kebede, Girma Temam, Gebreamlak Gidey. Writing – review & editing: Miruts Goshu, Hagos Godefay, Fantaw Bihonegn, Firew Ayalew,
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European Caricatures of Immigrants and their Ideological Loads as Determinants of Power Relations
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International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 4, July-August 2021 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 4, July-August 2021 www.ijassjournal.com ABDELKARIM BENLAAYOUNI Ibn Zohr University. Agadir/ Morocco The symbolic hegemony that stems from the problem of social production and the proliferation of social
class structures since Bourdieu and Althusser has attempted to understand the social structure and the
continuity of the social stratification wherein the interests of the majority and those who represent the lower
class are conflicting. In Europe, the failure of the proletarian revolution has made ideology more and more
central in the explanation of the reasons behind the continued stability of western capitalism despite the world
wars and the great depression. The two great concepts “hegemony” and “ideology” remain key factors in the
frameworks of consciousness and intellectuality. This has contributed in deepening and broadening the
sociological understanding of symbolic authority, power and power relations; by identifying the material and
non-mental elements in the dynamics of symbolic power mechanisms. Key words: Ideology.Power. Power Relations. Immigrants. Caricatures. Europe. The west 0. Introduction Critical Discourse Analysis, as a research tool,deals with the various social issues on the basis that it
considers discourse a social phenomenon making language a starting point for analyzing the power and
dominance. CDA stems from the idea that power and dominance are produced, used and treated in language and
via language. Language formulates the society and the culture and it is formulated through them. Language,
according to CDA, is not innocent as it is formulated according to the thought of its producer. Language varies
according to the embedded intentions of the sender and the impact he wants to conduct on the receiver. Therefore, in the same discourse the producer can exaggerate, complicate or simplify things. Consequently,
discourse is referred to in CDA as a social act and understanding it requires exploring the implicit and explicit
relations between language and the sociopolitical structures wherein it evolves and that it represents. Besides, CDA considers discourse as a changing historical truth. Language in CDA is an influential
social act that fulfills some ideological goals. Therefore, perception and conscious bridge the gap between the
text/ discourse and the world/ society. Ideology, power, history and critic are the fundamental bases of CDA. The connection between social psychology and discourse analysis, as two different but related disciplines,
clearly revealed the cognitive dimension that characterized the work of Wodak and Van Dijk throughout their
career. That was displayed in the introduction to the issue of Text related to the Third International Conference
on Language and Social Psychology as well as the conference about Discourse & Racism in 1987. They asserted
that “Discourse analysis has become sophisticated enough to allow a significant contribution to the analysis ABDELKARIMBENLAAYOUNI Page 212 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 4, July-August 2021 www.ijassjournal.com of serious problems, such as the dominance and inequality inherent in racism. This is but one of the many
directions a critical discourse analysis may (and in our view, should) take in the years ahead”. (Van Dijk
&Wodak, 1988 a: 4) So, in the mid 1980s the larger dimensions of critical discourse analysis were traced by the
three authorities: Fairlough (1985) and Van Dijk 1986 Racism and the press as well as Wodak (1986) Language
Behavior in Therapy Group. They made of power and ideology a starting point for their researches and
publication. 0. Introduction Wodak suggests four basic concepts for any critical discourse analysis: “ Four concepts figure
indispensably in all CDA: the concepts of critique, power, history, and ideology”(2007: 209). These concepts
are employed by the specialists in CDA in dealing with issues like dominance, injustice, gender differentiation,
social classes etc. Van Dijk (2001) also claims that these concepts are important because of the nature of topics
and the methodology of Critical Discourse Analysis. On another hand, the relationships between social groups are still governed by some ideologies and
relations of power that are manifested in different forms. Media are influential factors in the incarnation of these
ideologies, defending some and/ or refuting others. TV, newspapers, magazines and radio stations contribute in
the production of the collective opinion and they are governed by the ideologies of the politicians and decision
makers. Thus, they play an important role in framing, shaping and directing the public opinion. A quick overview of the historical reasons underlying the production of caricature as a revolutionary
model proves that it has reached a level of containment to the extent that analyzing a painting implies addressing
a group of factors including the cartoonist, the publisher, the recipient audience, the social structure, and the
historical era through which the painting was produced. Since its appearance, caricature has merged with a
variety of genres that form a force aligning with the pulse of society. Caricature confirms the problematic and suspicious relationship between art and authority. Since its
emergence, caricature carried the seeds of liberation of the artist from the subordination of the artist to the power
of money and politics. Thus, caricaturing underlined its dedication as an art that has a large audience
background and a great impact on people. The caricaturists’ mission, that draws its legitimacy and strength from
the popular background, became more and more oriented to mobilization, education, sensitizing and criticizing. Caricaturing benefited from the public’s increasing interest in political and social life and it became more
oriented to show exaggeration and distortion. Moreover, it focused on the reality and on events rather than on
individuals. Consequently, special interest arose from the political authority in this art to the extent that some
cartoonists began cooperating with politicians and decision- makers in promoting the policies intended to guide
the people and leading them to accept, confirm and coexist with some decisions and attitudes. 0. Introduction Caricaturists
started portraying some national symbols, as did Thomas Nast who created the initial form of Uncle Sam, which
was repainted later in 1917 in its familiar form by artist James Montgomery Flag. The First World War was a turning stage in the function of caricature. At that time the world witnessed
the emergence of political propaganda offices, and their coverage of the conflict in the various warring
countries. And that was marked with a change in the job of a cartoonist whose role used to be restricted to
criticizing government institutions, the army, and the church, to become an agent of propaganda. Caricaturists
later fell into great confusion whether they should pursue their criticism of state policies, or engage in the
national defense. Nevertheless, many factors stand as authoritarian in front of the caricaturists and their right to freedom
of expression. These barriers include political, religious and societal factors related to customs and conventions. ABDELKARIMBENLAAYOUNI Page 213 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com Thus appeared the red lines as the holocaust, which has always been considered forbidden to be approached. Similarly, mocking the sacred Christian symbols was always considered immoral. And this is going to be
confirmed later with the debate raised by the Danish cartoons that were considered offensive to the Muslims
sacred identity. Therefore, caricature acquired power and had a great authority as a means of expression. Caricature
became associated with power and became a means of perpetuating some ideologies and power relations in
society. The caricatures became loaded with messages and are driven by the vision and attitudes of some parties
that mean establishing some norms in society. For this reason we will restrict this article to explain the
embedded ideologies of caricatures about immigrants and how they contribute in creating and supporting some
power relations between immigrants and the host communities. The media landscape has witnessed great changes, which were manifested in the variety of tools used
to convey messages. Such variety has obviously affected the content and the way it is received by the audience. Political cartoons are one of the media tools used to convey political messages and they have been great markers
of ideologies. 0. Introduction Caricatures are based on specific ideologies and they aim at expressing the embedded messages
and the ideologies hidden between the lines and they are very influential in terms of both the content and the
delivery. aim of ideology is to “organize human masses and create the terrain on which men move, acquire
consciousness of their position and struggle” (Gramsci 1971:160). aim of ideology is to “organize human masses and create the terrain on which men move, acquire
consciousness of their position and struggle” (Gramsci 1971:160). Critical discourse analysis aims at revealing the embedded ideologies that are manifested in language. These ideologies are expressed through language and they are built into practices. Hence, they are
manifestations of the relations of power and control within a society. That is to say ideologies are "assumptions
which are built into practices which sustain relations of domination, usually in a covert way." (Fairclough:
2004: 112). Practitioners in Critical discourse Analysis have built on the above basis in analyzing the discourses
and they aimed at showing the embedded ideologies of discourses calling. Fairclough insisted on the role of
languages in constructing ideologies and he claims that texts are basically loaded to the extent that “meanings
are produced through interpretations of texts” (1992: 89). He explains the interrelationships between language
and society on the basis of an of power and ideology which justifies critical aim of CDA: “language connects
with the social through being the primary domain of ideology, and through being both a site of, and a stake
in, struggles for power.” (Fairclough, 1989: 12) Van Dijk connects critical discourse analysis with the dimensions of power abuse and inequality of
discourse: “we shall simply, and perhaps naively, summarize such criteria by saying that in our opinion CDA
should deal primarily with the discourse dimensions of power abuse and the injustice and inequality that
result from it.” (1993:252) He considers ideologies to be important worldviews that contribute in the
construction of the social cognition: “schematically organized complexes of representations and attitudes with
regard to certain aspects of the social world, e.g. the schema ... whites have about blacks’ (1993b: 258). Critical Discourse Analysts call for questioning and reconsidering these ideologies in discourse. They
don’t stop at the surface level of ideology as we stated above; but they deal with the deeply hidden aspects of
ideology that are related to people’s everyday beliefs. CDA is concerned with the mental representations of
different phenomena that are to be interpreted within the social and cultural background of these societies. Providing clear analysis to the texts and discourses that are ideologically embedded is a duty of the scholars
because neglecting the critical aspect of analyzing discourses risks them to reproduce the ideologies of the
language. aim of ideology is to “organize human masses and create the terrain on which men move, acquire
consciousness of their position and struggle” (Gramsci 1971:160). Fairclough insisted on this point stating that, “unless the analyst differentiates ideology from
knowledge, i.e. unless s/he is aware of the ideological dimensions of discourse, the chances are that s/he will
be unconsciously implicated in the reproduction of ideologies, much as the lay subject is.” (1985: 755) Notwithstanding, insisting on the importance of analyzing ideologies does not imply that the analysis is
for the sake of revealing and understanding their social effect only but also and primarily for enacting a change
in society because they do not represent society: "Ideologies are often false or ungrounded construction of
society." (Fairclough &Wodak: 1997:275) She also considers ideologies to be a vision to reality from one side
perspective and that they are shared by members of a group to enact unbalanced power relations via discourse:
“From the point of view of the DHA, ideology is defined as an (often) one-sided perspective or worldview
composed of related mental representations, convictions, opinions, attitudes, and evaluations. Ideologies are
shared by members of specific social groups. Ideologies serve as an important means of establishing and
maintaining unequal power relations through discourse” (Wodak 2015: 4) I.
Ideology in Critical Discourse Analysis: Moreover, the ABDELKARIMBENLAAYOUNI Page 214 International Journal of Arts and Social Science www.ijassjournal.com
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aim of ideology is to “organize human masses and create the terrain on which men move, acquire
consciousness of their position and struggle” (Gramsci 1971:160). International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 4, July-August 2021 www.ijassjournal.com www.ijassjournal.com aim of ideology is to “organize human masses and create the terrain on which men move, acquire
consciousness of their position and struggle” (Gramsci 1971:160). I.
Ideology in Critical Discourse Analysis: The concept “ideology” has undergone different uses since it was first coined by Destutt De Tracy in
1976. Napoleon Bonaparte, Marx and Althusser used the term ideology before the scholars of CDA do. Pêcheux, Van Dijk, Fairclough, Wodak and Billig went further than analyzing the surface level of ideology. Rather, they were concerned with the daily beliefs that determine discourse. Ideology is the concept or set of concepts embedded in what people say or write. It refers to the set of
beliefs and values shared by a group of people. Fowler insists that language is a means of restructuring
ideologies "language is not a clear window, but a refracting, structuring medium." (Fowler in Richardson:
2004: 53). These shared beliefs and values are represented by language and they are used to create some
relations between the different groups of society namely the dominating and dominated ones. According to
Fairclough and Wodak ideologies are “ways of representing and constructing society which reproduce
unequal relations of power, relations of domination and exploitation."(1997: 275) Ideology is one of the basic concepts in Critical Discourse Analysis and it occupies an important
position in the analysis of the different discourses. It is a set of idea or beliefs that individuals or groups of
people hold. These ideas are closely related to the awareness and the consciousness of people and they are like
intermediaries between the language and the surrounding world. Fairclough considers ideologies to be
“constructions of reality ... which are built into various dimensions of the forms/meanings of discursive
practices, and which contribute to the production, reproduction or transformation of relations of
domination”. (1992: 87) One of the basic objectives of analyzing discourse critically is to show the embedded ideologies of
discourses. The researchers in CDA benefit from the great contributions of the social theory about ideology
namely with Louis Althusser (1918- 1990) who claimed that individuals have a fake awareness of their reality
and this prevents them from perceiving the true nature of their social and economic situation. This hinders their
attempt to call for their rights. Besides Antonio Gramsci spoke about dominance and consensus are two
fundamental concepts to ideology. He claims that modern societies are not controlled by force; rather they are
dominated by some frames and patterns that are shared by individuals within a given society. II.
Ideology in Caricatural Discourse Investigating political cartoons dealing with immigrants in some influential European newspapers and
magazines (paper and online ones) shows that they target different issues related to a specific category of
immigrants each time. In some cases, the caricature is gender oriented; depicting Arab and Muslim men and/ or
women. In others, the subject matter of the caricature is the migration trip itself, while some other caricatures
deal with religion as a differentiating point of the immigrants’ identity etc. Immigrants in general and Islam as a
marker of these Arabs who move to live in European countries are commonly classified within the generalized ABDELKARIMBENLAAYOUNI Page 215 Page 215 International Journal of Arts and Social Science
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been shaped by the perception that Islamic culture represses women, encourages intolerant fundamentalism
(a term that was originally associated with a twentieth-century Protestant movement in favor of literal Bible
interpretation), and incites terrorism.” (Nathan etal. 2004: 9) For these reasons, analyzing caricatures about
immigrants should be conducted through the analysis of the different categories their subjects fall into. Therefore, they can be broadly classified into six main categories: the migration journey, politics, religion,
women and children’s right, sex and social issues and everyday behavior. But in our article, we are more
concerned with the issues where ideologies seem to be in conflict and more and new power relations are being
established through the drawings. The image and the context are two main factors that enable an understanding of the subject matter portrayed
by the drawing. The caricature creates satire through parodying the reality (individuals or events) in a particular
context and this is conducted in different ways such as exaggerating some features in artistic ways. The attitudes of the world leaders, including Arab and Muslim ones, towards immigration and the
immigrants’ issue are widely depicted in caricature. They have represented a considerable data when criticizing
how these immigrants die in the sea while leaders show no interest. Leaders are depicted in many cartoons as
being responsible for the crisis and they are accused by carelessness and irresponsibility towards immigrants. II.
Ideology in Caricatural Discourse Below (Figure 1) is a cartoon that is very specific in its relationship to reality that it maintains with history, time,
wars etc. Figure 1 Figure 1 This caricature depicts two European leaders in an unbalanced gambling scene: the French ex-president
FrançoisHollande playing cards with the German chancellor Angela Merkel. Refugees and immigrants, most of
them are head covered women and men with beards and turbans carrying their luggage and kids, are the stake in
the game. Merkel seems to have more (immigrants) near her while Hollande apparently has only one single
family. Similarly, he holds only one card; and Merkel is still holding many cards. Moreover, Hollande is
depicted with big bulging eyes and red cheeks. Both of them are sweating and FrançoisHollande is holding a
handkerchief and drying his sweat. With a big “F”, the caricature is entitled Full and an accompanying linguistic
message over Merkel’s head saying (Heu!... JeBluffais) meaning I am bluffing and one above Hollande’s
picture stating (Oh! Puree! préfère!); Oh I wish! ABDELKARIMBENLAAYOUNI Page 216 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com The above caricature is loaded with ideological connotations that are related to the time and place
dimensions. First of all, the repository of this caricature is the citizen who constructs its meanings: (German and
French) everyone engages in the reception of this caricature of multiple skills such as the vision of the world,
the perception of human values have become a game of vulgar language between two political leaders of two
European powers. The choice of the two countries is very meaningful and hints at the two socio-economic and
political movements: socialism of France and the nationalist socialism of Germany. The position as well as the
language and the visual icons of the cartoon state that the migration crisis exposes and unveils the fake figure of
the French socialism and the republic’s motto Liberty, Equality, Fraternity. The caricature favors Germany’s
human aspect namely in the immigration crisis. The two short sentences accompanying this caricatureare loaded with ideological-political
connotations: First the interjections HEU !!!!! then Merkel's first person pronoun “I ” in (JE BLEUFFAIS! I am
bluffing) implies that her words lack truth and credibility. II.
Ideology in Caricatural Discourse There is a sort of stupidity and absence of seriousness
that characterizes the discourse of political decision-makers namely when it comes to the lives of men, women,
children and old men . The lives of thousands of people become a card game for some leaders
. The same
interpretation for Holland when using the first person pronoun "I" in (Jepréfère I wish) implying that he wishes
Merkel was just bluffing because he is unable to fulfill his promises towards the immigrants. His words describe
the hypocritical socialism of which the French republic keeps only the label. Holland who seems to have lost all
his cards is in fact fantasizing about the serious decisions he is unable to take. His wish is that Merkel would be
incredible as he is already incredible. She is still holding many cards and she has lots of problems as well in
reference to the elections that were going on then. On the contrary, Hollande was unable even to run for the
second term of elections in France. The interjection “OH! PUREE!" is an informal jargon, which does not fit the situation as it comes from
a person who does not have the concepts to deliver a coherent and humane discourse on an alarming situation,
which has raised a great political and controversial problem. European caricatures insist on showing the Arab Muslim leaders in the same position as European ones
in the degree of carelessness, inhumanity and irresponsibility towards immigrants. Below is a caricature by the
Dutch cartoonist JoepBertrams about Greece Turkey EU and refugees. The Turkish president Tayyib Erdogan is shown in a Ping-Pong game while he is shooting a woman
with her baby towards Europe. Turkey is playing a political game with Europe through its representative
Greece, as the rackets show, and he seems to be in a position of attacking. The sport circuit is divided into two
halves with Barbed wires. ABDELKARIMBENLAAYOUNI Page 217 Page 217 International Journal of Arts and Social Science
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Figure 2 Figure 2 Figure 2 These ABDELKARIMBENLAAYOUNI Page 218 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com static representations of the Arab and Muslim immigrants in the 20
th
and 21
st
century represent a sort of
projection from the history of the western sense of superiority dating back to the colonial era. Caricatures of this
century are merely a continuity of the static stereotyped image of the west towards the east. static representations of the Arab and Muslim immigrants in the 20
th
and 21
st
century represent a sort of
projection from the history of the western sense of superiority dating back to the colonial era. Caricatures of this
century are merely a continuity of the static stereotyped image of the west towards the east. European caricatures do present Arabs and Muslims issues through their western European context and
ideology. This ideology has a premeditated conception of the Arabs and Muslims as inherent fanaticist,
fundamentalist, unreasoning and violent. Arabs and Muslims are automatically and instantly suspected even
though they are not involved in some terrorist acts. For instance the Oklahoma City bombing in 1995, the term
terrorism was used during the investigation and the whole act was everywhere described as a terrorist attack
conducted by a terrorist who wanted to spread terrorism etc. The terrorism character of the event continued until
the detention of the real attacker who was simply the American Citizen Alfred P. Murrah. It was proved that he
was neither Arab nor Muslim. It is then that the word terrorism was replaced by the term offense. And all media
replaced terrorism by violence, offence and similar light depictions. The Arab and Muslim immigrants have gone through a variety of issues that have been interpreted
beyond the literal meaning of the conflict between the host and the guests’ culture. Beside other issues, the
immigrant women and their freedom of clothing have raised lots of controversies. The burkini ban and the anti-
hijab campaigns are standpoints of the conflict between the east and the west; the Arab-Muslim ideology and the
European one. When France decided to ban the Islamic swimming suit (burkini), it was argued that the aim is to
protect the Muslim woman from the injustice of the patriarchal social customs. Yet, as Terrence G. Figure 2 The caricature above puts Turkey and Europe in the same position in front of their responsibility
towards immigrants. Both parties are treating immigrants as a game; a political game. While Turkey is
expelling; pushing immigrants with all his force and his facial expressions show the degree of anger and the
great efforts he is doing to push the lady and her baby, the European continent does not seem to be receiving
them. The Greek player is in the other side of the stadium letting the lady and her baby fall in the other side. Neither turkey nor Europe seem receiving the immigrant woman and baby who are about falling on the
dangerous iron fences on the borders. In this caricature, Turkey with its Islamic background is being attacked
more in the immigrants and refugees’ crisis namely that the lady shot is in Islamic attire and she refers to the
Syrian refugees. The blond lady who refers to Europe in general is helpless in front of the immigrants’ attacks
from turkey borders and this is a way of blaming the Arab and Muslim countries for the migration crisis in
Europe. The western view to the Arab world and the Arab Muslim identity has not changed since the first
contact between the two cultures and civilizations. The Arabs and Muslims are still referred to within the same
frames of the first ages of contact between the two civilizations. As is the case in cinema and literature,
caricaturists also tend to reproduce the same image of the Arab Muslim men and women, for instance, within
the same stereotypical frames wherein the males are depicted via some abusive images such as the pagan,
savage, rapist, kidnapper, fundamentalist and terrorist. Similarly, the Arab and Muslim woman is presented as a
sexualized belly dancer, a sexual fascination object, harem and now a terrorist whose mission is to give birth to
terrorists and satisfy their endless sexual thirst. These images are referring to the same thing and caricatures are
only changing labels in order to fit the historical context while the negative connotations are the same. Figure 2 Jews, in other 1https://www.nytimes.com/2016/08/19/world/europe/frances-burkini-bans-
are- are- about-more-than-religion-or-clothing.html?_r=1 (Last retrieved March 1oth, 2020) about-more-than-religion-or-clothing.html?_r=1 (Last retrieved March 1oth, 2020) ABDELKARIMBENLAAYOUNI Page 219 Page 219 International Journal of Arts and Social Science
ISSN: 2581-7922, www.ijassjournal.com Volume 4 Issue 4, July-August 2021 words, could never become German (or French, or English, etc.).” (Bunzl 2005: 502). The same thing is being
reproduced with the Arabs and Muslims immigrants who are considered “others” even if they were of the
second or third generations born and raised on European countries. words, could never become German (or French, or English, etc.).” (Bunzl 2005: 502). The same thing is being
reproduced with the Arabs and Muslims immigrants who are considered “others” even if they were of the
second or third generations born and raised on European countries. Another stance that reinforces this notion of superiority are the double standards of Europe concerning
the freedom of wearing clothes. The caricature below summarized the binary of attitudes in the west between
the Islamic veil and the Christian or Jewish head cover. Figure 3
As the caricaturist commented on the above image; “We run the risk of applying a double standard in Europe, a
standard where everything Muslim is oppressive and wrong, while everything Christian or Jewish is part of the
European tradition and history.” Figure 3 As the caricaturist commented on the above image; “We run the risk of applying a double standard in Europe, a
standard where everything Muslim is oppressive and wrong, while everything Christian or Jewish is part of the
European tradition and history.” As the caricaturist commented on the above image; “We run the risk of applying a double standard in Europe, a
standard where everything Muslim is oppressive and wrong, while everything Christian or Jewish is part of the
European tradition and history.” The case of the caricatures about the prophet Mohamed in the Danish newspaper is another flagrant
example of interest, political ideology and power of directing International public opinion about the issue. It is a
gross example of guiding the Muslim as well as the European public opinion in understanding and interpreting
the intended message of the cartoons and the whole issue. Figure 2 Peterson
called it: “These sorts of statements are a way to police what is French and what is not French,” (Cited in
Amanda Taub in The New York Times 2016)1. In fact, it is a matter of identity that is nurtured by the terrorist
attacks. This identity issue, incarnated in the Hijab and Burqa as well as burkini, dates back to the colonial
period when the veil was taken as a symbol of backwardness and idiocy compared to the European standard
dress of Europe that stands for superiority. It is all a matter of identity as explains Taub (2016) “The veil
remained a potent symbol of difference as colonialism collapsed after World War II and Muslims from
colonized countries flocked to France. But now, that difference was within a country trying to sort out its own
postcolonial identity.” (Ibid) The ideological differentiation between Europe and the other is historically embedded in the western
superiority not only through colonization campaigns as was the case in France, Italy and Spain but also in
Germany wherein the Nazi ideology played an important role in tightening the formulation of its fanatical
nationalist ideas by focusing on the “other” Jewish. In Germany, nationalism was defined as a return to race in
the sense that the Nazis viewed themselves as the protector of the nation- pure race. Consequently, the “other”
cannot belong to the national group, regardless of all the efforts made to reach cultural integration. This inherent
sense of purity and superiority kept being manifested in the German relationship with the other race including
Jews and the Arabs and Muslims who immigrate in Germany: “What was new about the late l9th-century
variant of Jew hatred was its anchoring in the notion of "race." A secular concept grounded in modernity's
striving toward rational classification, the idea of "race" gave Jews an immutable biological destiny. All of
this was connected to the project of nationalism, with the champions of anti-Semitism seeing themselves, first
and fore- most, as guardians of the ethnically pure nation-state. Given their racial difference, Jews could
never belong to this national community, no matter their strivings for cultural assimilation. Figure 2 So people and institutions are free to express their opinions, ideas and attitudes
towards all that happens around them. If freedom of expression is censored, trust is altered between the different
stakeholders and this would never contribute in the development of nations socially and economically. Media are largely concerned with the liberty of expression as the UNESCO Director-General Irina
Bokova said “Journalism thrives when media is free and independent, when journalists are safe to report,
when impunity is the exception,”2. Yet, freedom of expression comes at the intersection between what is legal
and what is ethical. Freedom of expression is and should never be a license to incite hatred and racist messages
as said the ex-UN General-Secretary, Kofi Annan. In the global world, information flow is limited neither by
national borders nor by the language specificity. This is more correct when it is related to pictures: the image
language is universal as it can be read and interpreted by everyone and everywhere. Sometime hate speech is
implicitly passed under the pretext of freedom of expression. Media sometimes do promote hate speech through
reporting the political discourse, which is filled with xenophobia, racism and intolerance against some peoples
or minorities in a particular society. That is why most countries that preserve the freedom of expression have
also passed some laws and regulations criminalizing hate speech. Lots of chapters of the criminal code of many
countries consider the dissemination of hate speech, racism, xenophobia and blasphemy a crime that should be
punished for. In Denmark for example freedom of expression is granted by the Danish constitution Grundloven and
exactly in section 77 wherein freedom of expression is determined as “Any person shall be at liberty to publish
their ideas in print, in writing, and in speech, subject to their being held responsible in a court of law. Censorship and other preventive measures shall never again be introduced.”3 Yet in the Danish criminal code,
exactly in section 140, blasphemy is prohibited and “Any person who, in public, ridicules or insults the
dogmas or worship of any lawfully existing religious community in this country shall be liable to a fine or to
imprisonment for any term not exceeding four months.” Moreover, the same code condemns any offender who
provokes violence and hatred speech among others with a fine or prison. Figure 2 The historical context wherein the Danish liberal- conservative alliance managed to replace the socio-
democrats, who had monopolized the political scene for 80 years, reveals the nature of the alliance and its
ideology based on privatization and liberal openness that was new to Danish people. An emergent need to shift
the interest of the public opinion from the social issues and shedding light on new issues was a smart strategy by
the politicians and the media in Denmark. Hence, immigrants/ foreigners were a suitable scapegoat to open a
new front of discussion on media and among people. The Dual rhetoric of the Jyllands-Posten newspaper and the Prime Minister Rasmussen as far as the
freedom of expression is concerned emphasizes the double attitudes towards immigrants in general and in the
cartoon affairs in specific. The same prime minister who refused meeting Muslim ambassadors to discuss the
cartoons crisis under the pretext of having no control over freedom of speech was the first to strongly condemn ABDELKARIMBENLAAYOUNI Page 220 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com www.ijassjournal.com the news that a Pakistani political party offered a reward to anyone who can assassinate any of the cartoonists of
the prophet which proved to be only a rumor. He had also supported the newspaper in March 2002 when it was
reproached by the Danish Press Council for violating the professional ethics when exposing the ethnic origins of
some people involved in a criminal case. Freedom of speech or freedom of expression comes among the fundamental human rights as it is
preserved in article 19 of the Universal declaration of Human rights. It is a fundamental component for
democracy and all peoples and governments are required to protect the right of expression both online and
offline. Freedom of speech is basic and it serves as a catalyst to other rights and to it are connected many rights
such as freedom of information, speech, press etc. it should not be referred to as a derivative right; rather it is a
right that really guarantees allthe other rights. Freedom of expression is compulsory for social, political as well
as economic growth of nations in the sense that the flow of information ensures innovation and supports
accountability and transparency. 2https://en.unesco.org/70years/freedom_of_expression 258
3The Criminal Code Order No. 909 of September 27, 2005, as amended by Act Nos. 1389 and 1400 of December
21, 2005
4 ABDELKARIMBENLAAYOUNI
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2https://en.unesco.org/70years/freedom_of_expression 258
3The Criminal Code Order No. 909 of September 27, 2005, as amended by Act Nos. 1389 and 1400 of December
21, 2005
4 Ibid International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com It’s clear that the legal supports against hate speech, xenophobia, racism and blasphemy in Denmark, as
in many European countries, is very rich and clear in terms of punishment and classifications of the violations. Besides, the Danish Media Liability Act of 1991 created the so-called “journalism ethics” which contains a set
of civil and criminal mandates to which media discourse must be consistent. The Danish Press Council is
allowed to punish violators with fines and imprisonment but that was not the case with the Jyllands-Posten
whose caricatures were full of hatred, accusation and xenophobia. In its annual report on Denmark, the European Commission against Racial Discrimination expressed
great concern about the prevalence of hate speech, xenophobia and intolerance regarding minorities. “The
general climate has continued to deteriorate in Denmark, with some politicians and parts of the media
constantly projecting a negative image of minority groups in general and Muslims in particular.”(3rd report. December 16th, 2005:4) Despite the big echoes and the violence that followed the Danish caricatures about the prophet of
Islam, many other newspapers and magazines, on top of which the French newspaper France Soir then Charlie
Hebdo, re-published the same cartoons loud-mouthing the right of expression and the freedom of speech. The
cartoons issue revealed that there was a sort of collusion from different parties in Europe: the media, politicians,
journalists and ordinary people who adopted the attitudes of the caricaturists. The debate was cleverly oriented and the whole issue was carefully structured so that the different
parties would excite each other. The editor-in-chief of the widely distributed Danish daily newspaper had lots of
difficulties decorating a book for children but he easily succeeded in publishing these cartoons that are not
definitely aimed at children. Flemming Rose, who was in charge of the cultural Corner of the newspaper, was responsible of the
alleged cartoons competition. He identifies himself as a Zionist Jew and he supports all the claims that the
Jyllands-Posten advocates Nazism and fascism since the thirties and forties. 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) Figure 2 The Danish law is very clear as far as
hate speech is concerned: it criminalizes any sort of dissemination of information threatening or insulting people
on their religions and beliefs among others. Section 266 b states that any person "who, publicly or with the
intention of wider dissemination, makes a statement or imparts other information by which a group of people
are threatened, insulted or degraded on account of their race, color, national or ethnic origin, religion, or
sexual inclination shall be liable to a fine or to imprisonment for any term not exceeding two years.”4 ABDELKARIMBENLAAYOUNI Page 221 Page 221 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 Flemming was a great supporter of
the anti- Islam theorist Daniel Pipes with his notorious quote “All Muslims are not Terrorists but all Terrorists
are Muslims” (2006: 1)5 Many observers claim that the cartoon crisis was avoidable if the prime minister of Denmark wished
but as it was not serving his political interests. He exploited the cartoons issue in supporting his strategy of
deepening the gap between civilizations and heightening the intensity of tensions in order to strengthen the
position of his political party internally and on the international level. It is true that the Danish economy was
affected by the boycott of the Arab Muslim world and it registered huge financial losses which were minor
compared to their political and ideological gains. The international public opinion and media divided between those who see the publication of these
drawings very normal and those who consider them provocative and hatred filled. Most of the European press
circles considered the caricatures about the prophet of Islam intended to embody the principle of intellectual and
journalistic freedom in Western societies rather than any hatred or offense. On the other hand, a large segment
of Arabs and Muslims as well as secular people found the caricatures loaded with provocation and xenophobia
from Islam and its sanctities. The surprising side of the issue is that the west has celebrated the anti- Muslim
caricatures not on the basis that they defended freedom of speech but because they approve the content of these
cartoons despite being filled with hatred and xenophobia. Defending freedom of speech is the pretext used in the
west to attack an individual or a marginalized group, as is the case of Arab and Muslim immigrants in Europe. 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) ABDELKARIMBENLAAYOUNI Page 222 Page 222 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com In light of this congested situation media managed to widen the gap between the two parts, which were
already far apart from each other. Supporters of each side conducted some pressure on the other: the Islamic
communities all over the world contested the caricatures even if they are deeply convinced that they do not
represent Islam. They are all aware that Islam is a religion of tolerance, peace and love. They know that the
prophet was a pioneer in dialogue and communication with his enemies before his supporters. Moreover, we are
all certain that the drawings were not reflecting the prophet, as they were ugly. The prophet Mohamed was
ranked second good-looking person after the prophet Joseph. On the other hand, the western media continued
supporting the Danish newspaper not from the principle of defending the caricatures per se or from the
significance and content they carry, but rather from the principle of defending intellectual freedom and the
liberty of expression. Nevertheless, the biased depictions of the European caricatures of immigrants
emphasizing the negative other have become markers of media reporting about immigrants in general claiming
the freedom of expression. Mazid concluded that the controversy made by these caricatures “epitomises the
struggle for control over discourse in that the debate is not overtly about anything other than the extent and
limits of freedom of expression” (2008: 35). The debate then shifted to whether the caricatures issue is a matter of clash of civilizations or a struggle
for liberties. Many parties from both sides renewed the old debate about cultural clash and the war of
civilizations while some media stand in the middle, as was the case of the Spanish newspaper "El Pais" which
warned against the exaggeration of criticism and despising as it expressed it support to the intellectual freedom
of expression. The issue was ambiguous: if the west considers criticizing a religion normal and inoffensive,
wouldn’t discriminating against practitioners of a particular religion and people affiliating to it racist and
offensive! It is for sur;e as is the case of Arab and Muslim immigrants in Europe. It is worth claiming that the caricatures’ issue was a culture, civilization and ideology issue in essence. 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) The Arab Muslim doctrines, that strictly forbid sculpture and any form of photography depicting religious
symbols namely prophets, was in confrontation with the west where no taboos and no limits should stop in the
way of freedom of expression including prophets and the companions. The debate was again directed and
presented as if the Arabs and Muslims are attempting to imposetheir vision on the west despite the cultural
differences. Yet, the real problem is not with the caricatures per se but with the way they present and draw the
personalities. In fact, the Muslims rejected not the caricatures of the prophet, but the connection between the
prophet, who was depicted with a turban that produced a bomb, and terrorism. The same contradiction can be found in different media in the west. For instance, the French Charlie
Hebdo is known for its biting satire. It basically targets governments, politicians and basically religion. The
magazine tends to tackle a variety of topics and issues. The background of Charlie Hebdo and the artists’
ideologies and orientation make of its drawings controversial. Charlie Hebdo is a magazine that is sort of
specialized in mocking institutions especially religion. It has specialized in mocking Islam and as its staff
always claims, their objective is to commodify Islam in the same manner as Christianity. The magazine defines
itself in its website6, as follows: “Charlie Hebdo is a punch in the face.... Against those who try to stop us thinking, against those who fear
imagination, against those who don’t like us to laugh. Charlie Hebdo is an angry magazine, a paper that
takes the piss. ... It’s a gazette of the grotesque – because that’s what so much of life and politics is... Charlie
Hebdo has no need of God, nor any need of Wall Street. Charlie doesn’t need two cars and three cellphones
to be happy. To be happy, Charlie Hebdo draws, writes, interviews, ponders and laughs at everything on this
earth which is ridiculous, giggles at all that is absurd or preposterous in life; which is to say - very nearly
everything... ” 6www.charliehebdo.fr/en/ ABDELKARIMBENLAAYOUNI Page 223 Page 223 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com Charlie’s satire is based on challenging taboos and offending under the label of “freedom of speech”. It
represents a tradition of satire that dates early in the French media. 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) And about its mission, Lebby Nelson wrote
in the Vox Magazine, “The magazine made fun of prominent politicians, religion, and pop culture, but it
lampooned Islam and Islamic extremists with particular zeal.”7 It is widely blamed for being racist,
islamophobic and it encourages hatred, homophobia and sexism. Most of its caricatures focus on the aspects of
the Arab Muslim society, which represent their identity. These aspects are negatively considered and they
include issues of the oppression of women, domestic violence, forced marriage, mandatory veiling and burqua. The cartoons shed special lights on phenomena such as the stoning of girls accused of adultery, homosexuality
and gays rights... these phenomena are decontextualized and being discussed from a European point of view that
undervalues the Arab Muslim identity and cultural specificity. Nevertheless, the issue is more than caricatures about a person or a group of people. It encompasses
politics, ideology and mainly economy. The great trick of Charlie Hebdo is that it markets a thought that it is
attacked because it targets religions. And it raises the emblem that it is making no limits to religious critics. Gerard Biard, the editor in chief who survived the attack, expresses his refusal of changing the policy of Charlie
Hebdo claiming that, "If we say to religion, 'You are untouchable,' we're fucked." But the problem is that it is
not criticizing religion in general but Islam in particular. France Soir was the first that dared to republish the cartoon about the crisis. This daily newspaper,
which was then in the process of bankruptcy, was the subject of big struggles between a group of capitalists
seeking to buy it and the journalists working in it and who had the final say about its fate. These journalists were
working under the aegis of ArcadiGaydamak“aRussian-born French-Israeli businessman, philanthropist, and
President of the Congress of Jewish Religious Communities and Organizations of Russia (KEROOR).”
(Wikipedia). He was also the head of the "Beitar" (militia of the Israeli Likud Party) and the football club of the
same name (Beitar), He was a candidate for the mayor of Jerusalem and he had legal issues against him on
charges of evading his tax duties. He used to hide his identity as long as possible by issuing international arrest
warrant against him on charges of evading his tax duties. 7Libby Nelson. https://www.vox.com/2015/1/7/7511001/charlie-hebdo-attack- paris. Jan 7, 2015,
8https://www.bbc.com/news/world-europe-30808284 (Last Retrieved February 23rd, 2020) 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) In 2016, the Italian designer Dolce & Gabbana announced the
New Line of Hijab and Abayas for Muslim woman and it was a step of the international brand to embrace hijab
and abaya through launching a set of images of glamorous Muslim women draped in silky robes ornamented
with bright scarves signed D&G. The debut line was basically targeting wealthy Muslim females namely those
from the Middle East. The event was widely covered in international media and the co-founder of D&G Stefano
Gabbana used the hashtag #dgabaya in his Instagram account. Not only the media that supported the fashion line
but also those who were against it found in it a good topic to attack the Muslim woman and the Islamic clothing. In the blog of Le Monde newspaper and on his social media pages, the French caricaturist Plantu
published a cartoon entitled “Dolce & Gabbana Launches a collection of Hidjabs” (Dolce & Gabbana Lances
une collection de hidjabs). In this caricature, Plantu shows two fashionable ladies, apparently Muslim women
wearing Hijab. They are taking gasses and each one is holding a fashionable bag. One of them is wearing a belt
of explosive that is commonly used by the jihadists in their terrorist attacks. One of them is asking the other
“when will the fashion belt be out?” (A quand la fashion ceinture?) Figure 4 g
The cartoon was largely criticized as being Islamophobic. It was considereddisgraceful, shameful and
disappointing as it was seen targeting Muslim women when connecting them to terrorists and terrorism. The
cartoon came in a time when Europe was target of many terrorist attacks (in Belgium and Paris etc.) that
nurtured the anti-Islamic sentiments. The ideological aspect of these caricatures resides in the effect they conduct on the public opinion. They contribute in creating a set of values and beliefs that are “representing and constructing society which
reproduce unequal relations of power, relations of domination and exploitation." As Fairclough and Wodak
explained (1997:275). These ideologies do in fact contribute in the construction of a reality, a reality that
establishes specific relations of dominance between the host Europeans and the Arab Muslim immigrants in the
west. Critical discourse analysis interest in these ideologies is in fact aiming at unveiling them namely that the
European citizens develop fake awareness of their reality. 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) Likewise, the chronology of popularity of Charlie Hebdo shows that reprinting the Danish Caricatures
represent a turning point in the life of the magazine (fame, tracking and sales). It has obtained more focus after
the murder of 12 of its staff in Paris. Before 2011 the magazine was not very popular and it had only 50.000
issues as circulation compared to 500.000 issues for its rival Le Canard Enchaîné . It was even about to go
bankrupt. However, with the issue "guest-edited" by the Prophet Mohammed ("100 lashes if you don't die of
laughter"), and the attacks to its office in 2011, it planned to raise its print from one million to three millions to
five millions copies of the same issue: “The normal print run of 60,000 was extended to five million - a week
after Islamist gunmen murdered 12 people at the magazine's offices and five others in subsequent attacks in
Paris” (BBC News website)8 For all these contradictions between the official version of the caricatures issue and the implications we
stated, we can assume neither the Danish the newspaper nor the French one published the cartoons to free
Danish book artists or the French caricaturists from painstaking self-censorship, but rather as an important
component of a broad campaign to incite hatred and xenophobia. And that has never been innocent as it always
goes in the direction of supporting some ideologies and visions of some politicians or journalists. As a matter of fact, the economical motives nurtured by the political interest and ideologies are strong
factors for redirecting the cartoons issue. The same concepts and the same depictions were repeated in different As a matter of fact, the economical motives nurtured by the political interest and ideologies are strong
factors for redirecting the cartoons issue. The same concepts and the same depictions were repeated in different 7Libby Nelson. https://www.vox.com/2015/1/7/7511001/charlie-hebdo-attack- paris. Jan 7, 2015
8https://www.bbc.com/news/world-europe-30808284 (Last Retrieved February 23rd, 2020) ABDELKARIMBENLAAYOUNI Page 224 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com issues such as the Hijab controversy in Europe. 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) Not only Europeans but also immigrants themselves
need to understand the embedded ideologies of caricatural discourses as a step towards emancipating themselves
from dominance, control, power abuse, inequality and racism. So, as “meanings are produced through
interpretations of texts” (Fairclough 1992: P.89), ideologies are understood through interpretation of the ABDELKARIMBENLAAYOUNI Page 225 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com embedded messages and the deep intentions of the caricaturists. Understanding these ideologies is in fact a first
step towards constructing a social cognition and then enacting change into society. Immigrants, through
analyzing the embedded ideologies of caricatures about their issues, do contribute in presenting the other side of
reality because these ideological stereotypes and representations of the west are only a one-side perspective of
reality. The debate about immigrants’ main issues wherein Arab and Islamic behaviors and style of life in the
west seems unpleasant and are rejected by Europeans hides a conflict between two broad ideologies. The refusal
of the west of the Islamic and Arabized signs of immigrants including the ones born and raised in Europe is
interpreted in the west as a failure of Europe to convince them with its values. In an interview with Hakim El
Karoui, Senior Fellow at Institut Montaigne and the author of the report “A French Islam is possible”, in
attempting to answer the question about the fact that “ there is a real French passion for the debate around the
veil, to the surprise of our neighbors” he replied, “The veil could therefore mean to French people that the
Republic has failed to make its values desirable, and has failed to share them with the immigrants’ children,
even though they were born on French soil.”9. Thus, the issue is more than a piece of cloth on some women’s
heads (headscarf) or a beard on men’s face, rather it is a style of life and a way of looking at things. The real
debate is not on the veil or the head cover, as there are different types of veils: the headscarf of Iran is not the
one of turkey nor is it the one of North Africa. Yet the western public opinion refers to all types as a veil; an
Islamic sign. 9https://www.institutmontaigne.org/en/blog/muslim-veil-france-why-so- controversial (Last retrieved
on February 25th,2020)
10 Statement by art historian NadeijeLaneyrie- Dagen, articulated during the hearings on the face veil in France.
Mission d’information sur la pratique du port du voile intégral sur le territoire national (December 8th, 2009,
Séance 4.30 Compte rendu no.16) Figure 5 The above cartoon by Signe Wilkinson in 2010 is a form of multiple-choice test about the image that
represents the greatest threat to the image of Islam. It shows three images of three people/ choices: A/ a bearded
man with an explosive belt around his waist and his thumb on the detonation button. B/ a masked man with a
scarf covering his head and face. He is carrying a big knife about to behead an innocent person who is sitting in
a state of peace of mind. C/ An artist sitting in his studio and he is holding a pencil to draw some pictures. The
first two people are Muslim (beards) while the third one is not (western) and they are presented as markers of
images representing Islam even though none of them is in fact related to Islam. The above caricature seems to
be defending Islam when claiming that the Jihadists Muslims who are killing people (al Qaida and ISIS...) are
spoiling the image of Islam more than the cartoons produced by Europeans. The discourse of this caricature is very blasphemous and offensive as it generalizes and summarizes the
image of Muslims and Islam in some fixed stereotypes that reduce it in terrorism, killing and violence. Moreover, the first two choices are very negative and they favor the last choice, which supports the ideology of
the caricature. The expected answer is inevitably choice C. Thus, the caricaturist aims at underestimating the
effect of the caricatures of the prophet Mohamed. There is a sort of reprimanding for the Muslims’ reactions to
the cartoons in the sense that these reactions should be to the real threats to Islam and its image rather than to the
drawings that have raised a lot of controversies. Finally, we can claim that European caricatures are manifestations of the ideology of right wing parties
whose beliefs revolve around the idea of order, punishment, superiority of the local authorities and hatred to
foreigners. These principles are summarized in Mudde’s claim “the ideological core of the populist radical
right – defined as a combination of nativism, authoritarianism, and populism” (2010: 1173) This ideological core has been clearly manifested in the European attitude towards immigrants and
Islamophobia in the sense that media and specifically caricatures have presented immigrants as a threat to the
nationalist identity. They were considered responsible for unemployment, crimes and social instability. 5http://www.danielpipes.org/comments/69868 (last retrieved on July12th, 2021) And here resides the essence of the problem: connecting the headscarf to religion and confusing
the public opinion to think that all Muslim women put a headscarf and all headscarves are hijab. The veil issue
has clearly exposed the contradictions and the double standard of the European community in the sense that
“while the modern secular nation state formally assigns itself a position of neutrality and distance on
religious matters, its authority is dependent on its capacity to determine which kinds of religious expressions
and practices are legitimate in public and which are not” (Amir- Moazami 2013: 91) The (il) legality of face covering is ideologically rooted in a debate between secularism and divine
perspectives. While the Muslim east considers covering women’s face a symbol of Piety, purity and religiosity,
“In our occidental societies the face is the part of the body which carries the heart of the individual, the soul,
the reason, the personality. For us this is a cultural secular heritage” (Laneyrie-Dagen 2009)10. The debate
about Muslim women’s hijab is strongly directed by ideologies. It is media oriented to frame immigrant women,
Arab and Muslim women and headscarves within the concept of hijab in its religious dimensions. Nevertheless,
wearing a headscarf by these women is a social issue that should that should be discussed in relation to the
women’s status in the family and in the whole Muslim society. Hijab or headscarf is more than a religious sign,
it is a cultural and anthropological expression as concluded the aforementioned Montaigne’s report of 2016 (A
French Islam is possible) which claims that “Muslim women wearing the veil are motivated by religious duty
(76%), issues of safety (35%) and the desire to display their Muslim faith (23%), with only 6% saying that
they are coerced or imitating others.” The conflict of ideologies becomes very sharp and obvious when the caricaturists tend to trivialize
some fundamental issues and put them in the same position with what is allowed and what is not. Take for
instance the caricature below (Figure 5) Page 226 Page 226 International Journal of Arts and Social Science
ISSN: 2581-7922, www.ijassjournal.com Volume 4 Issue 4, July-August 2021 Figure 5 These
stereotypes and generalized judgments were exploited by many politicians and political agendas that passed
anti-immigration and deportation laws. Hence, we can coin Adorno’s (1950) statement that the western
extremist thought stems from the psychological nature of individuals who cannot live in a multi-society and tend
to think according to a fixed, unchanging pattern and claim that the European tendency of rejecting immigrants ABDELKARIMBENLAAYOUNI Page 227 International Journal of Arts and Social Science
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Volume 4 Issue 4, July-August 2021 www.ijassjournal.com stems from their inability to accept the other who might lead a change in their culture and community to which
they do not see themselves ready. Nevertheless, Islamophobia as a term became significant of hatred of Islam
and Muslims. Those who were calling for fighting against racism were calling for fighting all sorts of
discrimination but speaking about fighting against Islamophobia implies fighting against the practitioners of that
religion. And this is very dangerous as it is filled with hatred and racism. Therefore, Islamophobia is a blatant
appeal to discrimination and racism on ideological basis. stems from their inability to accept the other who might lead a change in their culture and community to which
they do not see themselves ready. Nevertheless, Islamophobia as a term became significant of hatred of Islam
and Muslims. Those who were calling for fighting against racism were calling for fighting all sorts of
discrimination but speaking about fighting against Islamophobia implies fighting against the practitioners of that
religion. And this is very dangerous as it is filled with hatred and racism. Therefore, Islamophobia is a blatant
appeal to discrimination and racism on ideological basis. To conclude, we can claim that caricatures of Arabs and Muslim immigrants in Europe establish an image
of inferiority of this “other” on three distinctive levels: mental, ideological and economic. We can also claim
that the European cartoonists are not objective in their coverage of the issues related to immigrants namely those
from Arab and Muslim backgrounds. Figure 5 This subjectivity is due first to a lack of information; as they are inspired
mainly from the data presented in the media about those immigrants and also because European cartoonists
themselves are part of the whole system which feels the threat of Arabs and Muslims on their European identity
as well as their belief in the European supremacy and superiority over the other people. We cannot deny that
there are some exceptions that are almost invisible and have no direct effect on the socio-political life in their
countries. As any other discourse, caricature is governed by ideology. The caricaturist’s ideology is clear in the
subjects of his cartoons, which deal with some issues at the expense of others. The artist by nature is
supposed to tackle topics that defend the rights of the simple citizen against all forms of injustice, whether
by the institutions of the state or by the individuals in a given society. Yet this is not always the case in
European caricatures about immigrants and migration issues in general. International Journal of Arts and Social Science
ISSN: 2581-7922,
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Variable Virulence of Biotype 3 Vibrio vulnificus due to MARTX Toxin Effector Domain Composition
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RESEARCH ARTICLE
Host-Microbe Biology
crossm Variable Virulence of Biotype 3 Vibrio
vulnificus due to MARTX Toxin Effector
Domain Composition Solicited external reviewers: Carmen Amaro,
University of Valencia; Jonathon Audia,
University of South Alabama College of
Medicine. This paper was submitted via the
mSphereDirect™pathway. Vibrio vulnificus swap of its effector
proteins to alter virulence linked to outbreak Received 3 July 2017 Accepted 7 July
2017 Published 26 July 2017
Citation Kim BS, Gavin HE, Satchell KJF. 2017. Variable virulence of biotype 3 Vibrio vulnificus
due to MARTX toxin effector domain
composition. mSphere 2:e00272-17. https://doi
.org/10.1128/mSphereDirect.00272-17. Editor Sarah E. F. D’Orazio, University of
Kentucky
Copyright © 2017 Kim et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license. Address correspondence to Karla J. F. Satchell,
k-satchell@northwestern.edu. * Present address: Byoung Sik Kim, Metabolic
Regulation Research Center, Korea Research
Institute of Bioscience and Biotechnology,
Daejeon, Republic of Korea. Solicited external reviewers: Carmen Amaro,
University of Valencia; Jonathon Audia,
University of South Alabama College of
Medicine. This paper was submitted via the
mSphereDirect™pathway. Vibrio vulnificus swap of its effector
proteins to alter virulence linked to outbreak Received 3 July 2017 Accepted 7 July
2017 Published 26 July 2017 Received 3 July 2017 Accepted 7 July
2017 Published 26 July 2017
Citation Kim BS, Gavin HE, Satchell KJF. 2017. Variable virulence of biotype 3 Vibrio vulnificus
due to MARTX toxin effector domain
composition. mSphere 2:e00272-17. https://doi
.org/10.1128/mSphereDirect.00272-17. Editor Sarah E. F. D’Orazio, University of Editor Sarah E. F. D’Orazio, University of
Kentucky This paper was submitted via the
mSphereDirect™pathway. Vibrio vulnificus swap of its effector
proteins to alter virulence linked to outbreak IMPORTANCE Vibrio vulnificus is a serious infection linked to climate change. The
virulence capacity of these bacteria can vary by gene exchange, resulting in new
variants of the primary virulence toxin. In this study, we tested whether the emer-
gence of an epidemic strain of V. vulnificus with a novel toxin variant correlated with
a change in virulence. We found that restoring the biotype 3 toxin variant to the
putative progenitor-type toxin resulted in dramatically increased virulence, revealing
that the emergence of the biotype 3 strain could be linked to virulence reduction. This reduced virulence, previously found also in the biotype 1 strain, suggests that
reduced virulence may stimulate outbreaks, as strains have greater capacity to enter
the human food chain through reduced impact to environmental hosts. msphere.asm.org
1 Variable Virulence of Biotype 3 Vibrio
vulnificus due to MARTX Toxin Effector
Domain Composition Byoung Sik Kim,* Hannah E. Gavin,
Karla J. F. Satchell
Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago,
Illinois, USA Byoung Sik Kim,* Hannah E. Gavin,
Karla J. F. Satchell
Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago,
Illinois, USA ABSTRACT
Vibrio vulnificus is an environmental organism that causes septic human
infections characterized by high morbidity and mortality. The annual incidence and
global distribution of this pathogen are increasing as ocean waters warm. Clinical
strains exhibit variations in the primary virulence toxin, suggesting a potential for
the emergence of new strains with altered virulence properties. A clonal outbreak of
tilapia-associated wound infections in Israel serves as a natural experiment for the
sudden emergence of a new V. vulnificus strain. The effector domain content of the
multifunctional autoprocessing RTX (MARTX) toxin of the outbreak-associated bio-
type 3 (BT3) strains was previously shown to harbor a modification generated by re-
combination. The modification introduced an actin-induced adenylate cyclase effec-
tor domain (ExoY) and an effector domain that disrupts the Golgi organelle (DmX). Here, we report that the exchange of these effector domains for a putative progeni-
tor biotype 1 toxin arrangement produces a toxin that slows the lysis kinetics of tar-
geted epithelial cells but increases cellular rounding phenotypes in response to bac-
teria. In addition, replacing the biotype 3 toxin variant with the putative progenitor
biotype 1 variant renders the resulting strain significantly more virulent in mice. This
suggests that the exchange of MARTX effector domains during the emergence of
BT3 generated a toxin with reduced toxin potency, resulting in decreased virulence
of this outbreak-associated strain. We posit that selection for reduced virulence may
serve as a route for this lethal infectious agent to enter the human food chain by al-
lowing it to persist in natural hosts. Received 3 July 2017 Accepted 7 July
2017 Published 26 July 2017
Citation Kim BS, Gavin HE, Satchell KJF. 2017. Variable virulence of biotype 3 Vibrio vulnificus
due to MARTX toxin effector domain
composition. mSphere 2:e00272-17. https://doi
.org/10.1128/mSphereDirect.00272-17. Editor Sarah E. F. D’Orazio, University of
Kentucky
Copyright © 2017 Kim et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license. Address correspondence to Karla J. F. Satchell,
k-satchell@northwestern.edu. * Present address: Byoung Sik Kim, Metabolic
Regulation Research Center, Korea Research
Institute of Bioscience and Biotechnology,
Daejeon, Republic of Korea. KEYWORDS biotype 3, MARTX, Vibrio vulnificus, cytotoxins, mouse, recombination,
virulence factors KEYWORDS biotype 3, MARTX, Vibrio vulnificus, cytotoxins, mouse, recombination,
virulence factors T T
he seafood-associated pathogen Vibrio vulnificus causes severe wound and intes-
tinal infections that can progress to tissue necrosis, septicemia, organ failure, and
death, often within 48 h of pathogen exposure (1). The pathogen is spreading globally,
and more infections are occurring annually as its geographic distribution increases with
warming seawaters (2, 3). Biotype 1 (BT1) strains are most commonly associated with msphere.asm.org
1 July/August 2017
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e00272-17 Kim et al. FIG 1
Comparative structures of different V. vulnificus MARTX toxins. Effector arrangements of the BT3-type MARTX toxin in
representative strain BAA87 and the C-type toxin found in BT1 strains CMCP6 and LOS6966 and clade B strain V252. Each has five
effector domains, as shown by the labels, which are described in the text. The BAA87 hybrid strain modified to have a C-type toxin
is also shown. Notable amino acid changes in the clade B and BAA87 toxins are also noted. N-term, N-terminal; C-term, C-terminal. FIG 1
Comparative structures of different V. vulnificus MARTX toxins. Effector arrangements of the BT3-type MARTX toxin in
representative strain BAA87 and the C-type toxin found in BT1 strains CMCP6 and LOS6966 and clade B strain V252. Each has five
effector domains, as shown by the labels, which are described in the text. The BAA87 hybrid strain modified to have a C-type toxin
is also shown. Notable amino acid changes in the clade B and BAA87 toxins are also noted. N-term, N-terminal; C-term, C-terminal. clinical infections, while biotype 2 (BT2) strains cause infections in eels (1, 4, 5). From
1996 to 1999, pond-raised tilapia in Israel were linked to an outbreak of wound-
associated V. vulnificus infections that was due to a newly emerged clonal variant (6). These biotype 3 (BT3) strains are now in the seas of Israel, where they cause
occasional wound infections in fish handlers. The death rate from these infections
is 10%, with survivors suffering extreme morbidity, including amputations and long
hospitalizations (7). The primary virulence factor of V. vulnificus BT1, BT2, and BT3 strains is the multi-
functional autoprocessing RTX (MARTX) toxin (8–10). These large, secreted, polypeptide
toxins have long repeat regions that form pores in eukaryotic cell plasma membranes
for the delivery of catalytically active effector domains to cells (11, 12). Among all
V. KEYWORDS biotype 3, MARTX, Vibrio vulnificus, cytotoxins, mouse, recombination,
virulence factors vulnificus isolates, seven different MARTX variants with distinct effector domain
repertoires have been identified. In this naturally competent bacterium, the composi-
tion and organization of domains is altered by horizontal acquisition of DNA followed
by recombination within the rtxA1 toxin-encoding gene (13). Since the MARTX toxin is
a major virulence factor, we surmised that the exchange of effector content in the
MARTX toxin, resulting in altered toxin potency, could contribute to the emergence of
V. vulnificus strains with outbreak potential (14). The sudden emergence and clonality
of BT3 strains serves as a natural experiment for how changes in MARTX effector
contents affect disease progression and strain emergence (15). Therefore, in this study,
we asked how toxin type impacts virulence during strain emergence by generating a
strain in the BT3 background encoding a BT1 C-type toxin from a modified rtxA1 gene. Specifically, we hypothesized that the acquisition of new effector domains conferred
increased virulence on BT3 strains by generating MARTX toxins with increased potency
compared to that of the toxin types present in progenitors of BT3. However, our data
support the idea that the BT3 toxin type is actually less potent than the C type in this
background, suggesting that BT3 emerged in part due to selection for reduced viru-
lence that may enhance persistence in an environmental host. RESULTS Domain structure of BT3 V. vulnificus and putative progenitor strains. The
5,208-amino-acid (aa) MARTX toxin in representative BT3 strain BAA87 has five effector
domain regions: a domain of unknown function that binds prohibitin (DUF1), a cysteine
catalytic protein that inactivates Rho GTPases (RID), an alpha-beta hydrolase enzyme
that cleaves phosphatidylinositol-3-phosphate (ABH), an actin-stimulated adenylate
cyclase (ExoY), and a cysteine protease that disrupts the Golgi organelle (DmX) (Fig. 1A)
(11, 13, 16–18). This effector domain arrangement is unique to BT3 strains. Whole-
genome single-nucleotide polymorphism analysis indicates that BT3 strains emerged
from a BT1 clade B lineage (19). Analysis for this paper revealed that sequenced
representative clade B strain V252 (20) has an rtxA1 gene that encodes a 5,206-aa
MARTX toxin of equal length and 98.8% identity (5,146/5,206 aa) with that of the
representative BT1 isolate CMCP6. Unlike the BT3 MARTX toxin, CMCP6 and clade B July/August 2017
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2 Reduced Potency of BT3 Vibrio vulnificus MARTX Toxin TABLE 1 Plasmids and bacterial strains used in this study
Strain or plasmid
Relevant characteristic(s)a
Reference or source
V. vulnificus strains
LOS6966 (CDC9342-95)
BT1, clinical isolate, Texas/Louisiana, USA, 1995
A. DePaola, FDA
BAA87rif
BT3, human wound isolate, Afula, Israel, 1996, Rifr
16
KZ1
BAA87 rtxA1::kan (ΔrtxA1)
16
BS1313
BAA87 ΔvvhA
17
BS1314
KZ1 ΔvvhA
This study
BS1315
BS1313 rtxA1ΔexoY
This study
BS1316
BS1313 rtxA1ΔexoYΔdmx
This study
BS1317
BS1313 rtxA1BAA87rtxA1LOS6966 (rtxA1hyrid)
This study
BS1319
BS1313 rtxA1Δdmx
17
E. coli strains
DH5
F 80lacZ ΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK mK) phoA supE44
thi-1gyrA96 relA1
Laboratory collection
S17-1 pir
thi pro hsdR hsdM recA::RP4-2-Tc::Mu-km::Tn7; pir, Smr
Laboratory collection
Plasmids
pDS132
oriR6K sacB oriT RP4; Cmr
23
pBS1305
mcf::rrsp::cpdLOS6966 with flanking regions from BAA87 rtxA1 in pDS132; Cmr
This study
pBS1202
ΔexoY in pDS132; Cmr
This study
pBS1207
ΔexoYΔdmx in pDS132; Cmr
This study
aAntibiotic resistance: Apr, ampicillin; Kmr, kanamycin; Rifr, rifampin; Cmr; chloramphenicol. TABLE 1 Plasmids and bacterial strains used in this study
Strain or plasmid
Relevant characteristic(s)a aAntibiotic resistance: Apr, ampicillin; Kmr, kanamycin; Rifr, rifampin; Cmr; chloramphenicol. MARTX toxins have effector domain arrangements of DUF1, RID, ABH, a cysteine
protease that induces apoptosis (MCF), and a Ras/Rap1-specific endopeptidase (RRSP)
(Fig. 1) (11). This arrangement has been termed the C-type/type I toxin (14, 21). A notable difference identified between V252 and CMCP6 MARTX toxins is a point
mutation at the MCF catalytic cysteine residue of V252. RESULTS We suggest that the BT3 toxin
most likely arose from a C-type toxin as a recombination event that replaced an inactive
MCF and neighboring RRSP domains found in the clade B toxin with sequences for the
ExoY-DmX domains (Fig. 1). This exchange further incorporated a cysteine protease
domain (CPD) with the autoprocessing leucine changed to tryptophan. This likely
moves the initial autoprocessing site upstream to the next available Leu; autoprocess-
ing site flexibility has been experimentally demonstrated (22). Generation of BT3 strain BAA87 V. vulnificus reverted to carry the putative
progenitor C-type MARTX toxin. We sought to understand the impact of the effector
domain exchange by reverting the BT3 toxin back to an unmodified C-type toxin. Among sequenced C-type rtxA1 genes in our strain collection (14), the sequences
flanking the MCF-RRSP-CPD-coding sequence of BT1 strain LOS6966 were most similar
to the strain BAA87 rtxA1 (rtxA1BAA87) gene sequence, indicating that the LOS6966 rtxA1
(rtxA1LOS6966) gene would be a suitable source of DNA for amplification to mediate
the genetic exchange (97% nucleotide [nt] identity; 2,416/2,473 nt). The rtxA1
effector domain region was amplified from LOS6966 chromosomal DNA and cloned
into sacB counterselection vector pDS132 (23), and the LOS6966-derived gene
sequence was recombined into the rtxA1 gene of BT3 strain BAA87rif. The resulting
rtxA1BAA87rtxA1LOS6966 hybrid strain is referred to as the rtxA1hybrid strain hereinafter. In addition, variants of rtxA1 lacking for ExoY and DmX effector domain DNA sequences
(designated rtxA1ΔexoY, rtxA1Δdmx, and rtxA1ΔexoYΔdmx hereinafter) were generated
(Table 1). The ΔexoY and Δdmx arrangements were both confirmed to lack relevant
toxic activities (see Fig. S1 in the supplemental material; see also reference 17). To focus
our studies on rtxA1, the gene vvhA, encoding a second cytolysin known to impact
virulence and lyse cells in vitro (8–10), was deleted from all strains; thus, the BAA87
ΔvvhA strain is referred to here as the wild type (WT). BAA87 with hybrid toxin shows altered cytopathicity. All V. vulnificus strains lyse
epithelial cells in culture due to MARTX toxin repeat region pores (12, 24). When
incubated with HeLa cells, the rtxA1hybrid strain expressed, secreted, and delivered
MARTX toxin to target cells, as indicated by the release of lactate dehydrogenase (LDH) July/August 2017
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3 Kim et al. FIG 2
Function of BAA87 with hybrid toxin in cytotoxicity and cytopathicity. (A) HeLa cells were
coincubated with V. RESULTS vulnificus strains as indicated at an MOI of 20. At the indicated times, the LDH release
to cell culture supernatant was quantified using the Promega CytoTox96 nonradioactive cytotoxicity
assay, and the results are plotted as percent LDH release compared to the results for 100% lysis by 1%
Triton X-100. All strains have a deletion in vvhA, with BAA87 ΔvvhA indicated as the wild type. At t
225 min (arrow), the rtxA1hybrid, rtxA1ΔexoYΔdmx, and rtxA1 null strains induced significantly less lysis
than the wild type (P 0.01) as compared by analysis of variance (ANOVA). (B) HeLa cells were
coincubated with V. vulnificus at an MOI of 10, and cellular rounding was quantified from digital images
using the cell counter plug-in in NIH ImageJ. At both 60 and 120 min, the rtxA1hybrid strain induced
significantly more rounding than the other strains being compared (**, P 0.05; ***, P 0.01; ****, P
0.001). Data are the mean results standard deviations from three experimental sets. FIG 2
Function of BAA87 with hybrid toxin in cytotoxicity and cytopathicity. (A) HeLa cells were
coincubated with V. vulnificus strains as indicated at an MOI of 20. At the indicated times, the LDH release
to cell culture supernatant was quantified using the Promega CytoTox96 nonradioactive cytotoxicity
assay, and the results are plotted as percent LDH release compared to the results for 100% lysis by 1%
Triton X-100. All strains have a deletion in vvhA, with BAA87 ΔvvhA indicated as the wild type. At t
225 min (arrow), the rtxA1hybrid, rtxA1ΔexoYΔdmx, and rtxA1 null strains induced significantly less lysis
than the wild type (P 0.01) as compared by analysis of variance (ANOVA). (B) HeLa cells were
coincubated with V. vulnificus at an MOI of 10, and cellular rounding was quantified from digital images
using the cell counter plug-in in NIH ImageJ. At both 60 and 120 min, the rtxA1hybrid strain induced
significantly more rounding than the other strains being compared (**, P 0.05; ***, P 0.01; ****, P
0.001). Data are the mean results standard deviations from three experimental sets. (Fig. 2A). The hybrid strain revealed slower cell lysis than the wild-type control (Fig. 2A,
arrow at 225 min). Altered kinetics were also observed for rtxA1ΔexoY and rtxA1Δdmx
strains, with an additive effect for the rtxA1ΔexoYΔdmx strain. RESULTS This suggests that slowed
cell lysis by the hybrid is due to the structural or enzymatic changes brought about by
loss of ExoY and DmX from the BT3 toxin. In contrast, the rtxA1hybrid strain induced
more-rapid cell rounding than the WT strain. Interestingly, this indicates increased cell
cytopathicity despite delayed lysis. In contrast to the lysis phenotype, the rounding
effect is due to the gain of MCF and RRSP, not the loss of ExoY and DmX (Fig. 2B), since
accelerated rounding is seen with the hybrid strain but not with the double deletion
strain. BT3 V. vulnificus with C-type toxin is more virulent than BAA87. Noting that the
hybrid toxin conferred delayed lysis kinetics but accelerated rounding kinetics, we
tested the effect of the toxin swap on bacterial virulence. The dorsal side of outbred
female ICR mice was inoculated by subcutaneous (s.c.) injection with 5 106 CFU of
bacteria as previously detailed (16). This dose is approximately the 50% lethal dose
(LD50) for the wild-type strain (BAA87 ΔvvhA) (Fig. 2). An isogenic mutant lacking rtxA1
is significantly attenuated compared to the virulence of the parental strain. One
hundred percent of mice survived after rtxA1 null strain infection. In contrast, all mice
inoculated with the rtxA1hybrid strain died within 13 h, a significant acceleration
compared to the results for the parental strain (Fig. 3). msphere.asm.org
4 DISCUSSION In this study, we sought to test the hypothesis that naturally occurring effector
exchange in MARTX toxins can impact the emergence of epidemic strains. The clonal
outbreak of biotype 3 V. vulnificus associated with tilapia fish in Israel provided a natural
experiment to test this hypothesis. Contrary to our initial hypothesis, the hybrid strain
had increased virulence, indicating that the putative progenitor C-type toxin is actually
more potent, in terms of its virulence contribution, than the BT3 toxin of BAA87. This
increased virulence occurred despite slower cell lysis kinetics. These results are consis-
tent with recent observations that bacterial virulence outcomes are more likely linked
to MARTX effector domain activity than to the lytic cytotoxicity characteristic of MARTX
delivery in vitro (25). Moreover, these data reveal that a putative BT3 progenitor strain
harboring the C-type MARTX toxin could have been more virulent than the current msphere.asm.org
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e00272-17 Reduced Potency of BT3 Vibrio vulnificus MARTX Toxin FIG 3 The BT3 BAA87 hybrid with C-type toxin is hypervirulent. Female ICR mice (n 10; pooled data
from two experiments conducted with 5 mice per group) were infected s.c. with 5 106 CFU of the
indicated V. vulnificus strains, and survival was monitored for 48 h. The survival curve of the rtxA1hybrid-
strain-infected group is significantly different from that of the parental wild-type-strain-infected group,
as determined by Mantel-Cox log rank test. FIG 3 The BT3 BAA87 hybrid with C-type toxin is hypervirulent. Female ICR mice (n 10; pooled data
from two experiments conducted with 5 mice per group) were infected s.c. with 5 106 CFU of the
indicated V. vulnificus strains, and survival was monitored for 48 h. The survival curve of the rtxA1hybrid-
strain-infected group is significantly different from that of the parental wild-type-strain-infected group,
as determined by Mantel-Cox log rank test. BAA87 if the strains were completely isogenic. Therefore, this experiment suggests that
outbreak strains may arise from decreases in the potency of key virulence factors rather
than increased potency. In fact, a reduction of virulence leading to clinical emergence has been previously
suggested also for BT1 V. vulnificus strains. In mouse intragastric studies, strains with an
RRSP C-type toxin were 50-fold more virulent than isogenic strains with an RRSP
M-type toxin (14). DISCUSSION Loss of the RRSP-coding sequence from rtxA1 has occurred repeat-
edly, according to phylogenetic analysis, and RRSP M-type/type II toxin variants are
more common clinically. This occurs despite data showing that RRSP C-type toxins are
more common in oyster isolates (14). To date, RRSP clade B lineages likewise lack
clinical isolates (19). Unfortunately, no retrospective studies in humans have been
conducted to correlate strains with disease progression. Of course, the actual BT1/clade B and BT3 V. vulnificus strains existing today are not
isogenic, and this study cannot discount the possibility that other genetic changes may
have resulted in altered bacterial virulence potential during the divergence of BT1 and
BT3 lineages. Nonetheless, the data certainly reveal that MARTX toxin evolution is not
linear in the direction of increased potency, as the BT3 MARTX is notably less potent
than the putative progenitor-type toxin tested. Thus, we posit that movement of a
strain into human contact may be driven not by hypervirulence but by a reduction of
the potency of key virulence factors, including MARTX, that thereby reduces virulence
potential in an otherwise highly pathogenic background. July/August 2017
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e00272-17 MATERIALS AND METHODS The new
DNA arrangements were recombined into BAA87rif by conjugation, followed by sucrose counterselection
as detailed previously (17). The desired mutants were isolated and confirmed by PCRs using primer sets
ExoY_Lic_F and ExoY_Lic_R and Bio3X_1201 and Bio3X_1204. All strains were made isogenic with the previously generated strain BS1313 by deletion of vvhA to
remove this second cytolysin as previously described (17). Cytotoxicity assay. HeLa cells were seeded in a 6-well cell culture dish at a density of 1 105
cells/well in complete DMEM (supplemented as described above). On the following day, the cells were
washed twice with warmed phosphate-buffered saline (PBS) and the medium replaced with 3 ml of
DMEM lacking phenol red, fetal bovine serum (FBS), and antibiotic. Mid-log-phase bacteria were pelleted
and resuspended in PBS. One hundred microliters of bacterial suspension was added to each well of HeLa
cells to achieve a multiplicity of infection (MOI) of 20. The culture dish containing both HeLa and bacterial
cells was centrifuged at 500 g for 3 min and incubated at 37°C with 5% CO2. At each time point,
80 µl of cell supernatant was removed from the 3.1-ml total volume per well. Supernatant samples
were centrifuged at maximum speed (~15,000 g) for 60 s to pellet any bacteria or cell debris, and
50 µl was subsequently extracted for use in the LDH assay. Experiments were performed in
triplicate, with three wells of HeLa cells per bacterial strain. Each experiment also employed (i) three wells
of bacterium-free HeLa cells to serve as uninfected controls for spontaneous lysis and (ii) three wells of
bacterium-free HeLa cells that were lysed using Triton X-100, as indicated in the CytoTox 96 nonradio-
active cytotoxicity assay technical bulletin, to serve as maximum lysis controls. The results for all
bacterially infected samples were adjusted for spontaneous lysis and normalized to the percentage of
total lysis according to the equations given in the technical bulletin. Adenylate cyclase assay. The assay measuring the increase of adenylate cyclase in V. vulnificus-
treated Chinese hamster ovary cells was conducted as previously described (16). Mouse wound infection model. Five-week-old female ICR mice were purchased from Charles River
Laboratories, Inc., and allowed to adapt to the new environment for at least 24 h. V. MATERIALS AND METHODS Ethics statement. This work was performed in strict accordance with the recommendations in the
United States Public Health Service regulations and applicable federal and local laws. The methods for
modified toxin generation were approved by the Northwestern University Institutional Biosafety Com-
mittee. All mouse experiments were conducted in accordance with the protocols approved by the
Northwestern University Institutional Animal Care and Use Committee (protocol number IS00000318). y
Reagents, medium, and cell culture. The bacterial strains and plasmids used in this study are listed
in Table 1. Primers used for DNA amplification are listed in Table 2. HeLa cells were obtained directly from
the American Type Culture Collection (ATCC) and cultured at 37°C with 5% CO2 in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 µg/ml penicillin, and TABLE 2 Primers used in this study
Primer
Sequence (5= ¡ 3=)
LMPC_1301
CCAAGCGGTGTCTGGCTTATTGCTTGACCGTCCTATG
LMPC_1302
CTCATCCTTGGCAACAACTTCACCTTGCTCGTCCCAG
Los_1305
GCTAGAGAAGATGTACTCTGCGGCAG
Los_1306
CATCTGTTCATTAACAAAGCGTAGGCCG
ExoY_Lic_F
TACTTCCAATCCAATGCGGGGACAAATACAGATGCACCCCAT
ExoY_Lic_R
TTATCCACTTCCAATGCTAATTAATCGCTACCTGCTGGAAATTAGAAAAC
Bio3X_1201
GCTCTATCCAAACGTATTTCAATGTAAATG
Bio3X_1204
CTCCAGTCCACGCATCTTTCTG TABLE 2 Primers used in this study msphere.asm.org
5 Kim et al. 50 µg/ml streptomycin. Restriction and amplification enzymes and Gibson assembly reagents were
purchased from New England Biolabs, and DNA oligonucleotides and gBlocks gene fragments were from
Integrated DNA Technologies, Inc. Generation of mutant strains expressing modified MARTX toxin. All new V. vulnificus strains were
generated in a spontaneously rifampin-resistant isolate of BT3 strain BAA87. To generate the hybrid
rtxA1, a DNA fragment corresponding to the MCF-, RRSP-, and CPD-coding sequence of rtxA1 was
amplified from genomic DNA of LOS6966 using primers LMPC_1301 and LMPC_1302. At the same time,
two 500-bp gBlocks gene fragments corresponding to up- and downstream flanking regions of the
amplified fragment but containing the sequence matched to the BAA87 rtxA1 gene were synthesized. These three DNA fragments were assembled into SphI-SacI-digested pDS132 using Gibson assembly
master mix, generating plasmid pBS1305. The new DNA arrangement was recombined into BAA87rif by
conjugation followed by sucrose counterselection as detailed previously (16). The desired mutant was
isolated and confirmed by PCR using various primer sets, including LOS_1305 and LOS_1306, ExoY_Lic_F
and ExoY_Lic_R, and Bio3X_1201 and Bio3X_1204. The rtxA1Δdmx arrangement was previously described, as listed in Table 1. To generate rtxA1ΔexoY
and rtxA1ΔexoYΔdmx arrangements, 500-bp gBlocks gene fragment sets corresponding to up- and
downstream flanking regions of BAA87 rtxA1 bp 9703 to 9737 and 9703 to 12180 were assembled into
SphI-SacI-digested pDS132 by Gibson assembly, generating plasmids pBS1202 and pBS1207. MATERIALS AND METHODS vulnificus strains were
grown to mid-log phase (optical density at 600 nm [OD600] of approximately 0.5 to 0.8), harvested, and
diluted in PBS at 1 108 CFU/ml. After slight anesthesia with isoflurane, the mice were subcutaneously
injected with 50 µl of bacterial suspension under the dorsal skin. After 6 h postinfection, the mice
were monitored at least every 2 h until 24 h and then monitored occasionally after 24 h. Surviving mice
without any infection symptoms were euthanized at 96 h postinfection. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/
mSphereDirect.00272-17. FIG S1, PDF file, 0.1 MB. ACKNOWLEDGMENTS This work was supported by National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (grant number 2013R1A6A3A03024337 to
B.S.K.), the USDA National Institute of Food and Agriculture (a predoctoral fellowship to
H.E.G.), and the National Institutes of Health (grant numbers R01AI092825 and
R01AI098369 to K.J.F.S.). The funders had no role in study design, data collection and
interpretation, or the decision to submit the work for publication. emergence of Vibrio vulnificus disease in Israel. Environ Res 103:390–396.
https://doi.org/10.1016/j.envres.2006.07.002.
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genome sequence of the pathogenic bacterium vibrio vulnificus V252
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enhances the survival of Vibrio vulnificus during infection by protecting
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.org/10.1128/AEM.01806-10. 10. Lee CT, Pajuelo D, Llorens A, Chen YH, Leiro JM, Padrós F, Hor LI, Amaro
C. 2013. MARTX of Vibrio vulnificus biotype 2 is a virulence and survival
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(MARTX) toxins of vibrios. Microbiol Spectrum 3:VE-0002. https://doi.org/
10.1128/microbiolspec.VE-0002-2014. 14. Kwak JS, Jeong HG, Satchell KJ. 2011. Vibrio vulnificus rtxA1 gene
recombination generates toxin variants with altered potency during
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.org/10.1073/pnas.1014339108. 25. Gavin HE, Beubier NT, Satchell KJ. 2017. The effector domain region of
the Vibrio vulnificus MARTX toxin confers biphasic epithelial barrier
disruption and is essential for systemic spread from the intestine. PLoS
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Planetary Analog Field Operations as a Learning Tool
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PERSPECTIVE
published: 30 July 2020
doi: 10.3389/fspas.2020.00032 Planetary Analog Field Operations as
a Learning Tool
Gernot Groemer* and Seda Ozdemir
Austrian Space Forum, Spacesuit Laboratory, Innsbruck, Austria Mars and Moon analog field missions are established tools to investigate the
potential of instruments, workflows, materials, and human factors for characterizing
the astrobiological potential and geoscientific context of planetary surfaces. Historically,
there is a broad spectrum on both the scientific focus and the performance parameters
for analog missions. This applies specifically where performance parameters of
coordinated deployment of mission assets (e.g., rovers, human crewmembers, or
scientific instruments) are studied. We argue that scientific priorities and workflows shall
be consolidated at an early planning stage of deep space missions such as during phase-
0 or phase-A studies, while they can still impact the mission architecture design process. It is to be expected that a human-robotic mission to Mars or the Moon will include multiple
field assets such as human explorers, robotic vehicles including aerial reconnaissance,
mobility assets, habitat modules, stationary instruments, and engineering elements for
power, communication, and in-situ resource utilization. These require more complex
asset coordination compared to single-rover planetary missions. Therefore, we advocate
an “Exploration Cascade,” which helps to manage these multiple assets to optimize the
scientific return of planetary surface missions, to search for extinct and/or extant traces
of life, and to characterize the geoscientific context of the sites of interest. Edited by:
Tetyana Milojevic,
University of Vienna, Austria Reviewed by:
Felipe Gómez,
Centro de Astrobiología
(CSIC-INTA), Spain
Akos Kereszturi,
Research Centre for Astronomy and
Earth Sciences, Hungary
Jesús Martínez-Frías,
Consejo Superior de Investigaciones
Científicas (CSIC), Spain Keywords: planetary exploration, astrobiology, spacesuit simulators, exploration cascade, tactical planning *Correspondence:
Gernot Groemer
gernot.groemer@oewf.org Analog Missions as a Training Tool Specialty section:
This article was submitted to
Astrobiology,
a section of the journal
Frontiers in Astronomy and Space
Sciences g
g
Human-robotic Mars missions are likely to be launched within the next 2–3 decades. Some of these
missions will include surface sojourns with at least 1 month in duration (Davila et al., 2010; Drake
et al., 2010). Analog field campaigns have supported all previous planetary surface missions so far
(Preston and Dartnell, 2014) and contributed to planetary missions such as NASA-MSL’s Curiosity
Rover (e.g., Grotzinger et al., 2015; Kah and MSL Science Team, 2015) and ESA’s ExoMars (Vago
et al., 2018). It is generally understood that astrobiology constitutes one of the primary scientific
drivers for such missions (e.g., Belz et al., 2014; Domagal-Goldman et al., 2016; Fairén et al.,
2019). Analog studies are generally considered an efficient tool to prepare for future Mars missions,
complementing instrument validation campaigns. Various Mars and lunar analogs on Earth are
used to train for scientific operations on planetary surfaces, or study extraterrestrial processes (e.g.,
Preston and Dartnell, 2014, also see Figure 1). Additionally, they help to enlighten the logistics and
workflow-related aspects, focusing on human factors, engineering (e.g., human-robotic interaction)
constraints, as well as safety considerations. Received: 05 December 2019
Accepted: 22 May 2020
Published: 30 July 2020 INTRODUCTION Specialty section:
This article was submitted to
Astrobiology,
a section of the journal
Frontiers in Astronomy and Space
Sciences
Received: 05 December 2019
Accepted: 22 May 2020
Published: 30 July 2020
Citation:
Groemer G and Ozdemir S (2020)
Planetary Analog Field Operations as
a Learning Tool. Front. Astron. Space Sci. 7:32. doi: 10.3389/fspas.2020.00032 Analog Missions as a Training Tool Citation: July 2020 | Volume 7 | Article 32 Frontiers in Astronomy and Space Sciences | www.frontiersin.org Planetary Analog Research as a Tool Groemer and Ozdemir FIGURE 1 | Examples of analog missions. (A) NASA BASALT (USA), (B) D-MARS (Israel), (C) LUNARES (Poland), (D) ESA/PANGEA (Spain), (E) NASA/D-RATS
(USA), (F) HI-SEAS (USA), (G) Mars Desert Research Station (USA), (H) NASA/NEEMO underwater station (USA), (I) NDU Habitat Demonstrator (USA), (J)
OeWF/AMADEE-program (Austria, Oman), (K) Mars-500 (Russia), and (L) ESA/CAVES (Spain/Italy). Image credits: (A) photos with permission from Zara Mirmalek, (B)
photos with permission from Hilel Rubinstein/D-MARS, (C) photos with permission from Anna B. Gregorczyk, (D) photos from permission from ESA/S. Sirios, (E)
photos NASA, (F) photos with permission from Ross Lockwood, (G) photos with permissions from OeWF, (H) photos: NASA/Florida International University, (I) photos
with permission from Pablo de Leon, University of North Dakota, (J) photos with permission from OeWF, (K) photos: Roskosmos, (L) photos: European Space Agency. FIGURE 1 | Examples of analog missions. (A) NASA BASALT (USA), (B) D-MARS (Israel), (C) LUNARES (Poland), (D) ESA/PANGEA (Spain), (E) NASA/D-RATS
(USA), (F) HI-SEAS (USA), (G) Mars Desert Research Station (USA), (H) NASA/NEEMO underwater station (USA), (I) NDU Habitat Demonstrator (USA), (J)
OeWF/AMADEE-program (Austria, Oman), (K) Mars-500 (Russia), and (L) ESA/CAVES (Spain/Italy). Image credits: (A) photos with permission from Zara Mirmalek, (B)
photos with permission from Hilel Rubinstein/D-MARS, (C) photos with permission from Anna B. Gregorczyk, (D) photos from permission from ESA/S. Sirios, (E)
photos NASA, (F) photos with permission from Ross Lockwood, (G) photos with permissions from OeWF, (H) photos: NASA/Florida International University, (I) photos
with permission from Pablo de Leon, University of North Dakota, (J) photos with permission from OeWF, (K) photos: Roskosmos, (L) photos: European Space Agency. Besides these professional programs, there are relevant studies
performed at grassroots, mixed professional/citizen-science or
outreach-focused analog sites, such as the Mars Desert Research
Station MDRS in Utah (Kobrick et al., 2018), or NASA’s
Spaceward Bound Program (Allner et al., 2010; Rask et al.,
2011). Notably, a representative metasearch using the Google-
Scholar database on the number of publications focusing on
the keyword “Mars analog research” yields an increase from
611 results in 1997, to 1,890 in 2007, to 3,150 in 2017 (similar
increases were also observed on bibliographic databases NASA
ADS and Pubmed), indicating the emergence of a new scientific
field. Human Element Controversies and
Science as an Early Mission Design Driver and geoscientific performance indicators, should be represented
as an early-stage design driver for mission architectures. Current exploration frameworks, for instance, the NASA
ARTEMIS program, at first define the engineering border
conditions, including spacesuit designs, the Deep Space Gateway
infrastructure, etc., and then the science objectives are identified. Long lead times in developing deep space infrastructure tend
to be the first step in developing architecture and traditionally,
science is involved at a later point. So, for industrial policy
cycle considerations, capacity building needs to start prior to the
surface mission science being consolidated. Human Element Controversies and
Science as an Early Mission Design Driver
It could be argued that robotic exploration is a more efficient
tool than human explorers to study planetary surfaces concerning
the scientific aspect (as opposed to policy-driven programs like
Apollo). However, by looking into the exploration efficiency of
current and planned robotic missions, it becomes evident that a
human component adds significantly to the throughput of, e.g.,
sample selection, in-situ analysis, and procurement workflows
(Crawford, 2012). Glass and Briggs (2003) demonstrated in a field
test at Devon Island that humans can be up to 25 times more
scientifically productive than a rover under certain conditions. Another concern is the planetary protection aspect, as addressed
by the respective COSPAR recommendations (Kminek et al.,
2010), which requires a high level of cleanliness at the target
site or even access denial to the restricted areas. However, this
argument applies also to robotic elements, whereas the amount
of contamination (despite proper cleaning efforts on Earth)
increases with the mass of the rovers. Hence—besides ethical and
societal arguments (e.g., Dunér et al., 2018; Szocik, 2019)—we
argue that the expected benefits of including human explorers
outweigh the risks from a primarily scientific perspective. However, we suggest that in contrast to the Apollo missions,
a science-first principle will ultimately lead to a more effective
mission architecture. For instance, the Mars Sample Return
project IMOST exemplifies how significant it is to involve
detailed science work combined with engineering (Beaty
et al., 2018, 2019a,b). Hence, we favor a scientific consensus-
building before committing to specific mission architectures. Having multiple disciplines represented over multiple missions,
testing various instrumentation, facilitates the establishment of
“common trunk infrastructures.” Those provide the required
technological and operational baselines that provide high
flexibility for accommodating experiments and technologies. Human Element Controversies and
Science as an Early Mission Design Driver g
y
The bandwidth of analog missions ranges from non-scientific
initiatives with a focus on education, like the Chinese Plan-
C station in the northwestern Gobi desert, NASA’s Spaceward
Bound Program which aims to train the next generation of space
explorers by hosting students and teachers within some analogs
(see above for the references), and also highly focused projects
like the NASA D-RATS (Abercromby et al., 2013) or BASALT
missions (Lim et al., 2019), complemented by laboratory-type
analog studies (e.g., the Russian MARS-500 study; Ushakov
et al., 2014). In addition to that, analog missions vary in the
complexity of their scope. On the one end of the spectrum
there are direct instrument field validations and field data
acquisitions pertinent to geoscientific workflows such as the
AMASE expeditions verifying the performance of the ExoMars
PanCam (Amundsen et al., 2010). Managing Multiple Field Assets During
Analog Missions Balancing the needs and cultures of operational and scientific
teams during a mission can be crucial. For instance, during
the D-RATS missions (Abercromby et al., 2013), a discrepancy
between the space operations community (which had heritage
from operational branches of NASA with marginal experience
in geoscience field activities) and the scientific teams (stemming
from the academic community with less spaceflight operational
experience) was observed. This led to flight planning friction
losses resulting in both communities feeling under-served. Following the D-RATS lessons learned (Eppler et al., 2013;
Rader et al., 2013), as a best-practice example, the 2017 BASALT
missions included a Science Traceability Matrix (STM) and
operational concepts (“ConOps”; Lim et al., 2019) defining
the missions’ science objective. Additionally, they implemented
a near-real-time interaction between field personnel and the
science back rooms at Mission Control (Brady et al., 2019), as
well as selected operational considerations (Beaton et al., 2019). On the other side it features more complex surface-sojourn
suites of experiments, including human-factors and science
tactical decision making, such as the D-RATS studies (Litaker
and Howard, 2013). Notably, one of the underground campaigns,
MINAR 2017’s main focus is to carry out not only geoscientific
investigations and instrumental operations under planetary deep
subsurface conditions but also to develop technologies for
the mining industry (Payler et al., 2017). Therefore, due to
the variety in campaign goals, analog missions do not always
represent a realistic projection of planetary surface activities. For instance, only a few of them (such as D-RATS, BASALT,
or AMADEE) include operational remote science support teams
mimicking a major design driver for surface operations (e.g.,
Groemer et al., 2014, 2016): the modality of the decision-making
process, including constraints such as time-delay, bandwidth-
limitation, and segregated expertise. Hence, many operational
lessons learned of analog missions might be challenging to
implement in future flight missions. Although there was a lack of assets expected during an actual
mission, such as rovers, drones, or a physiological load, and
consumables modeling of astronauts, the BASALT field activities
were supported by the Minerva software suite (Marquez et al.,
2019), optimizing a traverse planning, timeline generation and
display (via the PLAYBOOK software, Marquez et al., 2017),
procedure management, execution monitoring, data archiving,
and visualization (Deans et al., 2017) and included a set of
codified flight rules, safety rules, and troubleshooting routines. Frontiers in Astronomy and Space Sciences | www.frontiersin.org Citation: Some examples of past analog campaigns include the
NASA DESERT-RATS field campaigns conducted between 1997
and 2010 (Abercromby et al., 2013), the MOONWALK project
(Imhof et al., 2015, 2017), the ESA CAVES missions (Bessone
et al., 2015), the NASA HI-SEAS long-duration missions
(Häuplik-Meusburger et al., 2017), an initiative by the UK Centre
for Astrobiology: the subsurface analog research MINAR (Payler
et al., 2017), ESA PANGEA (Bessone et al., 2018), the NASA
BASALT campaigns (Lim et al., 2019), or numerous stand-alone
expeditions studying highly specific astrobiological questions
(e.g., Schulze-Makuch et al., 2018) and others. Notably, in 2011,
the European Space Agency (ESA) created a topical team to
investigate recent analog activities (Martins et al., 2017) by using
the Earth as a tool for studying astrobiology, and to formulate
inputs and scientific needs for the improvement of ground-based
astrobiological research. The outcomes and lessons learned from
these and other analog missions constitute the building blocks for
OeWF’s field campaigns. Until now, the Austrian Space Forum (OeWF, German:
Österreichisches Weltraum Forum) has conducted 12 Mars
analog field campaigns, as part of the PolAres (2006–2017)
(Groemer, 2009) and the subsequent AMADEE Program
(since 2018). These missions included more than 750 h of
simulated EVA (extravehicular activity) operations and the
performance of more than 100 peer-review selected experiments. The aim of these initiatives is to identify research gaps in
the exploration roadmaps such as the Global Exploration
Roadmap (GER) (Crawford, 2014) of the International Space
Exploration Coordination Group (ISECG), the NASA Mars
Reference Architecture (DRM 5.0) (Drake, 2009), the Mars
Exploration Robotic Program (MREP) of ESA (Geelen et al.,
2013), as well as NASA’s upcoming ARTEMIS program
(Chavers et al., 2019). These programmatic roadmaps facilitated
the AMADEE program assumptions in terms of projected
crew composition, primary scientific objectives, and mission
architectural considerations. July 2020 | Volume 7 | Article 32 Frontiers in Astronomy and Space Sciences | www.frontiersin.org 2 Planetary Analog Research as a Tool Groemer and Ozdemir Human Element Controversies and
Science as an Early Mission Design Driver Managing Multiple Field Assets During
Analog Missions We argue that the planetary surface operations—once mission
safety criteria have been met—focusing on the astrobiological July 2020 | Volume 7 | Article 32 Frontiers in Astronomy and Space Sciences | www.frontiersin.org 3 Planetary Analog Research as a Tool Groemer and Ozdemir The EC visualizes and optimizes instrument workflows and
their required resources, environmental, and flight planning
border conditions, as well as the ground segment data processing
pipeline. In comparison to the established and well-tested
PLAYBOOK software, the EC is also used as one of the selection
criteria for experiment proposals at pre-mission phase, and it
allows for an inclusion of the ground segments’ remote science
support. Initially, it can be considered an empty roadmap
that is filled with mission aims, operational requirements
(e.g., safety rules for astronauts), scientific priorities (e.g.,
prioritizing of biomarker detection over media activities), and
finally with selected experiments during the mission preparation. Subsequently, after several Dress Rehearsals and map exercises, it
evolves into a web of dependencies identifying critical pathways
(aka sequence of experiment stages determining the minimum
duration for an operation) and susceptibilities to external
changes in the workflow as well as their potential alternatives by
providing strong networking between experiment PIs, mission
leaders, and team leaders, before and during the mission. Finally,
it offers lessons learned for future missions, e.g., identifying the
need for faster data processing pipelines or increasing instrument
robustness for critical pathways. Similarly, the Austrian Space Forum has established codified
standard operating procedures (SOPs), including workflows
analogous to Minerva, but also including physiological modeling
and monitoring and taking into account the limitations of
operating in high-fidelity spacesuit simulators. These procedures
are constantly trained and have evolved into an operational
toolkit independent of the technical framework deployed
(Groemer et al., 2016). It is to be expected that a human-robotic mission to Mars
will include multiple field assets ranging from human explorers,
aerial and surface robotic vehicles, human mobility, habitat
modules, stationary instruments and engineering elements for
power, communication, and in-situ resource utilization. Notably,
this variety will require a significantly more complex asset
coordination compared to single-rover planetary missions,
including the need for delay-tolerant networking and in-situ
high-performance computing (Geist et al., 2019). The NASA
MOSAIC initiative points out this challenge in hindsight of
multiple robotic assets on the surface (Hook et al., 2018) but is
not designed to address the scale and complexity of a human-
robotic mission. The Exploration Cascade Analog missions are tools to test permutations and the
decision-making trees of the EC. We argue that if the field
trials include representative scientific environments, such as a
time-delayed remote science support teams and realistic data
processing pipelines, multiple assets to be coordinated, and a
plausible rule set for the field operations, then uncertainties and
weaknesses in the flight mission planning can be substantially
reduced. Especially given the cost-benefit ratio of analog
missions, variants of the EC can be continuously field-tested with
a moderate effort along the mission planning up to the landing of
the actual flight missions. The “Exploration Cascade” (EC) is an OeWF-coined term
for tactically optimizing the sequence of measurements for
pursuing a pre-defined scientific question. Although the strategic
aims may be set well prior to the flight mission architecture
development, environmental dynamics (e.g., the Martian weather
or solar activity), infrastructure limitations (e.g., communication
ranges, safety rules), instrument anomalies (e.g., dust-induced
degradation), human factors (e.g., reduced productivity due to
isolation), and even (ground-based) data processing pipeline
limitations will have a significant impact on the modalities of
when and where to deploy which instruments. Building upon
established workflows and SOPs, the Austrian Space Forum has
devised the “Exploration Cascade” as an evolving algorithm,
taking into account the aforementioned border conditions. Generally, science operations in multidisciplinary campaigns
with a wide range of both requirements and expected data
products may present challenges in the coordination workflows,
but also offer synergistic effects, if properly applying the
EC as an operational tool. To exemplify this, under certain
circumstances, the high-res imagery obtained by an orbiting
telescope may need certain orbital parameters to be met
before the target of interest can be surveyed. Although aerial
vehicles may need significantly longer time for surveying a
site, they might still be the better choice as they would be
readily available. This workflow defines when and where to
deploy instruments, when their data are to be expected by
the Mission Support Center on Earth and how fast the data
processing can lead to knowledge influencing the decision
making of the flight planners. The exploration cascade was
first demonstrated in an early exploratory investigation during
the AMADEE-18 field campaign in Oman in February 2018,
bringing together 16 experiments (Garnitschnig, 2018), and will
be furthered during the AMADEE-20 field campaign in Israel in
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delivered to Earth by Mars sample return. Meteoritics Planet Sci. 54, 667–671. doi: 10.1111/maps.13232 Fairén, A. G., Schulze-Makuch, D., Whyte, L., Parro, V., Pavlov, A., Gómez-
Elvira, J., et al. (2019). Planetary protection and the astrobiological exploration
of Mars: proactive steps in moving forward. Adv. Space Res. 63, 1491–1497. doi: 10.1016/j.asr.2019.01.011 Belz, S., Buchert, M., Bretschneider, J., Nathason, E., and Fasoulas, S. (2014). Physicochemical and biological technologies for future exploration missions. Acta Astronaut. 101, 170–179. doi: 10.1016/j.actaastro.2014.04.023 Garnitschnig, S. (2018). Development of a supportive method for the detection
of biomarkers during future human-robotic Mars missions–A case study to
optimize the deployment modalities based on the results of the AMADEE-
18 analog mission in the Dhofar region in the Sultanate of Oman. (Bachelor
thesis). University of Innsbruck, Innsbruck, Austria, 47. Bessone, L., Sauro, F., Maurer, M., and Piens, M. (2018). “Testing technologies and
operational concepts for field geology exploration of the Moon and beyond:
the ESA PANGAEA-X Campaign,” in 20th EGU General Assembly, EGU. Abstract No: 4013. Available online at: https://meetingorganizer.copernicus. org/EGU2018/EGU2018-4013.pdf (accessed July 5, 2020). Geelen,
K.,
Vijendran,
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Rebuffat,
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Missions and Related Technology Developments. London: EPSC, EPSC2013-
1057. Available online at: https://meetingorganizer.copernicus.org/EPSC2013/
EPSC2013-1057.pdf (accessed July 5, 2020). Bessone, L., Sauro, F., and Stevenin, H. (2015). “Training safe and effective
spaceflight operations using terrestrial analogues,” in Space Safety Is No
Accident, eds T. Sgobba and I. Rongier (Cham: Springer), 313–318. REFERENCES Crawford, I. A. (2014). Science enabled by the global exploration roadmap. Astron. Geophysics 55, 3.20–3.23. doi: 10.1093/astrogeo/atu124 Abercromby, A. F. J., Chappell, S. P., and Gernhardt, M. L. (2013). Desert RATS
2011: human and robotic exploration of near-Earth asteroids. Acta Astronaut. 91, 34–48. doi: 10.1016/j.actaastro.2013.05.002 Davila, A. F, Skidmore, M., Fairén, A. G., Cockell, C., and Schulze-Makuch,
D. (2010). New priorities in the robotic exploration of Mars: the case for
in situ search for extant life. Astrobiology 10, 705–710. doi: 10.1089/ast. 2010.0538 Allner, M., McKay, C., Coe, L., Rask, J., Paradise, J., and Wynne, J. J. (2010). NASA’s
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programs designed to heighten public support for space science initiatives. Acta
Astronaut. 66, 1280–1284. doi: 10.1016/j.actaastro.2009.09.019 Deans, M., Marquez, J. J., Cohen, T., Miller, M., Deliz, I., Hillenius, S., et al. (2017). “Minerva: User-centered science operations software capability for
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research to investigate extravehicular activity science operations concepts
and capabilities for human planetary exploration. Astrobiology 19, 300–320. doi: 10.1089/ast.2018.1861 Drake, B. G., Hoffman, S. J., and Watts, K. D. (2010). “Human exploration of Mars
design reference architecture 5.0.,” in 2010 IEEE Aerospace Conference (Big Sky,
MT), 1–24. doi: 10.1109/AERO.2010.5446736 Beaty, D. W., Grady, M. M., McSween, H. Y., Sefton-Nash, E., Carrier, B. L.,
Altieri, F., et al. (2018). iMOST, The Potential Science and Engineering Value
of Samples Delivered to Earth by Mars Sample Return, Final Report August 14,
2018 (MEPAG), 186. Available online at: https://mepag.jpl.nasa.gov/reports/
iMOST_Final_Report_180814.pdf (accessed July 5, 2020). Dunér, D., Capova, K., Gargaud, M., Geppert, W., Kereszturi, A., and Persson,
E. (2018). CONCLUSIONS The proper implementation of the Exploration Cascade is yet to
be demonstrated, in particular, the robustness of the workflows
needs to be tested in representative environments. As such, the
AMADEE-20 mission in Israel in late 2020 will be a proving
ground for applying the EC. Mission teams have strived for a realistic projection of
the workflows involving both flight crews on “Mars” and the
remote science support teams on Earth. However, the multitude
and peculiarities of instruments and technologies available
to mission architects and researchers requires a plethora of
planning decisions, as in contrast to, e.g., rover missions, the
range of decision options rises exponentially with the number
of field assets deployed. Also, the bandwidth of scientific
priorities makes it challenging to identify patterns (e.g., perceived
optimal sequence of workflows) and preferably strategies beyond
anecdotal evidence. Therefore, we advocate for a deeper
understanding of the scaling effects of an increasing number
of field assets, considering long-duration surface sojourns with
engineering constraints along with low bandwidth and time-
delayed communication, as well as human factors. July 2020 | Volume 7 | Article 32 Frontiers in Astronomy and Space Sciences | www.frontiersin.org 4 Groemer and Ozdemir Planetary Analog Research as a Tool individually funded missions, are key to optimizing the science
return for future flight missions. The contribution of analog missions to flight mission
architectures is strengthened by a clear definition of scientific
priorities, awareness about mission architecture assumptions,
and a well-structured workflow that allows for an in-depth
analysis of the mission performance. Besides, a structured
lessons-learned process and emerging well-maintained science
data archives, that are open to the scientific community beyond AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication. REFERENCES doi: 10.1007/978-3-319-15982-9_37 y
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of Mars. Astrobiology 19, 245–259. doi: 10.1089/ast.2018.1869 Copyright © 2020 Groemer and Ozdemir. 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. Litaker,
H. L.,
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R. L. (2013). Social
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INDÚSTRIA CULTURAL E IDEOLOGIA
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* Universidade Federal de Rondônia (UNIR). Departamen
to de Ciências Sociais.
BR 364, Km 9,5. CEP: 76801-059. Porto Velho – Rondônia
– Brasil. humbertoalvesj9@gmail.com
https://orcid.org/0000-0002-5503-5484 INDÚSTRIA CULTURAL E IDEOLOGIA DOSSIÊ DOSSIÊ Humberto Alves Silva Junior* Humberto Alves Silva Junior* O trabalho parte da análise de conteúdo para abordar as discussões sobre o conceito de indústria cultural
cunhado e analisado por Theodor Adorno e Max Horkheimer e seu desdobramento no livro Teoria Estética
de Adorno, em especial sobre o cinema. O conceito compreende o caráter comercial e o modo de produção
industrial das produções culturais no capitalismo, tratadas, inclusive, como mercadorias e suas consequ
ências sobre o público, atuando principalmente como instrumento de manipulação ideológica na visão
adorniana. Entretanto, em Teoria Estética, o autor avança a discussão e admite que, apesar da presença da
ideologia, a indústria cultural poderia também desenvolver um espaço alternativo para produções massifi
cadas. Posteriormente, Frederic Jameson, inspirado no trabalho de Adorno, traçou linha semelhante, ao per
ceber que os produtos da indústria cultural não seriam apenas ideológicos. Para além de Adorno, afirmava
que eles também poderiam ser utópicos, pois a cultura de massa atrai o público com promessas coletivas e
individuais de um futuro melhor. Palavras-chave: Ideologia. Emancipação. Cinema. Cultura de massa. Escola de Frankfurt. INDÚSTRIA CULTURAL E IDEOLOGIA industrial, a nova complexidade do tráfego das
ruas, as intervenções e as demolições urbanas
e o aumento vertiginoso da população. dernidade, torna-se o contrário: dissemina ainda
mais seu caráter tenso. O gosto do grande públi
co pelas novas atividades culturais do século XIX
estava assentado na “impressão de realidade” e
nos estímulos e choques provenientes desses
novos meios de diversão. Muitos autores, como
Adorno, consideravam que esse mecanismo do
entretenimento mantinha o trabalhador preso ao
ritmo veloz das fábricas e dos centros urbanos
modernos, e, mesmo no ócio, o ritmo assemelha
-se ao da produção. De outro lado, os produtos ar
tísticos supostamente buscam semelhanças cada
vez mais estreitas com essa realidade, apenas me
diante um naturalismo. A profusão dessas imagens espelha,
num grau acentuado, o ritmo, a velocidade e
a mudança que marcam a experiência moder
na, o que, por sua vez, condiciona e transfor
ma inexoravelmente os aspectos psicológicos
e fisiológicos dos indivíduos expostos a essa
gama variada de estímulos, tornando-os seres
angustiados, ansiosos, ciclotímicos e nervosos. O cinema também contribui para a formação
desse estado neurológico. Especialmente em
Hollywood, a produção se assemelha à organi
zação de uma indústria, que inclui, dentre ou
tros elementos, uma divisão de trabalho defi
nida, cujo objetivo principal é o lucro. Ou seja,
a realização de um filme segue às exigências
do ritmo do capital, com a rapidez das produ
ções, que atinge toda a equipe de trabalhado
res: atores, diretores, montadores etc. Por outro
lado, o público experimenta sensações típicas
da vida moderna, como atentam Leo Charney
e Vanessa Schwartz: A “impressão de realidade” e os cho
ques intencionalmente veiculados pela indús
tria cultural são recursos frequentes, que se
tornaram norma nesse tipo de produção, em
especial no cinema. O que, de fato, está por
trás desse mecanismo é o padrão da cultura
de massa, que, assim como os outros produ
tos comerciais, necessitam dessa padronização
como meio imprescindível de garantir o lucro. Esse resultado advém, sobretudo, do ca
ráter da cultura de massa, fundamentado na
economia capitalista mais ampla. Desse modo,
assim como a indústria de eletrodomésticos
padroniza todos os âmbitos da produção, a
indústria cultural também necessita padroni
zar todo o seu sistema, seja na produção, na
distribuição, ou mesmo no próprio conteúdo
do produto. INTRODUÇÃO velocidade, a transformação, a efemeridade, a
instabilidade, a tecnologia, a ciência, o espetá
culo, o consumo e a perda da identidade, entre
outros. Esses signos, que comumente atuam na
vida cotidiana moderna de forma aguda, ex
cessiva, e de vários modos, estão inseridos no
cinema. Por esse motivo, Charney e Schawrtz
(2001) consideram o cinema como a arte que
melhor define e sintetiza a modernidade. Afir
mam que “a cultura moderna foi cinematogra
fia antes do cinema”, pois ele surgiu no final
do século XIX, seguindo as linhas da contur
bada realidade social do capitalismo moderno. O acelerado incremento científico e tec
nológico, principalmente a partir do século
XIX, com a Revolução Industrial e a crescente
difusão da cultura de massa no século XX, pro
piciou o crescimento vertiginoso do consumo
do lazer, não apenas facultando o nascimento
do cinema, mas sustentando-o até hoje como
meio de expressão artística, documental ou de
entretenimento comercial. Sob o aspecto cien
tífico, pode-se observar ainda que a “invenção”
do cinema tanto foi um produto desse tipo de
conhecimento, através do cruzamento de di
versas ciências – matemática, física, química,
mecânica óptica e eletricidade –, como tam
bém foi um instrumento destinado ao estudo,
dentre outros objetos, da fisiologia animal, dos
processos da visão e da fotografia. C
CRH S l
d
32
87
505 515 S
O cinema acompanhou a velocidade
dos novos tempos, apresentando várias regiões
do mundo em apenas alguns segundos, atra
vés da mudança rápida das imagens, compri
mindo, assim, as distâncias e o tempo, como
fazem os novos meios de transporte. A rápida
sucessão de imagens expostas ao espectador
provoca a sensação de não pertencimento a
lugar algum, apresentando, de forma cabal, a
identidade descentrada do sujeito moderno,
para o qual não há referências seguras dian
te da intensificação das mudanças. Esse ritmo
veloz acompanha também a pressa do trabalho Por outro lado, o cinema enfeixa, em
seu interior, vários signos fundamentais que
normalmente caracterizam a modernidade: a 505 http://dx.doi.org/ FFUKYL INDÚSTRIA CULTURAL E IDEOLOGIA Portanto, a inserção constante da
tensão, do choque e da chamada “impressão
de realidade” nos produtos artísticos da cultu
ra de massa segue o interesse calculado do em
presário da área cultural em padronizar seus
produtos como meio de apreender a atenção
do espectador e, assim, não arriscar os seus
negócios. O padrão estipulado pela indústria
cultural ocorre exatamente com o fito de agra
dar e acostumar o grande público ao modelo,
garantindo que os produtos culturais de hoje e
os de amanhã serão facilmente vendidos, des
de que sigam a fórmula. Os primeiros filmes de atualidades apresentavam com
frequência um simulacro de viagem não apenas ao
apresentar paisagens estrangeiras, mas também “pas
seios fantasmas, que eram filmados da parte dianteira
de trens ou da proa de barcos e que davam aos espec
tadores, sentados e parados, uma sensação palpável de
movimento (Charney; Schwartz, 2001, p.17). Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019 RH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. Esse estado neurológico provocado pela
modernidade é representado e incitado não
apenas pelo cinema, mas por vários modos de
entretenimento e meios de comunicação que,
na virada do século XIX, tinham como princi
pais motes o choque, o escândalo, o surpreen
dente, o sórdido, São exemplos dessas caracte
rísticas, observa Singer (2001), os cartuns de
revistas e de jornais sensacionalistas norte-a
mericanos, que comumente, em suas páginas,
apresentavam imagens de acidentes domésti
cos e automobilísticos. Caderno CR
Ao contrário do que se poderia imaginar,
o entretenimento, ao invés de servir como um
bálsamo para os problemas nevrálgicos da mo Para Adorno e Horkheimer (1985), o pa
drão da indústria cultural é consolidado pela 506 Humberto Alves Silva Junior técnica, responsável pelo poder de sedução que
imprime sobre os espectadores. Através do seu
aperfeiçoamento constante, a sensação do real
reproduzido pelo modelo é sempre renovada. repetem insistentemente os mesmo clichês –
conforma o espectador aos mecanismos de ma
nipulação, inculcando noções maniqueístas de
certo e de errado, de bom e de mau, as quais,
frequentemente, estão de acordo com as pers
pectivas da moral dominante. O padrão exerce
também uma função ideológica e estabelece
diretrizes não só para a forma do produto artís
tico comercial, para a qual o espectador deve
ser previamente preparado, mas igualmente
difunde ideias, valores, normas e regras de
conduta dominantes. Nesse sentido, a atração exercida pela
cultura de massa, principalmente o cinema co
mercial, está intimamente ligada ao poder de
levar os espectadores a um estado de superex
citarão, no qual eles passam a se sentir como
se, de fato, estivessem vivenciando aquilo que
é representado, entregando-se ao deleite das
sensações e da emoção. Esses estados “sensa
cionalistas” produzidos pela indústria cultural
tornam cada vez mais perfeita a ilusão de que
o produto artístico é similar ao mundo real, ou
que uma possível realidade futura, como no
caso dos filmes de ficção, encontra-se a nosso
alcance nos dias atuais. O conteúdo das obras da cultura de mas
sa está, portanto, impreterivelmente de acordo
com o establishment, pois seus produtores ten
tam convencer que a ordem social, defendida
tão bem nos filmes, é a única possível, elimi
nando qualquer fator que coloque em risco a
moral e os interesses econômicos do capitalis
mo. INDÚSTRIA CULTURAL E IDEOLOGIA entre alta cultura e cultura de massa, no âmbi
to mais amplo da indústria cultural. nome de um novo conceito de arte que, além de
estar em consonância com o dinamismo da mo
dernidade, deveria instaurar “originalidade e
espírito de pesquisa” como “elementos centrais
da produção artística ” (Xavier, 1978, p. 62). Esses movimentos pretendiam produ
zir filmes que escapassem da padronização
da narrativa clássica de Hollywood e que, ao
mesmo tempo, iniciassem uma nova expres
são artística. Estavam interessados em empres
tar ao cinema um caráter artístico, tal como se
verifica no teatro, na pintura, na música, na
escultura e na poesia. Seus membros passa
ram então a formular teorias estéticas para dar
fundamentação à nova arte, cujo suporte é a
estrutura cinematográfica. Os filmes, segundo essa concepção, de
veriam conduzir os espectadores, através da
imersão nas imagens, a um estado de liberta
ção dos mecanismos da lógica, percorrendo os
caminhos do fluxo imagético. Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019 Se a cultura de massa não aniquila os es
paços destinados à reflexão e ao livre pensar do
espectador, tais espaços ficam bastante reduzi
dos por conta das características do capital. Ao padrão inerente das produções co
merciais corresponde – presumem seus ide
alizadores – uma recepção também padroni
zada. Devido à difusão em massa do produto
comercial, busca-se construir uma espécie de
espectador adaptado a um estilo estereotipa
do, ansioso por se reconhecer na mesma forma
estética de sempre. No cinema, o padrão de
termina também o modo de olhar do público,
pois a lente normalmente é colocada em um
ponto no qual possa causar o efeito de se estar
vendo uma “realidade objetiva” (Xavier, 1978,
p. 22). Por isso, a narrativa se torna o modelo
mais comum nesse tipo de expressão. A racionalidade capitalista incorporada
ao fazer artístico interfere no conteúdo da obra
de arte, e é por esse motivo que surgem vários
movimentos com a pretensão de resgatar, ou
até mesmo fundar, novos princípios defini
dores da arte. Esses grupos normalmente vão
além desse objetivo ao criticarem a sociedade
capitalista e seus mecanismos de controle, em
princípio incompatíveis com a tendência liber
tadora da arte. O grande público da indústria cultural
e do cinema, acostumado com a linguagem
do cinema de Hollywood, rejeita o discurso
discrepante que possa destoar da linguagem
simplista dos vários gêneros comerciais, su
bordinando-se ao sensacionalismo repetitivo
que embala as produções da cultura de massa. O propósito é apenas o divertimento, através
da distensão emocional induzida por mecanis
mos técnicos padronizados que, aparentemen
te, aproximam a representação do real. C
C
S l
d
S
No caso do cinema, os primeiros movi
mentos artísticos surgem nas primeiras déca
das do século XX, como o Cinema Soviético. Teóricos, críticos e artistas, de um modo geral,
passam a elaborar filmes que tentam fugir das
padronizações impostas pela estética indus
trial, pretendendo trazer, também para o cine
ma, a aura artística já configurada em outras
expressões estéticas. É a partir de então que
se fomenta a oposição entre filme comercial e
filme de arte, ou filme de autor (termo elabo
rado pelos críticos da Cahiers du Cinéma na
década de 1950), que corresponde à oposição Há, por último, para Adorno e Horkhei
mer (1985), o padrão do próprio conteúdo
– com um número limitado de gêneros que 507 INDÚSTRIA CULTURAL E IDEOLOGIA ADORNO, HORKHEIMER E A CRÍ
TICA À INDÚSTRIA CULTURAL Os primeiros passos nesse sentido foram
dados ainda no cinema mudo. Grupos de intelec
tuais vinculados à renovação artística moderna
viram no cinema, até então considerado como
mero entretenimento, não só o meio ideal para a
expressão de uma nova arte, mas também a con
sideraram a arte moderna por excelência, pela
ampla capacidade expressiva do novo suporte
artístico, que possibilita ao artista experimentar
uma gama maior de recursos estéticos. No campo da teoria social o texto A In
dústria cultural – o esclarecimento como mis
tificação das massas, parte integrante do li
vro Dialética do esclarecimento, de Adorno e
Horkheimer, lançado em 1947, aposta numa vi
são pessimista em relação aos meios de expres
são representados pela indústria cultural, ape
sar da existência de uma crítica em relação às
produções da cultura de massa, principalmente
no interior do cinema. Como foi mostrado an
teriormente, esses autores aparentemente a ig
noraram, e elaboraram uma contundente crítica
aos “produtos artísticos industrializados”. Por outro lado, o cinema, que tem sua
origem na ciência e na técnica, é a arte que me
lhor traduz, através da imagem em movimen
to, a mudança, a efemeridade e a fragmentação
que formam a base material da vida moderna. O artista moderno tem, diante de si, um instru
mento privilegiado, que lhe permite conformar
sua obra às condições de vida produzida pelo
espírito na modernidade. Por conseguinte, para Adorno e Horkhei
mer, à medida que se ampliavam os eventos artís
ticos de um modo geral, difundia-se também um
tipo de produção artística voltada para o grande
público, tendo por meta principal o lucro. O cres
cimento dessa perspectiva promove a absorção
dos bens culturais pela lógica empresarial, que
organiza todas as etapas de construção da obra. É
a partir, portanto, desses elementos que se funda
menta o produto artístico comercial, tão caracte
rístico da cultura de massa. Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019 or, v. 32, n. 87, p. 505-515, Set./D
Dessa forma, o autor de cinema alcança
aquele ideal segundo Charles Baudelaire, que
definia o artista moderno como “alguém capaz
de concentrar a visão em elementos comuns da
vida da cidade”, compreendendo “suas qualida
des fugidias e, ainda assim, extrair, do momen
to fugaz, todas as sugestões da eternidade nele
contida” (Baudelaire, apud Harvey, 1999, p. 29). Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./D
Dessa forma, o autor de cinema alcança
aquele ideal segundo Charles Baudelaire, que
definia o artista moderno como “alguém capaz
de concentrar a visão em elementos comuns da
vida da cidade”, compreendendo “suas qualida
des fugidias e, ainda assim, extrair, do momen
to fugaz, todas as sugestões da eternidade nele
contida” (Baudelaire, apud Harvey, 1999, p. 29). A proposta dos primeiros teóricos do ci
nema segue, de perto, as orientações das van
guardas de outras expressões artísticas moder
nas, vinculadas, principalmente, às artes plás
ticas e à literatura, que combatiam os antigos
ideais de beleza e perfeição da arte clássica, em Em virtude disso, o status da arte muda
de perfil, devido ao impacto causado pela co
mercialização dos bens culturais. Um primei
ro aspecto a observar é que, no capitalismo, as
relações sociais que envolvem o fazer artísti
co passam, como outras relações sociais, a ser
mediadas pelo dinheiro; portanto, mesmo as 508 Humberto Alves Silva Junior invés de, inversamente, formar o público mais am
plo numa cultura intacta em substância (Habermas,
2003, p. 195). obras de arte que, de fato, pretendem testemu
nhar uma crítica à economia capitalista, de
vem passar pela chancela do capital. Com isso,
as obras de arte, que, outrora, eram considera
das como elementos de veneração e respeito,
perdem sua aura e tornam-se mercadorias. Um
segundo aspecto significativo é o fato de o ar
tista se tornar um trabalhador comum, atento
às vicissitudes do mercado para poder garan
tir sua própria sobrevivência. Como observam
Marx e Engels, no Manifesto Comunista: O caráter comercial dos produtos da cul
tura de massa acaba por determinar a forma
e o conteúdo desses mesmos produtos, com a
intenção de que eles sejam facilmente absorvi
dos por um número cada vez maior de espec
tadores (consumidores). É por esse motivo que
as produções comerciais seguem determinadas
fórmulas predefinidas, exatamente para atingir
o grande público, fazendo com que ele se fa
miliarize com o modelo. Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019 É nesse sentido que
Adorno e Horkheimer se referiam à importân
cia dos clichês nas produções cinematográfi
cas da cultura de massa: A burguesia despojou de sua auréola todas as ativida
des até então reputadas veneráveis e encaradas com
piedoso respeito. Transformou em seus trabalhado
res assalariados o médico, o jurista, o padre, o poeta,
o homem de ciência (Marx; Engels, 1986, p.24). As obras de arte, assim como a ciência,
já não são mais objetos de intensos debates
como ocorria antes nos cafés e salões; em seu
lugar, assume a cultura de massa, que não fa
vorece, como afirmam Adorno e Horkheimer
(1985), o livre curso do raciocínio político e ar
tístico, pois a informação é dada a partir de um
centro, sem que o espectador possa replicar. Uma prova de que, no capitalismo, a arte pas
sa a ser uma mercadoria como outra qualquer
e se insere na lógica mais ampla do mercado
está no fato de que sua difusão está atrelada
ao aparato do comércio geral, visando, funda
mentalmente, ao lucro. Não somente os tipos das canções de sucesso, os
astros, as novelas ressurgem ciclicamente como in
variantes fixos, mas o conteúdo do espetáculo é ele
próprio derivado deles e só varia na aparência. Os
detalhes se tornam fungíveis. A breve sequência de
intervalos, fácil de memorizar, como mostrou a can
ção de sucesso; o fracasso temporário do herói, que
ele sabe suportar como good sport que é; a boa pal
mada que a namorada recebe da mão forte do astro;
sua rude reserva em face da herdeira mimada são,
como todos os detalhes, clichês prontos para serem
empregados arbitrariamente aqui e ali e completa
mente definidos pela finalidade que lhes cabe no
esquema (Adorno; Horkheimer, 1985, p. 117-118). H Salvador v 32 n 87 p 505-515 Set /Dez 2019
A configuração da arte como um bem de
consumo a partir da indústria cultural, colo
ca em foco a tensão existente entre a esfera da
produção e da circulação eminentemente mer
cantil relacionada ao entretenimento, e uma
outra comumente chamada de modernismo,
“erudita”, de “arte”, ou ainda da alta cultura,
mais preocupada com os aspectos estéticos
das obras. A primeira se insere de forma mais
completa nos parâmetros da indústria cultural,
enquanto a segunda tenta se estabelecer como
uma resistência ao processo de mercantiliza
ção total da arte. INDÚSTRIA CULTURAL E IDEOLOGIA Por conseguinte, ocorre uma dupla
contradição em relação às produções da alta
cultura. Por um lado, a arte corre o risco de ter
seu valor intrínseco subjugado pelo valor mo
netário; por outro, isso torna vazio o protesto
político comumente relacionado a essa tendên
cia, vazio, pois as obras dependem do sistema
político e econômico que seus autores criticam. Há também uma outra similaridade en
tre os dois tipos de produção, que é a busca in
cessante pelo novo. Na cultura de massa, isso
aparece, por exemplo, na frequente criação
de gêneros e subgêneros: suspense, terror, fic
ção científica, pornografia, western. Contudo
a mudança é aparente, pois os estilos sempre
retornam com poucas modificações, para ape
nas chamar a atenção do público, que, assim,
se sente como estivesse, de fato, consumindo
um produto novo. Na alta cultura, a busca per
manente pelo novo também ocorre, mas com Portanto até mesmo as artes mais tradi
cionais, como a pintura, necessitam ser inter
mediadas pelo comércio até que alcancem o
público. Por conseguinte, ocorre uma dupla
contradição em relação às produções da alta
cultura. Por um lado, a arte corre o risco de ter
seu valor intrínseco subjugado pelo valor mo
netário; por outro, isso torna vazio o protesto
político comumente relacionado a essa tendên
cia, vazio, pois as obras dependem do sistema
político e econômico que seus autores criticam. Ainda que, de fato, essas afirmações se
refiram à cultura de massa, é preciso fazer al
gumas ressalvas, pois elas acabam conceden
do um poder absoluto à ideologia dominante,
compreendendo a indústria cultural apenas
como manipulação coletiva. Entretanto, em contraponto ao sentido
pessimista contido no texto Indústria Cultu
ral em relação à cultura de massa, Adorno, na
Teoria Estética, já teria apontado, em algumas
passagens, elementos utópicos na arte contem
porânea por ele considerada até então como
reificada. Adorno, nesse último texto, ressalva
que o segmento crítico da arte moderna repre
senta um lócus de contestação no interior das
relações reificadas do capital. Caderno CRH, Salvador, v. 32, n. 87, p. 505-51
Há também uma outra similaridade en
tre os dois tipos de produção, que é a busca in
cessante pelo novo. Na cultura de massa, isso
aparece, por exemplo, na frequente criação
de gêneros e subgêneros: suspense, terror, fic
ção científica, pornografia, western. Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019 Dessa forma, o fazer artístico, no capi
talismo, se destina prioritariamente às neces
sidades do capital, sendo controlado por ele e
não mais correspondendo às necessidades do
espírito, como afirma Habermas: […] as leis do mercado já penetram na substância
das obras, tornando-se imanentes a elas como leis
estruturais. Não mais apenas a difusão e escolha, a
apresentação e embalagem das obras – mas a pró
pria criação delas enquanto tais se orienta nos seto
res amplos da cultura dos consumidores, conforme
pontos de vista da estratégia de vendas no merca
do. Sim, a cultura de massa recebe o seu duvidoso
nome exatamente por conformar-se às necessidades
de distração e diversão de grupos de consumidores
com um nível de formação relativamente baixo, ao Entretanto a linha que delimita essas
duas tendências é extremamente precária, pois
existem pontos de convergência entre elas. Isso 509 INDÚSTRIA CULTURAL E IDEOLOGIA se deve ao fato de tanto as produções comer
ciais como as produções da alta cultura se en
contrarem inseridas no complexo mais amplo
da economia capitalista. Uma está mais direta
mente ligada ao caráter mercantil, fundamen
tada em uma estrutura burocrática capitalista,
a outra se estabelece como contraponto à pri
meira, na tentativa de garantir a preponderân
cia do valor artístico sobre o valor econômico. Contudo as duas vertentes estão relacionadas
à lógica capitalista; seus produtos precisam
ser elaborados, distribuídos e comercializados
dentro do processo econômico existente. uma outra intenção, a de criticar a sociedade
de consumo e romper com o formalismo ideo
lógico da cultura de massa. Entretanto as ino
vações alcançam um patamar-limite, devido
à própria banalização do recurso, estiolando
o processo criativo; a pretensão de causar es
cândalo se esgota, e o recurso é, em si, neutra
lizado. Assim, o projeto estético inovador da
cultura moderna é derrotado. Um último ponto a ser observado em
relação a uma possível interpenetração entre
alta cultura e cultura de massa se refere à ide
ologia. No texto Industria Cultural, Adorno e
Horkheimer definiam as produções da indús
tria cultural como ideológicas, apontando uma
suposta manipulação total do público, como
fica evidente na seguinte passagem: Uma segunda observação comumente
feita entre os estudiosos da cultura refere-se ao
aspecto mercantil, pois quase nenhuma pro
dução cultural, de “arte” ou “comercial”, pode
escapar da esfera da circulação na sociedade
capitalista. É esse segundo dilema que demar
ca a oposição entre alta cultura e cultura de
massa. Uma manifestação estética somente po
derá completar seu significado estético se for
colocada para a apreciação pública. Os consumidores são os trabalhadores e os empre
gados, os lavradores e os pequenos burgueses. A
produção capitalista os mantém presos em corpo e
alma e eles sucumbem sem resistência ao que lhes é
oferecido. Assim como os dominados sempre leva
ram mais a sério do que os dominadores a moral que
deles recebiam, hoje em dia, as massas logradas su
cumbem mais facilmente ao mito do sucesso do que
os bem-sucedidos. Elas têm os desejos deles. Obsti
nadamente, insistem na ideologia que as escraviza
(Adorno; Horkheimer,1985 p. 125). Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019
colocada para a apreciação pública. Portanto até mesmo as artes mais tradi
cionais, como a pintura, necessitam ser inter
mediadas pelo comércio até que alcancem o
público. 1 Benjamin foi um dos primeiros autores da Escola de
Frankfurt que concebia de forma positiva as novas ex
pressões artísticas fundamentadas na reprodução técnica
(principalmente no texto A obra de arte na época de sua
reprodutibilidade técnica) e, nesse sentido, avaliava que
os conceitos que definiam a arte em um período anterior
à modernidade teriam sido superados; ao mesmo tempo,
argumenta a favor de um suposto potencial político dessa
nova arte. INDÚSTRIA CULTURAL E IDEOLOGIA Contudo
a mudança é aparente, pois os estilos sempre
retornam com poucas modificações, para ape
nas chamar a atenção do público, que, assim,
se sente como estivesse, de fato, consumindo
um produto novo. Na alta cultura, a busca per
manente pelo novo também ocorre, mas com Depois de vinte e três anos entre a publi
cação de Indústria Cultural e o texto póstumo 510 Humberto Alves Silva Junior Teoria Estética, Adorno modificava em parte
suas impressões sobre a indústria cultural,
reconhecendo o pioneirismo de Walter Ben
jamin1 ao observar possíveis utilizações dos
meios de comunicação de massa modernos a
favor da emancipação humana. uma arte contemporânea compromete-se no
plano teleológico, político e prático com a rea
lidade social, mesmo que inserida na lógica da
indústria cultural, admitindo que isso é pos
sível através da conjunção dos meios técnicos
avançados com as experiências estéticas tam
bém avançadas, Adorno discute a resistência
da arte em relação aos aspectos reificantes da
indústria cultural: Toda obra, enquanto destinada a uma pluralidade, é
já, segundo sua ideia, a sua reprodução. Que Benja
min, na dicotomia da obra de arte aurática e da obra
de arte tecnológica, reprimisse este momento de
unidade em favor da diferença, que seria de fato a
crítica dialética de sua teoria (Adorno, p. 59, 2008). No entanto, quando a arte autônoma absorveu seria
mente os procedimentos técnicos industriais, estes
permaneceram-lhe exteriores. A reprodutibilidade
em massa de nenhum modo se tornou lei formal
imanente, como a identificação com o agressor se
compraz em afirmar. No próprio cinema, os momen
tos industriais e estético-artesanais divergem sob
pressão socioeconômica da industrialização radical
da arte, a sua adaptação integral aos padrões técni
cos alcançados colide com o que na arte se recusa à
integração (Adorno, 2008, p. 327). Assim, Adorno observava que o moder
nismo e o aspecto técnico de perfil industrial,
na arte contemporânea, eram fenômenos reais,
que não destruiriam a arte pelo fato de ela ter
sido impelida pelos condicionamentos técni
cos e econômicos da indústria cultural. É antes o postulado rimbaudiano mais progressista,
no qual os procedimentos técnicos mais avançados
e mais diferenciados se interpenetram com as ex
periências mais avançadas e mais diferenciadas. Mas estas, enquanto sociais, são críticas. Esta arte
moderna deve mostrar-se adulta à grande indústria,
não a manipulando apenas. INDÚSTRIA CULTURAL E IDEOLOGIA Além disso, ele também reconhece que a mar
ginalização da arte moderna radical seria um
sintoma da reação do status quo da sociedade
em relação às inovações propostas. da logicidade como elemento essencial na sua
definição. Para Adorno, as obras modernas ten
dem a ser abertas e, assim, dissociarem a forma
do conteúdo. Mas mesmo os arroubos revolu
cionários e inovadores dos mais variados tipos
de modernismo, na tentativa de estabelecer
uma arte inquieta, instável, desestabilizadora
da percepção, não são capazes de excluir a ló
gica do interior dessas novas formas. Ele cha
ma a atenção para definição da forma a partir
das ideias de simetria e repetição, mas que,
mesmo quando se tenta destruir essa “harmo
nia”, a lógica em si não desaparece, como ele
defende na Teoria Estética: O caráter técnico da arte moderna está
de acordo com o estilo de vida contemporâneo
e os meios de produção. No caso da arte mo
derna, segundo Adorno, os vestígios técnicos
se destacam e aderem a toda a obra nova, como
cicatrizes. O caráter técnico da arte moderna está
de acordo com o estilo de vida contemporâneo
e os meios de produção. No caso da arte mo
derna, segundo Adorno, os vestígios técnicos
se destacam e aderem a toda a obra nova, como
cicatrizes. No entanto, essa mesma obra se opõe ao
mundo por ela representado, exatamente por
configurá-lo de modo distinto, corresponden
do à interioridade dos homens como represen
tação, e, por conseguinte, representação de um
período. Portanto o conceito marxista de ideo
logia é capaz de dar pistas para decifrar o cará
ter concreto da relação entre a arte e a socieda
de, que constitui o estatuto das representações
sociais e das representações cinematográficas. As análises musicais, por exemplo, mostram que
mesmo nas obras mais desorganizadas e mais opos
tas à repetição existem analogias, que numerosas
partes correspondem a outras em quaisquer carac
terísticas e que apenas pela referência a elementos
idênticos é que se realiza a não identidade procura
da; sem nenhuma semelhança o caos permaneceria
por seu turno uma invariante (Adorno, 2008, p. 216). RH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019
Adorno também reconhecia que a arte
está imbuída do desejo de se construir um
mundo melhor. Entretanto ele era pessimis
ta em relação às possibilidades utópicas das
obras em relação à sociedade. INDÚSTRIA CULTURAL E IDEOLOGIA O seu próprio compor
tamento e a sua linguagem formal devem reagir es
pontaneamente à situação objetiva; a reação espon
tânea, enquanto norma, circunscreve em um para
doxo eterno da arte (Adorno, p. 59, 2008). Mesmo mantendo reservas em relação
ao cinema, Adorno considerava como exemplo
que não seria possível, mesmo na “industriali
zada”, uma integração completa entre o aspec
to técnico e o estético, apagando-se os rastros
desse último em uma arte relacionada com a
reprodutibilidade técnica. Pelo contrário, para
o autor, há uma resistência do aspecto estético
diante da técnica, como é caso do cinema, no
qual há uma tensão entre os fatores “estéticos”
e os “industriais”, a partir da pressão exercida
pelos interesses econômicos e políticos dos
grupos envolvidos na produção cinematográfi
ca, apesar de reconhecer, na Teoria Estética, as
possibilidades da arte em um novo contexto das
condições sociais e econômicas de produção. Adorno flexibilizava seu posicionamen
to no texto posterior, reconhecendo a interpe
netração da técnica com as experiências esté
ticas na arte contemporânea e a possibilidade
de aproveitar ao máximo dessa conjunção para
elaborar uma reação à própria indústria cul
tural, com sua tendência homogeneizadora e
manipuladora. Uma reação que se inscreve na
tradição da arte: “reagir à situação objetiva”. Adorno compreendia as relações intrínsecas
entre arte e a situação de seu tempo. Para ele, Hoje em dia, é já possível, na eletrônica, produzir
artisticamente a partir da natureza específica de
meios de origem extra-artística. O salto qualitativo
é evidente entre a mão que desenha um animal na
parede da caverna e a câmera, que permite o apare
cimento simultâneo das imagens em inúmeros luga
res (Adorno, 2008, p.59). Ca
o CR
Assim, o autor reconhece o caráter qua
litativo diferenciado dessa nova arte com a
reprodução das imagens em locais distintos. 511 INDÚSTRIA CULTURAL E IDEOLOGIA INDÚSTRIA CULTURAL E IDEOLOGIA Segundo o autor,
Freud defendia que as obras não são satisfa
ções imediatas do desejo, mas servem de meio
para transformar a pulsão libidinal em pro
dução social, apresentando, assim, o caráter
acrítico da obra na sociedade, e, desse modo,
concebendo a arte como aceitação conformis
ta. Assim, a psicanálise, que concebe a obra de
arte como um bem cultural agradável, acaba
por excluir a negatividade da arte, desperdi
çando os conflitos pulsionais que estão em sua
gênese e que poderiam indicar o seu potencial
de desvelação. Nesse sentido, Adorno conce
bia que uma arte com essas características faz
sumir a admiração para mergulhar o indivíduo
na obra, não mantendo a devida distância, o
desinteresse que permite estabelecer a diferen
ça entre a arte e a sociedade, e, assim, manter
o seu caráter de negatividade. Estendendo essa análise aos filmes pro
duzidos por algumas vanguardas, como o Ci
nema Soviético de 1920, é possível perceber
que, por mais que se utilizem recursos para
destruir a diegese do filme, sua forma mesma
não é destruída, e o esforço em apagar os li
mites tradicionais da representação fílmica se
torna uma constante; ele, por si só, estabele
ce uma constância, uma coerência, uma lógi
ca. Para Adorno, a associação entre a obra e o
real, que o artista e o crítico realizam, é uma
obrigação que faz parte da legitimação social
de qualquer arte. A intenção de desconstruir
do setor crítico da arte moderna e massificada
serve mais para cobrir do que para se destacar
como uma crítica verdadeira. JAMESON E AS POSSIBILIDADE
POLÍTICAS DA INDÚSTRIA CUL
TURAL A partir do texto clássico de Adorno so
bre a indústria cultural, o autor contemporâneo
Frederic Jameson elaborou reflexões fundamen
tais para a compreensão do fenômeno da cultura
de massa. Crítico do primeiro texto de Adorno,
Indústria cultural – o esclarecimento como mis
tificação das massas, esse autor defende uma
concepção mais pretensiosa para as produções
artísticas de caráter industrial. Passa a nutrir es
peranças em relação à arte contemporânea, de
fendendo que mesmo as obras mais massificadas,
inseridas por completo no sistema de produção
vigente, podem obter um grau de criatividade e
até mesmo um conteúdo político. A abordagem de Jameson relaciona es
sas duas instâncias opostas, ideologia e utopia,
como fundamentais para se compreender a
complexidade da relação entre a produção ar
tística na modernidade, em especial o cinema,
e sua recepção pelo público. O autor ressalta
também a relação entre, de um lado, cinema,
interesses dominantes do capital, produção de
ideologia no contexto cinematográfico, e, de
outro, suas implicações sociais. Assim, Jame
son percebia que a dimensão utópica da cons
ciência é indissociável da dimensão ideológi
ca, observando que há uma troca compensató
ria, uma gratificação, através de um vislumbre
positivo de um sentimento de comunidade em
troca da passividade. Jameson destaca que, na indústria cultu
ral, a arte política sobrevive concomitantemen
te com a ideologia dominante, inserida na pró
pria obra crítica. Ele afirma que o mesmo lugar
de atuação da ideologia na produção artística é
o lugar de sua crítica, defendendo que manipu
lação e utopia estão imbricadas no cinema. De
modo semelhante, Jameson ao se contrapor à
ideia da arte apenas como manipulação, colo
ca outro conceito freudiano, o de recalque. Ao
analisar as obras da cultura de massa, aponta
que elas exerceriam um poder de contenção em
relação aos sentimentos negativos, como: “trau
ma, memória culpada, desejo culpado ou inti
midador, angústia” (1995, p.25), em que o dese
jo recalcado é aplacado por um preenchimento
simbólico, como afirma na seguinte passagem
do livro O Inconsciente Político: Compreendendo desse modo essa jun
ção, vê-se que a ideologia não é apenas coer
ção, mas também sedução, e, por isso ela não
é algo mecânico, que surge apenas como um
epifenômeno da infraestrutura. Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019 Adorno, em Teoria Estética, defendia que
a arte crítica (parte do modernismo) tem de se
adequar às exigências do status quo, pois, se
gundo esse autor, as obras verdadeiramente au
tênticas são incompatíveis com a figuração que
a própria sociedade tem de si mesma, a pon
to de colocar em risco a sua autoconservação. Por isso cabe ao artista, sob essa perspectiva, ir Caderno CR
Outra observação adorniana sobre a arte
moderna, que é extremamente fecunda nas
análises cinematográficas, é sobre a presença 512 Humberto Alves Silva Junior jetos espúrios, então um passo preliminar também
deve ser teorizado em que esses mesmos impulsos
– na matéria prima sobre qual age o processo – são
inicialmente despertados dentro do próprio texto
que busca silenciá-los (Jameson, 1988, p. 297). além. Ele defende que o artista precisa ir ao ex
tremo em sua criação contando com o material
que possui, no interior nas condições de exis
tência em que se encontra, como a tecnologia. Jameson, referindo-se ao cinema, afirma
que o recalque e a satisfação dos desejos corres
pondem à unidade de um mesmo mecanismo
no interior do aspecto imaginário da obra de
arte. Mas não apenas isso: ele observa a dimen
são utópica no interior do próprio recalque, da
própria ideologia, pois esses mecanismos tam
bém estão associados às esperanças e fantasias
positivas da coletividade, que são expressas de
forma independente das distorções ideológicas
do cinema. INDÚSTRIA CULTURAL E IDEOLOGIA e do esmero estético que os caracteriza, eram
veiculados nos mesmos canais de divulgação
dos filmes comerciais. Adorno, antes de Jameson, concebia
que a arte não é desprovida de ideologia e de
verdade, como se fossem cão e gato. Por isso
mesmo, reforça a ideologia do material estéti
co da arte, pois ideologia e verdade aparecem
indivisíveis, na totalidade administrada pela
sociedade. Como elemento superestrutural, a in
dústria cultural é dependente das condições de
seu tempo, da economia e da história, e aponta
para elementos da realidade social contempo
rânea, tanto na técnica como no conteúdo. O
conceito adorniano demonstra sua capacidade
de suscitar novas discussões, tanto que o pró
prio Adorno o retoma vinte anos depois, em
uma abordagem mais ampla do significado da
indústria cultural. Por conseguinte, é possível perceber
que a representação fílmica se encontra no in
terior da representação ideológica. No sentido
social mais amplo, o cinema, como as outras
instituições sociais, são veículos da ideologia
dominante, mas, ao mesmo tempo, podem ser
espaços da crítica à ideologia. A respeito da cultura de massa e da in
dústria cultural, Jameson afirmava que esse
binômio continua como referência principal
de crítica por parte do cinema alternativo, no
esforço de cineastas que não concordam com o
modo industrial e a ideologia dessas produções
comerciais, apesar do constante crescimento
do entrecruzamento de aspectos dos dois tipos
de cinema, tornando mais aguda a dificuldade
em classificar um filme em uma das duas cate
gorias. O que não impede que elas sejam abor
dadas, discutidas e redefinidas ao se investigar
a realidade da arte contemporânea, em espe
cial o cinema e suas relações com a sociedade. É nesse sentido que assim afirma Jameson: Recebido para publicação em 10 de junho de 2019
Aceito em 18 de outubro de 2019 JAMESON E AS POSSIBILIDADE
POLÍTICAS DA INDÚSTRIA CUL
TURAL Para Jameson, analogamente, a consci
ência de classe é ideológica e utópica, pois car
rega em si a esperança, a alegria e o desejo de
se viver em comunidade, ao mesmo tempo em
que contém forte tendência à manipulação e à
acomodação. Do mesmo modo poderia ser per
cebida a recepção no cinema, pois ela é uma
expressão alegórica de uma solidariedade que
se encontra no imaginário coletivo. […] a função ideológica da cultura de massa é enten
dida como um processo pelo qual impulsos de outra
forma perigosos e protopolíticos são ‘administrados’
e desativados, racionalizados e se lhes oferecem ob 513 XAVIER, I. Sétima arte: um culto moderno. São Paulo:
Perspectiva, 1978. Recebido para publicação em 10 de junho de 2019
Aceito em 18 de outubro de 2019 REFERÊNCIAS ADORNO,
T.;
HORKHEIMER,
M. Dialética
do
esclarecimento. Rio de Janeiro: Jorge Zahar, 1985. ADORNO, T. W. Teoria estética. Lisboa: Edições 70, 2008. CHARNEY, L.; SCHWARTZ, V. O Cinema e a invenção da
vida moderna. São Paulo: Cosac & Naif, 2001. BENJAMIM, W. Magia e técnica, arte e política. São Paulo:
Brasiliense, 1994. HABERMAS, J. Mudança estrutural na esfera pública. Rio
de Janeiro: Tempo Brasileiro, 2003. HARVEY, D. Condição pós-moderna. São Paulo: Loyola,
1999. JAMESON, F. O inconsciente político. São Paulo: Ática,
1992. Humberto Alves Silva Junior Humberto Alves Silva Junior De l’analyse de contenu, analyse les discussions
sur le concept d’industrie culturelle inventé et
analysé par Theodor Adorno et Max Horkheimer et
son déploiement dans le livre La théorie esthétique
d’Adorno, en particulier le cinéma. L’industrie
culturelle ou culture de masse comprend le caractère
commercial et le mode de production industriel des
productions culturelles du capitalisme, même traitées
comme des marchandises et leurs conséquences
pour le public, agissant principalement comme
un instrument de manipulation idéologique selon
la vision adornienne. Cependant, dans la théorie
de l’esthétique, Adorno fait avancer la discussion
et admet que, malgré la présence d’une idéologie,
l’industrie culturelle pourrait également développer
un espace alternatif aux productions de masse. Plus tard, Frederic Jameson, inspiré par le travail
d’Adorno, tire un trait similaire dans lequel il réalise
que les produits de l’industrie culturelle ne seraient
pas seulement idéologiques. En plus d’Adorno,
il a affirmé qu’ils pourraient aussi être utopiques
promesses collectives et individuelles d’un avenir
meilleur. This article, based on content analysis, analyzes the
discussions about the concept of cultural industry
coined and analyzed by Theodor Adorno and Max
Horkheimer and its unfolding in the book Adorno’s
Aesthetic Theory, especially cinema. The cultural
industry or mass culture comprises the commercial
character and industrial mode of production of
cultural productions in capitalism, treated even as
commodities and their consequences to the public,
acting mainly as an instrument of ideological
manipulation in the adornian view. However, in
Aesthetic Theory, Adorno advances the discussion
and admits that despite the presence of ideology, the
cultural industry could also develop an alternative
space to mass productions. Later Frederic Jameson,
inspired by Adorno’s work, draws a similar line in
which he realizes that the products of the cultural
industry would not only be ideological, and in
addition to Adorno, he asserted that they could
also be utopian, as mass culture attracts the public
with collective and individual promises of a better
future. Mots-clés:
Idéologie. Émancipation. Cinéma. Culture de masse. École de Frankfurt. Mots-clés:
Idéologie. Émancipation. Cinéma. Culture de masse. École de Frankfurt. Mots-clés:
Idéologie. Émancipation. Cinéma. Culture de masse. École de Frankfurt. Keywords: Ideology. Emancipation. Movie theater. Mass culture. Frankfurt School. Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019
Humberto Alves Silva Junior – Doutor em Ciências Sociais pela UFBA. Professor do Departamento
de Ciências Sociais da Universidade Federal de Rondônia. Keywords: Ideology. Emancipation. Movie theater.
Mass culture. Frankfurt School. Caderno CRH, Salvador, v. 32, n. 87, p. 505-515, Set./Dez. 2019
t
t
c
q
p
g
r
C
m 5-515, Set./Dez. 2019
Tal aproximação exige que se leia a alta cultura e
a cultura de massa como fenômenos objetivamen
te relacionados e dialeticamente interdependentes,
como formas gêmeas e inseparáveis da fissão da
produção estética sob o capitalismo (Jameson, 1995,
p. 25). JAMESON, F. As marcas do visível. Rio de Janeiro: Graal,
1995. JAMESON, F. Pós-modernismo – a lógica cultural do
capitalismo tardio. Ática, 1996. MARX, K.; ENGELS, F. O manifesto do partido comunista. Petrópolis: Vozes, 1986. PIZZINI, J. Cinemais. Rio de Janeiro: Aeroplano, jan./fev. 2003. p.10-13. n. 33, Caderno CRH, Salvador, v. 32, n. 87, p. 5
A aproximação se estabelece na própria
tentativa de se classificar uma obra como per
tencente a um dos lados da oposição entre alta
cultura e cultura de massa, pois existem obras
que possuem tanto as características de um
produto comercial como aos traços que confi
guram uma produção “artística” da alta cultu
ra. Um exemplo disso são os filmes de Charles
Chaplin, que, apesar do tom denunciatório das
mazelas, seja do capitalismo, seja do fascismo, SINGER, B. Modernidade, hiperestímulo e o início do
sensacionalismo. In: CHARNEY, L.; SCHWARTZ, V. O
cinema e a invenção da vida moderna. São Paulo: Cosac
& Naif, 2001. XAVIER, I. Sétima arte: um culto moderno. São Paulo:
Perspectiva, 1978. 514 Humberto Alves Silva Junior Mots-clés:
Idéologie.
Émancipation.
Cinéma.
Culture de masse. École de Frankfurt. Humberto Alves Silva Junior Integra o Núcleo de Pesquisa em Sociologia
da Arte (NUCLEART/UFBA) Grupo de Pesquisa em Sociologia da Arte (SOAR/UNIR), desenvolvendo
pesquisas na área de Sociologia do Cinema. Humberto Alves Silva Junior – Doutor em Ciências Sociais pela UFBA. Professor do Departamento
de Ciências Sociais da Universidade Federal de Rondônia. Integra o Núcleo de Pesquisa em Sociologia
da Arte (NUCLEART/UFBA) Grupo de Pesquisa em Sociologia da Arte (SOAR/UNIR), desenvolvendo
pesquisas na área de Sociologia do Cinema. 515
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Serotonin-Mediated Tuning of Human Helper T Cell Responsiveness to the Chemokine CXCL12
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PloS one
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cc-by
| 9,002
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Serotonin-Mediated Tuning of Human Helper T Cell
Responsiveness to the Chemokine CXCL12 Elena Magrini1*¤, Ildiko` Szabo` 2, Andrea Doni1, Javier Cibella1, Antonella Viola1,3
1 Humanitas Clinical Institute IRCCS, Rozzano, Milan, Italy, 2 Department of Biology, University of Padua, Padua, Italy, 3 Department of Translational Medicine, University of
Milan, Rozzano, Milan, Italy Received March 22, 2011; Accepted June 22, 2011; Published August 10, 2011 Received March 22, 2011; Accepted June 22, 2011; Published August 10, 2011 Copyright: 2011 Magrini 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: The research leading to these results has received funding from the Italian Association for Cancer Research (AIRC) to I.S. (IG grant nu 5118) and to A.V. (IG grant nu5629 and RG 6278), the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement nu201106 and EMBO YIP to
A.V. 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 ¤ Current address: Department of Experimental Oncology, European Institute of Oncology, IFOM-IEO Campus, Milan, Italy ¤ Current address: Department of Experimental Oncology, European Institute of Oncology, IFOM-IEO Campus, Milan, Italy to naive T cells, thereby modulating T-cell activation
proliferation and differentiation [10]. [9], Introduction The 5-HT receptors have been divided into seven major classes
(5-HT1–5-HT7). The 5-HT3 receptor is the most phylogenetically
conserved; it is described as a ligand-gated ion channel, permeable
to monovalent and divalent cations [11], belonging to the Cys-
loop receptor super-family and sharing structural and functional
features with other members of this channel/receptor group [12]. The 5-HT3 receptor is constituted of five subunits organized in a
channel. To date, five different 5-HT3 receptor subunits were
identified (5-HT3A–E) and, among them, the 5-HT3A subunit is
essential to form a functional receptor, whereas the 5-HT3B
subunit is known to alter the pharmacological properties of the
channel [13,14]. The monoamine serotonin (5-HT) is known to mediate a
plethora of actions throughout the body. In the central and
autonomous nervous system, 5-HT is utilized by neurons to signal
both
volumic
and
fast
synaptic
neurotransmission. In
the
periphery, 5-HT can both contract and relax smooth muscle
(vascular and gastrointestinal), contract cardiac muscle, regulate
hepatic microcirculation and contribute to blood clotting [1]. Synthesis of 5-HT in the peripheral and central nervous system
is respectively operated by two different isoforms of tryptophan
hydroxylase (TPH), TPH-1 and -2 [2]. These enzymes catalyze
hydroxylation of tryptophan to 5-hydroxytryptophan, the imme-
diate precursor of 5-HT. Though conventionally considered a
neurotransmitter, 5-HT is primarily produced by the enterochro-
maffin cells in the gut [3] and it is taken up via an active transport
mechanism into platelets, which provide a rich reservoir of 5-HT
in the circulation. Subsequent release of platelet-stored 5-HT can
be rapidly triggered by platelet-activating factor, thrombin,
complement fragments C3a and C5a, or immunoglobulin E-
containing immune complexes. Thus, at sites of inflammation and
platelet activation, local concentrations of 5-HT can greatly
exceed the relatively low amounts found free in the serum [4]. Indeed, 5-HT is a known immunoregulator and it is implicated in
several inflammatory diseases [1,5–7]. In the context of the
adaptive immune response, 5-HT affects both T cell and dendritic
cell functions [6,8]. Interestingly, it has been recently reported that
dendritic cells uptake 5-HT at sites of inflammation and shuttle it In this study, we analyzed the possible role of 5-HT in T cell
migration. We found that 5-HT selectively inhibits migration
towards CXCL12 through the engagement of 5-HT3 receptor. We
propose that this process likely facilitates T cell infiltration in
inflamed tissues. August 2011 | Volume 6 | Issue 8 | e22482 Abstract In addition to its role as neurotransmitter, serotonin (5-HT) is an important modulator of inflammation and immunity. Here,
we report novel findings suggesting a 5-HT involvement in T cell migration. In particular, we show that 5-HT tunes the
responsiveness of human T lymphocytes to the broadly expressed chemokine CXCL12 in transwell migration assays. By real-
time PCR, western blot analysis and electrophysiological patch clamp experiments, we demonstrate that the type 3 5-HT
receptor (5-HT3) is functionally expressed in human primary T cells. In addition, specific 5-HT3 receptor agonists selectively
decrease T cell migration towards gradients of CXCL12 but not of inflammatory chemokines, such as CCL2 and CCL5. In
transmigration experiments, 5-HT3 receptor stimulation reverts the inhibitory effect of endothelial-bound CXCL12 on T cell
migration. Our data suggest that the reduced T cell responsiveness to CXCL12 induced by 5-HT may occur to facilitate T cell
extravasation and migration into inflamed tissues. o` I, Doni A, Cibella J, Viola A (2011) Serotonin-Mediated Tuning of Human Helper T Cell Responsiveness to the Chemokine CXCL12. PLoS
1371/journal.pone.0022482 Editor: Paul Proost, Rega Institute, University of Leuven, Belgium Editor: Paul Proost, Rega Institute, University of Leuven, Belgium Received March 22, 2011; Accepted June 22, 2011; Published August 10, 2011 5-HT3 receptor activity modulates CXCL12-induced T cell
migration In line with this, replacing sodium with N-methyl-
D-glucamine ions, which did not permeate through sodium
channels and 5-HT3 receptors, resulted in no agonist-induced
inward current (not shown). Next, to eliminate signals due to
Kv1.3 activity, in the following experiments we applied hyperpo-
larizing potentials (#250 mV) (Figure 3D–G) and cesium - a well-
known blocker of potassium channels - was the only cation present
in the pipette solution. Under these ionic conditions, the addition
of 2M-5HT to resting T cells, which did not display any current at
negative potentials under control condition (Figure 3D, left panel),
elicited activation of negative, inward currents (Figure 3D, right
panel). Moreover, in another representative experiment, addition
of 2M-5HT to cells at 290 mV holding potential in the whole-cell The 5-HT3 receptor subtype is expressed in human but not
mouse T cells [9,15] and thus we hypothesized that different
effects of 5-HT on human and mouse T cell migration might
depend on its specific expression. We evaluated T cell migration
after incubation with specific 5-HT3 receptor agonists, SR57227A
and 2-methyl-5-hydroxytryptamine (2M-5HT). As shown in
Figures 2A and 2B, 5-HT3 agonists reduced, in a dose-dependent
manner, CXCL12-induced migration of human resting T cells. We observed that 5-HT3 receptor agonists and 5-HT had a similar
inhibitory effect on T cell chemotaxis, suggesting that the effects of
5-HT on CXCL12-induced cell migration were most likely
mediated by 5-HT3 receptor engagement. Accordingly, the two
selective 5-HT3 receptor antagonists enhanced T cell chemotactic
response to CXCL12 in the range from 1 to 100 mM (Figure 2C–
D). Similar results were obtained when human T cells activated by
anti-CD3 and anti-CD28 antibodies (Abs) were used (Figure 2E). The use of different chemicals, including 5-HT, did not alter
neither spontaneous chemotaxis nor modified T cell chemokinesis
(not shown). In addition, in line with the absence of the receptor,
5-HT3 receptor agonist and antagonist did not affect the migration
of mouse T cells (Figure 2F). Human T cells express functional 5-HT3 receptor and
produce 5-HT 5-HT3 receptor activity modulates CXCL12-induced T cell
migration First, we investigated on the possible modulation of human and
mouse CD4+ primary T cells chemotaxis by 5-HT. The cells were
treated with 5-HT and allowed to migrate in a transwell assay
towards a gradient of CXCL12, a chemokine that binds and PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e22482 PLoS ONE | www.plosone.org Serotonin and T Cell Migration cell activation (Figure 3A). In contrast, no expression of the 5-
HT3B subunit was found (not shown). Similar results were
obtained by Western blot analysis, which showed that the 5-
HT3A subunit was constitutively expressed by CD4+ T cells and
that its expression was augmented upon cell activation (Figure 3B). triggers the chemokine receptor CXCR4. After stimulation with 5-
HT, human CD4+ T cells displayed decreased migration, whereas
the migration of mouse CD4+ T cells was not affected (Figure 1A). The inhibition of CXCL12-induced human T cell migration
exerted by 5-HT was dose-related in the range from 0.003 to
3 mM (Figure 1B). that its expression was augmented upon cell activation (Figure 3B). Next, we assessed the functional properties of the 5-HT3A
receptor in human CD4+ T lymphocytes through electrophysio-
logical patch clamp experiments performed in freshly-isolated T
cells in the whole-cell configuration (Figure 3C–G). In naive T
cells, Kv1.3, a voltage-gated, depolarization-activated potassium
channel, is responsible for maintenance of the resting membrane
potential (around 250 mV) [16]. In accordance, in primary T
lymphocytes we recorded Kv1.3-mediated potassium outward
currents,
displaying
typical
Kv1.3
inactivation
kinetics,
at
depolarizing voltages (Figure 3C, left panel) but no current was
observed at hyperpolarizing voltages. Upon addition of 5-HT to
the bath, an inward current appeared at negative voltages,
indicating that 5-HT alters ionic currents in T cells (Figure 3C,
right panel). In the experiments shown in Figure 3C, both pipette
and bath solutions contained gluconate, which did not permeate
chloride channels. Therefore, the outward current was due to the
exit of potassium via Kv1.3, while the 5-HT-elicited inward
current was due to the entrance of cations into the cell. Although
5-HT3A homomers also allow calcium permeation, in our
experimental
setting
the
concentration
of
calcium
in
the
extracellular solution was too low (1 mM) to give rise to a
significant current [16], suggesting that the major charge carrying
ion was sodium. Human T cells express functional 5-HT3 receptor and
produce 5-HT These results prompted us to verify and characterize the
expression of functional 5-HT3 receptors in human CD4+ T cells. We employed real-time PCR analysis to evaluate the expression of
5-HT3A and 5-HT3B subunits in primary, resting T lymphocytes
as well as in T cells activated by anti-CD3 and anti-CD28 Abs, or
phytohaemagglutinin (PHA). Activation of CD4+ T cell was
assessed by the up-regulation of activation surface markers, such as
CD69 and CD25 (not shown). As shown in Figure 3A, both resting
and activated human T cells expressed the 5-HT3A receptor
subunit. Moreover, the levels of 5-HT3A transcript increased upon Figure 1. 5-HT reduces human CD4+ T cell migration towards CXCL12 in a dose-dependent manner. Human primary (n = 3, Panel A;
n = 3, Panel B) or murine naive (n = 3, Panel A) CD4+ T cells were treated with the indicated concentrations of 5-HT for 30 min at RT and then allowed
to migrate towards CXCL12. Number of migrated human CD4+ T cells resulted 12326354 compared to 6856179 for untreated and 5-HT-treated cells,
respectively (mean 6 SEM; Panel A). Percentages of migrated cells for each condition were calculated as described in ‘‘Materials and Methods’’. Data
were statistically analyzed with either t-test (A) or Bonferroni’s multiple comparison test (B); *P,0.05, ***P,0.001. doi:10.1371/journal.pone.0022482.g001 Figure 1. 5-HT reduces human CD4+ T cell migration towards CXCL12 in a dose-dependent manner. Human primary (n = 3, Panel A;
n = 3, Panel B) or murine naive (n = 3, Panel A) CD4+ T cells were treated with the indicated concentrations of 5-HT for 30 min at RT and then allowed
to migrate towards CXCL12. Number of migrated human CD4+ T cells resulted 12326354 compared to 6856179 for untreated and 5-HT-treated cells,
respectively (mean 6 SEM; Panel A). Percentages of migrated cells for each condition were calculated as described in ‘‘Materials and Methods’’. Data
were statistically analyzed with either t-test (A) or Bonferroni’s multiple comparison test (B); *P,0.05, ***P,0.001. doi:10.1371/journal.pone.0022482.g001 August 2011 | Volume 6 | Issue 8 | e22482 PLoS ONE | www.plosone.org 2 Figure 2. 5-HT3 receptor activation reduces CXCL12-induced migration of resting and activated CD4+ T cells. August 2011 | Volume 6 | Issue 8 | e22482 Human T cells express functional 5-HT3 receptor and
produce 5-HT In the same
experimental conditions, with the exception that cesium was used in the pipette solution and NaCl in the bath solution (D–G), currents were also
recorded before (D, left panel) and after (D, right panel) addition of 3 mM of specific 5-HT3 agonist 2M-5HT to the bath solution. Cesium blocked
outward potassium currents and Kv1.3 activity was avoided by applying voltage pulses only from 2130 to 250 mV in 20 mV steps. Addition of 3 mM
2M-5HT to a cell held at 290 mV holding potential in the whole-cell configuration caused a significant inward current within few seconds (E). Currents were recorded at 270 mV holding potential in outside-out patch after addition of 3 mM 2M-5HT (F, upper trace) and after addition of Figure 3. Resting and activated human CD4+ T cells express functional 5-HT3 receptor. Human CD4+ T cells were stimulated with anti-CD3
and anti-CD28 Abs, PHA or PMA plus ionomycin for 72 h. (A) Gene expression for 5-HT3A receptor subunits was examined by real-time PCR. The graph
represents the quantitative analysis of the experiments. Values are expressed as fold increase over resting T cells. Data are mean of triplicates 6 SD of
the 22DDCt (n = 3); *P,0.05, **P,0.01, t-test. (B) T cell membranes were isolated by ultracentrifugation from total cell lysates and 20 mg/sample were
separated on SDS-PAGE gel. A representative immunoblot of three experiments performed showing the expression of 5-HT3A subunit is reported. Asterisk indicates the band corresponding to 5-HT3A monomer. (C–G) Freshly isolated CD4+ T cells were subjected to electrophysiological patch
clamp experiments in the whole-cell configuration. Cells were held at resting potential (250 mV) and voltage pulses of 400 ms duration were applied
in 20 mV steps ranging from 2130 to 210 mV. Currents were recorded before (C, left panel) and after (C, right panel) addition of 3 mM 5-HT to the
bath solution. Addition of 5-HT induced an inward current at hyperpolarizing voltages. Activity of Kv1.3 resulting in outward potassium current was
seen at depolarizing voltages 230 and 210 mV. Pipette: Kgluconate containing solutions; bath: Nagluconate containing solutions. In the same
experimental conditions, with the exception that cesium was used in the pipette solution and NaCl in the bath solution (D–G), currents were also
recorded before (D, left panel) and after (D, right panel) addition of 3 mM of specific 5-HT3 agonist 2M-5HT to the bath solution. Human T cells express functional 5-HT3 receptor and
produce 5-HT Hum
and anti-CD28 Abs, PHA or PMA plus ionomycin for 72 h. (A) Gene expression for 5-HT3A receptor subu
represents the quantitative analysis of the experiments. Values are expressed as fold increase over resti
the 22DDCt (n = 3); *P,0.05, **P,0.01, t-test. (B) T cell membranes were isolated by ultracentrifugation
separated on SDS-PAGE gel. A representative immunoblot of three experiments performed showing
Asterisk indicates the band corresponding to 5-HT3A monomer. (C–G) Freshly isolated CD4+ T cells
clamp experiments in the whole-cell configuration. Cells were held at resting potential (250 mV) and v
in 20 mV steps ranging from 2130 to 210 mV. Currents were recorded before (C, left panel) and afte
bath solution. Addition of 5-HT induced an inward current at hyperpolarizing voltages. Activity of Kv1
seen at depolarizing voltages 230 and 210 mV. Pipette: Kgluconate containing solutions; bath: N
experimental conditions, with the exception that cesium was used in the pipette solution and NaCl
recorded before (D, left panel) and after (D, right panel) addition of 3 mM of specific 5-HT3 agonist Figure 3. Resting and activated human CD4+ T cells express functional 5-HT3 receptor. Human CD4+ T cells were stimulated with anti-CD3
and anti-CD28 Abs, PHA or PMA plus ionomycin for 72 h. (A) Gene expression for 5-HT3A receptor subunits was examined by real-time PCR. The graph
represents the quantitative analysis of the experiments. Values are expressed as fold increase over resting T cells. Data are mean of triplicates 6 SD of
the 22DDCt (n = 3); *P,0.05, **P,0.01, t-test. (B) T cell membranes were isolated by ultracentrifugation from total cell lysates and 20 mg/sample were
separated on SDS-PAGE gel. A representative immunoblot of three experiments performed showing the expression of 5-HT3A subunit is reported. Asterisk indicates the band corresponding to 5-HT3A monomer. (C–G) Freshly isolated CD4+ T cells were subjected to electrophysiological patch
clamp experiments in the whole-cell configuration. Cells were held at resting potential (250 mV) and voltage pulses of 400 ms duration were applied
in 20 mV steps ranging from 2130 to 210 mV. Currents were recorded before (C, left panel) and after (C, right panel) addition of 3 mM 5-HT to the
bath solution. Addition of 5-HT induced an inward current at hyperpolarizing voltages. Activity of Kv1.3 resulting in outward potassium current was
seen at depolarizing voltages 230 and 210 mV. Pipette: Kgluconate containing solutions; bath: Nagluconate containing solutions. Human T cells express functional 5-HT3 receptor and
produce 5-HT Human resting (A–D),
human activated (E) or murine naive (F) CD4+ T cells were treated with the indicated concentration of 5-HT3 receptor agonists, 2M-5HT (A) and
SR57227A (B), or antagonists, ondansetron (C) and granisetron (D), for 30 min at RT and then used for migration assays towards CXCL12 (n = 3, from
Panel A to Panel D); number of human activated CD4+ T cells migrated in control condition resulted 610261033 compared to 45596846 and
820661407 for 2M-5HT and ondansetron treated cells, respectively (mean 6 SEM, n = 4, Panel E); (n = 3, Panel F). Percentages of migrated cells for
each condition were calculated as described in ‘‘Materials and Methods’’; *P,0.05, **P,0.01, ***P,0.001, Bonferroni’s multiple comparison test. doi:10.1371/journal.pone.0022482.g002
Serotonin and T Cell Migration Serotonin and T Cell Migration Figure 2. 5-HT3 receptor activation reduces CXCL12-induced migration of resting and activated CD4+ T cells. Human resting (A–D),
human activated (E) or murine naive (F) CD4+ T cells were treated with the indicated concentration of 5-HT3 receptor agonists, 2M-5HT (A) and
SR57227A (B), or antagonists, ondansetron (C) and granisetron (D), for 30 min at RT and then used for migration assays towards CXCL12 (n = 3, from
Panel A to Panel D); number of human activated CD4+ T cells migrated in control condition resulted 610261033 compared to 45596846 and
820661407 for 2M-5HT and ondansetron treated cells, respectively (mean 6 SEM, n = 4, Panel E); (n = 3, Panel F). Percentages of migrated cells for
each condition were calculated as described in ‘‘Materials and Methods’’; *P,0.05, **P,0.01, ***P,0.001, Bonferroni’s multiple comparison test. doi:10.1371/journal.pone.0022482.g002 receptors, currents were recorded either in the absence (Figure 3F,
upper trace) or in the presence of amiloride (Figure 3F, lower
trace). Amiloride did not reduce 2M-5HT-induced currents,
indicating that, upon addition of the 5-HT3 receptor agonist,
sodium did not enter the cells via classical amiloride-sensitive configuration caused a significant inward current within few
seconds (Figure 3E). T cells are known to express amiloride-
sensitive sodium channels [17]. To exclude the possibility that the
current recorded after addition of 2M-5HT was due to sodium
influx through amiloride-sensitive channels rather than 5HT3 August 2011 | Volume 6 | Issue 8 | e22482 August 2011 | Volume 6 | Issue 8 | e22482 PLoS ONE | www.plosone.org 3 Serotonin and T Cell Migration Figure 3. Resting and activated human CD4+ T cells express functional 5-HT3 receptor. August 2011 | Volume 6 | Issue 8 | e22482 Human T cells express functional 5-HT3 receptor and
produce 5-HT Cesium blocked
outward potassium currents and Kv1.3 activity was avoided by applying voltage pulses only from 2130 to 250 mV in 20 mV steps. Addition of 3 mM
2M-5HT to a cell held at 290 mV holding potential in the whole-cell configuration caused a significant inward current within few seconds (E). Currents were recorded at 270 mV holding potential in outside-out patch after addition of 3 mM 2M-5HT (F, upper trace) and after addition of August 2011 | Volume 6 | Issue 8 | e22482 PLoS ONE | www.plosone.org 4 4 Serotonin and T Cell Migration Serotonin and T Cell Migration 100 mM amiloride, in the presence of 2M-5HT, to the same patch (F, lower trace). Amiloride did not reduce the current. Whole-cell currents were
recorded in presence of ondansetron (100 mM) (G, left panel) and after addition of 3 mM 2M-5HT in the presence of 100 mM ondansetron (G, right
panel). Applied voltages ranged from 2130 to 210 mV. Data in Figures 3C–G are representative of at least five experiments in the same conditions
giving similar results. doi:10 1371/journal pone 0022482 g003 CXCL12 binding and CXCL12-induced actin polymerization in
T cells after stimulation or inhibition of 5-HT3 receptors. As
shown in Figure 6, neither 5-HT nor 5-HT3 receptor agonists or
antagonists had any detectable effect on CXCR4 expression (Panel
A), CXCL12 binding to its receptor (Panel B) or CXCR4-
mediated early signaling (Panel C). sodium channels. In accordance with this observation, 2M-5HT
failed to induce inward currents in cells pre-treated with
ondansetron (Figure 3G). CXCL12 binding and CXCL12-induced actin polymerization in
T cells after stimulation or inhibition of 5-HT3 receptors. As
shown in Figure 6, neither 5-HT nor 5-HT3 receptor agonists or
antagonists had any detectable effect on CXCR4 expression (Panel
A), CXCL12 binding to its receptor (Panel B) or CXCR4-
mediated early signaling (Panel C). Human T cells were found to express TPH-1 [18], thus we
asked whether T cells were able to synthesize 5-HT. To this end,
we performed an Ultra-Sensitive Serotonin enzyme immunoassay
(EIA) of supernatants obtained from resting and activated T cell
cultured in medium supplemented with fetal calf serum (FCS) that
was previously treated with charcoal-coated dextran, in order to
absorb the endogenous 5-HT. Human T cells express functional 5-HT3 receptor and
produce 5-HT Consistently with published data
[9], complete media supplemented with 10% FCS contained
276631 nM 5-HT, and this concentration was reduced to
1.160.3 nM following treatment with charcol-coated dextran
(mean 6 SEM, n = 3). Despite the intra-donor variability, in all
experiments performed we measured nanomolar concentrations of
5-HT released by both resting and activated cells (Figure 4A). However, the 5-HT release was reduced upon T cell activation
(P,0.01). These observations were confirmed by intracellular
staining and flow cytometric analysis (Figure 4B). CXCL12 is present on the surface of endothelial cells [19] and
thus we evaluated if 5-HT might influence T cell extravasation, by
regulating endothelial-bound CXCL12-chemoattraction. To ad-
dress this question, we compared the ability of human T
lymphocytes, treated or not with the specific CXCR4 antagonist
AMD3100 [20], to migrate towards an inflammatory stimulus,
such
as
CCL2,
through
CXCL12-coated
endothelial
cells. Endothelial-bound CXCL12 had an inhibitory effect on T cell
transendothelial migration and, interestingly, 5-HT3 receptor
stimulation by 2M-5HT enhanced transendothelial migration of
T cells, whereas 5-HT3 receptor inhibition through ondansetron
strongly reduced it (Figure 7). All these effects were specifically
determined by the presence of endothelial-bound CXCL12, as
they were abrogated by AMD3100 (Figure 7). Moreover, they did
not depend on the presence of CCL2, which was simply used as a
chemoattractive stimulus, given that 2M-5HT or ondansetron
exhibited no effect on migration towards this chemokine, as
previously demonstrated (Figure 5). 5-HT3 receptor stimulation enhances T cell
transmigration through CXCL12-coated endothelial cells We asked whether 5-HT modulates chemotactic responses
towards chemokines other than CXCL12. Thus, we evaluated the
effects of 5-HT3 receptor agonist (Figure 5A) and antagonist
(Figure 5B) on human T cell migration towards CCL2 or CCL5
gradients. Both 2M-5HT and ondansetron had no effect on T cell
migration towards the inflammatory chemokines CCL2 or CCL5,
indicating that the 5-HT/5-HT3 system did not alter T cell
chemotactic responses to all chemokines and did not act on
general mechanisms required for migration. Discussion doi:10.1371/journal.pone.0022482.g005 leukocytes to the luminal side of blood vessels, extravasation
(transendothelial migration) and cell migration. However, CXCR4 expression and its affinity for the chemokine
are not altered by 5-HT or by 5-HT3 receptor agonists or
antagonists. Moreover, 5-HT3 receptor stimulation or inhibition
do not alter actin polymerization in response to CXCL12,
indicating that the early events following CXCR4 stimulation
are not affected by 5-HT. The mechanism responsible for the
specific modulation of CXCL12-induced T cell migration by the
5-HT system is therefore still unclear and may involve pHi
regulation. Chemotaxis is indeed modulated by the contribution of
several different ion channels and by pHi changes [32–35],
although the precise mechanisms are still not clear. Platelets, at the sites of inflammation, play a crucial role as
amplifiers of leukocyte adhesion to the endothelium because of
their repertoire of adhesion molecules and soluble mediators [21]. 5-HT, one of the soluble mediators locally released by platelets, is
emerging as a key regulator of immunity, tuning in a very complex
way both innate and acquired responses [3,6,22]. In agreement, 5-
HT seems to be directly involved in the pathogenesis of several
inflammatory diseases, including irritable bowel syndrome [23],
colitis [24], contact allergy [25], atopic dermatitis [26], psoriasis
[27] and asthma [28]. Moreover, recent studies have demonstrat-
ed that platelet-derived 5-HT aggravates T-cell induced liver
damage in viral hepatitis [29,30]. CXCL12 regulates both homeostatic and pro-inflammatory
migration and homing of lymphocytes [36–38] and we suggest that
the selective control of T cell responses to CXCL12 by 5-HT may
be important in these contexts. Notably, it has been previously
observed that the neutrophil elastase cleaves CXCL12 expressed on
the bone marrow stroma, thus facilitating stem cell mobilization
from bone marrow into the peripheral circulation [39,40]. In
addition, another study has shown that elastase release by
transmigrating neutrophils deactivates endothelial-bound CXCL12
and attenuates subsequent T lymphocyte transendothelial migra-
tion [41]. Our data indicate that an additional mechanism has
evolved to selectively control responsiveness of lymphocytes to
CXCL12. We suggest that, reducing responsiveness to endothelial-
bound CXCL12 chemoattraction, 5-HT facilitates transendothelial
T cell migration towards gradients of inflammatory chemokines
released in inflamed tissues. Data obtained with 5-HT3 receptor
antagonists do not exclude the possibility that T cells themselves
may control their responsiveness to CXCL12 in an autonomous
manner. Discussion The attraction of leukocytes to the sites of inflammation and
infection is an essential component of the host response to diseases. The recruitment of circulating leukocytes from the blood stream
into inflamed tissues is driven by chemokines, produced either by
infiltrating inflammatory cells or by injured tissue cells. This
process depends on three distinct phases: vascular attachment of In order to investigate the mechanism of 5-HT effect on
CXCL12/CXCR4 response, we evaluated CXCR4 expression, Figure 4. 5-HT is produced and released by human CD4+ T cells. (A) Cell-released 5-HT was quantified by EIA. Data are mean of triplicate 6
SEM (n = 3). (B) Representative flowcytometric analysis of three experiments performed showing intracellular 5-HT in primary and activated human T
cells (MFI, normalized to isotype control staining, was 70.563.5 and 361 for resting and activated T cells, respectively; n = 3, P,0.01). doi:10.1371/journal.pone.0022482.g004 Figure 4. 5-HT is produced and released by human CD4+ T cells. (A) Cell-released 5-HT was quantified by EIA. Data are mean of triplicate 6
SEM (n = 3). (B) Representative flowcytometric analysis of three experiments performed showing intracellular 5-HT in primary and activated human T
cells (MFI, normalized to isotype control staining, was 70.563.5 and 361 for resting and activated T cells, respectively; n = 3, P,0.01). doi:10.1371/journal.pone.0022482.g004 PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e22482 5 Serotonin and T Cell Migration Figure 5. 5-HT3 receptor activity selectively modulates CXCL12-induced chemotaxis of human CD4+ T cells. Human resting CD4+ T cells
were treated with 2.5 mM 2M-5HT (n = 3, Panel A) or 100 mM ondansetron (n = 3, Panel B) for 30 min at RT, and then allowed to migrate towards
CXCL12, CCL2 or CCL5. Percentages of migrated cells for each condition were calculated as described in ‘‘Materials and Methods’’. **P,0.01,
***P,0.001, t-test. doi:10.1371/journal.pone.0022482.g005 Figure 5. 5-HT3 receptor activity selectively modulates CXCL12-induced chemotaxis of human CD4+ T cells. Human resting CD4+ T cells Figure 5. 5-HT3 receptor activity selectively modulates CXCL12-induced chemotaxis of human CD4+ T cells. Human resting CD4+ T cells
were treated with 2.5 mM 2M-5HT (n = 3, Panel A) or 100 mM ondansetron (n = 3, Panel B) for 30 min at RT, and then allowed to migrate towards
CXCL12, CCL2 or CCL5. Percentages of migrated cells for each condition were calculated as described in ‘‘Materials and Methods’’. **P,0.01,
***P,0.001, t-test. PLoS ONE | www.plosone.org Discussion On the other hand, since released local concentrations of
5-HT consequent to platelet activation at sites of damaged
endothelium greatly exceeds the relatively low amounts produced
by T cells, our straightforward interpretation is that during T cell
transendothelial migration the major source of 5-HT is most likely
produced by platelets. In addition, the fact that activated T cells
produce lower amounts of 5-HT than resting ones but up-regulate
expression of 5-HT3 receptor may indicate that, upon activation, T
cells become more dependent on external sources of 5-HT released,
for example, by platelets. The data reported in this study indicate that 5-HT inhibits
migration of human T cells towards CXCL12 gradients and that
this effect depends on the activity of 5-HT3 receptors, which are
functionally expressed in human T cells. The presence of functional 5-HT3 receptor has been already
described in the Jurkat T cell line using the fluorescent probe
SBFI/AM [31]. Here, through the patch clamp technique, we
show the presence of 5-HT3 receptor agonist-induced inward
currents, which are abolished by pre-incubation with 5-HT3
receptor antagonist, in human peripheral blood T cells, demon-
strating for the first time that 5-HT3 receptors are also active in
human primary T cells. Interestingly, our data indicate that T cells produce 5-HT and
suggest that they use it as an autocrine factor to reduce
responsiveness to CXCL12. Indeed, all our chemotaxis experi-
ments have been performed in serum-free conditions and hence in
the absence of external or contaminant sources of 5-HT. In these
conditions, the boosting effect of 5-HT3 receptor antagonists on T
cell migration suggests - but does not formally prove - that self-
produced 5-HT controls responsiveness to CXCL12. In addition,
T cells are able to sense an increase in the concentration of
extracellular 5-HT, and respond with a further reduction of
responsiveness to CXCL12 stimulation. One puzzling aspect of our findings concerns the molecular
basis for the selective action of the serotonin receptor 5-HT3 on
CXCL12/CXCR4. A trivial explanation for the selective control
of CXCL12-induced migration may be a direct modulation of the
expression of CXCR4 - the CXCL12 receptor - by 5-HT. In conclusion, our data unveil a novel connection between 5-
HT and chemokines that may play a crucial role during tissue
damage and inflammatory responses. PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e22482 6 Serotonin and T Cell Migration Figure 6. Ethics statement Human peripheral blood T (PBT) cells were isolated from Buffy
coats, a by-product of the isolation of blood coagulation factors,
obtained from healthy donors at the Centre for Blood Transfu-
sions of the Desio or Padua Hospital. The Centres for Blood
Transfusions collected blood donations after a written informed
consent, and did not release to our laboratory any personal data of
the blood donors. For all these reasons, our Institution did not
require approval from the local ethics committee. Discussion 5-HT3 receptor activity does not modify CXCR4 expression, CXCL12 binding and CXCL12-induced actin polymerization. (A) After treatment of resting human CD4+ T cells with 5-HT, the 5-HT3 receptor agonist 2M-5HT or the 5-HT3 receptor antagonist ondansetron for 0,
30 and 150 min, CXCR4 expression was analyzed by flow cytometry. Data refer to percentage of a representative experiment of three performed. (B)
Binding of CXCL12-AF647 on human CD4+ T cells. The cells were treated for 30 min at RT with 5-HT, 2M-5HT, ondansetron or the specific CXCR4
antagonist AMD3100 (ref. 20). The cells were then incubated with increasing concentrations of fluorescent CXCL12, in the presence of the indicated
chemicals. Data are expressed as MFI 6 SEM vs. CXCL12-AF647 concentrations (n = 4; ***P,0.001, Bonferroni’s multiple comparison test). (C) Human
CD4+ T cells were treated with 2M-5HT or ondansetron for 30 min at RT and then stimulated with CXCL12. Kinetics of CXCL12-induced F-actin
formation were analyzed by phalloidin staining and flow cytometric analysis. Data represent MFI 6 SEM vs. time (n = 3). doi:10.1371/journal.pone.0022482.g006 Figure 6. 5-HT3 receptor activity does not modify CXCR4 expression, CXCL12 binding and CXCL12-induced actin polymerization. (A) After treatment of resting human CD4+ T cells with 5-HT, the 5-HT3 receptor agonist 2M-5HT or the 5-HT3 receptor antagonist ondansetron for 0,
30 and 150 min, CXCR4 expression was analyzed by flow cytometry. Data refer to percentage of a representative experiment of three performed. (B)
Binding of CXCL12-AF647 on human CD4+ T cells. The cells were treated for 30 min at RT with 5-HT, 2M-5HT, ondansetron or the specific CXCR4
antagonist AMD3100 (ref. 20). The cells were then incubated with increasing concentrations of fluorescent CXCL12, in the presence of the indicated
chemicals. Data are expressed as MFI 6 SEM vs. CXCL12-AF647 concentrations (n = 4; ***P,0.001, Bonferroni’s multiple comparison test). (C) Human
CD4+ T cells were treated with 2M-5HT or ondansetron for 30 min at RT and then stimulated with CXCL12. Kinetics of CXCL12-induced F-actin
formation were analyzed by phalloidin staining and flow cytometric analysis. Data represent MFI 6 SEM vs. time (n = 3). doi:10.1371/journal.pone.0022482.g006 details of the study were approved by Istituto Clinico Humanitas
Animal Care and Use Committee (IACUC). PLoS ONE | www.plosone.org Cell culture and activation Human CD4+ PBT cells were isolated from the peripheral
blood of healthy donors by negative selection using RosetteSep kit
(StemCell Technologies) and cultured in RPMI 1640 medium
(Lonza) supplemented with non essential amino acids,1 mM
sodium pyruvate, 2 mM L-glutamine, 10 mM Hepes buffer
(Lonza) (complete) and 10% FCS (Euroclone). In some experi-
ments, human cells were activated by culture for 72 hours (h) in
the presence of 2.5 mg/ml plate-bound functional grade purified
anti-human CD3 Ab (OKT3; eBioscience) and 1 mg/ml of
purified NA/LE anti-human CD28 Ab (CD28.2; BD Biosciences)
in solution, with 2.5 mg/ml of PHA (Biochrom AG) or with a
combination of 10 nM of phorbol 12-myristate 13-acetate (PMA;
Sigma) and 1 mM of ionomycin (Sigma). T cell activation was
determined by labeling with anti-human CD25 Ab (2A3; BD Procedures involving animals and their care conformed with
institutional guidelines (authorisation n. 11/2006-A from the
Italian Ministry of Health) in compliance with national (4D.L. N.116, G.U., suppl. 40, 18-2-1992) and international law and
policies (EEC Council Directive 86/609, OJ L 358,1,12-12-1987;
NIH Guide for the Care and Use of Laboratory Animals, US
National Research Council 1996). All efforts were made to
minimize the number of animals used and their suffering. Full August 2011 | Volume 6 | Issue 8 | e22482 7 PLoS ONE | www.plosone.org Serotonin and T Cell Migration Figure 7. 5-HT3 receptor activation enhances T cell transmi-
gration through CXCL12-coated endothelial cells. Human resting
CD4+ T cells were treated with 2.5 mM 2M-5HT or 100 mM ondansetron
for 30 min at RT. Then, CD4+ T cells were incubated (+) or not (2) with
the CXCR4 antagonist AMD3100 (10 mM) and immediately used for a
transendothelial migration assay towards CCL2 through CXCL12-coated
endothelial cells. In absence of AMD3100, number of migrated control
cells resulted 15246270 compared to 18886252 and 12346398 for 5-
HT and ondansetron treated cells, respectively (mean 6 SEM). Percentages of migrated cells for each condition were calculated as
described in ‘‘Materials and Methods’’. n = 5, *P,0.05, Bonferroni’s
multiple comparison test. doi:10.1371/journal.pone.0022482.g007 with TURBO DNA-free Kit (Ambion). Total RNA from human
dorsal root ganglion (hDRG; Clontech) was used as positive
control. Reverse transcription was performed using the Super-
script III First-Strand Synthesis System (Invitrogen). Real-time
PCR was performed using SYBR Green PCR Master Mix
(Applied Biosystems) and detected by 7900HT Sequence Detec-
tion System (Applied Biosystems). Cell culture and activation The following primers were
used:
human
18S
(forward:
59-CGCCGCTAGAGGTGAA-
ATTC-39 and reverse: 59-CTTTCGCTCTGGTCCGTCTT-
39), human 5-HT3A (forward: 59-GCTTGCCAGAAAAGGTG-
AAATC-39 and reverse: 59-GGCGGATGACCACATAGAACT-
T-39), human 5-HT3B (forward: 59-CCCTACCTCTAAGTGC-
CATCT-39 and reverse: 59-GGCTTATAGTTCTCAATGGT-
CCC-39). Data were analyzed using the SDS2.2.2 software (Applied
Biosystems). The fold increase corresponded to 22DDCt, where the
DCt was the difference of the mean value of the Ct between the
studied and the housekeeping gene (18S ribosomal RNA), and the
DDCt was the difference of the DCt between activated and resting
cells for each gene. The standard deviation (SD) of the 22DDCt was
calculated applying the formula: SD of DDCt * 22DDCt * ln2. Figure 7. 5-HT3 receptor activation enhances T cell transmi-
gration through CXCL12-coated endothelial cells. Human resting
CD4+ T cells were treated with 2.5 mM 2M-5HT or 100 mM ondansetron
for 30 min at RT. Then, CD4+ T cells were incubated (+) or not (2) with
the CXCR4 antagonist AMD3100 (10 mM) and immediately used for a
transendothelial migration assay towards CCL2 through CXCL12-coated
endothelial cells. In absence of AMD3100, number of migrated control
cells resulted 15246270 compared to 18886252 and 12346398 for 5-
HT and ondansetron treated cells, respectively (mean 6 SEM). Percentages of migrated cells for each condition were calculated as
described in ‘‘Materials and Methods’’. n = 5, *P,0.05, Bonferroni’s
multiple comparison test. d i 10 1371/j
l
0022482 007 Western blotting
+ Human CD4+ T cells were collected on ice-cold 50 mM Tris-
HCl (pH = 7.5), 2 mM EDTA containing protease inhibitors
(Complete EDTA-free protease inhibitor cocktail tablets; Roche)
(hypotonic buffer). Nuclei were eliminated by centrifugation
(300 g, 10 min, 4uC) and membranes were isolated by further
centrifugation (48.000 rpm, rotor Type 90 Ti, 45 min, 4uC) and
solubilized in hypotonic buffer containing 1% NP40 (Calbiochem). Total protein concentration was determined by DC Protein assay
(Bio-Rad Laboratories). Specifically, 20 mg of total protein for each
sample were diluted in XT sample buffer (Bio-Rad Laboratories)
containing 10% b-mercaptoethanol (Bio-Rad Laboratories), boiled
for 10 min and stored at 220uC until use. Western blots were
immunolabeled using polyclonal rabbit anti-human 5HT3 recep-
tor Ab (1 mg/ml; Imgenex) and secondary HRP–conjugated goat
anti–rabbit IgG Ab (GE Healthcare). Membrane chemilumines-
cence was acquired using a ChemiDoc XRS system and the
Quantity One – 4.6.1 software (Bio-Rad Laboratories). p
p
doi:10.1371/journal.pone.0022482.g007 Biosciences) and anti-human CD69 Ab (FN50; BD Biosciences). The expression of CXCR4 was assessed by labeling with anti-
human CD184 (CXCR4) Ab (12G5; BD Biosciences). Samples
were analyzed using a FACS Canto flow cytometer and FACS
Diva software (BD Biosciences). Biosciences) and anti-human CD69 Ab (FN50; BD Biosciences). The expression of CXCR4 was assessed by labeling with anti-
human CD184 (CXCR4) Ab (12G5; BD Biosciences). Samples
were analyzed using a FACS Canto flow cytometer and FACS
Diva software (BD Biosciences). Murine naive CD4+ T cells were obtained from the lymph
nodes of C57BL/6 mice. Cells were sorted by negative selection
using a mixture of rat anti-mouse CD8 (TIB105), CD19 (1D3),
CD11b (A506) Abs and anti-rat IgG microbeads according to the
manufacturer’s instructions (Miltenyi Biotec). Murine CD4+ T
cells
were
cultured
in
complete
DMEM
medium
(Lonza)
supplemented with 50 mM b-mercaptoethanol (Gibco) and 10%
FCS. Percentage of CD4+ cells was determined by flow cytometric
analysis
after
labeling
with
anti-mouse
CD4
Ab
(GK1.5;
BioLegend). Chemicals 5-HT, 2M-5HT, SR57227A, ondansetron hydrochloride dehy-
drate, granisetron hydrochloride, amiloride hydrochloride hydrate
and AMD3100 were all purchased from Sigma. Stock solutions
and intermediate dilutions of 5-HT and 5-HT3 agonists or
antagonists were freshly prepared in water before each experiment
and kept on ice protected from direct light. Patch clamp Freshly isolated human CD4+ T lymphocytes were subjected to
electrophysiological patch clamp experiments in the whole-cell
configuration (or outside out patch in Figure 3F). Whole-cell
currents were recorded with an EPC-7 amplifier (List) as
previously described [43] and data were analyzed by using
pCLAMP8 program set (Axon Instruments). Leak currents were
not subtracted. The bath solution was composed of 156 mM
Nagluconate, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES
(pH = 7.3) and the intracellular solution contained 134 mM
Kgluconate,
1 mM CaCl2, 1 mM
MgCl2, 10 mM
EGTA,
10 mM HEPES (pH = 7.3) for experiments where Kv1.3 activity
and 5HT3 activities were recorded. In some control experiments,
to block inward cation current, in the bath solution Nagluconate
was replaced with N-methyl-D-glucamine chloride (NMDGCl). Where indicated, intracellular Kgluconate was replaced with
134 mM CsCl (pH to 7.3 with CsOH) to block outward potassium
current and the bath contained NaCl instead of Nagluconate. Human umbilical vein endothelial cells (HUVECs) were gently
gifted by M. Sironi (Milan, Italy) [42]. HUVECs were cultured in
Medium 199 (Lonza) supplemented with 2 mM L-glutamine,
100 U/ml penicillin/streptomycin (Lonza), 100 mg/ml heparin
(Sigma), 50 mg/ml EC growth supplement (ECGS; Biomedical
Technologies, Inc.) and 20% FBS in flasks coated with 1% gelatine
(Sigma) in PBS16 (Lonza) for at least 2 h at 37uC. 5-HT detection Real-time PCR
Total RNA from human CD4+ T cells was extracted using
RNAspin Mini RNA isolation Kit (GE Healthcare) and treated Real-time PCR To quantify 5-HT, CD4+ T cells (56106 cells/ml) were
incubated for 24 or 72 h at 37uC in complete RPMI 1640
medium supplemented with 10% charcoal/dextran-treated FBS, Total RNA from human CD4+ T cells was extracted using
RNAspin Mini RNA isolation Kit (GE Healthcare) and treated PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e22482 August 2011 | Volume 6 | Issue 8 | e22482 8 Serotonin and T Cell Migration 0,1% (w/v) L-ascorbic acid (Labor Diagnostika Nord), 30 mM
tranylcypromine (Sigma). FCS was pretreated with 0.25% (w/v)
charcoal-coated dextran (Sigma) overnight at 4uC. 5-HT released
into T cell culture supernatants was quantified using the Ultra-
Sensitive Serotonin enzyme immunoassay (EIA; Labor Diagnos-
tika Nord); same pretreated media was used as assay diluent. For
intracellular staining cells were fixed with paraformaldehyde 4%
for 15 min at 4uC and stained with monoclonal mouse anti-
serotonin (5HT-H209; Dako) and AlexaFluor488-goat anti-mouse
IgG (Molecular Probes) using the BD Cytofix/Cytoperm kit (BD
Biosciences) according to the manufacturer’s instructions. Mean of
Fluorescence Intensity (MFI) was measured by FACS Canto flow
cytometer and analyzed by Flow-Jo software. were present during the migration in both transwell compart-
ments. Human (1.5 nM) and murine (6.3 nM) CXCL12 were
purchased from PeproTech; human CCL2 (11.5 nM) and CCL5
(12.8 nM) were purchased from R&D; chemokines were all diluted
in migration medium. The number of living cells spontaneously
migrating through the filter was determined after 2 h at 37uC by
counting all the samples for the same time (1 min) at FACS Canto
flow cytometer. HUVECs were seeded at 15–206103/well and cultured to 90%
confluence on 24-well transwell tissue culture inserts (24-well plate
with inserts, 5 mm pore/6.5 mm, TC-treated; Costar) as previ-
ously described [42]. HUVECs were activated using IL-1b
(Peprotech) at 10 ng/ml final concentration for 4 h at 37uC in
HUVEC-culture medium and then incubated with CXCL12
20 ng/ml final concentration in migration medium for 15 min at
37uC. T cells were treated for 30 min at RT with the indicated
chemicals, then incubated with AMD3100 10 uM and immedi-
ately allowed to migrate towards CCL2 (11.5 nM) for 30 min at
37uC. AMD3100, 5-HT3 receptor agonists and antagonists were
present during the migration in both transwell compartments. Migration assays Cell migration in response to chemokine was tested using
Transwell assays (24-well plate with inserts, 3 mm pore/6.5 mm,
TC-treated; Costar). Briefly, after 2 h of serum starvation in
migration medium (serum-free RPMI 1640 supplemented with
0.1% BSA), 5-HT, 5-HT3 receptor agonists or antagonists were
added for 30 min at RT to cell suspension and then cell were used
for migration assays. 5-HT3 receptor agonists and antagonists Real-time PCR Migration experiments displayed an inter-donor variability in the
absolute number of migrating T cells, thus for each experiment
migration data have been normalized considering the number of
counted cells in control conditions as 100%. Statistical analysis After 5 hours of serum starvation in RPMI 1640 medium, T
cells were treated for 30 min at RT with the indicated chemicals
and then stimulated with CXCL12 (100 nM) at 37uC in a rotary
shaker. At indicated time points, stimulation was stopped by
adding 4% formaldehyde. Intracellular staining for F-actin was
performed by AlexaFluor488–phalloidin (Molecular Probes) using
the BD Cytofix/Cytoperm kit (BD Biosciences) according to the
manufacturer’s instructions. MFI was measured by FACS Canto
flow cytometer and analyzed with FACS Diva software. All experiments were performed at least three times. All results are
expressed as mean 6 the standard error of the mean (SEM). The
statistical significance was calculated using a Student’s t-test or a one-
way ANOVA, Bonferroni’s multiple comparison test (GraphPad
Prism 4). Differences were considered significant if P,0.05. Author Contributions Conceived and designed the experiments: EM IS AV. Performed the
experiments: EM IS JC. Analyzed the data: EM IS. Contributed reagents/
materials/analysis tools: AD. Wrote the paper: EM IS AV. Provided
technical help for most of the experiments: AD. Provided funds: AV. Conceived and designed the experiments: EM IS AV. Performed the
experiments: EM IS JC. Analyzed the data: EM IS. Contributed reagents/
materials/analysis tools: AD. Wrote the paper: EM IS AV. Provided
technical help for most of the experiments: AD. Provided funds: AV. Acknowledgments We thank Dr. Marina Sironi, Dr. Tihana Kasic and Dr. Anna Elisa
Trovato for providing reagents and assistance, Dr. Rita Lucia Contento,
Dr. Marinos Kallikourdis, Dr. Adelaida Sarukhan, Dr. Massimo Locati
and Dr. Ugo Cavallaro for critical reading of the manuscript. CXCL12 binding assay T cells were washed once with Hanks’ balanced salt solution
containing 20 mM Hepes and 0.2% BSA (Sigma) at pH = 7.4 and
resuspended in the same buffer. First, they were treated for 30 min
at RT with the indicated chemicals and then incubated for 30 min
at RT with different concentrations of CXCL12-AF647 (Almac
Sciences) in the range from 0 to 1000 ng/ml. Thereafter, cells
were washed twice in assay buffer and fixed in 1% paraformal-
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Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in
lymphocytes. Proc Natl Acad Sci U S A 105: 14861–14866. References PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e22482 August 2011 | Volume 6 | Issue 8 | e22482 9 Serotonin and T Cell Migration PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e22482 10
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https://openalex.org/W2885122465
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https://esurf.copernicus.org/articles/7/171/2019/esurf-7-171-2019.pdf
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English
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Systematic identification of external influences in multi-year microseismic recordings using convolutional neural networks
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Earth surface dynamics
| 2,019
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cc-by
| 15,206
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Systematic identification of external influences in
multi-year microseismic recordings using convolutional
neural networks Matthias Meyer1, Samuel Weber2,1, Jan Beutel1, and Lothar Thiele1
1Computer Engineering and Networks Laboratory, ETH Zurich, Zurich, Switzerland
2Department of Geography, University of Zurich, Zurich, Switzerland
Correspondence: Matthias Meyer (matthias.meyer@tik.ee.ethz.ch)
Received: 26 July 2018 – Discussion started: 6 August 2018
Revised: 16 November 2018 – Accepted: 17 December 2018 – Published: 4 February 2019 Matthias Meyer1, Samuel Weber2,1, Jan Beutel1, and Lothar Thiele1
1Computer Engineering and Networks Laboratory, ETH Zurich, Zurich, Switzerland
2Department of Geography, University of Zurich, Zurich, Switzerland Received: 26 July 2018 – Discussion started: 6 August 2018
Revised: 16 November 2018 – Accepted: 17 December 2018 – Published: 4 February 2019 Abstract. Passive monitoring of ground motion can be used for geophysical process analysis and natural haz-
ard assessment. Detecting events in microseismic signals can provide responsive insights into active geophysical
processes. However, in the raw signals, microseismic events are superimposed by external influences, for exam-
ple, anthropogenic or natural noise sources that distort analysis results. In order to be able to perform event-based
geophysical analysis with such microseismic data records, it is imperative that negative influence factors can be
systematically and efficiently identified, quantified and taken into account. Current identification methods (man-
ual and automatic) are subject to variable quality, inconsistencies or human errors. Moreover, manual methods
suffer from their inability to scale to increasing data volumes, an important property when dealing with very
large data volumes as in the case of long-term monitoring. In this work, we present a systematic strategy to identify a multitude of external influence sources, characterize
and quantify their impact and develop methods for automated identification in microseismic signals. We apply
the strategy developed to a real-world, multi-sensor, multi-year microseismic monitoring experiment performed
at the Matterhorn Hörnligrat (Switzerland). We develop and present an approach based on convolutional neural
networks for microseismic data to detect external influences originating in mountaineers, a major unwanted
influence, with an error rate of less than 1 %, 3 times lower than comparable algorithms. Moreover, we present
an ensemble classifier for the same task, obtaining an error rate of 0.79 % and an F1 score of 0.9383 by jointly
using time-lapse image and microseismic data on an annotated subset of the monitoring data. Earth Surf. Dynam., 7, 171–190, 2019
https://doi.org/10.5194/esurf-7-171-2019
© Author(s) 2019. This work is distributed under
the Creative Commons Attribution 4.0 License. Systematic identification of external influences in
multi-year microseismic recordings using convolutional
neural networks 1
Introduction With respect to unwanted
signal components, STA/LTA has also been used to detect
external influence factors such as footsteps (Anchal et al.,
2018) but due to its inherent simplicity, it cannot reliably
discriminate geophysical seismic activity from external (un-
wanted) influence factors such as noise from humans and nat-
ural sources like wind, rain or hail without manually super-
vising and intervening in the detection process on a case-by-
case basis. As a result, the blind application of STA/LTA will
inevitably lead to the false estimation of relevant geophysical
processes if significant external influences, such as wind, are
present (Allen, 1978). p
(
)
There exist several algorithmic approaches to mitigate
the problem of external influences by increasing the selec-
tivity of event detection. These include unsupervised algo-
rithms such as auto-correlation (Brown et al., 2008; Aguiar
and Beroza, 2014; Yoon et al., 2015), but these are ei-
ther computationally complex or do not perform well for
low signal-to-noise ratios. Supervised methods can find
events in signals with low signal-to-noise ratio. For exam-
ple, template-matching approaches such as cross-correlation
methods (Gibbons and Ringdal, 2006) use event examples
to find similar events, failing if events differ significantly
in “shape” or if the transmission medium is very inhomo-
geneous (Weber et al., 2018b). The most recent supervised
methods are based on machine learning techniques (Reynen
and Audet, 2017; Olivier et al., 2018) including the use of
neural networks (Kislov and Gravirov, 2017; Perol et al.,
2018; Li et al., 2018; Ross et al., 2018). These learning
approaches show promising results with the drawback that
large datasets containing ground truth (verified events) are
required to train these automated classifiers. In earthquake
research, large databases of known events exist (Kong et al.,
2016; Ross et al., 2018), but in scenarios like slope insta-
bility, analyses where effects are on a local scale and spe-
cific to a given field site such data are inexistent. Here, in-
homogeneities are present on a very small scale and field
sites differ in their specific characteristics with respect to
signal attenuation and impulse response. In order to apply
such automated learning methods to these scenarios, obtain-
ing a dataset of known events is required for each new field j
p
y
g
In general, event-based geoscientific investigations focus
on events originating from geophysical sources such as me-
chanical damage, rupture or fracture in soil, rock and/or ice. 1
Introduction 1
Introduction on the signals recorded. As a conclusion, it is a requirement
that external influences can be taken into account with an
automated workflow, including preprocessing, cleaning and
analysis of microseismic data. Passive monitoring of elastic waves, generated by the rapid
release of energy within a material (Hardy, 2003) is a non-
destructive analysis technique allowing a wide range of ap-
plications in material sciences (Labuz et al., 2001), engineer-
ing (Grosse, 2008) and natural hazard mitigation (Michlmayr
et al., 2012) with recently increasing interest into investiga-
tions of various processes in rock slopes (Amitrano et al.,
2010; Occhiena et al., 2012). Passive monitoring techniques
may be broadly divided into three categories, characterized
by the number of stations (single vs. array), the duration of
recording (snapshot vs. monitoring) and the type of analysis
(continuous vs. event-based). On the one hand, continuous
methods such as the analysis of ambient seismic vibrations
can provide information on internal structure of a rock slope
(Burjánek et al., 2012; Gischig et al., 2015; Weber et al.,
2018a). On the other hand, event-based methods such as the
detection of microseismic events (which are the focus of this
study) can give immediate insight into active processes, such
as local irreversible (non-elastic) deformation occurring due
to the mechanical loading of rocks (Grosse and Ohtsu, 2008). However, for the reliable detection of events irrespective of
the detection method, the signal source of concern has to be
distinguishable from noise, for example, background seis-
micity or other source types. This discrimination is a com-
mon and major problem for analyzing microseismic data. y
A frequently used example of an event detection mecha-
nism is an event detector called STA/LTA that is based on
the ratio of short-term average to long-term average (Allen,
1978). Due to its simplicity, this event detector is commonly
used to assess seismic activity by calculating the number of
triggering events per time interval for a time period of in-
terest (Withers et al., 1998; Amitrano et al., 2005; Senfaute
et al., 2009). It is often used in the analysis of unstable slopes
(Colombero et al., 2018; Levy et al., 2011) and is available
integrated into many commercially available digitizers and
data loggers (Geometrics, 2018). M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 172 Systematic identification of external influences in
multi-year microseismic recordings using convolutional
neural networks Applying these
classifiers to the whole experimental dataset reveals that approximately one-fourth of events detected by an event
detector without such a preprocessing step are not due to seismic activity but due to anthropogenic influences
and that time periods with mountaineer activity have a 9 times higher event rate. Due to these findings, we argue
that a systematic identification of external influences using a semi-automated approach and machine learning
techniques as presented in this paper is a prerequisite for the qualitative and quantitative analysis of long-term
monitoring experiments. Published by Copernicus Publications on behalf of the European Geosciences Union. Earth Surf. Dynam., 7, 171–190, 2019 1
Introduction The aim of this study is to use a
semi-automatic workflow to train a classifier which enables
the automatic identification of unwanted external influences
in real-world microseismic data. By these means, the geo-
physical phenomena of interest can be analyzed without the
distortions of external influences. Figure 1. Real-world measurement signals contain the phenom-
ena of interest superimposed with external influences. If directly
analyzed, the results are perturbed by the external influences. In
contrast to this approach (dashed lines), in this paper, we suggest
a systematic and automated approach to first identify a multitude
of external influence sources in microseismic signals using a classi-
fier. The classifier result data can then be used to quantify unwanted
signal components as well as drive more extensive and powerful
event detection and characterization methods leveraging combina-
tions of both the signals as well-labeled and classified noise data
(solid lines). To address these problems, this paper contains the follow-
ing contributions. We propose a strategy to identify and deal
with unwanted external influences in multi-sensor, multi-
year experiments. We compare the suitability of multiple al-
gorithms for mountaineer detection using a combination of
microseismic signals and time-lapse images. We propose a
convolutional neural network (CNN) for source identifica-
tion. We exemplify our strategy for the case of source iden-
tification on real-world microseismic data using monitoring
data in steep, fractured bedrock permafrost. We further pro-
vide the real-world microseismic and image data as an an-
notated dataset containing data from a period of 2 years as
well as an open-source implementation of the algorithms pre-
sented. time periods when mountaineers are present and when they
are not, which we use as a case study in the evaluation section
of this paper to exemplify our method. Figure 2 illustrates the overall concept in detail. In a first
step, the available data sources of a case study are assessed
and cataloged. Given a case study (Sect. 3) consisting of mul-
tiple sensors, one or more sensor signals are specified as pri-
mary signals (for example, the microseismic signals, high-
lighted with a light green arrow in the figure) targeted by
a subsequent domain-specific analysis method. Additionally,
secondary data (highlighted with dark blue arrows) are cho-
sen to support the classification of external influences con-
tained in the primary signal. 1
Introduction These sources originate, for example, in thermal stresses,
pressure variations or earthquakes (Amitrano et al., 2012). However, non-geophysical sources can trigger events as well:
(i) anthropogenic influences such as helicopter or moun-
taineers (Eibl et al., 2017; van Herwijnen and Schweizer,
2011; Weber et al., 2018b) and (ii) environmental influ-
ences/disturbances, such as wind or rain (Amitrano et al.,
2010). One way to account for such external influences is to
manually identify their sources in the recordings (van Herwi-
jnen and Schweizer, 2011). This procedure, however, is not
feasible for autonomous monitoring because manual identifi-
cation does not scale well for increasing amounts of data. An-
other approach is to limit to field sites far away from possible
sources of uncontrolled (man-made) interference or to focus
and limit analysis to decisively chosen time periods known
not to be influenced by, for example, anthropogenic noise
(Occhiena et al., 2012). In practice, both the temporal limita-
tion as well as the spatial limitation pose severe restrictions. First, research applications can benefit from close proximity
to man-made infrastructure since set up and maintenance of
monitoring infrastructure is facilitated (Werner-Allen et al.,
2006). Second, applications in natural hazard early warning
must not be restricted to special time periods only. Moreover,
they are specifically required to be usable close to inhabited
areas with an increasing likelihood for human interference www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings
173 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic 173 Figure 1. Real-world measurement signals contain the phenom-
ena of interest superimposed with external influences. If directly
analyzed, the results are perturbed by the external influences. In
contrast to this approach (dashed lines), in this paper, we suggest
a systematic and automated approach to first identify a multitude
of external influence sources in microseismic signals using a classi-
fier. The classifier result data can then be used to quantify unwanted
signal components as well as drive more extensive and powerful
event detection and characterization methods leveraging combina-
tions of both the signals as well-labeled and classified noise data
(solid lines). site requiring substantial expert knowledge for a very ardu-
ous, time-consuming task. 1
Introduction Conceptually, these secondary
data can be of different nature, either different sensor sig-
nals, e.g., time-lapse images or weather data, or auxiliary
data such as local observations or helicopter flight data. All
data sources are combined into a dataset. However, this re-
sulting dataset is not yet annotated as required to perform
domain-specific analysis leveraging the identified and quan-
tified external influences. 2
Concept of the classification method In this work, we present a systematic and automated ap-
proach to identify unwanted external influences in long-
term, real-world microseismic datasets and prepare these
data for subsequent analysis using a domain-specific anal-
ysis method, as illustrated in Fig. 1. Traditionally, the sig-
nal, consisting of the phenomena of interest and superim-
posed external influences, is analyzed directly as described
earlier. However, this analysis might suffer from distortion
through the external influences. By using additional sensors
like weather stations, cameras or microphones and external
knowledge such as helicopter flight plans or mountain hut
(Hörnlihütte) occupancy, it is possible to semi-automatically
label events originating from non-geophysical sources, such
as helicopters, footsteps or wind without the need of expert
knowledge. Such “external” information sources can be used
to train an algorithm that is then able to identify unwanted
external influence. Using this approach, multiple external
influences are first classified and labeled in an automated
preprocessing step with the help of state-of-the-art machine
learning methods. Subsequent to this classification, the addi-
tional information can be used for domain-specific analysis,
for example, to separate geophysical and unwanted events
triggered by a simple event detector such as an STA/LTA
event detector. Alternatively, more complex approaches can
be used, taking into account signal content, event detections
and classifier labels of the external influences. However, the
specifics of such advanced domain-specific analysis meth-
ods are beyond the scope of this paper and subject to future
work. A basic example of a custom domain-specific analysis
method is the estimation of separate STA/LTA event rates for In this work, we present a systematic and automated ap-
proach to identify unwanted external influences in long-
term, real-world microseismic datasets and prepare these
data for subsequent analysis using a domain-specific anal-
ysis method, as illustrated in Fig. 1. Traditionally, the sig-
nal, consisting of the phenomena of interest and superim-
posed external influences, is analyzed directly as described
earlier. However, this analysis might suffer from distortion
through the external influences. By using additional sensors
like weather stations, cameras or microphones and external
knowledge such as helicopter flight plans or mountain hut
(Hörnlihütte) occupancy, it is possible to semi-automatically
label events originating from non-geophysical sources, such
as helicopters, footsteps or wind without the need of expert
knowledge. Such “external” information sources can be used
to train an algorithm that is then able to identify unwanted
external influence. 2
Concept of the classification method Using this approach, multiple external
influences are first classified and labeled in an automated
preprocessing step with the help of state-of-the-art machine
learning methods. Subsequent to this classification, the addi-
tional information can be used for domain-specific analysis,
for example, to separate geophysical and unwanted events
triggered by a simple event detector such as an STA/LTA
event detector. Alternatively, more complex approaches can
be used, taking into account signal content, event detections
and classifier labels of the external influences. However, the
specifics of such advanced domain-specific analysis meth-
ods are beyond the scope of this paper and subject to future
work. A basic example of a custom domain-specific analysis
method is the estimation of separate STA/LTA event rates for Two key challenges need to be addressed in order to es-
tablish such an annotated dataset by automatic classification:
(i) suitable data types need to be selected for classification
since not every data type can be used to continuously clas-
sify every external influence (for example, wind sensors are
not designed to capture the sound of footsteps; flight data
may not be available for each time step) and (ii) a single
(preferred) or at least a set of suitable, well-performing clas-
sifiers has to be found for each type of external influence
source. Once these challenges have been solved, a subset of
the dataset is manually annotated in order to select and train
the classifier(s) in a “preparatory” phase required to be per-
formed only once, which includes manual data assessment
(Sect. 4) as well as classifier selection and training (Sect. 5). The trained classifier is then used in an automated setup to
annotate the whole dataset (Sect. 6). This “execution” phase www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 174 Figure 2. Conceptual illustration of the classification method to enable domain-specific analysis of a primary sensor signal (in our case,
microseismic signals denoted by the light green arrow) based on annotated datasets: a subset of the dataset, containing both sensor and
auxiliary data, is used to select and train a classifier that is subsequently applied to the whole dataset. By automatically and systematically
annotating the whole dataset of the primary signal of concern, advanced methods can be applied that are able to leverage both multi-sensor
data as well as annotation information. 2
Concept of the classification method Figure 2. Conceptual illustration of the classification method to enable domain-specific analysis of a primary sensor signal (in our case,
microseismic signals denoted by the light green arrow) based on annotated datasets: a subset of the dataset, containing both sensor and
auxiliary data, is used to select and train a classifier that is subsequently applied to the whole dataset. By automatically and systematically
annotating the whole dataset of the primary signal of concern, advanced methods can be applied that are able to leverage both multi-sensor
data as well as annotation information. tion as well as some backdrop areas further away on the
mountain ridge. Figure 3 shows an overview of the field
site including the location of the seismometer and an ex-
ample image acquired with the camera. The standard image
size is 1424 × 2144 pixels captured every 4 min. The Vaisala
WXT520 weather data as well as the rock surface tempera-
ture are transmitted using a custom wireless sensor network
infrastructure. A new measurement is performed on the sen-
sors every 2 min and transmitted to the base station, resulting
in a sampling rate of 30 samples h−1. can be performed in a one-shot fashion (post-processing all
data in one effort) or executed regularly, for example, on a
daily or weekly basis if applied to continuously retrieved
real-time monitoring data. This additional information can be
used to perform a subsequent domain-specific analysis. This
study concludes with an evaluation (Sect. 7) and discussion
(Sect. 8) of the presented method. 3
Case study Significant data gaps are prevented by using solar panels,
durable batteries and field-tested sensors, but given the cir-
cumstances on such a demanding high-alpine field site, cer-
tain outages of single sensors, for example, due to power
failures or during maintenance, could not be prevented. Nev-
ertheless, this dataset constitutes an extensive and close-to-
complete dataset. The data used in this paper originate from a multi-sensor
and multi-year experiment (Weber et al., 2018b) focus-
ing on slope stability in high-alpine permafrost rock walls
and understanding the underlying processes. Specifically,
the sensor data are collected at the site of the 2003 rock-
fall event on the Matterhorn Hörnligrat (Zermatt, Switzer-
land) at 3500 m a.s.l., where an ensemble of different sen-
sors has monitored the rockfall scar and surrounding environ-
ment over the past 10 years. Relevant for this work are data
from a three-component seismometer (Lennartz LE-3Dlite
MkIII), images from a remote-controlled high-resolution
camera (Nikon D300, 24 mm fixed focus), rock surface tem-
perature measurements, net radiation measurements and am-
bient weather conditions, specifically wind speed from a co-
located local weather station (Vaisala WXT520). The recordings of the case study were affected by external
influences, especially mountaineers and wind. This reduced
the set of possible analysis tools. Auxiliary data which help
to characterize the external influences are collected in addi-
tion to the continuous data from the sensors. In the presented
case, the auxiliary data are non-continuous and consist of lo-
cal observations, preprocessed STA/LTA triggers from We-
ber et al. (2018b), accommodation occupancy of a nearby
hut and a non-exhaustive list of helicopter flight data from a
duration of approximately 7 weeks provided by a local heli-
copter company. The seismometer applied in the case study presented is
used to assess the seismic activity by using an STA/LTA
event detector, which means for our application that the seis-
mometer is chosen as the primary sensor and STA/LTA trig-
gering is used as the reference method. Seismic data are
recorded locally using a Nanometrics Centaur digitizer and
transferred daily by means of directional Wi-Fi. The data are
processed on demand using STA/LTA triggering. The high-
resolution camera’s (Keller et al., 2009) field of view cov-
ers the immediate surroundings of the seismic sensor loca- In following, we use this case study to exemplify our
method, which was presented in the previous sections. Earth Surf. Dynam., 7, 171–190, 2019 3
Case study 4
Manual data assessment A ground truth is often needed for state-of-the-art classifiers
(such as artificial neural networks). To establish this ground www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic reco 175 Figure 3. The field site is located on the Matterhorn Hörnligrat at 3500 m a.s.l. which is denoted with a red circle. The photograph on the
right is taken by a remotely controlled DSLR camera at the field site on 4 August 2016, 12:00:11 CET. The seismometer of interest (white
circle) is located on a rock instability which is close to a frequently used climbing route. Figure 3. The field site is located on the Matterhorn Hörnligrat at 3500 m a.s.l. which is denoted with a red circle. The photograph on the
right is taken by a remotely controlled DSLR camera at the field site on 4 August 2016, 12:00:11 CET. The seismometer of interest (white
circle) is located on a rock instability which is close to a frequently used climbing route. ability for classification given the task and the data. A se-
lection of classifiers is therefore trained and tested with the
annotated subset and optimized for best performance, which
can, for example, be done by selecting the classifier with the
lowest error rate on a defined test set. The classifier selection,
training and optimization is repeated until a sufficiently good
set of classifiers has been found. This suitability is defined
by the user and can, for example, mean that the classifier is
better than a critical error rate. These classifiers can then be
used for application in the automatic classification process. truth while reducing the amount of manual labor, only a sub-
set of the dataset is selected and used in a manual data as-
sessment phase, which consists of data evaluation, classifier
training and classifier selection as depicted in Fig. 4. Data
evaluation can be subdivided into four parts: (i) character-
ization of external influences in the primary signal (that is
the relation between primary and secondary signals), (ii) an-
notating the subset based on the primary and secondary sig-
nals, (iii) selecting the data types suitable for classification
and (iv) performing a first statistical evaluation with the an-
notated dataset, which facilitates the selection of a classifier. Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ 4.1
Characterization sources on a larger time frame. Mountaineers, for example,
show characteristic patterns of increasing or decreasing loud-
ness, and helicopters have distinct spectral patterns, which
could be beneficial to classify these sources. Additionally, the
images captured on site show when a mountaineer is present
(see Fig. 3), but due to fog, lens flares or snow on the lens,
the visibility can be limited. The limited visibility needs to be
taken into account for when seismic data are to be annotated
with the help of images. Another limiting factor is the tempo-
ral resolution of one image every 4 min since mountaineers
could have moved through the camera’s field of view in be-
tween two images. The seismometer captures elastic waves originating from dif-
ferent sources. In this study, we will consider multiple non-
geophysical sources, which are mountaineers, helicopters,
wind and rockfalls. Time periods where the aforementioned
sources cannot be identified are considered as relevant, and
thus we will include them in our analysis as a fifth source,
the “unknown” source. The mountaineer impact will be char-
acterized on a long-term scale by correlating with hut ac-
commodation occupancy (see Fig. 10) and on a short-scale
by person identification on images. Helicopter examples are
identified by using flight data and local observations. By ana-
lyzing spectrograms, one can get an intuitive feeling for what
mountaineers or helicopters “look” like, which facilitates the
manual annotation process. In Fig. 5, different recordings
from the field site are illustrated, which have been picked by
using the additional information described at the beginning
of this section. For six different examples, the time domain
signal, its corresponding spectrogram and STA/LTA triggers
are depicted. The settings for the detector are the same for all
the subsequent plots. It becomes apparent in Fig. 5b–c that
anthropogenic noise, such as mountaineers walking by or he-
licopters, is recorded by seismometers. Moreover, it becomes
apparent that it might be feasible to assess non-geophysical The wind sensor can directly be used to identify the impact
on the seismic sensor. Figure 6 illustrates the correlation be-
tween tremor amplitude and wind speed. Tremor amplitude
is the frequency-selective, median, absolute ground velocity
and has been calculated for the frequency range of 1–60 Hz
according to Bartholomaus et al. (2015). 3
Case study Characterization and statistical evaluation are the only steps
where domain expertise is required while it is not required
for the time- and labor-intensive annotation process. In the following, the previously explained method will be
exemplified for wind and mountaineer detection using micro-
seismic, wind and image data from a real-world experiment. The required steps of subset creation, characterization, anno-
tation, statistical evaluation and the selection of the data type
for classification are explained. Before an annotated subset
can be created the collected, data must be characterized for
their usefulness in the annotation process, i.e., which data
type can be used to annotate which external influence. The classifier selection and training phase presumes the
availability of a variety of classifiers for different input data
types, for example, the broad range of available image clas-
sifiers (Russakovsky et al., 2015). The classifiers do not per-
form equally well on the given task with the given subset. Therefore, classifiers have to be selected based on their suit- Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic record M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 176 Figure 4. The manual preparation phase is subdivided into data evaluation (a) and classifier selection and training (b). First, the data
subset is characterized and annotated. This information can be used to do a statistical evaluation and select data types which are useful for
classification. Domain experts are not required for the labor-intensive task of annotation. The classifiers are selected, trained and optimized
in a feedback loop until the best set of classifiers is found. Figure 4. The manual preparation phase is subdivided into data evaluation (a) and classifier selection and training (b). First, the data
subset is characterized and annotated. This information can be used to do a statistical evaluation and select data types which are useful for
classification. Domain experts are not required for the labor-intensive task of annotation. The classifiers are selected, trained and optimized
in a feedback loop until the best set of classifiers is found. Earth Surf. Dynam., 7, 171–190, 2019 4.1
Characterization By manually an-
alyzing the correlation between tremor amplitude and wind
speed, it can be deduced that wind speeds above approxi-
mately 30 km h−1 have a visible influence on the tremor am-
plitude. Rockfalls can best be identified by local observations since
the camera captures only a small fraction of the receptive
range of the seismometers. Figure 5e illustrates the seis- Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 177 Figure 5. Microseismic signals and the impact of external influences. (a) During a period of little anthropogenic noise, the seismic activity is
dominant. (b) In the spectrogram, the influence of mountaineers becomes apparent. Shown are seismic signatures of (c) a helicopter in close
spatial proximity to the seismometer, (d) wind influences on the signal and (e) a rockfall in close proximity to the seismometer. The red dots
in the signal plots indicate the timestamps of the STA/LTA triggers from Weber et al. (2018b). Figure 5. Microseismic signals and the impact of external influences. (a) During a period of little anthropogenic noise, the seismic activity is
dominant. (b) In the spectrogram, the influence of mountaineers becomes apparent. Shown are seismic signatures of (c) a helicopter in close
spatial proximity to the seismometer, (d) wind influences on the signal and (e) a rockfall in close proximity to the seismometer. The red dots
in the signal plots indicate the timestamps of the STA/LTA triggers from Weber et al. (2018b). the geophysical application (Colombero et al., 2018; Weber
et al., 2018b). mic signature of a rockfall. The number of rockfall observa-
tions and rockfalls caught on camera is however very limited. Therefore, it is most likely that we were unable to annotate
all rockfall occurrences. As a consequence, we will not con-
sider a rockfall classifier in this study. www.earth-surf-dynam.net/7/171/2019/ 4.2
Annotation Consecutive segments are 2 min segments sequentially
extracted from the continuous microseismic signal. Figure 6. Impact of wind (light orange) on the seismic signal. The
tremor amplitude (dark blue) is calculated according to Bartholo-
maus et al. (2015). The effect of wind speed on tremor amplitude
becomes apparent for wind speeds above approximately 30 km h−1. Note the different scales on the y axes. For mountaineer classification, the required label is
“mountaineer” but additional labels will be annotated which
could be beneficial for classifier training and statistical analy-
sis. These labels are “helicopters”, “rockfalls”, “wind”, “low
visibility” (if the lens is partially obscured) and “lens flares”. The “wind” label applies to segments where the wind speed
is higher than 30 km h−1, which is the lower bound for no-
ticeable wind impact as resulted from Sect. 4.1. Figure 8 depicts the availability of image-linked segments
per week during the relevant time frame. A fraction of the
data is manually labeled by the authors, which is illustrated
in Fig. 8. Two sets are created: a training set containing 5579
samples from the year 2016, and a test set containing 1260
data samples from 2017. The test set has been sampled ran-
domly to avoid any human prejudgment. For each day in
2017, four samples have been chosen randomly, which are
then labeled and added to the test set. The training set has
been specifically sampled to include enough training data for
each category. This means, for example, that more moun-
taineer samples come from the summer period where the
climbing route is most frequently used. The number of ver-
ified rockfalls and helicopters is non-representative, and al-
though helicopters can be manually identified from spectro-
grams, the significance of these annotations is not given due
to the limited ground truth from the secondary source. There-
fore, for the rest of this study, we will focus on mountaineers
for qualitative evaluation. For statistical evaluation, we will
however use the manually annotated helicopter and rockfall
samples to exclude them from the analysis. The labels for all
categories slightly differ for microseismic data and images
since the types of sources which can be registered by each
sensor differ. This means, for instance, that not every classi-
fier uses all labels for training (for example, a microseismic
classifier cannot detect a lens flare). 4.2
Annotation It also means that for the
same time instance one label might apply to the image but
not to the image-linked microseismic segment (for example,
mountaineers are audible but the image is partially obscured
and the mountaineer is not visible). This becomes relevant in
Sect. 5.3.4 when multiple classifiers are used for ensemble
classification. Figure 6. Impact of wind (light orange) on the seismic signal. The
tremor amplitude (dark blue) is calculated according to Bartholo-
maus et al. (2015). The effect of wind speed on tremor amplitude
becomes apparent for wind speeds above approximately 30 km h−1. Note the different scales on the y axes. Figure 7. Illustration of microseismic segmentation. Event-linked
segments are 10 s segments starting on event timestamps. Image-
linked segments are 2 min segments centered around an image
timestamp. Consecutive segments are 2 min segments sequentially
extracted from the continuous microseismic signal. Figure 7. Illustration of microseismic segmentation. Event-linked
segments are 10 s segments starting on event timestamps. Image-
linked segments are 2 min segments centered around an image
timestamp. Consecutive segments are 2 min segments sequentially
extracted from the continuous microseismic signal. quentially extracted from the continuous microseismic sig-
nal. Only the image-linked segments are used during annota-
tion; their label can, however, be transferred to other seg-
mentation types by assigning the image-linked label to over-
lapping event-linked or consecutive segments. Image-linked
and event-linked segments are used during data evaluation
and classifier training. Consecutive segments are used for au-
tomatic classification on the complete dataset. Here, falsely
classified segments are reduced by assigning each segment a
validity range. A segment classified as mountaineer is only
considered correct if the distance to the next (or previous)
mountaineer is less than 5 min. This is based on an estima-
tion of how long the mountaineers are typically in the audible
range of the seismometer. Earth Surf. Dynam., 7, 171–190, 2019 4.2
Annotation The continuous microseismic signals are segmented for an-
notation and evaluation. Figure 7 provides an overview of
the three segmentation types, event-linked segments, image-
linked segments and consecutive segments. Image-linked
segments are extracted due to the fact that a meaningful re-
lation between seismic information and photos is only given
in close temporal proximity. Therefore, images and micro-
seismic data are linked in the following way. For each image,
a 2 min microseismic segment is extracted from the contin-
uous microseismic signal. The microseismic segment’s start
time is set to 1 min before the image timestamp. Event-linked
segments are extracted based on the STA/LTA triggers from
Weber et al. (2018b). For each trigger, the 10 s following
the timestamp of the trigger are extracted from the micro-
seismic signal. Consecutive segments are 2 min segments se- It can be seen in Fig. 5a that during a period which is not
strongly influenced by external influences the spectrogram
shows mainly energy in the lower frequencies with occa-
sional broadband impulses. The red circles in the subplots in Fig. 5 indicate the times-
tamps of the STA/LTA events for a specific geophysical anal-
ysis (Weber et al., 2018b). By varying the threshold of the
STA/LTA event trigger, the number of events triggered by
mountaineers can be reduced. However, since the STA/LTA
event detector cannot discriminate between events from geo-
physical sources and events from mountaineers, changing the
threshold would also influence the detection of events from
geophysical sources. This fact would affect the quality of
the analysis since the STA/LTA settings are determined by www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 178 Figure 6. Impact of wind (light orange) on the seismic signal. The
tremor amplitude (dark blue) is calculated according to Bartholo-
maus et al. (2015). The effect of wind speed on tremor amplitude
becomes apparent for wind speeds above approximately 30 km h−1. Note the different scales on the y axes. Figure 7. Illustration of microseismic segmentation. Event-linked
segments are 10 s segments starting on event timestamps. Image-
linked segments are 2 min segments centered around an image
timestamp. 4.3
Data type selection After the influences have been characterized, the data type
which best describe each influence needs to be selected. The
wind sensor delivers a continuous data stream and a direct
measure of the external influence. In contrast, mountaineers,
helicopters and rockfalls cannot directly be identified. A data
type including information about these external influences
needs to be selected. Local observations, accommodation oc-
cupancy and flight data can be discarded for the use as clas-
sifier input since the data cannot be continuously collected. According to Sect. 4.1, it seems possible to identify moun-
taineers, helicopters and rockfalls from microseismic data. Moreover, mountaineers can also be identified from images. Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 179 Figure 8. Number of image/microseismic data pairs in the dataset
(dark blue) and in the annotated subset (light orange) displayed over
the week number of the years 2016 and 2017. Note the logarithmic
scale on the y axis. where ∗denotes the convolution operator, g(·) is a nonlinear
function, Ij is an input channel, bk is the bias related to the
feature map FH,k, and wk,j is the kernel related to the input
image Ij and feature map FH,k. Kernel and bias are trainable
parameters of each layer. This principle can be applied to
subsequent convolutional layers. Additionally, a strided con-
volution can be used which effectively reduces the dimen-
sion of a feature map as illustrated by L1 in Fig. 9. In an
all-convolutional neural network (Springenberg et al., 2014),
the feature maps of the output convolutional layer are aver-
aged per channel. In our case, the number of output channels
is chosen to be the number of event sources to be detected. Subsequent scaling and a final (nonlinear) activation func-
tion are applied. If trained correctly, each output represents
the probability that the input contains the respective event
source. In our case, this training is performed by calculat-
ing the binary cross-entropy between the network output and
the ground truth. The error is back-propagated through the
neural network and the parameters are updated. The training
procedure is performed for all samples in the dataset and is
repeated for multiple epochs. Figure 8. Number of image/microseismic data pairs in the dataset
(dark blue) and in the annotated subset (light orange) displayed over
the week number of the years 2016 and 2017. 5
Classifier selection and training 5
Classifier selection and training The following section describes the classifier preselection,
training, testing and how the classifiers are used to annotate
the whole data stream as illustrated in Fig. 4b. First, a brief
introduction to convolutional neural networks is given. If the
reader is unfamiliar with neural networks, we recommend to
read additional literature (Goodfellow et al., 2016). 5.2
Classifier selection As a consequence, the data types selected to perform clas-
sification are microseismic data, images and wind data. The
microseismic data used are the signals from the three com-
ponents of the seismometer. As a consequence, the data types selected to perform clas-
sification are microseismic data, images and wind data. The
microseismic data used are the signals from the three com-
ponents of the seismometer. Multiple classifiers are available for the previously selected
data types: wind data, images and microseismic data. yp
g
For wind data, a simple threshold classifier can be used,
which indicates wind influences based on the wind speed. For simplicity, the classifier labels time periods with wind
speed above 30 km h−1 as “wind”. For images, a convolu-
tional neural network is selected to classify the presence of
mountaineers in the image. The image classifier architecture
is selected from the large pool of available image classifiers
(Russakovsky et al., 2015). For microseismic data, three dif-
ferent classifiers will be preselected: (i) a footstep detector
based on manually selected features (standard deviation, kur-
tosis and frequency band energies) using a linear support
vector machine (LSVM) similar to the detector used in An-
chal et al. (2018), (ii) a seismic event classifier adopted from
Perol et al. (2018) and (iii) a non-geophysical event classi-
fier which we call MicroseismicCNN. We re-implemented
the first two algorithms based on the information from the
respective papers. The third is a major contribution in this
paper and has been specifically designed to identify non-
geophysical sources in microseismic data. 4.3
Data type selection Note the logarithmic
scale on the y axis. Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ 5.1
Convolutional neural networks Convolutional neural networks have gained a lot of interest
due to their advanced feature extraction and classification
capabilities. A convolutional neural network contains mul-
tiple adoptable parameters which can be updated in an iter-
ative optimization procedure. This fact makes them generi-
cally applicable to a large range of datasets and a large range
of different tasks. The convolutional neural network consists
of multiple so-called convolutional layers. A convolutional
layer transforms its input signal with ci channels into ch fea-
ture maps as illustrated in Fig. 9. A hidden feature map FH,k
is calculated according to The proposed convolutional neural network (CNN) to
identify non-geophysical sources in microseismic signals
uses a time–frequency signal representation as input and con-
sists of 2-D convolutional layers. Each component of the time
domain signal, sampled at 1 kHz, is first offset compensated
and then transformed with a short-time Fourier transforma-
tion (STFT). Subsequently, the STFT output is further pro-
cessed by selecting the frequency range from 2 to 250 Hz
and subdividing it into 64 linearly spaced bands. This time–
frequency representation of the three seismometer compo- FH,k = g
ci
X
j=1
I ∗
j wk,j + bk
! , www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 180 Figure 9. Simplified illustration of a convolutional neural network. An input signal, for example, an image or spectrogram, with a given
number of channels ci is processed by a convolutional layer LH. The output of the layer is a feature map with ch channels. Layer LO takes
the hidden feature map as input and performs a strided convolution which results in the output feature map with reduced dimensions and
number of channels co. Global average pooling is performed per channel and additional scaling and a final activation are applied. Figure 9. Simplified illustration of a convolutional neural network. An input signal, for example, an image or spectrogram, with a given
number of channels ci is processed by a convolutional layer LH. The output of the layer is a feature map with ch channels. 5.1
Convolutional neural networks Layer LO takes
the hidden feature map as input and performs a strided convolution which results in the output feature map with reduced dimensions and
number of channels co. Global average pooling is performed per channel and additional scaling and a final activation are applied. Table 1. Layout of the proposed non-geophysical event classifier,
consisting of multiple layers which are executed in sequential order. The convolutional neural network consists of multiple 2-D convolu-
tional layers (Conv2D) with batch normalization (BatchNorm) and
rectified linear unit (ReLU) activation. Dropout layers are used to
minimize overfitting. The sequence of global average pooling layer,
a scaling layer and the sigmoid activation compute one value be-
tween 0 and 1 resembling the probability of a detected mountaineer. nents can be interpreted as 2-D signal with three channels,
which is the network input. The network consists of multiple
convolutional, batch normalization and dropout layers, as de-
picted in Table 1. Except for the first convolutional layer, all
convolutional layers are followed by batch normalization and
rectified linear unit (ReLU) activation. Finally, a set of global
average pooling layer, dropout, trainable scaling (in the form
of a convolutional layer with kernel size 1) and sigmoid ac-
tivation reduces the features to one value representing the
probability that a mountaineer is in the microseismic signal. In total, the network has 30 243 parameters. In this architec-
ture, multiple measures have been taken to minimize over-
fitting: the network is all-convolutional (Springenberg et al.,
2014), batch normalization (Ioffe and Szegedy, 2015) and
dropout (Srivastava et al., 2014) are used and the size of the
network is small compared to recent audio classification net-
works (Hershey et al., 2016). Layer
Stride
Output
channels
Conv2D + BatchNorm + Linear
1
32
Conv2D + BatchNorm + ReLU
2
32
Dropout
–
32
Conv2D + BatchNorm + ReLU
2
32
Dropout
–
32
Conv2D + BatchNorm + ReLU
1
32
Conv2D + ReLU
1
32
Dropout
–
32
Conv2D + ReLU
1
1
Global average pooling
1
1
Dropout
–
1
Conv2D
1
1
Sigmoid activation
–
1 5.3
Training and testing We will evaluate the microseismic algorithms in two scenar-
ios in Sect. 7.1. In this section, we describe the training and
test setup for the two scenarios as well as for image and en-
semble classification. In the first scenario, event-linked seg-
ments are classified. In the second scenario, the classifiers on
image-linked segments are compared. The second scenario
stems from the fact that the characterization from Sect. 4.1
suggested that using a longer temporal input window could
lead to a better classification because it can capture more
characteristics of a mountaineer. Training is performed with
the annotated subset from Sect. 4, and a random 10 % of the
training set are used as the validation set, which is never used
during training. For the non-geophysical and seismic event
classifiers, the number of epochs has been fixed to 100 and
for the image classifier to 20. After each epoch, the F1 score of the validation set is calculated, and based on it, the best
performing network version is selected. The F1 score is de-
fined as F1 score =
2 · true positive
2 · true positive + false negative + false positive. Earth Surf. Dynam., 7, 171–190, 2019 5.3.3
Image classification Since convolutional neural networks are a predominant tech-
nique for image classification, a variety of network archi-
tectures have been developed. For this study, the MobileNet
(Howard et al., 2017) architecture is used. The number of
labeled images is small in comparison to the network size
(approximately 3.2 million parameters) and training the net-
work on the Matterhorn images will lead to overfitting on the
small dataset. To reduce overfitting, a MobileNet implemen-
tation which has been pre-trained on ImageNet (Deng et al.,
2009), a large-scale image dataset, will be used. Retraining
is required since ImageNet has a different application fo-
cus than this study. The climbing route, containing the sub-
ject of interest, only covers a tiny fraction of the image, and
rescaling the image to 224 × 224 pixels, the input size of the
MobileNet, would lead to vanishing mountaineers (compare
Fig. 3). However, the image size cannot be chosen arbitrarily
large since a larger input requires more memory and results
in a larger runtime. To overcome this problem, the image has
been scaled to 448 × 672 pixels, and although the input size
differs from the pre-trained version, network retraining still
benefits from pre-trained weights. Data augmentation is used
to minimize overfitting. For data augmentation, each image
is slightly zoomed in and shifted in width and height. The
network has been trained to detect five different categories. In this paper, only the metrics for mountaineers are of inter-
est for the evaluation and the metrics for the other labels are
discarded in the following. However, all categories are rel-
evant for a successful training of the mountaineer classifier. These categories consist of “mountaineer”, “low visibility”
(if the lens is partially obscured), “lens flare”, “snowy” (if
the seismometer is covered in snow) and “bad weather” (as
far as it can be deduced from the image). It is common to do multiple iterations of training and test-
ing to get the best-performing classifier instance. We per-
form a preliminary parameter search to estimate the number
of iterations. The estimation takes into account the number
of training types (10 different classifiers need to be trained)
given the limited processing capabilities. As a result of the
search, we train and test 10 iterations and select the best clas-
sifier instance of each classifier type to evaluate and compare
their performance in Sect. 7. 5.3.3
Image classification The input of the microseismic classifiers must be variable
to be able to perform classification on event-linked segments
and image-linked segments. Due to the principle of convolu-
tional layers, the CNN architectures are independent of the
input size, and therefore no architectural changes have to be
performed. The footstep detector’s input features are aver-
aged over time by design and are thus also time invariant. 5.3.2
Image-linked segments experiment In the second microseismic experiment, the image-linked
segments will be used. Each classifier is trained and evalu-
ated on the image-linked segments. The training parameters
for training the classifiers on image-linked segments are as
before but additionally data augmentation is used to mini-
mize overfitting. Data augmentation includes random circu-
lar shift and random cropping on the time axis. Moreover, to
account for the uneven distribution in the dataset, it is made
sure that during training the convolutional neural networks
see one example of a mountaineer every batch. The learning
rate is set to 0.0001, which was determined with a prelimi-
nary parameter search. The classifiers are then evaluated on
the image-linked segments. 2 · true positive 2 · true positive + false negative + false positive. The threshold for the network’s output is determined by run-
ning a parameter search with the validation set’s F1 score as
the metric. Training was performed in batches of 32 sam-
ples with the ADAM (Kingma and Ba, 2014) optimizer and
cross-entropy loss. The Keras (Chollet, 2015) framework Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 181 with a TensorFlow backend (Abadi et al., 2015) was used
to implement and train the network. The authors of the seis-
mic event classifier (Perol et al., 2018) provide TensorFlow
source code, but to keep the training procedure the same, it
was re-implemented with the Keras framework. The footstep
detector is trained with scikit-learn (Pedregosa et al., 2011). Testing is performed on the test set which is independent of
the training set and has not been used during training. The
metric error rate and F1 score are calculated. metrics can be calculated with the following assumption: if
any of the event-linked segments which are overlapping with
an image-linked segment are classified as mountaineer, the
image-linked segment is considered to be classified as moun-
taineer as well. The results will be discussed in Sect. 7.1. 5.3.1
Event-linked segments experiment Literature suggests that STA/LTA cannot distinguish geo-
physical sources from non-geophysical sources (Allen,
1978). Therefore, the first microseismic experiment investi-
gates if the presented algorithms can distinguish events in-
duced by mountaineers from other events in the signal. The
event-linked segments are used for training and evaluation. The results will be discussed in Sect. 7.1. www.earth-surf-dynam.net/7/171/2019/ 6
Automatic classification 0.0952 on image-linked segments. Both convolutional neural
networks score a lower error rate on image-linked segments
of 0.0096 (MicroseismicCNN) and 0.0313 (seismic event
classifier). For the given dataset, our proposed Microseis-
micCNN network outperforms the seismic event classifier
in both the event-linked segment experiment as well as the
image-linked segment experiment. The MicroseismicCNN
using a longer input window (trained on image-linked seg-
ments) is comparable to classification on images and outper-
forms the classifier trained on event-linked segments. When
combining image and microseismic classifiers, the best re-
sults can be achieved. In Sect. 7.1, it will be shown that the best set of classifiers
is the ensemble of image classifier and MicroseismicCNN. Therefore, the trained image classifiers and Microseismic-
CNN classifier are used to annotate the whole time period
of collected data to quantitatively assess the impact of moun-
taineers. The image classifier and the MicroseismicCNN will
be used to classify all the images and microseismic data, re-
spectively. The consecutive segments and images are used
for prediction. To avoid false positives, we assume that a
mountaineer requires a certain amount of time to pass by the
seismometer as illustrated in Fig. 7; therefore, a mountaineer
annotation is only considered valid if its minimum distance
to the next (or previous) mountaineer annotation is less than
5 min. Subsequently, events within time periods classified as
mountaineers are removed and the event count per hour is
calculated. The number of training/test iterations that were run for
each classifier has been set to 10 through a preliminary pa-
rameter estimation. To validate our choice, we have evalu-
ated the influences of the number of experiments for only
one classifier. The performance of the classifier is expected
to depend on the number of training/test iterations (more iter-
ations indicate a better chance of selecting the best classifier). However, the computing time is increasing linearly with in-
creasing number of iterations. Hence, a reasonable trade-off
between the performance of the classifier and the comput-
ing time is desired to identify the ideal number of iterations. Figure 11 represents the statistical distribution of the clas-
sifier’s performance for the different number of training/test
iterations. Each box plot is based on 10 independent sets of
training/test iterations. 5.3.4
Ensemble In certain cases, a sensor cannot identify a mountaineer al-
though there is one; for example, the seismometers cannot
detect when the mountaineer is not moving or the camera
does not capture the mountaineer if the visibility is low. The usage of multiple classifiers can be beneficial in these
cases. In our case, microseismic and image classifiers will be
jointly used for mountaineer prediction. Since microseismic
labels and image labels are slightly different, as discussed in
Sect. 4.2, the ground truth labels must be combined. For a
given category, a sample is labeled true if any of microseis-
mic or image labels are true (logical disjunction). After indi- In certain cases, a sensor cannot identify a mountaineer al-
though there is one; for example, the seismometers cannot
detect when the mountaineer is not moving or the camera
does not capture the mountaineer if the visibility is low. The usage of multiple classifiers can be beneficial in these
cases. In our case, microseismic and image classifiers will be
jointly used for mountaineer prediction. Since microseismic
labels and image labels are slightly different, as discussed in
Sect. 4.2, the ground truth labels must be combined. For a
given category, a sample is labeled true if any of microseis-
mic or image labels are true (logical disjunction). After indi- To be able to compare the results of the classifiers trained
on image-linked segments to the classifiers trained on event-
linked segments (Sect. 5.3.1), the classifiers from Sect. 5.3.1
will be evaluated on the image-linked segments as well. The www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 182 Table 2. Results of the different classifiers. The addition of
“(events)” labels the classifier versions trained on event-linked seg-
ments vidual prediction by each classifier, the outputs of the clas-
sifiers are combined similarly and can be compared to the
ground truth. Error rate
F1 score
Event-linked segments
Footstep detector (events)
0.1702
0.7692
Seismic event classifier (events)
0.1250
0.8291
MicroseismicCNN (events)
0.0641
0.9062
Image-linked segments
Footstep detector (events)
0.0706
0.5389
Seismic event classifier (events)
0.0540
0.6047
MicroseismicCNN (events)
0.0309
0.731
Footstep detector
0.0952
0.52
Seismic event classifier
0.0313
0.7383
MicroseismicCNN
0.0096
0.9167
Image classifier
0.0088
0.9134
Ensemble
0.0079
0.9383 6
Automatic classification While the box indicates the interquar-
tile range (IQR) with the median value in orange, the whisker
on the appropriate side is taken to 1.5 × IQR from the quar-
tile instead of the maximum or minimum if either type of
outlier is present. Beyond the whiskers, data are considered
outliers and are plotted as individual points. As can be seen 7
Evaluation In the following, the results of the different classifier exper-
iments described in Sect. 5.3 will be presented to determine
the best set of classifiers. Furthermore, in Sect. 7.2 and 7.3,
results of the automatic annotation process (Sect. 6) will used
to evaluate the impact of external influences on the whole
dataset. 7.1
Classifier evaluation 5.4
Optimization Due to potential human errors during data labeling, the train-
ing set has to be regarded as a weakly labeled dataset. Such
datasets can lead to a worse classifier performance. To over-
come this issue, a human-in-the-loop approach is followed,
where a preliminary set of classifiers is trained on the training
set. In the next step, each sample of the dataset is automat-
ically classified. This procedure produces a number of true
positives, false positives and true negatives. These samples
are then manually relabeled and the labels for the dataset are
updated based on human review. The procedure is repeated
multiple times. However, this does not completely avoid the
possibility of falsely labeled samples in the dataset, since the
algorithm might not find all human-labeled false negatives,
but it increases the accuracy significantly. The impact of false
labels on classifier performance will be evaluated in Sect. 7.1. Earth Surf. Dynam., 7, 171–190, 2019 7.1
Classifier evaluation The results of the classifier experiments (Table 2) show that
the footstep detector is the worst at classifying mountaineers,
with an error rate of 0.1702 on event-linked segments and www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 183 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings
183
Figure 10. Event count, hut occupancy and rock temperature over time: (a) for the years 2016 and 2017 and (b) for a selected period during
defreezing of the rock. The event rate from Weber et al. (2018b) is illustrated in light blue and the rate after removal of mountaineer-induced
events in dark blue. The strong variations in event rates correlate with the presence of mountaineers and hut occupancy and in panel (b) with
the total net radiation. The impact of mountaineers is significant after 9 July and event detection analysis becomes unreliable. in Fig 11 the F1 score saturates at nine iterations Therefore
ence on the classification performance since the mean per Figure 10. Event count, hut occupancy and rock temperature over time: (a) for the years 2016 and 2017 and (b) for a selected period duri
defreezing of the rock. The event rate from Weber et al. (2018b) is illustrated in light blue and the rate after removal of mountaineer-induc
events in dark blue. The strong variations in event rates correlate with the presence of mountaineers and hut occupancy and in panel (b) wi
the total net radiation. The impact of mountaineers is significant after 9 July and event detection analysis becomes unreliable. Figure 10. Event count, hut occupancy and rock temperature over time: (a) for the years 2016 and 2017 and (b) for a selected period during
defreezing of the rock. The event rate from Weber et al. (2018b) is illustrated in light blue and the rate after removal of mountaineer-induced
events in dark blue. The strong variations in event rates correlate with the presence of mountaineers and hut occupancy and in panel (b) with
the total net radiation. The impact of mountaineers is significant after 9 July and event detection analysis becomes unreliable. ence on the classification performance since the mean per-
formance is worse for a high percentage of false labels. in Fig. 11, the F1 score saturates at nine iterations. Therefore,
our choice of 10 iterations is a reasonable choice. 7.2
Statistical evaluation 7.2
Statistical evaluation The annotated test set from Sect. 4.2 and the automatically
annotated set from Sect. 6 are used for a statistical evaluation
involving the impact of external influences on microseismic
events. Only data from 2017 are chosen, since wind data are
not available for the whole training set due to a malfunction
of the weather station in 2016. The experiment from Weber
et al. (2018b) provides STA/LTA event triggers for 2017. Ta-
ble 4 shows statistics for several categories, which are three
external influences and one category where none of the three
external influences are annotated (declared as “unknown”). For each category, the total duration of all annotated seg-
ments is given and how many events per hour are triggered. It
becomes apparent that mountaineers have the biggest impact
on the event analysis. Up to 105.9 events per hour are de-
tected on average during time periods with mountaineer ac-
tivity, while during all other time periods the average ranges
from 9.09 to 13.12 events per hour. This finding supports our
choice to mainly focus on mountaineers in this paper and
shows that mountaineers have a strong impact on the analy-
sis. As a consequence, a high activity detected by the event
trigger does not correspond to a high seismic activity; thus,
relying only on this kind of event detection may lead to a
false interpretation. From the automatic section in Table 4, it Figure 10b focuses on the defreezing period. The zero
crossing of the rock temperature has a significant impact on
the event count variability. A daily pattern becomes visible
starting around the zero crossing. Since few mountaineers are
detected in May, these can be discarded as the main influence
for these patterns. The total net radiation, however, indicates
an influence of solar radiation on the event count. Further in-
depth analysis is needed but this example shows the benefits
of a domain-specific analysis, since the additional informa-
tion gives an intuitive description of relevant processes and
their interdependencies. After 9 July, the impact of moun-
taineers is significant and the event detection analysis be-
comes unreliable. Different evaluation methods are required
to mitigate the influence of mountaineers during these peri-
ods. Figure 13 depicts that mountaineer predictions and hut oc-
cupancy correlate well, which indicates that the classifiers
work well. The discrepancy in the first period of each sum-
mer needs further investigation. 7.1
Classifier evaluation In Sect. 5.4, the possibility of falsely labeled training sam-
ples has been discussed. As expected, our evaluation in Ta-
ble 3 indicates that falsely labeled samples have an influ- Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 184 can be deduced that the average number of triggered events
per hour for times when the signal is influenced by moun-
taineers increases by approximately 9 times in comparison to
periods without annotated external influences. The effect of
wind influences on event rate is not as clear as the influence
of mountaineers. The values in Table 4 indicate a decrease
of events per hour during wind periods, which will be briefly
discussed in Sect. 8.2. As can be seen in Fig. 12, events are triggered over the
course of the whole year, whereas events that are annotated as
coming from mountaineers occur mainly during the summer
period. The main increase in event count occurs during the
period when the rock is unfrozen, which unfortunately coin-
cides with the period of mountaineer activity. Therefore, it
is important to account for the mountaineers. However, even
if the mountaineers are not considered, the event count in-
creases significantly during the unfrozen period. The inter-
pretation of these results will not be part of this study but
they are an interesting topic for further research. Figure 11. The statistical distribution of the classifier’s perfor-
mance for the different number of training/test iterations is illus-
trated. Each box plot is based on 10 independent sets of training/test
iterations. The F1 score saturates after 9 iterations and validates our
choice of 10 iterations. 7.3
Automatic annotation in a real-world scenario Table 3. Influence of falsely labeled data points on the test perfor-
mance. Shown are the mean values over 10 training/test iterations. The result of applying the ensemble classifier to the whole
dataset is visualized for two time periods in Fig. 10. The
figure depicts the event count per hour before and after re-
moving periods of mountaineer activity, as well as the rock
temperature, the overnight stays at the Hörnlihütte and the
total net radiation. From Fig. 10a, it becomes apparent that
the mountaineer activity is mainly present during summer
and autumn. An increase is also visible during increasing hut
overnight stays. During winter and spring, only few moun-
taineers are detected but some activity peaks remain. By
manual review, we were able to discard mountaineers as
cause for most of these peaks; however, further investigation
is needed to explain their occurrence. False labels (%)
25
12.5
6.25
3.125
0
F1 score (mean)
0.7953
0.8633
0.8761
0.8835
0.8911
Error rate (mean)
0.0208
0.0149
0.0139
0.0129
0.0122 Earth Surf. Dynam., 7, 171–190, 2019 7.2
Statistical evaluation With the annotations for the
whole time span, it can be estimated that from all events de- Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ r et al.: Systematic identification of external influences in multi-year microseismic recordings 185 Table 4. Statistics of the manually and automatically annotated sets of 2017 per annotation category. “Unknown” represents the category
when none of the other categories could have been identified. Given are the total duration of annotated segments per category and the mean
number of STA/LTA events per category. number of STA/LTA events per category. Unknown
Mountaineer
Wind
Helicopter
Manual
Duration (hours)
28.87
1.9
6.6
3.73
Mean number of events per hour
10.6
95.26
11.21
13.12
Automatic
Duration (hours)
6832.3
296.53
1364.2
–
Mean number of events per hour
11.76
105.9
9.09
–
Figure 12. Illustration of the cumulative number of events triggered by the STA/LTA event detector for all events, for events triggered by
mountaineers and for events triggered by unknown sources. The results presented in this paper were used to annotate the events. The time
period during which the rock temperature in 1 m depth is above 0 ◦C is shaded in gray. Figure 12. Illustration of the cumulative number of events triggered by the STA/LTA event detector for all events, for events triggered by
mountaineers and for events triggered by unknown sources. The results presented in this paper were used to annotate the events. The time
period during which the rock temperature in 1 m depth is above 0 ◦C is shaded in gray. notating the external influences (negative examples). Posi-
tive examples, used in Yuan et al. (2018), Ruano et al. (2014)
and Kislov and Gravirov (2017), inherently contain a limita-
tion as this approach requires that events as well as influenc-
ing factors must be identified and identifiable in the signal
of concern. This is especially hard where no ground truth
information except (limited) experience by professionals is
available. Therefore, the strategy presented in this work to
create an annotated dataset using negative examples is ad-
visable to be used. It offers to perform cross-checks if cer-
tain patterns can be found in different sensor/data types, and
in many cases the annotation process can be performed by
non-experts. Also, additional sensors allow to directly quan-
tify possible influence factors. The detour required by first
classifying negative examples and then analyzing the phe- tected in Weber et al. 8
Discussion 8.1
Classification of negative examples Earth Surf. Dynam., 7, 171–190, 2019 7.2
Statistical evaluation (2018b), approximately 25 % originate
in time periods with mountaineer activity and should there-
fore not be regarded as originating from geophysical sources. 8
Discussion 8
Discussion 8.1
Classification of negative examples The previous section has shown that a certain degree of un-
derstanding of the scenario and data collected is nevertheless
necessary in order to achieve a significant analysis. The effort
in creating an annotated data subset, despite being time and
labor consuming, is an overhead but as we show can be out-
weighed by the benefits of better analysis results. For data
annotation, two distinct approaches can be followed: anno-
tating the phenomena of interest (positive examples) or an- Earth Surf. Dynam., 7, 171–190, 2019 www.earth-surf-dynam.net/7/171/2019/ M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 186 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings Figure 13. Correlation of mountaineer activity and hut occupancy. The normalized number of mountaineer segments per week and the
normalized number of overnight stays at the Hörnlihütte per week is plotted over time. Figure 13. Correlation of mountaineer activity and hut occupancy. The normalized number of mountaine
normalized number of overnight stays at the Hörnlihütte per week is plotted over time. Figure 13. Correlation of mountaineer activity and hut occupancy. The normalized number of mountaineer segments per week and the
normalized number of overnight stays at the Hörnlihütte per week is plotted over time. sifier to increase the overall accuracy. The different modali-
ties strengthen the overall meaningfulness and make the clas-
sifier more robust. Table 4 shows that during windy seg-
ments less events are triggered than in periods that cannot be
categorized (“unknown” category). A possible explanation
is that the microseismic activity is superimposed by broad-
band noise originating in the wind. For these time segments,
a variable trigger sensitivity (Walter et al., 2008) or a dif-
ferent event detection algorithm can improve the analysis. Better shielding the seismometer from the wind would re-
duce these influences significantly but the typical approach
in seismology to embed it into the ground under a substan-
tial soil column is next to impossible to implement in steep
bedrock and perennially frozen ground as found on our case-
study field site. Nonetheless, Table 4 gives an intuitive feel-
ing that our method performs well since the statistical dis-
tribution of manually and automatically annotated influences
sources is similar. We therefore conclude that with our pre-
sented method it is possible to quantify the impact of external
influences on a long-term scale and across variable condi-
tions. 8.2
Multi-sensor classification In Sect. 5, multiple classifiers for different sensors have been
presented. The advantages of classifying microseismic sig-
nals are that continuous detection is possible and that no
additional sensors are required. The classification accuracy
of the convolutional neural network and the image classi-
fier presented are comparable. Classification of time-lapse
images, however, has the disadvantage of a low time res-
olution proportional to the capture frequency, for example,
a maximum of 15 images per hour in our example. Con-
tinuous video recording could close this gap at the cost of
requiring a more complex image classifier, the size of the
data and higher processing times, which are likely infeasi-
ble. The main advantage of images is that they can be used
as additional independent sensors to augment and verify mi-
croseismic recordings. First, images can be used for anno-
tations, and second, they can be used in an ensemble clas- 8.1
Classification of negative examples nomena of interest offers further benefits. In cases where the
characteristics of the phenomena of interest are not known
in advance (no ground truth available) and in cases where a
novel analysis method is to be applied or when treating very-
long-term monitoring datasets, working only on the primary
signal of concern is hard and error margins are likely to be
large. In these cases, it is important to take into account all
knowledge available including possible negative examples,
and it is significant to automate as much as possible using
automatic classification methods. Earth Surf. Dynam., 7, 171–190, 2019 8.3
Feature extraction In Sect. 4.1, the different characteristics of event sources
have been discussed. The characteristic features can be used
to identify and classify each source type. The convolutional
neural network accomplishes the task of feature extraction
and classification simultaneously by training on an extensive www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 187 annotated dataset. An approach without the requirement of
an annotated dataset would be to manually identify the char-
acteristics and then design a suitable algorithm to extract the
features. For example, the helicopter pattern in Fig. 5c shows
distinct energy bands indicating the presence of a fundamen-
tal frequency plus the harmonics. These features could be
traced to identify the model and possibly localize a helicopter
(Eibl et al., 2017) with the advantage of a relaxed dataset
requirement. The disadvantage would be the requirement of
further expertise in the broad field of digital signal processing
and modeling as well as more detailed knowledge on each
such phenomena class of interest. Also, it is likely that such
an approach would require extensive sensitivity analysis to
be performed alongside modeling. Moreover, if the algorithm
is handcrafted by using few examples, it is prone to overfit-
ting based on these examples (see also the next subsection). This problem of overfitting exists as well for algorithm train-
ing and can be solved by using more examples; however, it is
easier to annotate a given pattern (with the help of additional
information) than understanding its characteristics, and thus
the time- and labor-consuming task of annotation can be out-
sourced in the case of machine learning. Figure 5 indicates
that little anthropogenic noise (Fig. 5a) has less broadband
background noise than wind (Fig. 5d) and the impulses oc-
cur in a different frequency band. However, the signal plots
show a similar pattern. To identify wind from microseismic
data manually, one could utilize a frequency-selective event
detector, although it is not clear if this pattern and frequency
range are representative of every occurrence of wind and if
all non-wind events could be excluded with such a detector. 8.3
Feature extraction Using a dedicated wind sensor for identification of wind peri-
ods as presented in this study overcomes these issues with the
drawback of an additional sensor which needs to be installed
and maintained, and that during failure of the additional sen-
sor no annotation can be performed. specific location, type or frequency response. This will be an
important study for the future since it will reduce the dataset
collection and training time significantly if a new seismome-
ter is deployed. 9
Conclusions In this paper, we have presented a strategy to evaluate the im-
pact of external influences on a microseismic measurement
by categorizing the data with the help of additional sensors
and information. With this knowledge, a method to classify
mountaineers has been presented. We have shown how ad-
ditional sensors can be beneficial to isolate the information
of interest from unwanted external influences and provide a
ground truth in a long-term monitoring setup. Moreover, we
have presented a mountaineer detector, implemented with a
convolutional neural network, which scores an error rate of
only 0.96 % (F1 score: 0.9167) on microseismic signals and a
mountaineer detector ensemble which scores an error rate of
0.79 % (F1 score: 0.9383) on images and microseismic data. The classifiers outperform comparable algorithms. Their ap-
plication to a real-world, multi-sensor, multi-year microseis-
mic monitoring experiment showed that time periods with
mountaineer activity have an approximately 9 times higher
event rate and that approximately 25 % of all detected events
are due to mountaineer interference. Finally, the findings of
this paper show that an extensive, systematic identification of
external influences is required for a quantitative and qualita-
tive analysis on long-term monitoring experiments. 8.5
Outlook This work has only focused on identifying external influ-
ences, what we have shown to be a prerequisite for micro-
seismic analysis. Future work lies in finding and applying
specific analytic methods, especially finding good parame-
ter sets and algorithms for each context. Additionally, the
classifier could be extended to include helicopters as well as
geophysical sources such as rockfalls. A disadvantage of the
present method is the requirement of a labeled dataset. Semi-
supervised or unsupervised methods (Kuyuk et al., 2011) as
well as one- or few-shot classification methods (Fei-Fei et al.,
2006) could provide an alternative to the presented training
concept without the requirement of a large annotated dataset. Competing interests. The authors declare that they have no con-
flict of interest. Competing interests. The authors declare that they have no con-
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the X-Sense 2 project. It was financed by Nano-Tera.CH (NTCH)
(ref. no. 530659). We would like to thank Tonio Gsell and the rest of
the PermaSense team for technical support. We acknowledgement
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Air Zermatt helicopter company for providing us with helicopter
flight data. We thank Lukas Cavigelli for insightful discussions. Reviews from Marine Denolle and two anonymous referees
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due to too few data examples. Instead of learning representa-
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cept. MM and SW developed the code and maintained field site and
data together with JB. MM prepared and performed the experiments www.earth-surf-dynam.net/7/171/2019/ Earth Surf. Dynam., 7, 171–190, 2019 Earth Surf. Dynam., 7, 171–190, 2019 M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings M. Meyer et al.: Systematic identification of external influences in multi-year microseismic recordings 188 and evaluated the results with SW. MM prepared the manuscript as
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Reduction in the population prevalence of hepatitis C virus viraemia among people who inject drugs associated with scale‐up of direct‐acting anti‐viral therapy in community drug services: real‐world data
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Citation for published version (APA):
Palmateer, N. E., McAuley, A., Dillon, J. F., McDonald, S., Yeung, A., Smith, S., Barclay, S., Hayes, P.,
Shepherd, S. J., Gunson, R. N., Goldberg, D. J., Hickman, M., & Hutchinson, S. J. (2021). Reduction in the
population prevalence of hepatitis C virus viraemia among people who inject drugs associated with scale-up of
direct-acting anti-viral therapy in community drug services: REAL WORLD DATA. Addiction, 116(10), 2893-
2907. https://doi.org/10.1111/add.15459 University of Dundee
Reduction in the population prevalence of hepatitis C virus viraemia among people
who inject drugs associated with scale-up of direct-acting anti-viral therapy in
community drug services
Palmateer, Norah E.; McAuley, Andrew; Dillon, John F.; McDonald, Scott; Yeung, Alan;
Smith, Shanley
Published in:
Addiction University of Dundee Document Version
Peer reviewed version Link to publication in Discovery Research Portal Citation for published version (APA):
Palmateer, N. E., McAuley, A., Dillon, J. F., McDonald, S., Yeung, A., Smith, S., Barclay, S., Hayes, P.,
Shepherd, S. J., Gunson, R. N., Goldberg, D. J., Hickman, M., & Hutchinson, S. J. (2021). Reduction in the
population prevalence of hepatitis C virus viraemia among people who inject drugs associated with scale-up of
direct-acting anti-viral therapy in community drug services: REAL WORLD DATA. Addiction, 116(10), 2893-
2907. https://doi.org/10.1111/add.15459 General rights
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and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Palmateer Norah (Orcid ID: 0000-0003-4493-0173)
Yeung Alan (Orcid ID: 0000-0001-5226-3695)
Hickman Matthew (Orcid ID: 0000-0001-9864-459X) Palmateer Norah (Orcid ID: 0000-0003-4493-0173)
Yeung Alan (Orcid ID: 0000-0001-5226-3695)
Hickman Matthew (Orcid ID: 0000-0001-9864-459X) Palmateer Norah (Orcid ID: 0000-0003-4493-0173)
Yeung Alan (Orcid ID: 0000-0001-5226-3695)
Hickman Matthew (Orcid ID: 0000-0001-9864-459X) REDUCTION IN THE POPULATION PREVALENCE OF HCV VIRAEMIA AMONG PEOPLE WHO INJECT
DRUGS ASSOCIATED WITH SCALE-UP OF DIRECT-ACTING ANTIVIRAL THERAPY IN COMMUNITY
DRUG SERVICES: REAL WORLD DATA Authors: Norah E Palmateer1,2, Andrew McAuley1,2, John F Dillon3, Scott McDonald1, Alan Yeung1,2,
Shanley Smith1,2, Stephen Barclay1,4, Peter Hayes5, Samantha J Shepherd6, Rory N Gunson6, David J
Goldberg2,1, Matthew Hickman7, Sharon J Hutchinson1,2 1Glasgow Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, UK
2Public Health Scotland, 5 Cadogan Street, Glasgow, G2 6QE, UK
3University of Dundee, Nethergate, Dundee, DD1 4HN, UK
4Glasgow Royal Infirmary, 84 Castle Street, Glasgow, G4 0SF, UK
5Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh, EH16 4SA, UK
6West of Scotland Specialist Virology Centre, 8-16 Alexandra Parade, G31 2ER, Glasgow, UK
7University of Bristol, Tyndall Avenue, Bristol, BS8 1TH, UK 1Glasgow Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, UK
2Public Health Scotland, 5 Cadogan Street, Glasgow, G2 6QE, UK
3University of Dundee, Nethergate, Dundee, DD1 4HN, UK
4Glasgow Royal Infirmary, 84 Castle Street, Glasgow, G4 0SF, UK
5Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh, EH16 4SA, UK
6West of Scotland Specialist Virology Centre, 8-16 Alexandra Parade, G31 2ER, Glasgow, UK
7University of Bristol, Tyndall Avenue, Bristol, BS8 1TH, UK Declarations of competing interests SJH has received honoraria from Gilead, outside of the submitted work. JFD declares research grants,
in part supporting the scale-up of HCV treatment in Tayside, from AbbVie, Bristol-Myers Squibb,
Gilead, Janssen, Merck Sharpe & Dohme, and Roche and other research grants and honoraria for
lectures form AbbVie, Abbott, Bristol-Myers Squibb, Gilead, Janssen, Merck Sharpe & Dohme and
Roche, outside of the submitted work. PH has spoken or been on advisory boards for AbbVie, BMS,
Eisai Ltd, Falk, Ferring, Gilead, Gore, Janssen, Lundbeck, MSD, Norgine, Novartis, ONO
Pharmaceuticals, Pfizer and Roche. All other authors declare no competing interests. Corresponding author: Norah Palmateer; Email: norah.palmateer@nhs.net; Tel: (44)7979004363 Running head: HCV among people who inject drugs ABSTRACT Background and aims: There has been little empirical evidence to show the ‘real world’ impact of
scaling-up direct-acting antiviral (DAA) treatment among people who inject drugs (PWID) on
hepatitis C virus (HCV) viraemia at a population level. We aimed to assess the population impact of
rapid DAA scale-up to PWID delivered through community services – including drug treatment,
pharmacies, needle exchanges, and prisons – in the Tayside region of Scotland, compared with
Greater Glasgow & Clyde (GGC) and the Rest of Scotland (RoS). Background and aims: There has been little empirical evidence to show the ‘real world’ impact of
scaling-up direct-acting antiviral (DAA) treatment among people who inject drugs (PWID) on
hepatitis C virus (HCV) viraemia at a population level. We aimed to assess the population impact of
rapid DAA scale-up to PWID delivered through community services – including drug treatment,
pharmacies, needle exchanges, and prisons – in the Tayside region of Scotland, compared with
Greater Glasgow & Clyde (GGC) and the Rest of Scotland (RoS). Design: Natural experiment, evaluated using data from national biennial surveys of PWID and
national clinical data. Setting: Services providing injecting equipment (2010-2018) and HCV treatment clinics (2017-2018)
across Scotland. Participants: 12,492 PWID who completed a questionnaire and provided a blood spot (tested for
HCV-antibodies and RNA); 4,105 individuals who initiated HCV treatment. Intervention and comparator: The intervention was rapid DAA scale-up among PWID, which
occurred in Tayside. The comparator was GGC/RoS. Measurements: Trends in HCV viraemia and uptake of HCV therapy over time; sustained viral
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Findings: Uptake of HCV therapy (last year) among PWID between 2013-14 and 2017-18 increased
from 15% to 43% in Tayside, 6% to 16% in GGC and 11% to 23% in RoS. Between 2010 and 2017-18,
the prevalence of HCV viraemia (among antibody-positives) declined from 73% to 44% in Tayside,
67% to 58% in GGC and 64% to 55% in RoS. The decline in viraemia was greater in Tayside (2017-18
AOR 0·47, 95% CI 0·30-0·75, p=0.001) than elsewhere in Scotland (2017-18 AOR 0·89, 95% CI 0·74-
1·07, p=0.220) relative to the baseline of 2013-14 in RoS (including GGC). Per-protocol SVR rates
among PWID treated in community sites did not differ from those treated in hospital sites in Tayside
(97·4% vs 100·0%, p=0·099). Manuscript word count: 3,716 This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/add.15459 This article is protected by copyright. All rights reserved. ABSTRACT Conclusions: Scale-up of direct-acting antiviral treatment among people who inject drugs can be
achieved through hepatitis C virus (HCV) testing and treatment in community drug services whilst
maintaining high sustained viral response rates and, in the Tayside region of Scotland, has led to a
substantial reduction in chronic HCV in the population. INTRODUCTION In many countries, people who inject drugs (PWID) comprise the largest population of people
infected with hepatitis C virus (HCV) as well as the largest group at ongoing risk of infection.1 In
recognition of viral hepatitis as a global problem, the World Health Organization (WHO) has set
targets to eliminate HCV, including a 80% reduction in HCV incidence by 2030. 2 To achieve this
target, countries will need to provide optimal coverage of harm reduction services (including needle
and syringe programmes and opioid agonist therapy), but will also need to prioritise major scale-up
(i.e. expansion) of HCV treatment for PWID.3–6 Uptake of HCV therapy among PWID has been very
limited until the recent introduction of direct-acting antiviral therapies (DAAs) – highly effective,
tolerable and simple-to-administer therapies for the treatment of HCV infection.7,8,9 – Further, there
has been little empirical evidence to show the ‘real world’ impact of scaling-up HCV DAA treatment
among PWID on HCV viraemia at a population level.10,11,12 In the Tayside region of Scotland, an evaluation of the impact of major and rapid scale-up of DAAs
among PWID is underway, involving the provision of HCV testing and treatment across the full range
of community services engaged with this population – including drug treatment, pharmacies, needle
exchanges and prisons.13 The scale-up in Tayside, compared to other areas of Scotland, provides a
unique opportunity to evaluate a natural experiment of the scale-up of DAAs on HCV viraemia
prevalence in the PWID population. Scotland is one of the few countries world-wide with comprehensive national surveillance systems,
including large serial cross-sectional bio-behavioural surveys of PWID to monitor HCV infection and
an HCV clinical database to monitor treatment outcomes.14,15 Using these data, we assess the early
impact of DAA scale-up in Tayside, compared to other regions in Scotland. We aim to test the
hypothesis that rapid scale-up of DAAs in community-based services translates into a decline in HCV
viraemia among a population of PWID, and to test whether this rapid-scale up leads to any
differences in sustained viral response (SVR) rates to therapy. Specifically, we examined: (i) the
population-level prevalence of HCV therapy uptake, HCV viraemia and treatment-induced viral
clearance among PWID over time and by region , (ii) the change in uptake of HCV therapy, HCV
viraemia and treatment-induced viral clearance among PWID over time and by region, and (iii) SVR
rates by region, treatment setting, and PWID status. METHODS The current study forms one component of the “Evaluating the population impact of hepatitis C
direct acting antiviral treatment as prevention for people who inject drugs (EPIToPe)” study, a mixed
methods evaluation of a natural experiment of HCV ‘Treatment as Prevention’ (TasP) among PWID.13
The ‘intervention’ consists of the scale-up of DAAs and expansion of care pathways in National
Health Service (NHS) Tayside (an administrative health region in Scotland), with other sites in
Scotland acting as the ‘controls’: NHS Greater Glasgow & Clyde (GGC) and the Rest of Scotland (RoS). NHS Tayside has an estimated 2,700 PWID, which compares to 10,000 and 17,300 in GGC and RoS,
respectively.13,16,17 Prevalence of antibodies to HCV among PWID in Tayside in 2017-18 was 56%, as
compared to 68% in GGC and 48% in RoS.14 The scale-up in Tayside involves the treatment of at least
500 PWID over a 2-3 year period beginning in 2017; mathematical modelling has indicated that this
has the potential to reduce the chronic HCV prevalence among PWID in Tayside from approximately This article is protected by copyright. All rights reserved. 30% to 10% or less. In the rest of Scotland, financial considerations precluded rapid scale-up of
treatment beyond those eligible via the fibrosis-based prioritisation in place at the time. 30% to 10% or less. In the rest of Scotland, financial considerations precluded rapid scale-up of
treatment beyond those eligible via the fibrosis-based prioritisation in place at the time. The data analysed here relate to a time-point midway through scale-up, when approximately 200
PWID (i.e. 40% of the target) had been treated in community settings in Tayside. This study
therefore evaluates the early impact of the DAA scale-up in Tayside. To address the aims described
in the introduction, two sources of data were utilised, which are described further below and
summarised in Box 1. Needle Exchange Surveillance Initiative (NESI) NESI is a national bio-behavioural survey of PWID that has been undertaken approximately bienially
since 2008-09 and covers an estimated 10-15% of the active PWID population in Scotland.14
Recruitment takes place at sites that provide sterile injecting equipment across Scotland (some of
which may also provide opioid agonist therapy); sites are chosen to be broadly geographically
representative and roughly 100 sites are included per sweep, representing approximately 50% of all
sites that provide injecting equipment in Scotland. Trained interviewers facilitate the completion of a
questionnaire and take a blood spot sample from all consenting participants. Dried blood spots (DBS)
are tested for antibodies to HCV and HCV RNA; methods have been described in detail
previously.14,18 Written informed consent was obtained from each patient included in the study and
the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected
in approval by the West of Scotland Research Ethics Committee (reference 08/S0709/46). As part of EPIToPe, approximately 200 PWID had been treated in community settings in Tayside
during 2017. As part of the NESI 2017-18 sweep, interviews in Tayside were carried out in early
2018. Thus, four sweeps of NESI (2010, 2011-12, 2013-14 and 2015-16) were undertaken prior to,
and one (2017-18) following, the early scale-up of DAAs. This article is protected by copyright. All rights reserved. Clinical database This article is protected by copyright. All rights reserved. g
p
g
inics in Scotland (consisting of clinics in hospitals, as well as nurse-led clinics in prisons and
ommunity settings).15 We extracted data (age, sex assigned at birth, injecting status, treatment
ates, SVR achievement, treatment setting and clinic) on patients who commenced therapy between
Box 1. Summary of data sources
Data source
Needle Exchange Surveillance Initative (NESI)
Hepatitis C Clinical database
Research aim
addressed
(i) To determine the population-level prevalence
of uptake of HCV therapy, HCV viraemia and
treatment-induced clearance among PWID over
time, comparing NHS Tayside with NHS GGC/RoS
(ii) To examine the associations between
region/year and uptake of HCV therapy, HCV
viraemia and treatment-induced clearance among
PWID. To determine SVR rates by region,
treatment setting, and PWID status. Brief
description
Serial cross-sectional bio-behavioural surveys of
PWID recruited at sites that provide sterile
injecting equipment across Scotland
National database holding information
on patients attending 17 out of the 18
HCV treatment clinics in Scotland
(includes hospital, prison and
community settings)
Dates data
collected
Survey sweeps undertaken in 2010, 2011-12,
2013-14, 2015-16, 2017-18
Jan 2017 – Dec 2018 (date of
treatment commencement)
Measurements
HCV viraemia (HCV antibody positive and HCV
RNA positive based on DBS results)
HCV therapy (self-reported and only available
for 2013-14 onwards)
Treatment-induced viral clearance
(combination of DBS result and self-reported HCV
therapy, therefore only available for 2013-14
onwards)
ITT and per-protocol SVR
Comparators
Region: NHS Tayside vs. NHS GGC vs. RoS
Time: survey year
PWID status
Region: NHS Tayside vs. NHS
GGC vs. RoS
Treatment setting: hospital vs. prison vs. community
Analyses
(i)
Proportions of respondents who
reported ever/last year uptake of
therapy, with HCV viraemia
(imputed), and with treatment-
induced viral clearance (imputed) by
region and survey year
(ii)
Multi-level logistic regression to
examine associations between
region/survey year and uptake of
therapy in the last year, HCV
viraemia, and treatment-induced
viral clearance
Proportions of respondents who
achieved SVR by PWID status, region
and treatment setting. This article is protected by copyright. All rights reserved. DBS = dried blood spot; GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; ITT = intention-to-treat; NHS = National Health
Service; PWID = people who inject drugs; RNA = ribonucleic acid; RoS = Rest of Scotland; SVR = sustained viral response clinics in Scotland (consisting of clinics in hospitals, as well as nurse-led clinics in prisons and
community settings).15 We extracted data (age, sex assigned at birth, injecting status, treatment
dates, SVR achievement, treatment setting and clinic) on patients who commenced therapy between
January 2017 and December 2018. Data were complete to 31st March 2019, so patients who This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. commenced treatment at the end of December 2018 had sufficient time to complete treatment and
have a follow-up SVR test. commenced treatment at the end of December 2018 had sufficient time to complete treatment and
have a follow-up SVR test. NESI Based on the results of DBS testing, participants were categorised as HCV antibody positive or
negative. Antibody positives were further classified as viraemic (HCV antibody positive and HCV RNA
positive) or cleared (HCV antibody positive and HCV RNA negative). In the latest three surveys (2013-
14, 2015-16 and 2017-18), participants were asked if they had commenced drug therapy for their
HCV infection ever or in the last year. Participants were further categorised into the groups indicated
in Box 2 by combining their HCV infection status from their DBS test with their self-reported uptake
of therapy. Those with cleared infection who reported receiving HCV therapy in the past (i.e. Group
A in Box 2) were defined as having “treatment-induced viral clearance”. In order to generate appropriate denominators for HCV therapy uptake, we created definitions of
individuals who were ‘eligible for therapy’. The denominator for those who had ever received
therapy consisted of those with viraemia (i.e. Groups B and D in Box 2) plus those who had cleared
infection with evidence of therapy (i.e. Group A). The denominator for therapy uptake in the last
year consisted of those with viraemia (i.e. Groups F and H in Box 2) plus those who had cleared
infection with evidence of therapy in the last year (i.e. Group E). This article is protected by copyright. All rights reserved. Clinical database
A SVR was defined as testing PCR negative for HCV RNA at either 24 weeks (pre-2014) or 12 weeks
(2014 onwards) following completion of therapy, according to clinical guidelines.19 The time period is
Box 2. NESI Classification of respondents into groups based on HCV antibody/RNA status combined with self-
reported history of HCV therapy (ever and in the last year)
HCV antibody positive & HCV RNA
negative (Cleared infection)
HCV antibody positive & HCV RNA
positive (Viraemic infection)
Reported ever receiving
therapy for HCV
Cleared infection through therapy
(Group A)
Viraemic (relating to either failed
therapy or re-infection following
therapy)
(Group B)
Did not report ever receiving
therapy for HCV*
Cleared infection spontaneously
(Group C)
(Group D)
Reported receiving therapy
for HCV in the last year
Cleared infection through recent
therapy (Group E)
Viraemic (relating to either failed
therapy or re-infection following
recent therapy) (Group F)
Did not report receiving
therapy for HCV in the last
year*
Cleared infection spontaneously or
cleared through therapy more than
one year ago
(Group G)
Viraemic (Group H)
*Includes those who said they had not received treatment, as well as those who were unaware of their
HCV infection sification of respondents into groups based on HCV antibody/RNA status combined with self-
istory of HCV therapy (ever and in the last year) This article is protected by copyright. All rights reserved. from the confirmed end of treatment (rather than the expected end of treatment) if a patient
prematurely discontinues. Only patients who had a documented SVR at 24+ weeks (pre-2014) or 12+
weeks (2014 onwards) were considered to have a SVR. Intention-to-treat (ITT) SVR rates were
calculated as the number of patients who achieved SVR as a proportion of the number of patients
who commenced treatment. Per-protocol SVR rates were calculated as the number of patients
achieving SVR as a proportion of the number of patients with an SVR outcome recorded (i.e. excluding unknown results). Statistical analysis To describe the NESI participants, initial analyses looked at demographic and behavioural variables,
by survey year and region: categorical variables were expressed as numbers and percentages
whereas continuous variables were presented as means or medians. To test for differences across
surveys, chi-square tests were performed for categorical variables and Wilk’s lambda was calculated
for continuous variables. The main statistical analyses undertaken are described below under headings that relate to the
three aims described in the introduction. SVR rates by region, treatment setting, and PWID status (clinical database) Where individuals had multiple courses of treatment, the SVR relating to the treatment episode with
the most recent completion date was used in the analysis. ITT and per-protocol SVR rates were
calculated by PWID status, region (Tayside/GGC/RoS), and treatment setting
(hospital/prison/community). Analyses were undertaken in Stata version 13·1.21 Graphs were produced in Excel 2016 and R version
3.5.1.22,23 The analysis was not pre-registered on a publicly available platform and therefore the results should
be considered exploratory. Population-level prevalence of uptake of HCV therapy, HCV viraemia and treatment-induced viral
clearance (NESI data) Population-level prevalence of uptake of HCV therapy, HCV viraemia and treatment-induced viral
clearance (NESI data) First, multiple imputation by chained equations (MICE) was applied to the NESI data to impute
missing HCV antibody and HCV RNA results.20 Missing data was assumed to be missing at random
(MAR). This assumption, while not testable, was deemed plausible as there was no reason to believe
that there was any systematic relationship between the absence of HCV antibody or HCV RNA
measurement and the missing value. Survey year, region and time since onset of injecting were used
as predictors in the imputation model and twenty imputed datasets were generated. Sensitivity
analyses were performed by comparing the results from MICE to results generated from complete
case analyses. Where survey years were presented separately, NESI respondents who had participated more than
once within a given survey year were identified (on the basis of initials, sex assigned at birth, date of
birth, and NHS Board of recruitment) and only their first interview/DBS was included in the analysis. (When pooling data across the surveys, participants who had participated multiple times across
survey years were considered – see multilevel logistic regression). The proportions of respondents in NESI who reported ever/last year uptake of therapy, with
viraemia, and with treatment-induced clearance were compared across survey sweeps and NHS
Board regions (Tayside/GGC/RoS). GGC was presented separately because it is the largest NHS board
in Scotland (representing approximately 33% of individuals with problem drug use in Scotland and
40% of all NESI respondents in the included survey years).17 Additionally, the setting where HCV therapy was initiated among respondents in the 2017-18 survey
was examined and a chi-square test was performed to test for a difference in the proportion
reporting community-initiated therapy by region. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Associations of region and survey year with uptake of HCV therapy, HCV viraemia and treatment-
induced viral clearance (NESI data) Associations of region and survey year with uptake of HCV therapy, HCV viraemia and treatment-
induced viral clearance (NESI data) We used logistic regression to examine the association between the main predictors region and
survey year and the outcomes uptake of therapy (in the last year), HCV viraemia (among antibody
positives) and treatment-induced viral clearance (among antibody positives) in the NESI data
collected from 2013-14 through 2017-18 (the models were restricted to these years because these
were the only surveys in which the HCV treatment questions were asked). GGC was combined with
RoS in these analyses to simplify the analysis of interactions between region and year. Univariable
models were constructed for each predictor and outcome, followed by multivariable models
adjusted also for sex (assigned at birth), age, time since onset of injecting, homelessness in the last
six months (yes/no), receipt of methadone in the last six months (yes/no) and imprisonment in the
last year (yes/no). Multivariable models were fitted with interactions between survey year and
region and a likelihood ratio test (LRT) was conducted to test for evidence of the interaction. A multi-
level regression modelling was used to take clustering and non-independence of observations
among individuals into account, given some individuals had participated more than once across the
surveys (identified on the basis of initials, sex (assigned at birth), date of birth, and NHS Board of
recruitment). Role of the funding source The funders had no role in the study design, data collection, data analysis, data interpretation,
writing of the report, or the decision to submit for publication. RESULTS A total of 12,492 PWID who participated in the NESI survey and 4,105 individuals who initiated HCV
treatment were included in this study. A further breakdown of the sample sizes that were included
in the different analyses is given in Figures 1a and 1b. An overview of the NESI samples recruited in
Tayside, GGC, and RoS are presented in supplementary tables S1-S3, respectively. The prevalence of
HCV antibodies in Tayside (n=818), GGC (n=5,105), and RoS (n=6,358) ranged between 41-56%, 63-
69% and 44-52%, respectively, between 2010 and 2017-18. Approximately 70-80% of the sample
were male, and this was consistent across the surveys and regions. Average age and time since onset
of injecting increased across the surveys in all regions, suggestive of an ageing cohort effect. Excessive alcohol consumption was generally lower in Tayside compared with GGC and RoS (ranging Excessive alcohol consumption was generally lower in Tayside compared with GGC and RoS (ranging This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. from 10-19%, 23-30% and 19-30%, respectively). Cocaine injection in the last six months was also
generally lower in Tayside and showed evidence of an increase across the surveys in all regions (from
4% to 14% in Tayside, from 16% to 50% in GGC, and from 7% to 19% in RoS). from 10-19%, 23-30% and 19-30%, respectively). Cocaine injection in the last six months was also
generally lower in Tayside and showed evidence of an increase across the surveys in all regions (from
4% to 14% in Tayside, from 16% to 50% in GGC, and from 7% to 19% in RoS). In Scotland overall, the proportion of those who said they had ever received therapy or received
therapy in the last year increased from 19% (167/863) and 9% (72/800) in 2013-14 to 38% (301/785)
and 21% (144/680) respectively in 2017-18 (Table 1). In Tayside, uptake of therapy in the last year
increased from 15% (12/81) to 43% (26/61) between 2013-14 and 2017-18, as compared with 6%
(22/378) to 16% (50/322) in GGC and 11% (38/341) to 23% (68/297) in RoS. Sensitivity analyses comparing the results from data imputation to results generated from complete
case analyses are presented in supplementary table S4. Results were robust to these changes. RESULTS Between 2010 and 2017-18, the prevalence of HCV viraemia in Tayside decreased from 30% (95% CI
24-36%) in 2010 to 24% (95% CI 18-30%) in 2017-18 among all respondents and from 73% (95% CI
64-82%) to 44% (95% CI 34-55%) among antibody positives (Table 1 and Figure 2). The latter
compares to declines from 67% (95% CI 64-70%) to 58% (95% CI 54-63%) and 64% (95% CI 61-68%)
to 55% (95% CI 50-59%) among antibody-positives in GGC and RoS, respectively. The prevalence of treatment-induced clearance (among antibody positives) increased in Tayside
from 16% (95% CI 9-23%) to 40% (95% CI 30-49%), in comparison to 5% (95% CI 3-6%) to 14% (95%
CI 11-17%) in GGC and 10% (95% CI 8-13%) to 22% (18-25%) in RoS. Figure 3 displays the setting where HCV therapy was initiated among respondents in the 2017-18
survey. A much larger proportion of respondents (74%, 62/84) in Tayside reported starting
treatment in the community, as compared with GGC (11%, 15/137) and RoS (27%, 52/190)
(χ2=108·86, p<0·001). DISCUSSION In this study, we found that rapid scale-up of DAAs among PWID in community settings in Tayside
led to a greater decline in the prevalence of viraemic infection among PWID than elsewhere in
Scotland. There was also evidence that HCV viraemia declined, to a lesser extent, in other sites in
Scotland correlated with increases in HCV treatment. Further, we found that the increase in
community treatment in Tayside was not associated with any reduction in SVR rates in per-protocol
analyses. While numerous mathematical modelling studies have examined the hypothetical impact of scaling
up HCV therapy on chronic HCV prevalence, there has been little empirical evidence to corroborate
these models.11-13 One study has examined the impact of DAAs on chronic HCV prevalence at a sub-
national level (in prisons).24 Other “real world” evidence published focuses on scaling up treatment
and SVR, rather than impact at a population level.25-27 To our knowledge, only one other study to
date has used national data to examine the impact of treatment scale-up among PWID on viraemic
prevalence at a population level. They found that treatment uptake (ever) among Australian PWID
increased from 10% to 41%, and that viraemic prevalence decreased from 43% to 25% overall,
between 2015 and 2017.10 However, direct comparisons with Australia cannot be drawn, given the
different interventions: they evaluated the impact (pre vs. post) of the introduction of completely
unrestricted access to DAAs across the country, whereas we evaluated an intervention (that had
been demonstrated in mathematical models to deliver population-level change in HCV viraemia)
involving scale-up of DAAs in a specific PWID population, compared with a natural control group,
through delivery of therapy across multiple community-based settings. Further, a limitation
recognised in the Australian study was the amount of missing RNA results from DBS testing (44%-
60% of antibody positive samples across the three years of data), which may have led to bias within
their analysis, compared in our study to 4%-11% missing RNA results. It is notable treatment interventions had been occurring in Tayside prior to EPIToPe, albeit to a much
lesser extent, with approximately 100 PWID treated with interferon-based therapies during 2012-
2016.28 . These treatment interventions may account for the higher uptake of therapy at ‘baseline’
(i.e. pre-EPIToPe scale-up) in Tayside as compared with other regions. SVR rates by region, treatment setting, and PWID status (clinical database) There was a difference of approximately 6 to 8 percentage points in ITT rates between PWID and
non-PWID in GGC (74% vs 81%, p=0·007), RoS (66% vs. 72%, p=0·008) and Tayside (78% vs. 86%,
p=0·119) (Table 3, Figure 4, Figure S1). In all Scottish regions, per-protocol SVR rates did not differ
significantly between PWID and non-PWID. There was evidence that ITT SVR rates were lower in
community settings as compared with hospital settings in GGC (70% vs. 80%, p<0·001), RoS (65% vs. 72%, p=0·01) and Tayside (78% vs. 85%, p=0·121). Per-protocol SVR rates did not differ significantly
across treatment settings in any Scottish regions. Associations of region and survey year with uptake of HCV therapy, HCV viraemia and treatment-
induced viral clearance (NESI data) PWID recruited in Tayside were more likely to have reported uptake of therapy, after adjustment for
survey year and other variables (Table S5: adjusted odds ratio [AOR] 3·13, 95% CI 2·22-4·41). While
there was insufficient evidence for an interaction between year and region (likelihood ratio test/LRT
p=0·112), stratified analyses were suggestive of a larger increase in uptake of therapy in Tayside
(AOR 5·19, 95% CI 2.87-9.37) as compared with RoS (AOR 2·35, 95% CI 1·67-3·31), where the
baseline for comparison was 2013-14 RoS (Table 2). After adjustment, respondents in Tayside had lower odds of viraemia (AOR 0·66, 95% CI 0·52-0·84)
as compared with those in the rest of Scotland, with evidence of an interaction between region and
year (LRT p=0·034), indicating that there was a larger reduction in HCV viraemia in Tayside (AOR
0·47, 95% CI 0·30-0·75) as compared with RoS (AOR 0·89, 95% CI 0·74-1·07), when compared to RoS
2013-14 (Tables 2 and S5). After adjustment, respondents in Tayside were more likely to have treatment-induced clearance
(Table S5: AOR 3·57, 95% CI 2·65-4·81) as compared with those in the rest of Scotland. There was This article is protected by copyright. All rights reserved. insufficient evidence of an interaction between region and year (LRT p=0·254) but some evidence of
a larger increase in treatment-induced clearance in Tayside (AOR 4·12, 95% CI 2·56-6.61) as
compared with RoS (AOR 2·49, 95% CI 1·88-3·30) (Table 2). insufficient evidence of an interaction between region and year (LRT p=0·254) but some evidence of
a larger increase in treatment-induced clearance in Tayside (AOR 4·12, 95% CI 2·56-6.61) as
compared with RoS (AOR 2·49, 95% CI 1·88-3·30) (Table 2). SVR rates by region, treatment setting, and PWID status (clinical database) DISCUSSION However, while these early
interventions did not constitute ‘rapid’ scale-up, they can nevertheless be considered as part of the
package of treatment interventions delivered to PWID in Tayside and their impact can be seen pre- This article is protected by copyright. All rights reserved. 2017-18. We have demonstrated the impact of partial scale-up; future sweeps of NESI will allow us
to measure further progress towards reducing viraemia to 10% or lower. 2017-18. We have demonstrated the impact of partial scale-up; future sweeps of NESI will allow us
to measure further progress towards reducing viraemia to 10% or lower. DAA scale-up is one of the key tools for countries to achieve HCV elimination. In their strategy for
elimination of viral hepatitis, the WHO have set targets relating to incidence of HCV infection, but
not for prevalence viraemia/chronic infection.2 We believe that monitoring impact on prevalence of
viraemia is a necessity to evaluate the direct, short-term impact of DAA scale-up as any increase in
recent therapy uptake would be directly observable in viraemic prevalence. We acknowledge that is
nevertheless important, in the longer term, to monitor incidence, as this can also impact on viraemic
prevalence. In particular, incidence of HCV reinfection is a key consideration with regard to HCV
elimination among PWID: if population-level viremia is not rapidly reduced to sufficiently low levels,
then reinfections will cause a rise in the HCV viraemic prevalence.29 Future analyses examining the
impact of full scale-up in Tayside will consider both primary incident infections and re-infections. We have also provided evidence that rapidly scaling up treatment to people through community
drug services (and who are therefore likely continuing to inject either during and/or after therapy)
does not compromise SVR rates. Though the intention-to-treat SVR rates were lower in community
sites (as compared with hospital) and among PWID, this is likely a result of PWID not attending their
appointment for confirmation of SVR and is therefore a reflection of lack of follow-up rather than
lack of SVR attainment.30 The latter is corroborated by NESI data from Tayside in 2017-18, which
show that, of those who said they had been initiated on treatment in the last year (who we can
assume are a mixture of individuals who did/did not return for a confirmatory SVR test), 96% were
PCR negative (95% CI 81-99%). DISCUSSION Non-compliance is nevertheless a concern, as there is evidence to
suggest poor health outcomes among non-compliant individuals; however, they represent a small
minority of the patients that commence therapy.31 Although the DAAs are highly tolerable, easy to
take/administer and involve a much shorter course of treatment than the interferon-based
therapies, compliance can nevertheless be challenging for individuals with complex personal and
social circumstances.30 A major strength of our analysis is the “natural experiment” design, whereby Tayside represents the
“intervention” group and GGC/RoS are the “control” group. A further strength of our study is the
data sources, which are comprehensive and national: few countries are in a position to be able to
measure population-level changes in HCV viraemia among PWID, NESI being one of only four serial
bio-behavioural studies of its kind internationally.10,32,33 However, we acknowledge several A major strength of our analysis is the “natural experiment” design, whereby Tayside represents the
“intervention” group and GGC/RoS are the “control” group. A further strength of our study is the
data sources, which are comprehensive and national: few countries are in a position to be able to
measure population-level changes in HCV viraemia among PWID, NESI being one of only four serial
bio-behavioural studies of its kind internationally.10,32,33 However, we acknowledge several
limitations. First, we relied on self-report for uptake of HCV therapy, which may be subject to recall
and social desirability biases. Second, there is sampling variation between the serial surveys and
could be a potential reason for differences between the survey sweeps. However, most sample
characteristics were relatively stable across the period examined or followed expected trends - such
as the increase in cocaine injecting.34 Third, there were variations in prevalence of HCV antibody in
Tayside, which was 41%-42% in 2010-2012 and increased to 56% in 2013-14. These changes may
result from selection bias because, as part of the intervention, treatment became available at some
of the needle exchange and drug treatment settings where NESI recruitment takes place, potentially
attracting more hepatitis C infected clientele, thereby artificially increasing the prevalence.28,35 To
account for this issue, we analysed and presented the viraemia data among HCV antibody positives,
which removes any variation in overall antibody prevalence (we have also presented the prevalence
of viraemia among all respondents for comparison purposes). We acknowledge that this selection This article is protected by copyright. All rights reserved. Financial support This study was funded by Public Health Scotland (PHS) and the National Institute for Health Research
(NIHR). NIHR funding was through the Programme Grants for Applied Research programme (Grant
Reference Number RP-PG-0616-20008). Research grants from AbbVie, Bristol-Myers Squibb, Gilead,
Janssen, Merck Sharpe & Dohme, and Roche supported, in part, the scale-up of HCV treatment in
Tayside. Acknowledgments We would like to acknowledge the NIHR Health Protection Research Unit in Behavioural Science and
Evaluation, HCV specialist nurses and HCV treatment centres, the NESI Steering Group, the HCV
Prevention Leads Group, NESI researchers, staff from the West of Scotland Specialist Virology
Centre, Avril Taylor for her role in the conception of the NESI study, and the respondents who
volunteered their time to participate in the NESI studies. DISCUSSION bias could also have affected the rates of treatment uptake and viraemia itself for the same reasons. However, if we examine the distribution of settings where individuals initiated treatment from the
clinical database (which captures a comprehensive picture of all those receiving treatment), 24%, 9%
and 67% of treatment initiations in Tayside in 2017/2018 occurred in hospitals, prisons and
community settings, respectively. Comparing that to the distribution obtained from NESI
respondents recruited in Tayside shows that the oversampling from community settings, while not
insignificant, is not vast (with 11%, 10% and 79% of treatment initiations in 2017-18 in hospitals,
prisons and community settings, respectively). A fourth limitation of the study relates to the assays
used to detect HCV antibody and RNA on DBS. The sensitivity and specificity of the antibody test are
99% and 100%, respectively; the corresponding values for the PCR test are 100% and 96%.36,37 There
is therefore a very small chance of false negatives or false positives on both tests. We may have
missed individuals who had very recently been infected given that antibody and RNA levels may fall
below the lower limits of detection during this time.38 However, these are likely to be very small
numbers and should therefore not affect our viraemic prevalence estimates.18 Finally, the
incomplete treatment data was a limitation and is reflected in the ITT SVR rates as they include
missing confirmatory tests. In conclusion, we have demonstrated the feasibility and effectiveness of an approach that involves
HCV testing and treatment in a range of community services engaged with PWID, in increasing HCV
treatment uptake and thereby reducing the prevalence of HCV viraemia. Our findings provide
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Page-Shafer K, Pappalardo BL, Tobler LH, Phelps BH, Edlin BR, Moss AR, et al. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table 1. Trends in uptake of HCV therapy and estimated prevalence of HCV viraemia and treatment-induced viral clearance among people who inject drugs
attending injection equipment provision services across Scotland, 2010-2018 (data from the Needle Exchange Surveillance Initiative/NESI)
Survey sweep able 1. Trends in uptake of HCV therapy and estimated prevalence of HCV viraemia and treatm
ttending injection equipment provision services across Scotland, 2010-2018 (data from the Nee Table 1. This article is protected by copyright. All rights reserved. GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; N/A = not applicable
*among therapy–eligible respondents; see methods for definitions
**Missing antibody and RNA data have been imputed; see methods. This article is protected by copyright. All rights reserved. Logistic regression analyses of a) uptake of therapy in the last year, b) HCV viraemia (among HCV antibody positives) and c) treatment-induced viral clearance
(among HCV antibody positives), by region and survey year, among people who inject drugs attending injection equipment provision services across Scotland, 2013-201
(data from the Needle Exchange Surveillance Initiative/NESI)
AORs*
Outcome
Predictor
AOR (95% CI)
p-value
Uptake of therapy (in last year)†
(n=2,680)
Rest of Scotland (including GGC)
2013-14
1
2015-16
0·65 (0·44-0·97)
0·033
2017-18
2·35 (1·67-3·31)
<0·001
Tayside
2013-14
2.12 (1.10-4.08)
0.025
2015-16
2.87 (1.67-4.92)
<0.001
2017-18
5.19 (2.87-9.37)
<0.001
HCV viraemia (among HCV antibody positives)
(n=3,681)
Rest of Scotland (including GGC)
2013-14
1
2015-16
1·50 (1·27-1·77)
<0·001
2017-18
0·89 (0·74-1·07)
0·220
Tayside
2013-14
0.96 (0.64-1.44)
0.853
2015-16
0.57 (0.38-0.84)
0.005
2017-18
0.47 (0.30-0.75)
0.001
Treatment-induced viral clearance (among HCV antibody positives)
(n=3,681)
Rest of Scotland (including GGC)
2013-14
1
2015-16
0·70 (0·51-0·96)
0·027
2017-18
2·49 (1·88-3·30)
<0·001
Tayside
2013-14
2.46 (1.40-4.33)
0.002
2015-16
4.37 (2.60-7.35)
<0.001
2017-18
4.12 (2.56-6.61)
<0.001
AOR = adjusted odds ratio; CI = confidence interval; HCV = hepatitis C virus; GGC = Greater Glasgow & Clyde
*AORs derived from multivariable models with interaction effects (see Table S5 for univariable and main effects models). Multivariable models are also adjusted for sex, age, time since onset of injecting,
homelessness (last six months), receipt of methadone (last six months) and imprisonment (last year)
†among therapy–eligible respondents; see methods for definitions stic regression analyses of a) uptake of therapy in the last year, b) HCV viraemia (among HCV antibody positives) and c) treatment-induced viral clearance
ntibody positives), by region and survey year, among people who inject drugs attending injection equipment provision services across Scotland, 2013-2018*
N
dl E
h
S
ill
I iti ti
/NESI) Table 2. Logistic regression analyses of a) uptake of therapy in the last year, b) HCV viraemia (among HCV antibody positives) and c) treatment-induced viral clearance
(among HCV antibody positives), by region and survey year, among people who inject drugs attending injection equipment provision services across Scotland, 2013-2018*
(data from the Needle Exchange Surveillance Initiative/NESI) Table 2. This article is protected by copyright. All rights reserved. Trends in uptake of HCV therapy and estimated prevalence of HCV viraemia and treatment-induced viral clearance among people who inject drugs
attending injection equipment provision services across Scotland, 2010-2018 (data from the Needle Exchange Surveillance Initiative/NESI)
Survey sweep
Region
2010
2011-12
2013-14
2015-16
2017-18
Therapy uptake*
Ever
All Scotland
N/A
N/A
19% (167/863)
17% (171/1006)
38% (301/785)
Tayside
N/A
N/A
31% (28/90)
35% (31/89)
65% (49/75)
GGC
N/A
N/A
14% (54/399)
14% (57/422)
31% (111/360)
Rest of Scotland
N/A
N/A
23% (85/374)
17% (83/495)
40% (141/350)
Last year
All Scotland
N/A
N/A
9% (72/800)
7% (66/949)
21% (144/680)
Tayside
N/A
N/A
15% (12/81)
23% (18/79)
43% (26/61)
GGC
N/A
N/A
6% (22/378)
3% (13/406)
16% (50/322)
Rest of Scotland
N/A
N/A
11% (38/341)
8% (35/464)
23% (68/297)
Prevalence of HCV viraemia**
All
All Scotland
37% (35%-39%)
33% (31%-35%)
35% (33%-37%)
38% (36%-40%)
32% (29%-34%)
Tayside
30% (24%-36%)
24% (16%-32%)
34% (27%-40%)
32% (26%-38%)
24% (18%-30%)
GGC
45% (42%-47%)
39% (36%-43%)
45% (41%-48%)
47% (43%-50%)
40% (36%-43%)
Rest of Scotland
31% (28%-33%)
28% (25%-31%)
27% (25%-30%)
34% (31%-36%)
27% (24%-29%)
HCV antibody positives
All Scotland
66% (64%-68%)
61% (58%-64%)
60% (57%-63%)
67% (64%-69%)
55% (52%-58%)
Tayside
73% (64%-82%)
59% (45%-73%)
60% (51%-69%)
58% (48%-67%)
44% (34%-55%)
GGC
67% (64%-70%)
62% (58%-67%)
64% (60%-68%)
72% (68%-76%)
58% (54%-63%)
Rest of Scotland
64% (61%-68%)
61% (56%-65%)
57% (53%-61%)
65% (61%-68%)
55% (50%-59%)
Prevalence of treatment-induced viral
clearance**
All
All Scotland
N/A
N/A
5% (4%-6%)
4% (3%-5%)
11% (10%-13%)
Tayside
N/A
N/A
9% (5%-13%)
10% (6%-14%)
22% (16%-27%)
GGC
N/A
N/A
3% (2%-4%)
3% (2%-4%)
10% (8%-12%)
Rest of Scotland
N/A
N/A
5% (4%-6%)
4% (3%-5%)
10% (9%-12%)
HCV antibody positives
All Scotland
N/A
N/A
8% (7%-10%)
7% (6%-8%)
20% (18%-22%)
Tayside
N/A
N/A
16% (9%-23%)
18% (12%-25%)
40% (30%-49%)
GGC
N/A
N/A
5% (3%-6%)
4% (3%-6%)
14% (11%-17%)
Rest of Scotland
N/A
N/A
10% (8%-13%)
7% (5%-9%)
22% (18%-25%)
GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; N/A = not applicable All GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; N/A = not applicable
*among therapy–eligible respondents; see methods for definitions
**Missing antibody and RNA data have been imputed; see methods. This article is protected by copyright. All rights reserved. Table 2. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Logistic regression analyses of a) uptake of therapy in the last year, b) HCV viraemia (among HCV antibody positives) and c) treatment-induced viral clearance
(among HCV antibody positives), by region and survey year, among people who inject drugs attending injection equipment provision services across Scotland, 2013-2018
(data from the Needle Exchange Surveillance Initiative/NESI)
AORs*
Outcome
Predictor
AOR (95% CI)
p-value
Uptake of therapy (in last year)†
(n=2,680)
Rest of Scotland (including GGC)
2013-14
1
2015-16
0·65 (0·44-0·97)
0·033
2017-18
2·35 (1·67-3·31)
<0·001
Tayside
2013-14
2.12 (1.10-4.08)
0.025
2015-16
2.87 (1.67-4.92)
<0.001
2017-18
5.19 (2.87-9.37)
<0.001
HCV viraemia (among HCV antibody positives)
(n=3,681)
Rest of Scotland (including GGC)
2013-14
1
2015-16
1·50 (1·27-1·77)
<0·001
2017-18
0·89 (0·74-1·07)
0·220
Tayside
2013-14
0.96 (0.64-1.44)
0.853
2015-16
0.57 (0.38-0.84)
0.005
2017-18
0.47 (0.30-0.75)
0.001
Treatment-induced viral clearance (among HCV antibody positives)
(n=3,681)
Rest of Scotland (including GGC)
2013-14
1
2015-16
0·70 (0·51-0·96)
0·027
2017-18
2·49 (1·88-3·30)
<0·001
Tayside
2013-14
2.46 (1.40-4.33)
0.002
2015-16
4.37 (2.60-7.35)
<0.001
2017-18
4.12 (2.56-6.61)
<0.001
AOR = adjusted odds ratio; CI = confidence interval; HCV = hepatitis C virus; GGC = Greater Glasgow & Clyde
*AORs derived from multivariable models with interaction effects (see Table S5 for univariable and main effects models). Multivariable models are also adjusted for sex, age, time since onset of injecting,
homelessness (last six months), receipt of methadone (last six months) and imprisonment (last year)
†among therapy–eligible respondents; see methods for definitions AOR = adjusted odds ratio; CI = confidence interval; HCV = hepatitis C virus; GGC = Greater Glasgow & Clyde
*AORs derived from multivariable models with interaction effects (see Table S5 for univariable and main effects models). Multivariable models are also adjusted for sex, age, time since onset of injecting,
homelessness (last six months), receipt of methadone (last six months) and imprisonment (last year)
†among therapy–eligible respondents; see methods for definitions This article is protected by copyright. All rights reserved. Table 3. This article is protected by copyright. All rights reserved. CI = confidence interval; GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; PWID = people who inject drugs, SVR = sustained viral response
*Patient had indicated ever injecting drugs
**Community sites include needle exchanges, pharamacies, drug treatment centres, and other outreach clinics This article is protected by copyright. All rights reserved. Intention-to-treat/per-protocol SVR rates by region, PWID status and treatment setting among patients initiated on HCV therapy in Scotland du
2017-2018 (data from the Hepatitis C Clinical Database)
Region
PWID*
Non-PWID*
Χ2, p-value
n/N
% (95% CI)
n/N
% (95% CI)
Intention-to-
treat
All Scotland
2227/3111
71·6%
(70·0%-73·2%)
765/994
77·0%
(74·2%-79·5%)
11·021, p=0·001
Tayside
343/441
77·8%
(73·6%-81·6%)
61/71
85·9%
(75·6%-93·0%)
2·433, p=0·119
GGC
1099/1477
74·4%
(72·1%-76·6%)
345/427
80·8%
(76·7%-84·4%)
7·379, p=0·007
Rest of Scotland
785/1193
65·8%
(63·0%-68·5%)
359/496
72·4%
(68·2%-76·3%)
6·937, p=0·008
Per-protocol
All Scotland
2227/2267
98·2%
(97·6%-98·7%)
765/777
98·5%
(97·3%-99·2%)
0·167, p=0·683
Tayside
343/351
97·7%
(95·6%-99·0%)
61/61
100%
(94·1%-100%)
1·418, p=0·234
GGC
1099/1112
98.8%
(98·0%-99·4%)
345/352
98·0%
(96·0%-99·20%)
1·333, p=0·248
Rest of Scotland
785/804
97·6%
(96·3%-98·6%)
359/364
98·6%
(96·8%-99·6%)
1·219, p=0·270
Region
Hospital
Prison
Community**
Χ2, p-value (comparing
community vs. hospital)
n/N
% (95% CI)
n/N
% (95% CI)
n/N
% (95% CI)
Intention-to-
treat
All Scotland
1923/2507
76·7%
(75·0%-78·3%)
207/349
59·3%
(54·0%-64·5%)
841/1194
70·4%
(67·8%-73·0%)
16·815, p<0·001
Tayside
104/123
84·6%
(76·9%-90·4%)
34/38
70·8%
(55·9%-83·1%)
266/341
78·0%
(73·2%-82·3%)
2·398, p=0·121
GGC
1038/1297
80·0%
(77·8%-82·2%)
84/144
58·3%
(49·8%-66·5%)
322/463
69·6%
(65·1%-73·7%)
21·356, p<0·001
Rest of Scotland
781/1087
71·9%
(69·1%-74·5%)
89/157
56·7%
(58·6%-64·6%)
253/390
64·9%
(59·9%-69·6%)
6·655, p=0·010
Per-protocol
All Scotland
1923/1957
98·3%
(97·6%-98·8%)
207/211
98·1%
(95·2%-99·5%)
841/855
98·4%
(97·3%-99·1%)
0·035, p=0·851
Tayside
104/104
100·0%
(96·5%-100·0%)
34/35
97·1%
(85·1%-99·9%)
266/273
97·4%
(94·8%-99·0%)
2·717, p=0·099
GGC
1038/1054
98·5%
(97·6%-99·1%)
84/87
96·6%
(90·3%-99·3%)
322/323
99·7%
(98·3%-100·0%)
2·961, p=0·085
Rest of Scotland
781/799
97·8%
(96·5%-98·7%)
89/89
100·0%
(95·9%-100·0%)
253/259
97·7%
(95·0%-99·2%)
0·004, p=0·952 Table 3. Intention-to-treat/per-protocol SVR rates by region, PWID status and treatment setting among patients initiated on HCV therapy in Scotland during
2017-2018 (data from the Hepatitis C Clinical Database) This article is protected by copyright. All rights reserved. CI = confidence interval; GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; PWID = people who inject drugs, SVR = sustained viral response
*Patient had indicated ever injecting drugs
**Community sites include needle exchanges, pharamacies, drug treatment centres, and other outreach clinics CI = confidence interval; GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; PWID = people who inject drugs, SVR = sustained viral response
*Patient had indicated ever injecting drugs
**Community sites include needle exchanges, pharamacies, drug treatment centres, and other outreach clinics **Community sites include needle exchanges, pharamacies, drug treatment centres, and o This article is protected by copyright. All rights reserved. Figure 1a. This article is protected by copyright. All rights reserved. Number of participants included in the analysis of population-level prevalence of uptake
of HCV therapy, HCV viraemia and treatment-induced viral clearance and the analysis of associations
of region and survey year with uptake of HCV therapy, HCV viraemia and treatment-induced viral
clearance (data from the Needle Exchange Surveillance Initiative/NESI)
2,680/3,681/3,681
Included in logistic
regression analyses
withoutcomes therapy
uptake/HCV
viraemia/treatment-
induced viral clearance
(the latter two
restricted to antibody
positives)
2,654/2,429
included in analysis
of trends in therapy
uptake ever/in the
last year
13,012
interviews completed
12,492
individuals
(3,168 in 2010; 2,154 in 2011-12;
2,344 in 2013-14; 2,696 in 2015-16;
2,130 in 2017-18)
520 within-survey
duplicates excluded
(147 in 2010; 40 in
2011-12; 118 in 2013-
14; 127 in 2015-16; 74
in 2017-18; 14 with
insufficient identifiers)
12,492/6,893 (all/HCV
antibody positives)
included in analysis of
trends in prevalence of
HCV viraemia
(1,063 of these were
missing HCV antibody
and/or PCR result;
these were imputed)
7,170/3,979 (all/HCV
antibody positives)
Included in analysis of
trends in prevalence of
treatment-induced viral
clearance
(579 of these were
missing HCV antibody
and/or PCR result;
these were imputed)
All survey years
Restricted to 2013-14 onwards
(date from which therapy
uptake data is available)
Duplicates not excluded
(accounted for by
multi-level model) and
restricted to 2013-14
onwards 13,012
interviews completed 520 within-survey
duplicates excluded
(147 in 2010; 40 in
2011-12; 118 in 2013-
14; 127 in 2015-16; 74
in 2017-18; 14 with
insufficient identifiers) Restricted to 2013-14 onwards
(date from which therapy
uptake data is available) Duplicates not excluded
(accounted for by
multi-level model) and
restricted to 2013-14
onwards All survey years 12,492/6,893 (all/HCV
antibody positives)
included in analysis of
trends in prevalence of
HCV viraemia
(1,063 of these were
missing HCV antibody
and/or PCR result;
these were imputed) 12,492/6,893 (all/HCV
antibody positives)
included in analysis of
trends in prevalence of
HCV viraemia
(1,063 of these were
missing HCV antibody
and/or PCR result;
these were imputed) 7,170/3,979 (all/HCV
antibody positives)
Included in analysis of
trends in prevalence of
treatment-induced viral
clearance
(579 of these were
missing HCV antibody
and/or PCR result;
these were imputed) 2,680/3,681/3,681
Included in logistic
regression analyses
withoutcomes therapy
uptake/HCV
viraemia/treatment-
induced viral clearance
(the latter two
restricted to antibody
positives) 7,170/3,979 (all/HCV
antibody positives)
Included in analysis of
trends in prevalence of
treatment-induced viral
clearance
(579 of these were
missing HCV antibody
and/or PCR result;
these were imputed) 2,680/3,681/3,681
Included in logistic
regression analyses
withoutcomes therapy
uptake/HCV
viraemia/treatment-
induced viral clearance
(the latter two
restricted to antibody
positives) 2,654/2,429
included in analysis
of trends in therapy
uptake ever/in the
last year Figure 1a. This article is protected by copyright. All rights reserved. Number of participants included in the analysis of population-level prevalence of uptake
of HCV therapy, HCV viraemia and treatment-induced viral clearance and the analysis of associations
of region and survey year with uptake of HCV therapy, HCV viraemia and treatment-induced viral
clearance (data from the Needle Exchange Surveillance Initiative/NESI) This article is protected by copyright. All rights reserved. Figure 1b. Participants included in the analysis of SVR rates (data from the Scottish Hepatitis C
4,168
treatment initiations between
Jan 2017 – Dec 2018
4,105
Individuals initiating treatment
between Jan 2017 – Dec 2018
63 duplicates excluded
(episode with most
recent completion date
was retained)
4,105
individuals included in
intention-to-treat (ITT) analyses
1,061 individuals with
unknown treatment
outcome
3,044
Individuals included in per-
protocol analyses Figure 1b. Participants included in the analysis of SVR rates (data from the Scottish Hepatitis C
Clinical Database). 4,168
treatment initiations between
Jan 2017 – Dec 2018
4,105
Individuals initiating treatment
between Jan 2017 – Dec 2018
63 duplicates excluded
(episode with most
recent completion date
was retained)
4,105
individuals included in
intention-to-treat (ITT) analyses
1,061 individuals with
unknown treatment
outcome
3,044
Individuals included in per-
protocol analyses 4,168
treatment initiations between
Jan 2017 – Dec 2018 63 duplicates excluded
(episode with most
recent completion date
was retained) 4,105
Individuals initiating treatment
between Jan 2017 – Dec 2018 1,061 individuals with
unknown treatment
outcome 3,044
Individuals included in per-
protocol analyses 4,105
individuals included in
intention-to-treat (ITT) analyses individuals included in
ntion-to-treat (ITT) analyses Figure 1b. Participants included in the analysis of SVR rates (data from the Scottish Hepatitis C
Clinical Database). Figure 1b. Participants included in the analysis of SVR rates (data from the Scottish Hepatitis C This article is protected by copyright. All rights reserved. Figure 2. Prevalence of viraemic and cleared HCV infection among people who inject drugs attending
injection equipment provision services across Scotland, 2013 to 2018. Figures present a) all
respondents and b) HCV antibody positives only (data from the Needle Exchange Surveillance
Initiative/NESI). Ab = antibody; GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; RNA = ribonucleic acid
34%
32%
24%
45%
47%
40%
27%
34%
27%
9%
10%
22%
3%
3%
10%
5%
4%
10%
14%
14%
9%
21%
15%
18%
16%
15%
11%
43%
44%
45%
30%
35%
32%
51%
47%
52%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
Tayside
GGC
Rest of Scotland
Chronic HCV (Ab pos, RNA pos)
Cleared HCV, with evidence of treatment
Cleared HCV, no evidence of treatment
Ab negative
60%
58%
44%
64%
72%
58%
57%
65%
55%
16%
18%
40%
5%
4%
14%
10%
7%
22%
24%
24%
16%
31%
23%
27%
33%
28%
23%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
Tayside
GGC
Rest of Scotland
Chronic HCV (Ab pos, RNA pos)
Cleared HCV, with evidence of treatment)
Cleared HCV, no evidence of treatment
a)
b) 34%
32%
24%
45%
47%
40%
27%
34%
27%
9%
10%
22%
3%
3%
10%
5%
4%
10%
14%
14%
9%
21%
15%
18%
16%
15%
11%
43%
44%
45%
30%
35%
32%
51%
47%
52%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
Tayside
GGC
Rest of Scotland
a) Cleared HCV, with evidence of treatment
Ab negative Cleared HCV, with evidence of treatment
Ab negative Cleared HCV, no evidence of treatment Cleared HCV, no evidence of treatment Cleared HCV, no evidence of treatment
Ab negative
60%
58%
44%
64%
72%
58%
57%
65%
55%
16%
18%
40%
5%
4%
14%
10%
7%
22%
24%
24%
16%
31%
23%
27%
33%
28%
23%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
Tayside
GGC
Rest of Scotland
b) Figure 2. Prevalence of viraemic and cleared HCV infection among people who inject drugs attending
injection equipment provision services across Scotland, 2013 to 2018. This article is protected by copyright. All rights reserved. Figures present a) all
respondents and b) HCV antibody positives only (data from the Needle Exchange Surveillance
Initiative/NESI). Ab = antibody; GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; RNA = ribonucleic acid
,
g
60%
58%
44%
64%
72%
58%
57%
65%
55%
16%
18%
40%
5%
4%
14%
10%
7%
22%
24%
24%
16%
31%
23%
27%
33%
28%
23%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
2013-14 2015-16 2017-18
Tayside
GGC
Rest of Scotland
Chronic HCV (Ab pos, RNA pos)
Cleared HCV, with evidence of treatment)
Cleared HCV, no evidence of treatment
b) Figure 2. Prevalence of viraemic and cleared HCV infection among people who inject drugs attending
injection equipment provision services across Scotland, 2013 to 2018. Figures present a) all
respondents and b) HCV antibody positives only (data from the Needle Exchange Surveillance
Initiative/NESI). Ab = antibody; GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; RNA = ribonucleic acid /
)
Ab = antibody; GGC = Greater Glasgow & Clyde; HCV = hepatitis C virus; RNA = ribonucleic aci This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 3. Site of initiation of most recent course of HCV therapy* among people who inject drugs
attending injection equipment provision services across Scotland, 2017-18 (data from the Needle
Exchange Surveillance Initiative/NESI). GGC = Greater Glasgow & Clyde; GP = general practice; HCV = hepatitis C virus
*among those who had ever received HCV therapy
†includes needle exchanges, drug treatment sites, community health centres and at an individual’s home
70%
11%
52%
16%
10%
16%
11%
74%
27%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
GGC
Tayside
Rest of Scotland
Don’t know
GP
Community†
Prison
Hospital 70%
11%
52%
16%
10%
16%
11%
74%
27%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
GGC
Tayside
Rest of Scotland
Don’t know
GP
Community†
Prison
Hospital Figure 3. Site of initiation of most recent course of HCV therapy* among people who inject drugs
attending injection equipment provision services across Scotland, 2017-18 (data from the Needle
Exchange Surveillance Initiative/NESI). GGC = Greater Glasgow & Clyde; GP = general practice; HCV = hepatitis C virus
*among those who had ever received HCV therapy
†includes needle exchanges, drug treatment sites, community health centres and at an individual’s home Figure 3. Site of initiation of most recent course of HCV therapy* among people who inject drugs
attending injection equipment provision services across Scotland, 2017-18 (data from the Needle
Exchange Surveillance Initiative/NESI). GGC = Greater Glasgow & Clyde; GP = general practice; HCV = hepatitis C virus
*among those who had ever received HCV therapy
†includes needle exchanges, drug treatment sites, community health centres and at an individual’s home g
/
)
GGC = Greater Glasgow & Clyde; GP = general practice; HCV = hepatitis C virus
*among those who had ever received HCV therapy among those who had ever received HCV therapy
†includes needle exchanges, drug treatment sites, community health centres and at an individual’s hom This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 4. Intention-to-treat/per-protocol SVR rates by region and treatment setting among patients attending specialist liver clinics in Scotland, 2017-2018
(data from the Hepatitis C Clinical Database). GGC = Greater Glasgow & Clyde; SVR = sustained viral response
80.0
58.3
69.6
71.9
56.7
64.9
84.6
70.8
78.0
98.5
96.6
99.7
97.8
100.0
97.7
100.0
97.1
97.4
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
Hospital
Prison
Community
Hospital
Prison
Community
Hospital
Prison
Community
Hospital
Prison
Community
Hospital
Prison
Community
Hospital
Prison
Community
GGC
Rest of Scotland
Tayside
GGC
Rest of Scotland
Tayside
Intention-to-treat
Per protocol Figure 4. Intention-to-treat/per-protocol SVR rates by region and treatment setting among patients attending specialist liver clinics in Scotland, 2017-2018
(data from the Hepatitis C Clinical Database). Figure 4. Intention-to-treat/per-protocol SVR rates by region and treatment setting among patients attending specialist liver clinics in Scotland
(data from the Hepatitis C Clinical Database). eat/per-protocol SVR rates by region and treatment setting among patients attending specialist liver clinics in Scotland, 2017-2018
C Clinical Database) GGC = Greater Glasgow & Clyde; SVR = sustained viral response This article is protected by copyright. All rights reserved.
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Units of Language Mixing
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cc-by
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Units of Language Mixing: A
Cross-Linguistic Perspective Artemis Alexiadou1,2*† and Terje Lohndal3,4† 1 Department of English and American Studies, Humboldt University of Berlin, Berlin, Germany, 2 Leibniz-Zentrum Allgemeine
Sprachwissenschaft, Berlin, Germany, 3 Department of Language and Literature, NTNU Norwegian University of Science
and Technology, Trondheim, Norway, 4 Department of Language and Culture, UiT The Arctic University of Norway, Tromsø,
Norway Language mixing is a ubiquitous phenomenon characterizing bilingual speakers. A frequent context where two languages are mixed is the word-internal level,
demonstrating how tightly integrated the two grammars are in the mind of a speaker
and how they adapt to each other. This raises the question of what the minimal unit of
language mixing is, and whether or not this unit differs depending on what the languages
are. Some scholars have argued that an uncategorized root serves as a unit, others
argue that the unit needs to have been categorized prior to mixing. We will discuss
the question of what the relevant unit for language mixing is by studying word-internal
mixing in Cypriot Greek-English, English-Norwegian, Greek-English, Greek-German,
and Spanish-German varieties that have been reported in the literature based on data
from judgment experiments and spoken corpora. By understanding and modeling the
units of language mixing across languages, we will gain insight into how languages adapt
to each other word-internally, and what some possible outcomes of language contact
are in the minds of speakers. Reviewed by: Keywords: English, German, Greek, Norwegian, Spanish, language mixing, distributed morphology David W. Lightfoot,
Georgetown University, United States Specialty section: This article was submitted to
Language Sciences,
a section of the journal
Frontiers in Psychology This article was submitted to
Language Sciences,
a section of the journal
Frontiers in Psychology This article was submitted to
Language Sciences,
a section of the journal
Frontiers in Psychology (1) Q’aya suya-wa-nki
[las cuatro-ta]. (Quechua/Spanish)
tomorrow wait-1OB-2SG at four-ACC
Qo-yku-sqa-sun-ña
[bukis]
give-ASP-ASP-1PL-FUT-CON box
‘Tomorrow you wait for me at four. We’ll have a go at
boxing.’ (Muysken, 2000, based on Urioste, 1966, p. 7) (1) Q’aya suya-wa-nki
[las cuatro-ta]. (Quechua/Spanish)
tomorrow wait-1OB-2SG at four-ACC
Qo-yku-sqa-sun-ña
[bukis]
give-ASP-ASP-1PL-FUT-CON box
‘Tomorrow you wait for me at four. We’ll have a go at
boxing.’ (Muysken, 2000, based on Urioste, 1966, p. 7) Received: 06 May 2018
Accepted: 24 August 2018
Published: 27 September 2018 INTRODUCTION *Correspondence:
Artemis Alexiadou
artemis.alexiadou@hu-berlin.de Much work on code-switching/code mixing/language mixing has aimed to provide a typology of
possible mixing patterns across different languages. One example of this can be found in Muysken
(2000, 2013) work. He defines mixing as lexical items and grammatical features from two (or more)
languages that appear in one sentence. In Muysken (2000), he proposes a three-way typology which
consists of insertion, alternation, and congruent lexicalization. Insertion involves insertion of well-
defined chunks of language B into a sentence that otherwise belongs to language A. An example of
this is provided in (1). †Both authors have contributed
equally to this work Edited by: Edited by:
Enoch Oladé Aboh,
University of Amsterdam, Netherlands ORIGINAL RESEARCH
published: 27 September 2018
doi: 10.3389/fpsyg.2018.01719 Keywords: English, German, Greek, Norwegian, Spanish, language mixing, distributed morpholog The Nature of Word-Internal Mixing In Muysken (2013), a fourth type is added to the typology,
namely backflagging. An example is given in (4). Since Poplack (1980), there has been significant work on the issue
of word-internal language mixing, or code-switching as most
of the relevant literature labels it. Her work can in many ways
be seen as one of the initiators to what MacSwan (2014, p. 4)
calls the ‘constraint-based research program.’ She proposed two
constraints, given in (5) and (6). (4) Q: What will you be when you grow up? (Dutch/Moroccan
Arabic) A:
Ik ben doctor wella ik ben ingenieur. A:
Ik ben doctor wella ik ben ingenieur. I am doctor or I am engineer. ‘I will become a doctor or an engineer.’ (Nortier, 1990,
p. 142) I am doctor or I am engineer. ‘I will become a doctor or an engineer.’ (Nortier, 1990,
p. 142) (5) The Equivalence Constraint
Codes will tend to be switched at points where the surface
structures of the languages map onto each other. Backflagging is defined as insertion of heritage language
discourse marker in L2 discourse. (6) The Free Morpheme Constraint
A switch may occur at any point in the discourse at which it
is possible to make a surface constituent cut and still retain
a free morpheme. This typology is largely confined to word-level units and
beyond. Recent work has begun to use language mixing as a probe
into which basic units are put to use in both monolingual and
bilingual speakers (González-Vilbazo and López, 2011; Alexiadou
et al., 2015; Alexiadou, 2017a; Riksem et al., in press; Riksem,
2018). As we will see, these and other works argue that there are
grammatical differences between varieties in terms of how mixing
takes place. Our concern is precisely these differences rather
than being able to predict exactly which mixing strategy a given
speaker decides to use in a specific context; see Wohlgemuth
(2009) for an extensive and typological investigation of the latter
in the context of mixing involving verbs. We won’t discuss the first constraint (5), but the second
constraint is quite important for the present paper. Sankoffand
Poplack (1981) developed (6) further and stated it as follows: (6) The Free Morpheme Constraint Revisited
A switch may not occur between a bound morpheme and
a lexical form unless the latter has been phonologically
integrated into the language of the bound morpheme. Citation: Alexiadou A and Lohndal T (2018)
Units of Language Mixing:
A Cross-Linguistic Perspective. Front. Psychol. 9:1719. doi: 10.3389/fpsyg.2018.01719 In the case of alternation, two different languages A and B alternate within the same sentence,
as shown in (2). September 2018 | Volume 9 | Article 1719 1 Frontiers in Psychology | www.frontiersin.org Alexiadou and Lohndal Units of Language Mixing (2) a. maar’t hoeft niet li-’anna ida šeft ana... but it need not for-when I see I
‘but it need not be, for when I see, I...’ (Moroccan
Arabic/Dutch; Muysken, 2000, based on Nortier, 1990,
p. 213) (2) a. maar’t hoeft niet li-’anna ida šeft ana... but it need not for-when I see I
‘but it need not be, for when I see, I...’ (Moroccan
Arabic/Dutch; Muysken, 2000, based on Nortier, 1990,
p. 213) consider an entirely different variety typologically speaking,
namely Telugu. The section “Discussion and Analysis” will
discuss and compare the patterns across the different varieties
and also comment on recent work by López (2018). Lastly, the
section “Conclusion” concludes the paper. p
b. Andale pues and do come again. (Spanish/English)
‘That’s all right then, and do come again.’ (Muysken,
2000, based on Gumperz and Hernández-Chavez, 1971,
p. 118) b. Andale pues and do come again. (Spanish/English)
‘That’s all right then, and do come again.’ (Muysken,
2000, based on Gumperz and Hernández-Chavez, 1971,
p. 118) BACKGROUND This section will provide some context and relevant background
for the present paper. In the Section “The Nature of Word-
Internal Mixing,” we discuss the notion of word-internal mixing,
situating it within a long research history in work on language
mixing. We then also discuss why word-internal mixing is
important for modeling multilingual’s linguistic competence. Congruent lexicalization is defined as the use of elements from
either language in a structure that is wholly or partly shared by
languages A and B. An example is provided in (3). (3) Weet jij [whaar] Jenny is? (Dutch: Waar Jenny is)
‘Do you know where Jenny is?’ (English/Dutch; Crama and
van Gelderen, 1984) Frontiers in Psychology | www.frontiersin.org The Nature of Word-Internal Mixing The formulation in (6) does not allow examples like in (7), but
it allows examples like (8). The goal of the present paper is to synthesize and compare the
current findings from various bilingual populations, in particular
heritage language speakers. We will focus on word-internal
mixing in Cypriot Greek-English, English-Norwegian, Greek-
English, Greek-German, and Spanish-German varieties based on
data from judgment experiments and spoken corpora. (7) a. ∗eat-iendo
eat-ing
‘eating’
(Poplack, 1980, p. 586)
b. run-eando
run-ing
‘running’
(Sankoffand Poplack, 1981, p. 5)
(8) a. flip-eando
flip-ing
‘flipping’
(Sankoffand Poplack, 1981, p. 5)
b. parqu-eando
park-ing
‘parking’
(MacSwan, 2005, p. 7) (7) a. ∗eat-iendo
eat-ing
‘eating’
(Poplack, 1980, p. 586) (10) PF Interface Condition 92–93) makes clear:1 Code-switching (CS) is not an entity which exists out there in the
objective world, but a construct which linguists have developed
to help them describe their data. It is therefore pointless to argue
about what CS is, because, to paraphrase Humpty Dumpty, the
word CS can mean whatever we want it to mean. Words are linguistic units, but they are not phonetic units: no
merely phonetic analysis of a string of spoken sounds can reveal
to us the number of words it is made up of, or the division
between word and word. [. . .] As, consequently, neither sound
nor meaning in itself shows us what is one word and what is
more than one word, we must look out for grammatical (syntactic)
criteria to decide the question. As is to be expected given this situation, the asymmetry
between (7) and (8) is controversial in the literature. Several
scholars have argued for it (e.g., Bentahila and Davies, 1983; Berk-
Seligson, 1986; Clyne, 1987; MacSwan, 1999), whereas others
have presented counterexamples (e.g., Nartey, 1982; Bokamba,
1989; Myers-Scotton, 1993; Halmari, 1997; Chan, 1999; Hlavac,
2003; Grimstad et al., 2014, 2018; Grimstad, 2017; Riksem, 2018). MacSwan and Colina (2014, p. 203) note that ‘[i]n some cases,
researchers have not adequately documented the phonological
characteristics of items to permit us to judge their level of
integration, and in others assumptions about the morphological
status of elements have not been made explicit.’ This suggests that
mixing or switching is primarily a phonological phenomenon: Poplack (2013, p. 12), in a discussion of what we have learned
from Poplack (1980) till today also highlights the following:
‘Phonological and morphosyntactic integration are independent. Phonology of both CS and B, is variable, and thus cannot
reliably be used to distinguish between them.’ Again, relying on
phonological criteria alone is problematic. The second reason is related to the concept of the lexicon
that is assumed (see also Grimstad, 2017). As (9) makes clear,
the distinction between borrowing and mixing generally invokes
the question of whether a given unit is part of the lexicon of
the recipient language or the donor language. This assumes,
as in e.g., Muysken (2000) model, that a multilingual speaker In an important respect, then, language switching is phonological
switching. 1The literature is ripe with discussions of how to define words, an issue we won’t
delve further into. See, among many, Katamba (2004), Katamba and Stonham
(2006), and Anderson (2015) for good discussions. (10) PF Interface Condition (i) Phonological input is mapped to the output in one
step with no intermediate representations. In the language contact literature, there are two distinct
positions concerning switching and borrowing. The first position
sees code switching and borrowing as part of a continuum (e.g.,
Myers-Scotton, 1993, 2002, 2006; van Coetsem, 2000; Thomason,
2003). The second position rather views code switching and
borrowing as two distinct processes (Sankoffand Poplack, 1981;
MacSwan, 1999; Poplack and Dion, 2012; MacSwan and Colina,
2014; Poplack, 2018). In a thorough review of the debate,
Grimstad (2009, pp. 6–7), based on Matras (2009, p. 106) and
Muysken (2000, p. 69), characterizes the two positions as in (9). (ii) Each set of internally ranked constraints is a
constraint dominance hierarchy, and a language-
particular phonology is a set of constraint dominance
hierarchies. (iii) Bilinguals
have
a
separately
encapsulated
phonological system for each language in their
repertoire in order to avoid ranking paradoxes, which
result from the availability of distinct constraint
dominance hierarchies with conflicting priorities. (iv) Every syntactic head must be phonologically parsed
at Spell Out. Therefore, the boundary between heads
(words) represents the minimal opportunity for code-
switching. (iv) Every syntactic head must be phonologically parsed
at Spell Out. Therefore, the boundary between heads
(words) represents the minimal opportunity for code-
switching. (9) a. Borrowing is the diachronic process by which languages
enhance
their
vocabulary
(or
other
domains
of
structure),
while
code-switching
is
instances
of
spontaneous language mixing in the conversation
of bilinguals. Borrowed items originate as code-switches. (10) builds on the PF Disjunction Theorem in MacSwan
(1999, 2000). MacSwan (1999,2009,2013) further argues that
mixing in contexts of head movement is prohibited, which
(10iv) derives given that a word is defined as ‘a lexical head
(X0) whose morphological composition has been determined
internally within the lexicon’ (MacSwan, 2005, p. 11). b. Code-switching
involves
inserting
alien
words
or
constituents into a clause; borrowing involves entering
alien elements into a lexicon. However, as Grimstad also points out, when dealing with these
notions, it is worth keeping the following quote from Gardner-
Chloros (2009, pp. 10–11) in mind: However, there are several issues with MacSwan’s and others’
approach in terms of banning word-internal mixing. Here, we
will highlight two. To begin with, a phonological definition is problematic if
not simply because words are hard to define simply based
on phonological criteria, as the following quote by Jespersen
(1924/1963, pp. (7) a. ∗eat-iendo
eat-ing
‘eating’ (7) a. ∗eat-iendo
eat-ing
‘eating’ g
b. run-eando
run-ing
‘running’ b. run-eando
run-ing
‘running’
(Sankoffand Poplack, 1981, p. 5) This paper is organized as follows. The section “Background”
provides some background, in particular concerning the nature
of word-internal language mixing. Then we move on to the
case studies reviewed in the paper. The section “Word-Internal
Mixing in Varieties Involving Greek” will consider word-internal
mixing in Greek-German and Cypriot Greek-English, whereas
the section “Word-Internal Mixing in German-Spanish” will
look into Spanish-German. The section “Word-Internal Mixing
in Varieties Involving Norwegian” is devoted to word-internal
mixing in varieties involving English and Norwegian. In the
section “Word-Internal Mixing in Telugu,” we zoom out and (8) a. flip-eando
flip-ing
‘flipping’ (8) a. flip-eando
flip-ing
‘flipping’
(Sankoffand Poplack, 1981, p. 5) b. parqu-eando
park-ing
‘parking’
(MacSwan, 2005, p. 7) The reason is that run is has a clear English phonology with a
mid-central vowel [∧] which is not part of Spanish phonology. In September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org 2 Alexiadou and Lohndal Units of Language Mixing (8), on the other hand, flip and parqu have been accommodated
to Spanish phonology. Since then, the distinction between (7)
and (8) has often been referred to as one of code switching vs. borrowing. A borrowed word is phonologically integrated into
the recipient language, whereas code switches are taken from two
different lexicons. MacSwan proposes the PF Interface Condition as a way of
modeling this claim. The condition is given in (10) (MacSwan,
2009, p. 331; MacSwan and Colina, 2014, p. 19). (10) PF Interface Condition When we stop speaking one language and begin
speaking another, the shift is prominently characterized by a
change in the way we say words. So conceived, the relevant
research question would appear to revolve around discovering the
conditions under which one can switch from one phonological
system to another (MacSwan and Colina, 2014, p. 188). September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org 3 Alexiadou and Lohndal Units of Language Mixing distractions, shifts of attention and interest, and errors (random or
characteristic) in applying his knowledge of the language in actual
performance. has an individual mental lexicon for every language she knows. However, a contemporary speaker has no access as such to
information about which lexicon a particular element belongs to
or how it became a member of that lexicon. MacSwan (2005,
p. 11) makes this point clear in the context of distinguishing
between borrowing and borrowing for the nonce:2 This idealization also relates to the distinction between
competence and performance outlined in Chomsky (1965). Competence is the tacit linguistic knowledge a speaker has,
whereas performance is the employment of this knowledge
in actual production. In formal grammar, the focus has been
on developing competence models based on the linguistic
performance of speakers. For the purposes of a synchronic theory of language contact,
the distinction between BORROWING and NONCE BORROWING
is unimportant: The difference in meaning depends on a word’s
history – inaccessible to a linguistic system represented in the
mind/brain of an individual. This has been a successful research strategy insofar as it
has uncovered a range of new generalizations and theoretical
proposals concerning the unique human ability for language. However, if the models are going to be ecologically valid,
they clearly need to generalize beyond monolinguals. Chomsky
(1986) emphasizes the notion of I-language, which connotes an
individual, internal, and intensional language. A core task has
been to try to answer the question of what a possible mental
grammar is. The hypothesis is that there are constraints on what
can be a human mental grammar, constraints that may or may
not hold cross-linguistically. The theories and models that are
developed should simultaneously include the possible structure
and exclude the impossible ones. The same argument could be made for mixing more generally. In the present paper, we will make such an argument as part
of developing a non-lexicalist approach to language mixing (cf. Multilingual Individuals and Their The challenge is to account for the patterns found in terms of
general properties of the grammar. Notice that only in this way
can the phenomena of code-mixing help refine our perspective on
general grammatical theory. If there were a special and separate
theory of code-mixing, it might well be less relevant to general
theoretical concerns. For a long time, formal approaches to grammar have explicitly or
implicitly followed the idealization set forth in Chomsky (1965,
p. 3): Linguistic theory is concerned primarily with an ideal speaker-
listener, in a completely homogeneous speech-community,
who knows its language perfectly and is unaffected by such
grammatically irrelevant conditions as memory limitations, In what follows, we will adopt this perspective and make use of
it in our comparative analysis of word-internal language mixing. (10) PF Interface Condition González-Vilbazo and López, 2011; Pierantozzi, 2012; Bandi-
Rao and den Dikken, 2014; Alexiadou et al., 2015; Grimstad
et al., 2014, 2018; Merchant, 2015; Lillo-Martin et al., 2016;
Alexiadou, 2017b; Grimstad, 2017; Riksem, 2017, 2018). This
work tries to respond to the following challenge posed by
MacSwan (2013,2014, pp. 347: 18): ‘Whether a sufficiently
rich non-lexicalist theory involving late insertion, such as
distributed morphology [. . .], could achieve similar results [to
lexicalist approaches] has not been investigated.’ An explicit
such model has been provided in López (2018), the aim being
to develop a minimalist Distributed Morphology model of
code-switching, labeled MDM in his work. From an MDM
perspective, bilinguals have only one list containing the roots
from their two languages, List 1 in Distributed Morphology,
and only one list containing the vocabulary insertion rules of
their two languages, List 2 in Distributed Morphology. Put
differently, multilinguals have more vocabulary items at their
disposal to realize a particular syntactic structure. We will
come back to this, in particular in the section “Discussion and
Analysis.” From this perspective, it is obvious why studying multilingual
individuals is crucial if you want to discover the potential range
of human grammars. The current contribution focuses on word-
internal language mixing, which is but one of many aspects of
multilinguals’ knowledge and use of language. Within the literature studying language mixing, it is either
argued that special mechanisms are needed to account for
language mixing or that such mechanisms are not needed. Null
theories or constraint-free theories are theories of the latter type,
they assume that mixing is not constrained by special rules
unique to mixing (cf. Mahootian, 1993; MacSwan, 1999, 2000,
2005, 2014; González-Vilbazo and López, 2011, 2012; Pierantozzi,
2012; Bandi-Rao and den Dikken, 2014; Grimstad et al., 2014,
2018 Alexiadou, 2017a). Rather, ‘exactly the same principles
which apply to monolingual speech apply to code-switching’
(Mahootian, 1993, p. 3). This aligns with the following quote from
Muysken (2000, p. 3): The current contribution is intended to further the work
done in the emergent non-lexicalist program, in particular by
contributing a larger cross-linguistic picture of patterns of word-
internal mixing. The Verbal Domain Alexiadou (2017a) discusses word internal mixing in two Greek
varieties, English-Cypriot Greek, and German-Greek. Both these
varieties make use of two different patterns when it comes to
mixing: What is typically labeled the light verb construction
(LVC) pattern, and the affixal pattern. These are illustrated in (11)
for Greek-German. (14)
a. b. More concretely, for a string like (15a), from Embick (2004,
p. 365), the structure is provided in (15b). (14)
a. b. a. b. a. b. (14)
a. (11) a. kano
abschalten
do.1SG
kick.back.INF
‘I am kicking back.’ (Alexiadou, 2011: ex. [12])
b. skan-ar-o
scan-AFF-1SG
‘I am scanning.’ (Alexiadou, 2011: ex. [13]) More concretely, for a string like (15a), from Embick (2004,
p. 365), the structure is provided in (15b). More concretely, for a string like (15a), from Embick (2004,
p. 365), the structure is provided in (15b). (15) a. The metal flatt-en-ed. b. (15) a. The metal flatt-en-ed. Alexiadou (2017a, p. 174) provides further details about the
sociolinguistic context of these data; see her paper for further Alexiadou (2017a, p. 174) provides further details about the
sociolinguistic context of these data; see her paper for further
discussion and references. She also proposes an analysis of the
LVC pattern, a pattern that we won’t focus on in the current
paper. As shown in (12), the affixal pattern also exists in the Cypriot
Greek-English variety. (12) a. muv-ar-o
move-AFF-1SG
‘I am moving.’
b. kansel-ar-o
cancel-AFF-1SG
‘I
am
canceling.’
(Gardner-Chloros,
2009,
pp. 50–51) In languages like Greek, there are many verbalizing affixes
(see Alexiadou, 2017a, p. 179 and references therein). These are
typically taken to realize little v. (12) a. muv-ar-o
move-AFF-1SG
‘I
i
’ ‘I am moving.’ ypically taken to realize little v. Alexiadou (2017b, p. 182) proposes the following structure for
he affixal pattern. (16) Alexiadou (2017b, p. 182) proposes the following structure for
the affixal pattern. (16) In these examples, we see that the Greek affix attaches to the
German and/or English root. A dedicated affix, -ar-, is used to
verbalize the root. This particular affix triggers stress shift to the
penultimate syllable. Even though it is used less frequently than
many other verbalizing affixes in Modern Greek, it is the default
verbalizer in these mixing varieties. In Greek, there is a kind of default affix that is used to
verbalize non-native roots. This affix can be used in a range
of contexts. WORD-INTERNAL MIXING IN VARIETIES
INVOLVING GREEK 2Other scholars do not agree, arguing that speakers can distinguish between
nonce borrowing, established loans, and (multiword) code switches (Poplack and
Dion, 2012; Poplack, 2018). In particular, Poplack (2018, pp. 156–157) argues that
differential patterns of language use related to these three categories demonstrate
their psychological reality. We do not disagree that they may behave differently, but
the question is whether or not this reflects the underlying linguistic competence
of the speaker, at the fine-grained level of minimal syntactic units. The current
contribution suggests that they may not differ. In this section, we will consider word-internal mixing in
languages that are mixed with Greek, notably English and
German. The section “The Verbal Domain” discusses the verbal September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org 4 Alexiadou and Lohndal Units of Language Mixing We will now consider how Alexiadou (2017a) accounts for the
asymmetries. Her analysis is based on Distributed Morphology
(Halle and Marantz, 1993; Embick and Noyer, 2007; Embick,
2015), where syntactic categorization takes place in the syntax
via the presence of functional heads. Roots are category-neutral
and need to be categorized in the syntax by way of merging
with a functional head (see Alexiadou and Lohndal, 2017b for
more on various analytical possibilities). Little v turns a root
into a verb whereas little n turns a root into a noun, as shown
in (14). domain whereas the section “The Nominal Domain” is concerned
with the nominal domain. Our goal is not to develop and
motivate previous in-depth analyses of the data, rather to
present the data and the gist of the analysis for the sake of
the cross-linguistic comparison in the section “Discussion and
Analysis.” The Verbal Domain Put differently, this default affix (v) can be seen
as incorporating into a non-native root, cf. Bandi-Rao and den
Dikken (2014). Alexiadou (2011) observes that the affixal patterns are
not in free distribution in Greek-German. (11a) and (12b)
contain a monosyllabic root, yet not all monosyllabic roots
can take part in the affixal pattern. As an example, (13)
contains a monosyllabic root, yet only the light verb strategy is
licit. Frontiers in Psychology | www.frontiersin.org The Nominal Domain (13) na kanun kämpfen
SUBJ do.3PL fight.INF
‘They should fight.’
(Alexiadou, 2011: ex. [25]) (13) na kanun kämpfen
SUBJ do.3PL fight.INF
‘They should fight.’
(Alexiadou, 2011: ex. [25]) Alexiadou
(2011,
2017b)
and
Alexiadou
et
al. (2015)
discuss cases of word internal mixing in the noun phrase
involving
Greek-German
and
Greek-English
pairs. All
the
examples
provided
are
of
the
following
form:
the
involve a German or English stem to which a Greek affix is
added. Furthermore, it is always an English/German root which
combines with a Greek affix. The combination of a Greek root
with a German inflection is rejected by speakers. September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org 5 Alexiadou and Lohndal Units of Language Mixing (17)
Mixing
German
Greek
a. to
matrátz-i
die
Matratze
to.strom-a
the.N
mattress.N
the.F
mattress
the
mattress.N
b. to
regál-i
das
Regal
to raf-i
the.N shelf.N
the.N shelf
the shelf-N
c. o. Vetrét-as
der
Vertreter
o
andiprosopos
the.M
representative-
M
the.M
representative
the
representative.M
d. i
Káss-a
die
Kasse
to tami-o
the.F
cashpoint.F
the.F
cashpoint
the
cashpoint.N
e. i
Kél-a
der
Keller
to kelar-i
the.F cellar.F
the.M cellar
the
cellar.N roots, as argued for in detail in Kramer (2015) nor inflection class,
as they can be freely assigned structurally. (17) (21)
word-internal (21) word-internal The Verbal Domain González-Vilbazo and López (2011) show that mixing happens
word-internally, as depicted in (22). (22)
Wir
utilisieren
spanische
Wörter, die
We
use
Spanish
words
than
dann
alemanisiert
werden
y
then
Germanized
are
and
hacen
klingen
un
poco
raro
do
sound
a
bit
strange Fotopoulou (2004) cites Greek-German examples where no
inflection is added to the German noun and the determiner
comes from Greek: (19) mu pire tin
Ausfahrt
me took the.F.ACC exit (19) mu pire tin
Ausfahrt
me took the.F.ACC exit ‘We use Spanish words, that are then Germanized and sound
a bit strange.’ Goula (2017) observes similar cases of mixing in Greek-
English, which she tested experimentally. When the morphology
of the noun is not adapted, the determiner may come from Greek. She moreover notes that sometimes the determiner bears default
gender, e.g., neuter for inanimates, see Tsimpli and Hulk (2013)
and Anagnostopoulou (2017b) for recent discussion, or carries
over the gender of its Greek translation equivalent, masculine in
the example below. This the so-called analogical gender strategy,
see López (2018) for further discussion. g
(González-Vilbazo and López, 2011, p. 833) In word internal mixing, speakers are able to combine a
Spanish root with a German verbal inflection, as shown in (23). However, speakers cannot in general combine a German root
with a Spanish infinitival inflection (24) (González-Vilbazo and
López, 2011, p. 835). (23)
a. cos-ier-en
b. utilis-ieren
‘sew’
‘use’
(24)
a. ∗benutz-ear b. ∗näh-ear
‘use’
‘sew’ (23)
a. cos-ier-en
b. utilis-ieren
‘sew’
‘use’
(24)
a. ∗benutz-ear b. ∗näh-ear
‘use’
‘sew’ (20)
a. to
map
b. o
map
the.N map
the.M map (20)
a. to
map
b. o
map
the.N map
the.M map (20)
a. to
map
b. o
map
the.N map
the.M map (20)
a. to
map
b. o
map
the.N map
the.M map Goula (2017) shows that the translation equivalence choice
was preferred in the comprehension task, while the default choice
was preferred in the production task. As González-Vilbazo and López (2011, p. 835) emphasize, in
the German-Spanish variety “German/Spanish bilinguals accept
(and produce) nonce words created by joining together a Spanish
root and a German verbal inflection. However, these same
bilinguals reject a word made up of a German root and a Spanish Alexiadou (2011, 2017b) proposes the structure in (21): gender
and inflection class information are on n. WORD-INTERNAL MIXING IN
GERMAN-SPANISH In this section, we will consider word-internal mixing in a variety
whereby German and Spanish are mixed. Before we turn to
the verbal and nominal domains, a brief note about the data
is in order. The data are taken from González-Vilbazo (2005)
and González-Vilbazo and López (2011). They report that the
data come from the German School of Barcelona. This school
consists of between 1,000 and 1,400 students who from an
early age generally have a high exposure to both languages. Their informants belong to a homogenous socio-economic
community whereby the majority are Spanish/German bilinguals. In this section, we will consider word-internal mixing in a variety
whereby German and Spanish are mixed. Before we turn to
the verbal and nominal domains, a brief note about the data
is in order. The data are taken from González-Vilbazo (2005)
and González-Vilbazo and López (2011). They report that the
data come from the German School of Barcelona. This school
consists of between 1,000 and 1,400 students who from an
early age generally have a high exposure to both languages. Their informants belong to a homogenous socio-economic
community whereby the majority are Spanish/German bilinguals. This multilingual environment, much like other multilingual
environments, make use of language mixing. As González-
Vilbazo and López (2011, p. 837) report, the students are proud
of their mixing practices. Gardner-Chloros (2009, p. 50) reports the following Greek-
English mixing cases in the variety of British born Cypriot Greek
speakers: (18)
BBC Mixing
English
Greek
a. marketa (F)
market
agora (F)
b. hoteli (N)
hotel
ksenodohio (N)
c. kuka (F)
cooker
furnos (M)
d. fishiatiko (N)
fish and chip
shop
–
e. kitsi (N)
kitchen
kuzina (F)
f. ketlos (M)
kettle
–
g. haspas (M)
husband
andras (M) This multilingual environment, much like other multilingual
environments, make use of language mixing. As González-
Vilbazo and López (2011, p. 837) report, the students are proud
of their mixing practices. Frontiers in Psychology | www.frontiersin.org (28) German/English root + Greek affix According to González-Vilbazo and López (2011), every
Spanish verb carries a specification for its conjugation class. Furthermore, v bears unvalued features for conjugation class. In order to value this feature, V-to-v movement needs to take
place. Crucially, German verbs do not carry a specification
for conjugation class. Therefore, it cannot be the case that a
German verbal root could incorporate into a v that is specified
for conjugation class. However, a Spanish verbal root can be
embedded and incorporate into a German v because this v is
unspecified for conjugation class, as in (25b). The light v is always
realized with the Spanish verb in (25a). González-Vilbazo (2005) further cites examples which do
not involve affixation, but as we have seen above for Greek, a
determiner from Spanish in combination with a German noun. Put differently, Spanish functional material is able to combine
with a German root. (29)
a. la Stunde
b. el Lehrer
the hour
the teacher
‘the hour’
‘the teacher’ (29)
a. la Stunde
b. el Lehrer
the hour
the teacher
‘the hour’
‘the teacher’ (29)
a. la Stunde
b. el Lehrer the teacher ‘the teacher’ (25) a. b. (25) a. b. In (29), the gender of the article corresponds to the gender of the
German noun. As Spanish lacks neuter, all German nouns that
are neuter are preceded by the Spanish masculine article, which
is the default gender in the language. In (29), the gender of the article corresponds to the gender of the
German noun. As Spanish lacks neuter, all German nouns that
are neuter are preceded by the Spanish masculine article, which
is the default gender in the language. (25) a. The reverse pattern is also found: (30) die ley
the.F law
‘the law’ (30) die ley
the.F law
‘the law’ (30) die ley
the.F law
‘the law’ This latter case is more complex. As González-Vilbazo (2005)
details, feminine Spanish nouns are preceded by the feminine
German determiner no matter the gender of the German
translation equivalent (in this case, neuter). In other words, this
group preserves its gender. In the case of masculine Spanish
nouns, the only articles that are allowed are those that are
syncretic for masculine and neuter. As a result, indefinite
determiners are preferred, as shown in (31a). This is the reverse in the Greek-German/English cases: This is the reverse in the Greek-German/English cases: The Verbal Domain In fact, the nominal
mixing data support the view that neither gender is a property of September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org 6 Alexiadou and Lohndal Units of Language Mixing (27) Spanish root + German affix (27) Spanish root + German affix verbal inflection.” As we saw above, the reverse is observed in
mixing varieties of Greek: A German or English root always
combines with a Greek affix, and the combination of a Greek root
with a German inflection is rejected by speakers. This is the reverse in the Greek-German/English cases: Frontiers in Psychology | www.frontiersin.org Mixing in American Norwegian American Norwegian is a heritage language of Norwegian. It
is spoken in North America, mainly in the United States. Its speakers today are descendants of immigrants who came
from Norway approximately from the 1850s until the 1920s. This makes American Norwegian a minority language which
exists in a language community significantly dominated by
English. All American Norwegian speakers share the following
characteristics: American Norwegian is their L1, and in many
cases this was their only language up until school age. In recent
decades, all speakers of American Norwegian have been heavily
English-dominant, resulting in significant lexical access issues
when speaking American Norwegian. This means that they often
display a mixture of the two languages, making their speech ideal
for studying language mixing (Grimstad, 2018; Riksem, 2018). (35)
a. spend-e
b. rais[e]-er
c. catch-a
spend-INF
raise-PRES
catch-PAST
‘to spend’
‘raise(s)’
‘caught’
(blair_
(blair_WI_
(sunburg_
WI_02gm)
01gm)
MN_03gm)
(36)
a. road-en
b. voting-a
c. fenc[e]-a
road-
voting-
fenc[e]-
DEF.SG.M
DEF.SG.F
DEF.PL.N
‘the road’
‘the voting’
‘the fences’
(webster_
(westby_
(coon_valley_
SD_02gm)
WI_01gm)
WI_06gm) In the verbal cases, we see that an English item can acquire
both the infinitival form, the present tense and the past tense
(see Eide and Hjelde, 2015 for more on tense in American
Norwegian). In the nominal forms, the nouns can be inflected
according to definiteness, number, and gender/declension
class. Haugen (1953) conducted the first large-scale investigation of
American Norwegian. He provides examples like the following. (32) Så play-de
dom game-r
then play-PAST they game-PL
‘Then, they played games’ (32) Så play-de
dom game-r
then play-PAST they game-PL
‘Then, they played games’
(33) Så happ[e]n-a de så at e kåm inn på office-en te
so happen-PAST it so that I came in to office-DEF to y p y
g
(33) Så happ[e]n-a de så at e kåm inn på office-en te
so happen-PAST it so that I came in to office-DEF to
statskasserar-en då
national.treasurer-DEF then
‘So it happened that I came into the office of the national
treasurer.’ Riksem et al. (in press) analyze the mixing cases in (35)
and (36) as cases whereby an English root is embedded into a
Norwegian grammatical structure. Crucially, the English roots do
not have any grammatical features. Rather, features are merged
in the functional spine and morphophonological exponents
come to realize them. (28) German/English root + Greek affix September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org 7 Units of Language Mixing Alexiadou and Lohndal (28) German/English root + Greek affix The only exception
discussed involves genitive case, where no switch is allowed,
although both masculine and neuter indefinite articles bear the
same form, (31b). According to López (2018), this is so as German
nouns often bear genitive morphology themselves, i.e., there is
a concord effect between the determiner and the noun, (31c). As Spanish lacks case morphology, genitive German inflection
is blocked from appearing on a Spanish noun. This contrast
suggests to us that not only gender but also case inflection
is on n, as argued for Greek in Alexiadou (2017b), and see
Anagnostopoulou (2017a) for a general claim on the relationship
between n and case. Since n is Spanish, no case morphology can
appear there and concord is blocked: The Nominal Domain González-Vilbazo (2005, p. 141 f.) notes that nominal word
internal mixing is less frequent in the Spanish-German variety,
but it appears to obey a morphological restriction similar to that
of verbal mixing: a Spanish affix cannot attach to a German stem,
while the reverse is allowed: (26) a. ∗Stuhl-o
chair.MASC
b. Segurat-en
security.man-PL González-Vilbazo (2005) notes that such nouns end in -e in
the singular, while they take the affix -en in the plural. The
singular marking suggests that they are not Spanish nouns as they
should end in -a. He argues that this case is different from that
of verbal mixing: In the verbal mixing there is overt verbalizing
morphology, e.g., ier- that enables the further suffixation of
German inflectional material. This affix creates a German verbal
stem to which further German affixes can be added. This is not
the case in the nominal domain. The Greek mixing data seen
in the previous section further support this. There is no overt
nominalizing morphology present. To this end González-Vilbazo
(2005) suggests that a covert affix is present that creates a German
base to which further affixation is possible. We note that within
Distributed Morphology, this intermediate step is not necessary:
Little n is the nominalizer and carries all inflection. From this
perspective, in Spanish-German the direction of affixation is as
shown in (27). (31)
(Spanish = masculine, German = neuter)
a. ?der/ein
cuaderno
DEF/INDEF
notebook
b. ∗eines
tenedor
INDEF.GEN
fork
c. eines
Mannes
INDEF.GEN
man.GEN In the next section, we turn to a different pair of languages,
namely English and Norwegian. WORD-INTERNAL MIXING IN VARIETIES
INVOLVING NORWEGIAN where L stands for lexical item and INFL for inflectional
morphology. (34) a. LSEC
+
INFLMAIN
b. LMAIN
+
INFLMAIN
c. ∗LSEC
+
INFLSEC
d. ∗LMAIN
+
INFLSEC (34) a. LSEC
+
INFLMAIN
b. LMAIN
+
INFLMAIN
c. ∗LSEC
+
INFLSEC
d. ∗LMAIN
+
INFLSEC In this section, we will consider mixing of varieties where
Norwegian is one of the languages. We will consider two
varieties: the heritage language American Norwegian in the
section “Mixing in American Norwegian,” and then Norwegians
in Norway who mix English into their Norwegian in the section
“Mixing of English and Norwegian in Spoken Norwegian.” As we
will see, the same patterns are found in both varieties. (34c) does not hold for bigger mixed chunks, and some other
cases studied by Grimstad (2017); see her work for details.3 Riksem et al. (in press) provide a range of examples of verb-
internal and noun-internal mixing. The former is illustrated in
(35) and the latter in (36). Frontiers in Psychology | www.frontiersin.org Mixing in American Norwegian know that I have left before know that I have left before (39) (39) ‘I know that I have left before.’ (39) Turning to word-internal mixing in the nominal domain,
Sunde (2016, p. 143) provides examples such as (42). (42) a. Fant traden
found trade.DEF
‘I found the trade.’ (42) a. Fant traden
found trade.DEF ‘I found the trade.’ b. Jeg kommer til å holde den scouten
I come to to holde that scout.DEF
‘I will hold that scout.’ In general, then, we see that English roots can combine with
Norwegian functional material to yield instances of word-internal
mixing. c. Inspect kniven i inventoryen min
inspect knife.DEF in inventory.DEF my
‘Inspect the knife in my inventory.’ Mixing of English and Norwegian in
Spoken Norwegian Again, we see that the lexical items can come from English
whereas the morphology comes from Norwegian. The same analysis as Riksem et al. (in press) develop for
American Norwegian can also be used for the data seen in this
sub-section: English roots are merged into structures based on
Norwegian features. No further assumptions need to be made. Norwegians generally have a high proficiency in English, in
particular the younger generations. In Norway, it is well-known
that they often mix English words into their Norwegian. A recent
study by Sunde (2016) investigates a gaming community, which
is a community where English is especially important. Based
on oral and spoken data, Sunde (2016) argues that Norwegian
is clearly the matrix language in Myers-Scotton (1993) sense,
as it is the language contributing the morphosyntactic frame. That is, Norwegian determines the order of morphemes and
functional morphology. This corresponds to what we in the
section “The Verbal Domain” called main language, which is
a more general label not specifically associated with Myers-
Scotton’s implementation. One example of this is provided
in (40); the translation into English is ours (Sunde, 2016,
p. 138). Mixing in American Norwegian .]
overwhelemed by all terrorists.DEF
‘Even if he traded himself out, he still let his team mate,
who remained on A, behind, and he was overwhelmed
by all the terrorists.’ (40) Selv om han trada seg sjøl ut [. . .] så lot han fortsatt
even if he traded REFL self out so let he still
team maten sin som
team mate his who
var på A værende igjen aleine, og han blei
was on A remaining again alone and he was
overwhelma av alle terroristene [. . .]
overwhelemed by all terrorists.DEF
‘Even if he traded himself out, he still let his team mate,
who remained on A, behind, and he was overwhelmed
by all the terrorists.’ In this structure, definiteness, number, and gender are all
encoded on one functional projection. It could also be that gender In this structure, definiteness, number, and gender are all
encoded on one functional projection. It could also be that gender
is encoded on n (Alexiadou, 2004, 2017a; Kramer, 2015), this
particular choice does not matter for present purposes. The
features then combine with the root to yield the actual exponent,
as shown in (38). is encoded on n (Alexiadou, 2004, 2017a; Kramer, 2015), this
particular choice does not matter for present purposes. The
features then combine with the root to yield the actual exponent,
as shown in (38). (38) (38) overwhelemed by all terrorists.DEF Sunde (2016, p. 140) shows that instances of infinitival, present
and past tense forms occur. Some of her examples are given in
(41). (41) a. De har ikke tid til å defuse bomben. they have not time to to defuse bomb.DEF
‘They do not have time to defuse the bomb.’ ‘They do not have time to defuse the bomb.’ b. Carryer deg lett ut. carry you easily out
‘I easily carry you out A similar logic underlies verbal mixing. An abstract structure
is provided in (39), and here, the root again combines with
the tense morpheme to yield the exponent renter ‘rents.’
Again, the structure is taken from Riksem et al. (in press);
see that paper for additional justification of this particular
structure. c. Nå overextenda de veldig. now overextended they a.lot c. Nå overextenda de veldig. d. Vet at jeg har leavet før. d. Vet at jeg har leavet før. Mixing in American Norwegian An abstract structure for the American
Norwegian noun phrase can be illustrated in (37) (Riksem et al.,
in press). More recently, the establishment of the Corpus of American
Nordic Speech (Johannessen, 2015) has generated a lot of
new work on American Norwegian (see e.g., the summary in
Riksem, 2018). In particular, Grimstad et al. (2014), Riksem
(2018), Riksem et al. (in press), and Grimstad et al. (2018)
have studied language mixing based on corpus data from
50 speakers of American Norwegian. These speakers are all
between 70 and 100 years of age and constitute probably
the last generation of American Norwegian speakers. The
following discussion will be based on data from Riksem et al. (2017). (37)
3Grimstad et al. (2014) provide an analysis of how these bigger mixed chunks can
be analyzed within this framework (see also Grimstad, 2017). Another similar type
of exception involves the use of the English plural marker -s with an English noun
in an otherwise Norwegian noun phrase. See Riksem (2018) on this phenomenon. (37) ) In general, Norwegian is the main language while the other
is the secondary language (Åfarli, 2015). The main language can
be argued to provide the overall grammatical structure, including
more or less all derivational and inflectional morphology. In
many cases, the lexical items also come from the main language,
but when they do not, they come from the secondary language. Åfarli, 2015 and Riksem et al. (2018) depict this as in (34) 3Grimstad et al. (2014) provide an analysis of how these bigger mixed chunks can
be analyzed within this framework (see also Grimstad, 2017). Another similar type
of exception involves the use of the English plural marker -s with an English noun
in an otherwise Norwegian noun phrase. See Riksem (2018) on this phenomenon. September 2018 | Volume 9 | Article 1719 8 Alexiadou and Lohndal Units of Language Mixing (40) Selv om han trada seg sjøl ut [. . .] så lot han fortsatt
even if he traded REFL self out so let he still
team maten sin som
team mate his who
var på A værende igjen aleine, og han blei
was on A remaining again alone and he was
overwhelma av alle terroristene [. . WORD-INTERNAL MIXING IN TELUGU In this section, we will consider data from Classical Telugu
(a South-Central Dravidian language) reported by Bandi-Rao
and den Dikken (2014). The data are based on acceptability
judgments. They observe an asymmetry similar to the one we
have observed for other pairs discussed above when looking
at a mixing variety of English-Telugu: Only Telugu roots can
combine with English -ify. It is not possible for an English root to September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org 9 Units of Language Mixing Alexiadou and Lohndal TABLE 1 | Possible and impossible patterns of word-internal mixing. Language pair
Possible mixing
Impossible mixing
root
inflection
root
inflection
German-Spanish
Spanish
German
German
Spanish
German-Greek
German
Greek
Greek
German
English-Greek
English
Greek
Greek
English
English-Norwegian
English
Norwegian
Norwegian
English
English-Telugu
Telugu
English
English
Telugu combine with the Telugu -inc affix, as the contrast between (43)
and (44) shows. (43)
a. My sister kal(i)p-ified
the curry. kalp ‘stir’
b. You have to
kar(i)g-ify the butter
karg ‘melt’
c. The teacher made the
child
Ed(i)c-ify in school. Edc ‘cry’
(Bandi-Rao and den Dikken, 2014, p. 163)
(44) ∗vaaDu nanni love-inc-EEDu. he.NOM me.ACC love-DO-PST.AGR
‘He loved me.’ (Bandi-Rao and den Dikken, 2014, p.165)
TABLE 1 | Possible and impossible patterns of word-internal mixing. Language pair
Possible mixing
Impossible mixing
root
inflection
root
inflection
German-Spanish
Spanish
German
German
Spanish
German-Greek
German
Greek
Greek
German
English-Greek
English
Greek
Greek
English
English-Norwegian
English
Norwegian
Norwegian
English
English-Telugu
Telugu
English
English
Telugu
One potential answer to this question is to suggest that TABLE 1 | Possible and impossible patterns of word-internal mixing. combine with the Telugu -inc affix, as the contrast between (43)
and (44) shows. TABLE 1 | Possible and impossible patterns of word-internal mixing. Language pair
Possible mixing
Impossible mixing combine with the Telugu -inc affix, as the contrast between (43)
and (44) shows. T
L combine with the Telugu -inc affix, as the contrast between (43)
and (44) shows. (43)
a. My sister kal(i)p-ified
the curry. kalp ‘stir’ b. You have to
kar(i)g-ify the butter
karg ‘melt’ c. The teacher made the
child
Ed(i)c-ify in school. Edc ‘cry’ (Bandi-Rao and den Dikken, 2014, p. 163) (44) ∗vaaDu nanni love-inc-EEDu. (44) ∗vaaDu nanni love-inc-EEDu. WORD-INTERNAL MIXING IN TELUGU he.NOM me.ACC love-DO-PST.AGR he.NOM me.ACC love-DO-PST.AGR ‘He loved me.’ (Bandi-Rao and den D He loved me.’ (Bandi-Rao and den Dikken, 2014, p.165) One potential answer to this question is to suggest that
the asymmetries we observe are simply an effect of the main
language. In other words, the morphosyntactic spine comes from
the language whose affixes the speakers employ, i.e., Greek,
English, Norwegian and German, respectively (cf. also Grosjean,
2008, 2013 on the notion of ‘language mode’). However, it
is important to clarify what we mean by main language. For
instance, Myers-Scotton (1993, 2002) and Jake et al. (2002) argue
that language mixing necessitates a distinction between matrix
language and embedded language. A matrix language is the
main language of the speaker and it has a grammatical correlate:
It is responsible for word order and for providing functional
morphemes. The embedded language can provide lexical items. Scholars have extensively discussed the predictions and factual
accuracy of the matrix language model (see MacSwan, 2014,
pp. 14–16 and references therein). We follow Åfarli (2015) and
Riksem et al. (in press) in arguing that main and secondary
languages, to the extent that they are valid, are observational
phenomena, not theoretical primitives. The authors attribute this to the fact that Telugu affix is
an incorporator, while the English affix is not. They relate this
to English systematically disallowing incorporation into verbal
heads. For example, English does not allow (45) but instead
makes use of (46). (45) a. ∗John meat-eats. b. ∗John up-looked the number. (46) a. John eats meat. b. John looked up the number. If incorporation were to take place in (44), an ill-formed head
at PF would be the result, assuming that mixing below the head-
level is banned (MacSwan, 1997, 2000), a claim Bandi-Rao and
den Dikken (2014) endorse. Thus, it is avoided. By contrast, the
Telugu root and the English affix only come together as a unit
at PF, i.e., the Telugu root never incorporates into -ify because
principles of English grammar do not license it. Evidence that this may be problematic as a general answer
is provided by the Telugu cases since there, it is the secondary
language that provides the functional morphology. Furthermore,
as González-Vilbazo (2005) observes, speakers reject forms where
a Spanish affix attaches to a German stem. Such data suggest that
the relevant factor cannot be the division of labor between main
language and secondary language. WORD-INTERNAL MIXING IN TELUGU Importantly, though, a caveat
is in order. The crucial data from both Telugu and German-
Spanish are based on acceptability judgments (see Toribio,
2001a,b on the latter in studying mixing). In other work on
language mixing, it has been observed that judgments are not
always reliable indicators of the underlying grammar. Let us
consider one such example. September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org DISCUSSION AND ANALYSIS The sections “Word-Internal Mixing in Varieties Involving
Greek,” “Word-Internal Mixing in German-Spanish,” “Word-
Internal
Mixing
in
Varieties
Involving
Norwegian,”
and
“Word-Internal Mixing In Telugu” demonstrate that there are
some interesting differences between the various varieties. As
González-Vilbazo and López (2011, p. 835) emphasize, mixing
between a Spanish root and a German verbal inflection is fine,
but the same individuals reject an element consisting of a
German root and a Spanish verbal inflection. Alexiadou (2017a,
p. 167) notes that the asymmetry is the reverse for Greek mixing
varieties: It is always a German/English root combining with a
Greek affix. For American Norwegian, the root can be either
Norwegian or English, but we generally do not find a Norwegian
root with English inflection (Grimstad, 2017; Grimstad et al.,
2018). This is also the case in the nominal domain. Furthermore,
Telugu displays an asymmetric pattern whereby Telugu roots can
combine with English functional morphology but English roots
cannot appear together with Telugu functional morphology. The
following table offers an overview of the patterns seen in our
survey. In mixing between English and Spanish, Moro (2014) reports
that whereas speakers accept the pattern in (47), they reject the
pattern in (48). (47) a. el employer
‘the employer’
b. la washing machine
‘the washing machine’
(48) a. ∗the casa
‘the house’
b. ∗the vecina
‘the neighbor’ b. la washing machine
‘the washing machine’ (48) a. ∗the casa
‘the house’ (48) a. ∗the casa
‘the house’ The variation displayed in Table 1 raises the question of what
the source behind these various asymmetries are. b. ∗the vecina b. ∗the vecina
‘the neighbor’ ‘the neighbor’ Frontiers in Psychology | www.frontiersin.org 10 Alexiadou and Lohndal Units of Language Mixing This asymmetry would suggest that an English determiner
cannot appear together with a Spanish noun. As Liceras et al. (2008) make clear, such an asymmetry is not factually attested. Examples like (48) are attested in spontaneous production
(see also Liceras et al., 2005, 2008; Pierantozzi, 2012; López,
2018). Liceras et al. (2005, 2008) also show that the Spanish
determiner is preferred in language use. The scholars suggest
that such a preference can be accounted for by what they
label the Grammatical Features Spell-out Hypothesis (GFSH). The GFSH holds that functional categories containing highly
‘grammaticized’ features will be chosen. Because they have
gender, Spanish determiners contain more features than English
determiners, and therefore Spanish determiners will be preferred. DISCUSSION AND ANALYSIS This competition is determined on
the basis of the available VIs for the individual language pairs:
e.g., -ar- is the default realization of v in the case of Greek and
Germanic pairs, while -isier- the default realization of v in the case
of Spanish and German pairs, as Spanish has no overt realization
of v. Now, scholars do not always have large corpora available
to make the comparison that was just made. However, such
findings as in the English-Spanish mixing case may caution
us to draw too big conclusions based on acceptability data
alone. Judgments involving mixing are often negative due to
sociolinguistic reasons, suggesting that they often should be
combined with corpus evidence when such evidence is available
(see e.g., González-Vilbazo et al., 2013 for relevant discussion). However, assuming that the data reported are adequate, a
further option would be to appeal to morpho-phonology in
accounting for why some data points do not fit the overall
generalization. That is, in the spirit of MacSwan (1999, 2000,
2005, 2013), Bandi-Rao and den Dikken (2014), and MacSwan
and Colina (2014) and research cited there, those cases where a
root and functional morphology are not able to combine must
be ill-formed due to some PF-rule. This would be a language
specific rule that would hopefully relate to other properties in the
grammar, e.g., as to whether or not a specific functional element
is able to incorporate with a root (cf. Bandi-Rao and den Dikken,
2014). Approaching this problem from the perspective of Distributed
Morphology (Embick and Noyer, 2007; Embick, 2015; Alexiadou,
2017a,b), we assume that nouns and verbs are syntactically
derived. In particular, they emerge when a-categorial roots
combine with categorizing heads (v and n): b. b. (49)
a. Let us illustrate this idea in some detail. We begin with the
observation that in the nominal domain, as also stated in López
(2018), two options are available to speakers: either to make
use of the default marking in, e.g., Spanish (masculine), Greek
(masculine for animates, neuter for in-animates) or to associate
with the gender of the translation equivalent, i.e., adopt the
analogical transfer strategy. By contrast, it is not clear what the
default gender is in German, for reasons that have to do with
gender shift in the history of the language (neuter to masculine;
Steinmetz, 1997). DISCUSSION AND ANALYSIS As Grimstad et al. (2018) point out in their discussion,
the GFSH is a hypothesis about production preferences
which is guided by a grammatical mechanism on the PF
side. (see Grimstad, 2017 for extensive discussion of this point). Speakers are able to shift from mode to mode, suggesting that in
the monolingual mode alternative realizations are blocked. In the
bilingual mode, matters are more complex. Let us illustrate this
by discussing two of our patterns: (50)
Spanish root +
German affix
vs. ∗German root + Spanish
affix
(51)
German/English root
+ Greek affix
vs. ∗Greek root + German/
English affix (51)
German/English root
+ Greek affix
vs. ∗Greek root + German/
English affix Both patterns involve cases where a root in combination
with v or n create the vP and nP phase, respectively (cf. Marantz, 2001, 2007 and Arad, 2003, 2005). In both cases, the
complement of the phase head comes from one language, while
the realization of n, v from the other language. We have rejected
above the GFSH, which appeals to visibility, though at first sight
our data seem compatible with this, as in (50) and (51), the
realization of v an n seems to come from the language that
makes more distinctions within a domain (e.g., case, gender,
declension classes, conjugation classes, etc.). But note that it
is not the case that all possible realizations of v and n are
found in the data. This is particularly clear in the Greek case
of mixing in the verbal domain, where the default verbalizer -
ar- is used, although the language has a very rich system of
verbalizers. Similar observations can be made for the nominal
domain, where mixing does not distribute nouns equally across
the 8 declension classes of Greek but instead picks class 2 for
masculine, class 3 for feminine, and class 6 for neuter nouns
[assuming Ralli (1994) classification, see also Alexiadou and
Müller (2008)]. Thus, it seems to us there is a default mechanism
to integrate Germanic roots into Greek morphology: speakers
pick the default/underspecified realization, if such a realization
is available. That is, the bilingual speaker in view of the fact that
she has more VIs at her disposal will pick an overt realization,
if a default such realization is available. The default realization is
the one that is compatible with the largest number of roots, i.e.,
the roots of both languages. Frontiers in Psychology | www.frontiersin.org September 2018 | Volume 9 | Article 1719 DISCUSSION AND ANALYSIS This would explain why it would not
be able to merge with an English root: Assuming that the
combination between the verbalizer and the root is local, inc
might simply be a realization of a v head in a higher phase,
and thus it cannot combine with the English root. Moreover,
note that the ungrammatical examples cited in Bandi-Rao and
den Dikken (2014) involve a stative verb combining with inc. That might very well be accidental. If not, however, and if inc
is more like English do/make, we may get a different flavors
of v effect, meaning that there may be incompatibility between
stative psych roots and causative/eventive semantics. A more
in-depth investigation of Telugu would be required in order to
investigate this, which goes beyond the scope of the current
paper. Matters are different in the verbal domain. We hold that -ify-
and its cognates across languages, i.e., Greek -ar-, German -isiere-
are realizations of v. In languages where v is overtly realized by
these forms, which are the default ones, roots combine with these
to form the word-internal mixed cases, just as we have seen in the
data reviewed in this paper. Spanish lacks verbalizers, although it has verbal conjugations. We assume that the features related to conjugation are
attached post-syntactically (Oltra-Massuet, 1999). Nevertheless,
the features need to match the root in order for the appropriate
conjugation to appear, ruling out Spanish inflection with German
roots, again a PF effect. Alexiadou (2017a, p. 183) provides additional examples here
given in (52), which are similar to the data in (30) showing that
German roots cannot combine with Spanish inflection. (52) a. ∗kämpf-ar-o
fight-AFF-1SG
b. ∗schwim-ar-o
swim-AFF-1SG
c. ∗lauf-ar-o
run-AFF-1SG (52) a. ∗kämpf-ar-o
fight-AFF-1SG The fact that speakers pick overt default realizations seems
to suggest that all illicit combinations are filtered-out at PF. We
assume as in Embick (2010) that the phase head determines
also the phonology of the whole phase, see also López et al. (2017) for further evidence. We mentioned earlier that several
word-internal switches in Greek are filtered out because of
phonotactics (cf. MacSwan, 1999, 2000; MacSwan and Colina,
2014). Therefore, it seems plausible to assume that if speakers
can pick among different types of n/v to combine with roots,
they pick those that will fit the general phonology/properties of
the phase head. Put differently, the phonology within a phase
head needs to be uniform. DISCUSSION AND ANALYSIS Thus, in the case of the Spanish-German pairs,
the system treats masculine and neuter alike, while feminine
nouns are always marked feminine. In the Greek mixing pairs,
the blocking of Germanic affixes on Greek roots is probably
a PF effect, as we will show below for the verbal domain as As already mentioned, we further assume that information
about inflectional class, gender, and case is realized on n, for
nouns (e.g., Alexiadou, 2004, 2017b, Kramer, 2015), and v
hosts verbalizers across languages. Once categorized, nP and
vPs become part of extend projections, which we assume to be
identical across languages. When it comes to bilingual speakers
it is important to distinguish between utterances in monolingual
mode and those in bilingual mode. Assuming that the abstract
clausal structure is universal, these productions will differ in
terms of realization and flavors of heads present in the structure September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org 11 Alexiadou and Lohndal Units of Language Mixing with English roots (see Haugen, 1953 and in particular Riksem,
2017). We do not find cases of Norwegian roots appearing with
English functional morphology, which may be due to Norwegian
being a heritage language and therefore, when English functional
morphology is used, speakers will not insert a Norwegian root (as
we know that they have quite significant problems with lexical
access, see again Grimstad, 2017, 2018 and Riksem, 2018). well. For instance, a Greek root cannot appear ending in a
consonant, thus bearing zero (German or English) morphology. A German/English root can, however, and this is why (17) is also
possible. Support for our view on how VIs are chosen comes
from another set of data discussed in López (2018) involving
Swahili/English word-internal mixing: English nouns are used in
such contexts, and they always have a Swahili noun class prefix. As noun classes play a role similar to gender and are associated
with n, this pair behaves similar to the other varieties we have
been discussing here. Telugu is a bit more complex basically because inc is not
exactly identical to ify, as it can be used to form non-lexical
causatives as well. This means that it might very well be
that inc realizes something higher than our verbalizing v, i.e.,
it is the realization of a make type v head, which takes a
vP as its complement. Frontiers in Psychology | www.frontiersin.org REFERENCES Arad, M. (2003). Locality constraints on the interpretation of roots: the case of
Hebrew denominal verbs. Nat. Lang. Linguist. Theory 21, 737–778. doi: 10.1023/
A:1025533719905 Åfarli, T. A. (2015). “A syntactic model for the analysis of language mixing
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A. (2017a). Building
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varieties. Z. Sprachwissenschaft 36, 165–192. doi: 10.1515/zfs-2017-0008 Chan, B. H.-S. (1999). Aspects of the Syntax, Production and Pragmatics of Code-
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London, University College London. Chan, B. H.-S. (1999). Aspects of the Syntax, Production and Pragmatics of Code-
Switching with Special Reference to Cantonese-English. Doctoral dissertation,
London, University College London. Chomsky, N. (1965). Aspects of the Theory of Syntax. Cambridge, MA: MIT Press. Alexiadou, A. (2017b). “Gender and nominal ellipsis,” in A Schrift to Fest Kyle
Johnson, Vol. 1, eds N. LaCara, K. Moulton, and A.-M. DISCUSSION AND ANALYSIS This is a far more refined role
of phonology than an across-the-board ban on word-internal
mixing. b. ∗schwim-ar-o
swim-AFF-1SG b. ∗schwim-ar-o swim-AFF-1SG c. ∗lauf-ar-o
run-AFF-1SG These examples show that a Greek verbalizer cannot combine
with a German root. Alexiadou (2017a, p. 184) argues that these
examples are ruled out for morphophonological reasons. She
points out that word-internal and word-initial consonant clusters
are dis-preferred in Greek. For that reason, Greek speakers
instead make use of the light verb strategy, as seen in (5a),
repeated here as (53). Finally, note that what we discuss here is largely compatible
with López’s (2018) view and model. There are, however,
several issues and questions that we would like to raise. A first issue relates to the problem of root-equivalence, i.e.,
the question of whether roots from two different languages are
interpreted identically by the Encyclopedia, List 3 in Distributed
Morphology. We agree with López (2018) that most likely
this is rarely the case (see also Grimstad, 2018 and Riksem,
2018 for discussion). Does this suggest that the two forms
have very different contexts of use or is it simply an issue of
retrieval? Moreover, it is not the case that languages have the
same inventory of roots (Alexiadou and Lohndal, 2017a), and
the implications of this should be examined in the context of
language mixing. (53) kano
abschalten
do.1SG kick.back.INF
‘I am kicking back.’ Furthermore, it should be noted that the examples in (52)
contain either an umlaut or a diphthong. Neither of these exist
in Greek phonology. Since Greek supplies the v, the output of
word formation via incorporation needs to adhere to Greek
phonotactics. Considering English-Norwegian, speakers combine English
roots with Norwegian functional morphology, they generally
do not combine Norwegian roots with English functional
morphology. This is arguably because they are in a ‘Norwegian’
language mode. However, as Grimstad (2017) shows, they do use
English morphology in the verbal domain, though importantly,
only in combination with English roots. In the nominal domain,
there are cases of English functional morphology appearing In addition, we think that his system predicts a lot more of free
variation than it is actually found in the data, a point López (2018)
himself also acknowledges. Moreover, the system predicts the September 2018 | Volume 9 | Article 1719 12 Alexiadou and Lohndal Units of Language Mixing morpho-phonological reasons rule out the particular mixing in
question. CONCLUSION Parts of this research has been supported by grant AL554/8-1 to
AA. In this paper, we have surveyed instances of word-internal
language mixing across several different language pairs. In
general, a root from one language can combine with functional
morphology from another. In cases where such a combination
is not licit, we have argued that there may be two reasons
why this is the case: Either because the language mode of the
speaker suggests that the functional morphology should come
from the language with overt default realization or because DISCUSSION AND ANALYSIS We have also shown how a decompositional model like
Distributed Morphology can be utilized to analyze the patterns. possibility of double realization of a particular feature. Though
such cases do exist, they are certainly limited. Finally, it is not
entirely clear how the competition between different realizations
of a particular feature is resolved. In other words, assuming the
subset principle (Halle, 1997), how do we decide which form is
more specific, the L1 or the L2 one? López (2018) argues that the
competition does not take place, as the conditions for insertion
of vocabulary items are very different. We have outlined above a
system that favors overt realizations but picks default forms, thus
blocking double realization. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication. ACKNOWLEDGMENTS We are grateful to reviewers for detailed feedback on this paper
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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. Riksem, B. R., Grimstad, M. B., Lohndal, T., and Åfarli, T. A. (in press). Language Riksem, B. R., Grimstad, M. B., Lohndal, T., and Åfarli, T. A. (in press). Language
mixing within verbs and nouns in American Norwegian. J. Compar. Ger. Syntax Riksem, B. R., Grimstad, M. B., Lohndal, T., and Åfarli, T. A. (in press). Language
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Sankoff, D., and Poplack, S. (1981). A formal grammar for code-switching. Pap. Linguist. 14, 3–45. doi: 10.1080/08351818109370523 Steinmetz, D. (1997). “The great gender shift and the attrition of neuter nouns
in West Germanic: the example of German,” in New Insights in Germanic
Linguistics II, eds I. Rauch and G. Carr (New York, NY: Peter Lang), 201–224. Copyright © 2018 Alexiadou and Lohndal. 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. September 2018 | Volume 9 | Article 1719 Frontiers in Psychology | www.frontiersin.org REFERENCES No use,
distribution or reproduction is permitted which does not comply with these terms. Sunde, A. M. (2016). Inspect kniven i inventoryen min.” Språklig praksis i et nytt
domene. [“Inspect kniven i inventoryen min.” Linguistic practices in a new
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IIASS VOLUME 15 (2022)
Social Sciences IIASS VOLUME 15 (2022) Innovative Issues and Approaches in Social Sciences IIASS is a double blind peer review academic journal published 3
times yearly (January, May, September) covering different social
sciences:
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the headlines were typeset in 14 pt. Arial, Bold 1 Thesara V.P. Jayawardane, PhD, is a Senior Lecturer and Head of the Department
of Industrial Management, University of Moratuwa, Sri Lanka (thesaraj@uom.lk)
2 Vathsala M. Wickramasinghe, PhD, is a Professor at the Department of
Management of Technology, University of Moratuwa, Sri Lanka.
(Vathsala@uom.lk) Abstracting and Indexing services Abstracting and Indexing services
COBISS, International Political Science Abstracts, CSA Worldwide
Political Science Abstracts, CSA Sociological Abstracts, PAIS
International, DOAJ, Google scholar. FINANCIAL EFFECT ON THE ADJUSTMENT OF
SPOUSES DURING COVID 19 LOCKDOWN
Thesara V.P. Jayawardane1, Vathsala M. Wickramasinghe2 Thesara V.P. Jayawardane1, Vathsala M. Wickramasinghe2 Objectives 1) To investigate whether individuals suffer from fears on
financial situation and uncertainty 2) To investigate the level of adjustment of spouses 3) To investigate support extended by the other family members 4) To analyse whether the support extended by the other family
members moderates the relationship between individuals’
fears on financial situation/uncertainty and the level of
adjustment of spouses This research has extracted evidence from former studies done on
the same issues through the literature review. As a result the study
has identified numerous ways how couples have been affected due
to financial fears and uncertainties and the way they adjust
themselves with the support of families. This research developed a
conceptual framework with the models of human behaviour and the
ways these couples would function. Abstract The objectives of this study was to investigate whether individuals
suffer from fear of financial situation and uncertainty, to investigate
the level of adjustment of spouses, to investigate support extended
by the other family members and, to analyse whether the support
extended by the other family members moderates the relationship
between individuals’ fears on financial situation/uncertainty and, the
respective level of adjustment of spouses. The data collected from an
online survey, through a random sample of 300 adults aged 25 years
and older residing in the Western Province, Sri Lanka were analysed. A conceptual model was developed and variables such as fear of
financial situation, uncertainty and extended family support available
for spouses was reviewed in relation to the adjustment of spouses. Four hypotheses were established. Statistical analysis wer performed
to identify moderator effect of resources available for spouses. It
were identified that COVID-19 created fear of financial situation and
uncertainty among married couples and, it was concluded that
support provided from their respective families contributed to
adjustment of Sri Lankan spouses. A key limitation was that there
was no equal participation from both genders. Future research may
include replicating the same study with a more diverse sample. Future researchers can conduct a follow-up study to identify long-
term effects of COVID-19 on Sri Lankan married couples. Key Words: Adjustment of Spouses, Uncertainty, Financia
Situation, Family Support, Covid-19 Key Words: Adjustment of Spouses, Uncertainty, Financial
Situation, Family Support, Covid-19 Introduction As one of the most talked about pandemics in the past few centuries,
Corona Virus disease (COVID-19) will have a special place in
historical documents over the immense impact it instigated up on
human lives globally. The difficulties it brought upon married couples,
the challenges it effectuated needs to be studied to identify the
impact COVID-19 had on spouses and families. Factors such as fear
of financial situation and uncertainty of the future, has caused many
couples to rift apart and the stability and the harmony of their
relationships had been maintained due to the support received from
their respective relatives and other family members. The results of
these situations will have an effect on the future generation and
therefore, a thorough understanding will benefit when analysing
functions of marital couples after COVID-19. The following objectives
were derived in order to identify the above-mentioned information. Literature Review
Adjustment of Spouses j
p
Adjustment of Spouses can be defined as “a condition in which there
is an overall feeling in husband and wife of pleasure and contentment
with their marriage and with each other” (Thomas, 1977) or the
accommodation of spouses to each other (Kendrick and Drentea,
2016). Many studies have identified that there is a relationship between adjustment of spouses and marital satisfaction (Bhaskar-
Shrinivas et.al, 2005). Some studies have identified that the inability
of partners to adjust in global crisis results in failure of marriage
(Schütter and Boerner, 2013). Lauring and Selmer (2010) have found
that couples who are adjustable in any crisis or disastrous situation
stay together through a life time. between adjustment of spouses and marital satisfaction (Bhaskar-
Shrinivas et.al, 2005). Some studies have identified that the inability
of partners to adjust in global crisis results in failure of marriage
(Schütter and Boerner, 2013). Lauring and Selmer (2010) have found
that couples who are adjustable in any crisis or disastrous situation
stay together through a life time. As Bakker (2005) points out, the perceptions of one spouse gets
transferred to other spouse whether it is a negative or a positive
influence and hence the adjustment level of one spouse will most
certainly influence the other. This is significant when the spouses are
experiencing an extreme condition like the COVID-19 pandemic. The
negativities of one spouse such as fears and uncertainties will affect
the adjustment level of the other. When consigned to isolation
without movement due to lockdown, the spouses will become highly
dependent on each other and, as a result the frequency and level of
interaction between the spouses also increase (Takeuchi et al.,
2002). Therefore, the adjustment of spouses also gets affected. Studies have found that the spouses, who start living under the same
roof for a longer duration, come across many differences in the way
they perceive values and beliefs (Andreason, 2008). This is
accelerated with financial fears and other uncertainties that they face
due to COVID-19. In a study done by Shaffer and Harrison (2001) it was identified that
the adjustment of spouses is smoother when there were no financial
barriers or uncertainties present. Accordingly, when the spouses
were employed and had no fear or uncertainty of the future, they
adjust well in their marriages. Literature Review
Adjustment of Spouses Shaffer and Harrison (2001) also
identified that spouses who had support from their respective
extended families were better in adjustment than compared to those
without such support. This proposes that the external support such
as the family values, cultural beliefs and social pressure brought by
the extended families ensured the relationship was maintained firmly
(Cerdin and Selmer, 2013). All these studies have commonly identified that the adjustment of
spouses gets affected by factors such as financial fears and
uncertainties which are commonly seen among couples during
COVID-19 pandemic. Similarly couples with external support have
found it easier to adjust in spite of these obstacles during COVID-19. In the following section variables of fear of financial situation,
uncertainty and resources available for spouses are reviewed in
relation to the adjustment of spouses and propose our hypotheses of
the study. The conceptual model developed for the study is shown in
figure 1. Figure 1: Conceptual Model Figure 1: Conceptual Model Fear of Financial Situation Fear of Financial Situation COVID-19 has brought about many challenges to the lives of
spouses and, fear of financial situation has been one of the dominant
obstacles among them. With downsizing and closing of businesses,
the unemployment rate has gone up causing an economic collapse
all around the country due to COVID-19. This has made many
spouses feel threatened about their financial security and the fear of
losing employment or income has caused a strain on many
relationships. Schneider et al. (2017, p.4) states that “During times of
widespread economic upheaval, financial stress impacts families
directly via individual job loss, as well as indirectly through
uncertainty about the national economy and/or local unemployment
rates”. This fear is worse for daily income earners and couples with
low income rates who require more adjustment in their relationships. Therefore, the COVID-19 pandemic has brought many financial fears
among spouses and caused an additional stress in their marriages. Such fears of financial situations needs to be addressed and dealt
with in order to adjust in their marital relationships and, these
spouses would require external support in order to do so. A study
done by Millet et al. (2020) identifies that the adverse effects of COVID-19 brings fears of financial situation and stress causing
additional strain on couples and their marital adjustment. Another
extensive study done by Conger et.al (1999) identifies how fear of
finances and economic strain causes emotional distress which
directly affects the marital relationships of spouses. Balzarini et al. (2020) also suggests that COVID-19 brings stresses such as
financial fear which directly causes poor relationship functioning. Amato and Rogers (1977) states financial reasons as one of the
most prominent reasons for failure of marriage among couples. Therefore, it is evident that the fear of financial situation caused by
COVID-19 would bring many stressors for couples and their
relationship adjustments. Based on the above reviewed literature, it
is hypothesised: yp
H1: Fear of Financial situation of spouses influences the
adjustment of spouses. Uncertainty y
Uncertainty is when couples can no longer predict the future or what
is to happen (Berger and Bradac, 1982). When faced with a COVID-
19 type of a pandemic situation couples has no prior experience to
rely on which increases the level of uncertainty (Senatore, 2013). Therefore, uncertainty causes couples unable in making decisions or
adjustments within their relationships. Even though the vaccines are
in effect, since there is no real cure for COVID-19, uncertainty
persists among couples. As couples influence each other with
adjustments in their relationships, it is inevitable to see the impact of
uncertainty on that. Even when one partner gets affected due to
uncertainty the other will also find it difficult to adjust, as a result
(Brown, 1995). A study done by Cimprich et al. (2005) identified that many health
crisis situations bring uncertainty about the future among couples. The study further reiterated that repetition of such health disasters
such as COVID-19 waves shall increase the uncertainty which will
affect the adjustment of couples. Downe-Wamboldt et al. (2006)
stated how uncertainty increased negativities in couple’s marital
relationships. Uncertainty increases the level of adjustment of
couples during any health disaster (Northouse et.al, 1998). They
have also identified how uncertainty affects levels of adjustment in
couples when facing health diseases. This study concludes that high
level of uncertainty causes poorer adjustment in couples. In another study, Northouse et al. (2001) identified that couples
facing fatal health crisis claimed to have high uncertainty which
caused adjustment issues among the spouses. In a similar study,
Northouse et al. (2007) claimed that while uncertainty created a
significant impact on adjustment of couples, over time these couples
managed to overcome these uncertainties with external resources. In the COVID-19 pandemic type of health disasters many
uncertainties come to place such as fear of the health of loved ones,
the future of the family and the income and well-being of each other. As a result couples require additional adjustment to maintain
harmony in their relationships (Karney et al., 2005). In this study the
role of uncertainty was investigated to identify how it affects the
adjustment of couples during the COVID-19. Based on the above reviewed literature, it is hypothesised:
H2: Uncertainty influences the adjustment of spouses. Based on the above reviewed literature, it is hypothesised: H2: Uncertainty influences the adjustment of spouses. Resources available for spouses p
With the COVID-19 and the lockdown which followed, many couples
required assistance when dealing with the fear of financial situation
and uncertainties. The resources available for the couples have
assisted in the way the couples adjusted during these obstacles
caused by the COVID-19. The extended family has been defined by Taylor (2013) as biological
members of the couples who are beyond the immediate family
considered. The parents, in laws, cousins and the siblings of the
couples can be identified as such external family. According to
Wilson (1999) what is important than the actual definition is the
support offered by them to the couple. Family Support during Fear of Financial Situation y
pp
g
Wilson (1999) further reiterates that having such external family
support shall ease the financial fears and uncertainties a couple may
have. The support extended by external family has made it viable for
couples to adjust to the fear of financial situation. A study done by Walsh (2015) identifies how family support eases
financial fears during crisis situations as the couples can rely upon
the relations in an emergency fund requirement situation. With the
new technology, even during lockdown the finances can be assisted
with using the internet and online banking; therefore, having family support would certainly have a positive effect in the fear of financial
situation for couples. Falicov et al. (2020) observes that families who
struggle with financial difficulties cope better during COVID-19 as
they are used to financial hardships. As a result he finds that such
couples are not too concerned with the family support available with
regards to fear of financial situation. In another study by Adams et al. (1996), it was identified that couples
with no family support found additional financial strain which caused
stress in their marital relationships. Pearlin and Schooler (1978) has
pointed out the importance of family support when coping with
financial fears. He emphasises how having extended family support
eases the financial burdens a couple undergo during stressful times
which helps them with adjustment. Engstrom (2012) states how the
external families have more resources in hand which can be
contributed to the couples during the financial fears. Family support extended from relations will bring emotional security
and stability among couples who can then easily face the fears of
financial situation caused by the COVID-19 (Walsh, 2015). Many
couples with children found that COVID-19 caused extra strain on the
marriages due to the children being isolated at homes without going
to school and the couples having to work from home. This additional
stress intensifies the fear of financial situation which affects the
adjustment of couples. The lack of support offered by the extended
family during this traumatic period would clearly escalate the
challenges the couples face, Fraenkel (2019). Based on the above
reviewed literature, it is hypothesised: H3: Family support available for spouses moderates the
relationship between Fear of financial situation and the
adjustment of spouses. Family Support during Uncertainty Family Support during Uncertainty Walsh (2015) speaks of the negative effects of isolation. With the
lock downs or travel restrictions due to COVID-19, many couples
could not associate or interact with families. Thus, the support
extended from family was broken which brings a negative effect in
the adjustment of spouses. Waites (2009) points out how when
couples receive family support they act as a collaborative team which
assists when facing uncertainties. In a study done by Dressler (1985), it is specified how the lack of
support from extended family creates stress which affects the
harmony and adjustment of couples. He further concludes that
people with better support from extended families perceive their
marriages to be more adjustable and peaceful. Walen and Lachman
(2000) identified the relationship of extended family support with
uncertainties and adjustment of spouses. They concluded that
among couples with family support, the adjustment and wellbeing
was always observable. This type of support is essential when
couples face uncertainties due to COVID-19. In a study done by Waites (2009) it was identified that extended
families are seen mostly in collective cultures like in Sri Lanka, where
couples depend up on relations and family members to share
responsibilities. Strong et al. (2008) states that external family
members tend to appreciate the idea of group relationships than
couples and therefore cause a rift between couples when interfered
unnecessarily. Therefore, it can be seen that not having the family
support during COVID-19 and the couples who go through the
lockdowns alone may feel more harmony as a result. In a study done by Bester and Malan-Van Rooyen (2015), it was
identified that when relations or other family lives with the couples,
the uncertainties are less as there is a strong mental support system
taking place. They further reiterated that couples were less stressed
and displayed more strength when such family members were
providing support during uncertain times. Imber-Black (2020) has identified how the use of new technology
allowed the families to support couples even during social distancing
taking place. Therefore even during COVID-19 many families still
provided support for couples which helped to ease their worries and
uncertainties. Based on the above reviewed literature, it is
hypothesised: H4: Family support available for spouses moderates the
relationship between Uncertainty and the adjustment of
spouses. Measures To measure the Fear of Financial situation and Uncertainty, a 14-
item measure was used, which can be seen in Appendix 1. These
measures were taken using the questionnaire by Deguglielmo
(1973), named ‘The Development of an Instrument for Measuring
Financial Adjustment. These items were on a five-point Likert scale
ranging from always (1) to never (5). To measure the Adjustment of spouses, a 2-item measure was used,
which is shown in Appendix 2. These measures were taken using the
questionnaire by Hansen (1978), named ‘Marital adjustment,
idealization, and conventionalization’. These items are on a four-point
Likert-type scale ranging from always agree (4) to disagree
frequently (1). To measure the Resources available for spouses, a 11-item measure
was used, which are shown in Appendix 3. These measures were
taken using the questionnaire by Deguglielmo (1973), named ‘The
Development of an Instrument for Measuring Financial Adjustment. These items were on a five-point Likert scale ranging from always (1)
to never (5). H4: Family support available for spouses moderates the
relationship between Uncertainty and the adjustment of
spouses. With the above literature review, it can be seen that the family
support available for married spouses that affect their adjustment during COVID-19 impacts their fear of financial situation and
uncertainty. during COVID-19 impacts their fear of financial situation and
uncertainty. Method As seen in the literature review many couples have faced challenges
due to COVID-19 and fear of financial situation and uncertainty has
threatened the well being and harmony of their marriages. The
adjustment of these couples was influenced by the support extended
by their families and the survey was created to identify the actual
results of these factors. Participants
300
i i 300 participants took part in the survey and they were male and
female adults of the ages 25-60 years residing in the Western
Province of Sri Lanka. The survey which was in English medium took
25 minutes on average to complete. Initially the respondents were
briefed about the purpose of the survey and the google link to the
survey was sent only after receiving the consent from them. All the
participants took the survey voluntarily and remained anonymous. Method of data collection The online survey started on the 12th of March 2021 and ended on
the 12th of June 2021. This was a period where Sri Lankan
government had imposed a partial lockdown all across the country. Travelling was strictly limited to essential services and mostly all
work was done from home unless it is essential work such as health
or military services. The questionnaires were prepared and uploaded
on google form. 25 – 60 year old adults whose contact emails were
extracted from government institutes, private companies, social
media and other web groups were sent the details of the survey
explaining the purpose. Once the demographics and the consent
were confirmed the link was sent for their anonymous participation. Population and Sample p
p
The total population of Sri Lanka comes close to 21.5million people
and 48% of the population consists of adults of the ages 25-60 years
(worldpopulationreview.com 24/09/21) which is equivalent to roughly
10 million people. In the Western province the total number of adults
in the ages 25-60 comes to roughly 1 million. In order to calculate a
“95% confidence level with only a 5% chance of the sample results
differing from the true population average, confidence interval of the margin of error is calculated by 1/√N. Here N is considered as the
number of participants or sample size” (Niles, 2006). Therefore, the
300 participants who contributed in the survey would suffice in
justifying the total population that was studied. margin of error is calculated by 1/√N. Here N is considered as the
number of participants or sample size” (Niles, 2006). Therefore, the
300 participants who contributed in the survey would suffice in
justifying the total population that was studied. Method of data analysis y
Validity and reliability of the measures were evaluated. Principle
component factor analysis was conducted using SPSS software. Factor analysis yielded two factors for fear of financial situation and
uncertainty; these were named as fear of financial situation and fear
of uncertainty. Factor analysis yielded two factors for resources
available for spouses; these were named as family resources and
cultural pressure. The fit measures were given in Table 1. Results of
these tests were shown in Appendix 1 to 3. Moderation analysis was
conducted using PROCESS programme developed by Hayes (2013). Indirect effects were assessed based on 5,000 bootstrapped
samples using bias-corrected 95% confidence intervals (CI) for the
size and significance of the effects. Table 1. Fit measures Table 1. Fit measures Cronbach’s
alpha
Explained
variation
Eigenvalue
AVE Construct
reliability
Fear
of
financial
situation
and
uncertainty:
.932
.932
-
-
-
Fear
of
financial
situation
.958
47.47
7.695
.728
.956
Fear
of
uncertainty
.906
27.31
2.774
.698
.920
Adjustment
of
spouses
.800
83.349
1.667
.834
.909
Support
from
other
family
members
.890
69.488
3.474
.695
.919 Results Since fear of financial situation and fear of uncertainty yielded two
factors, we analysed two separate models. ,
y
p
Results relating to the fear of financial situation and adjustment of
spouses moderated by support from other family members are
shown in Table 2. As can be seen in Table 2, the effect of fear of
financial situation (IV) on adjustment of spouses is not significant (p >
.05). The effect of support from other family members (M) on
adjustment of spouses is significant (B = -.7316, p < .001). The effect
of interaction on adjustment of spouses is also significant (B = .1624,
p < .001). Relationships between fear of financial situation (IV) and
adjustment of spouses are significant for all low (b = .2667, p < .001),
average (b = .3665, p < .001), and high (b = .4663, p < .001) values
of support from other family members (M). Overall, support from
other family members (M) moderates the relationship between fear of
financial situation (IV) and adjustment of spouses. Figure 2 shows
this relationship figuratively. Table 2. Fear of financial situation and adjustment of spouses
moderated by support from other family members. Adjustment of spouses (DV)
B(SE)
Fear
of
financial
situation (IV)
-.1170 (.1157)
Support
from
other
family members (M)
-.7316 (.1387)***
Interaction (IVxM)
.1624 (.0378)***
R2
.1786
F (df1, df2)
21.3782 (3, 295)***
∆R2
.0513
∆F(df1, df2)
18.4183 (1, 295)***
Conditional effects:
-SD
Mean
+SD
Support
from
other
family members (M)
2.3634
2.9779
3.5924
Effect (t)
.2667 (5.1558)
***
.3665
(7.3168)***
.4663
(7.9698)***
Notes: Unstandardized regression coefficients are reported; standard
errors = SE. Bootstrap sample size = 5000. ***p < .001 (two-tailed). Figure 2. Moderation Graph- fear of financial situation and
adjustment of spouses moderated by support from family members
1,8
1,9
2
2,1
2,2
2,3
2,4
2,5
2,6
2,7
2,8
low
med
high
Adjustment of spouses
Fear of finantial situation
other fam
support
high
med
low
Family
resources Table 2. Fear of financial situation and adjustment of spouses
moderated by support from other family members. Results Adjustment of spouses (DV)
B(SE)
Fear
of
financial
situation (IV)
-.1170 (.1157)
Support
from
other
family members (M)
-.7316 (.1387)***
Interaction (IVxM)
.1624 (.0378)***
R2
.1786
F (df1, df2)
21.3782 (3, 295)***
∆R2
.0513
∆F(df1, df2)
18.4183 (1, 295)***
Conditional effects:
-SD
Mean
+SD
Support
from
other
family members (M)
2.3634
2.9779
3.5924
Effect (t)
.2667 (5.1558)
***
.3665
(7.3168)***
.4663
(7.9698)***
Notes: Unstandardized regression coefficients are reported; standard
errors = SE. Bootstrap sample size = 5000. ***p < .001 (two-tailed). Figure 2. Moderation Graph- fear of financial situation and
adjustment of spouses moderated by support from family members
1,8
1,9
2
2,1
2,2
2,3
2,4
2,5
2,6
2,7
2,8
low
med
high
Adjustment of spouses
Fear of finantial situation
other fam
support
high
med
low
Family
resources Table 2. Fear of financial situation and adjustment of spouses
moderated by support from other family members. 1,8
1,9
2
2,1
2,2
2,3
2,4
2,5
2,6
2,7
2,8
low
med
high
Adjustment of spouses
Fear of finantial situation
other fam
support
high
med
low
Family
resources med
Fear of finantial situation Figure 2. Moderation Graph- fear of financial situation and
adjustment of spouses moderated by support from family members Results relating to the fear of uncertainty and adjustment of spouses
moderated by support from other family members are shown in Table
3. As can be seen in Table 3, the effect of fear of uncertainty (IV) on
adjustment of spouses is significant (B = -.7966, p < .001). The effect
of support from other family members (M) on adjustment of spouses
is significant (B = -.4030, p < .001). The effect of interaction on
adjustment of spouses is also significant (B = .1915, p < .01). Relationships between fear of uncertainty (IV) and adjustment of
spouses are significant for low (b = -.3439, p < .001) and average (b
= -.2262, p < .001) values of support from other family members (M). Overall, support from other family members (M) moderates the
relationship between fear of uncertainty (IV) and adjustment of
spouses. Figure 3 shows this relationship figuratively. Table 3. Fear of uncertainty and adjustment of spouses moderated
by support from other family members. Results Adjustment of spouses (DV)
B(SE)
Fear of uncertainty
(IV)
-.7966 (.1374) ***
Support from other
family members (M)
-.4030 (.1219)**
Interaction (IVxM)
.1915 (.0389) ***
R2
.1046
F (df1, df2)
11.4840 (3, 295)***
∆R2
.0736
∆F(df1, df2)
24.2438(1, 295) ***
Conditional effects:
-SD
Mean
+SD
Support from other
family members (M)
2.3634
2.9779
3.592
4
Effect (t)
-.3439
(-4.9324) ***
-.2262
(-3.5819)***
-.1085
(-1.6629)
Notes: Unstandardized regression coefficients are reported; standard
errors = SE. Bootstrap sample size = 5000. **p < .01, ***p < .001 (two-
tailed). Table 3. Fear of uncertainty and adjustment of spouses moderated
by support from other family members. Notes: Unstandardized regression coefficients are reported; standard
errors = SE. Bootstrap sample size = 5000. **p < .01, ***p < .001 (two-
tailed). Notes: Unstandardized regression coefficients are reported; standard
errors = SE. Bootstrap sample size = 5000. **p < .01, ***p < .001 (two-
tailed). Figure 3. Moderation Graph- fear of uncertainty and adjustment of
spouses moderated by support from other family members
2
2,1
2,2
2,3
2,4
2,5
2,6
low
med
high
Adjustment of spouses
Fear of uncertainty
other fam support
high
med
low
Cultural pressure Figure 3. Moderation Graph- fear of uncertainty and adjustme
spouses moderated by support from other family members
2
2,1
2,2
2,3
2,4
2,5
2,6
low
med
high
Adjustment of spouses
Fear of uncertainty
other fam support
high
med
low
Cultural pressure Cultural pressure Figure 3. Moderation Graph- fear of uncertainty and adjustment of
spouses moderated by support from other family members Discussion of Results COVID-19 has created many challenges for couples and they have
undergone many adjustments in order to remain in harmonious
relationships. The fear of financial situation due to unemployment
and lack of business transactions and uncertainties created with not
knowing of the expiration of the pandemic and the health issues
attached to it, has enhanced the level of adjustment of couples. Thus, this study has identified the factors that moderate the effects of
the financial fear and uncertainties couples go through in relation to
their adjustment level. The literature has identified how the social
distancing and other non-pharmaceutical interventions such as
wearing face masks and following hygiene measures makes people
feel safe but at the same time makes couples feel stressed and
isolated. The findings of this study affirm that and, further emphasises that
having thoughts of stress and uncertainty amplifies their level of
adjustment and brings disharmony in marital relationships. Therefore,
it is necessary to address these issues when identifying the impact of
COVID-19 has had on couples emotionally, Qiu et.al. (2020). A study done by Xiang et. al (2020) identifies how COVID-19
pandemic has brought uncertainties in married couples. This along
with fear of financial situation causes many psychological distresses
among couples and according to Ahorsu (2020), this affects the
adjustment level in such couples. Harper et.al (2020) has found how
majority of couples who face uncertainties and financial fears require
psychological support to adjust in their relationships. Therefore while
identifying the relationship between fear of financial situation and
uncertainty on adjustment of couples in Sri Lanka, this study also
investigated the moderation effect of family resources had on the
above relationships. The study identified that many participants felt
moderate to high levels of uncertainty throughout the COVID-19
lockdown periods. These participants declared that the level of
support extended by the external families moderated these
adjustments. This is in line with a similar study done by Van Bortel
et.al (2016) and another study done by Brooks et al. (2020) where it
is evident that the support given by the external families moderates
the effects of the pandemics on couples. During uncertain times such
as pandemics when couples go through many financial hardships the
support of external families acts as a resource in coping with these
situations, Rubin et.al (2010). Discussion of Results Therefore, by observing the study
results it is evident that the external family support moderates the
effects of the couples’ adjustment due to fear of financial situation
and uncertainty caused by COVID-19. Conclusion Conclusion
The COVID-19 has caused many disruptions in Sri Lanka including
closing down of businesses and schools. Many couples were
consigned to their homes due to periods of lockdown and working
from home measures implemented by the government. The effect of
spending lengthy periods of time exclusively with each other has
brought
many
tensions
among
married
couples. With
the
uncertainties and the fear of the financial situation in the future
caused by the doubts and obstacles of COVID-19 has worsened
these worries. Thus, this study was conducted with the aim of identifying the couples’ well being and adjustment level during the
state regulated lockdown periods. identifying the couples’ well being and adjustment level during the
state regulated lockdown periods. The specified main objectives of this study was to investigate
whether individuals suffer from fears on financial situation and
uncertainty, to investigate the level of adjustment of spouses, to
investigate support extended by the other family members and to
analyse whether the support extended by the other family members
moderates the relationship between individual’s fears on financial
situation/uncertainty and the level of adjustment of spouses. The study was conducted initially with the development of a
conceptual model. In here, the three variables fear of financial
situation, uncertainty and resources available for spouses were
studied in relation to the adjustment of spouses. Thereafter, four
hypotheses were developed. These are H1: Fear of Financial
situation of spouses influences the adjustment of spouses, H2:
Uncertainty influences the adjustment of spouses, H3: Family
support available for spouses moderates the relationship between
Fear of financial situation and the adjustment of spouses and H4:
Family support available for spouses moderates the relationship
between Uncertainty and the adjustment of spouses. The methodology of the study began with the data collection done
with a survey. Over 300 male and female adults of the ages 25-60
years took part in the survey questionnaire which had 82 questions. Since fear of financial situation and uncertainty yielded two factors
and resources available for spouses yielded one factor, the study
analysed two separate models. Results relating to the fear of
financial situation and adjustment of spouses moderated by family
resources conclude that family resources moderates, the relationship
between fear of financial situation and adjustment of spouses. Limitations and Future Research Limitations and Future Research
As in any study, there are a few imitations that can be identified in
this. Main limitation arises with the fact that the survey questionnaire
was self-administered. As a result the participants may at times
respond with a more socially acceptable response than voicing the
actual truth. Another limitation is the number of participants who
responded to the questionnaire. The anticipation of the study was to
obtain an equal number of participation from both genders but the
majority of the respondents were females. The language and
computer/technical literacy were another two limitations as the
questionnaire was compiled in English and the mode of responding
required internet and the ability to navigate through a computer or a
mobile device. The respondents with such language and technical
ability represented a certain demographic and may not necessarily
represent all the married couples in the country as a whole. To avoid these limitations, in the future more studies can be
conducted with a diverse group with more methods of collecting data
such as face to face interviews and case studies. In order to identify
the actual impact of COVID-19 on couples, these types of studies
should be conducted time to time in the future through and post the
pandemic. Additional research can be conducted in the future to
make recommendations to these couples in further adjustments and
moving forward in the new normal way of life amidst COVID-19. Conclusion Results relating to the uncertainty and adjustment of spouses
moderated by family resources conclude that the family resources
moderates, the relationship between fear of uncertainty and
adjustment of spouses. Overall, the results of this study conclude that the challenges brought
by the COVID-19 pandemic caused many strains and tensions
among couples who required adjustment in order to maintain
harmony in their relationships. The married couples who were estranged with each other due to lockdown faced fear of the financial
situation and uncertainties which has increased this effect. Having
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Psychiat. 7(3):228–9. Appendix Appendix 1. Fear of financial situation and uncertainty
Item
Fear
of
financial
situation
Uncertainty
My spouse’s attempt to control my
spending
money
causes
disagreement. .764
For me, it has been difficult to adjust
to the economic needs of my spouse
.813
I feel that if my spouse had a better
education we would have more money
.825
Fights over money often occur
.882
My spouse and I find it difficult to
communicate when expressing views
on monetary needs
.903
Disagreements over money offer an
easy way to release hostility
.923
Disagreements over what to spend
money on have occurred in my
marriage
.800
My spouse and I have disagreements
over who will handle the family money
.901
My spouse and I disagree about which
bills need to be paid at the first of the
month
.798
I consider budgeting money carefully
to be important
.855
I
would
respect
my
spouse's
occupation if he did not earn an
average salary
.880
My spouse's spending habits are
agreeable with me and efficient
.798
I feel ·financially capable to take care
of myself in cases of crisis
.873
I feel that education guarantees a
stable income
.765
Eigenvalue
7.695
2.774
Explained variation (rotation sum of 47.47
27.31 Appendix 1. Fear of financial situation and uncertainty squared Loadings)
Cronbach’s alpha
.958
.906
AVE
.728
.698
Construct reliability
.956
.920 Appendix 2. Adjustment of spouses
Item
Adjustment
Handling Family Finances
.913
Matters of Recreation
.913
Eigenvalue
1.667
Explained
variation
(rotation
sum
of
squared
Loadings)
83.349
Cronbach’s alpha
.800
AVE
.834
Construct reliability
.909 Appendix 3. Support extended by other family members Appendix 3. Support extended by other family members
Item
Support
My spouse and children always assist with me with
household chores
.792
My
mother
in-law
and
father
in-law
have
Babysat/looked after my children when I am
occupied with my career duties
.867
My
other
in-laws
(eg. Sisters
in-law)
have
Babysat/looked after my children when I am
occupied with my career duties
.817
My own parents have Babysat/looked after my
children when I am occupied with my career duties
.866
The day to day contribution of my husband in the
home-front is a great support for my career
advancement
.824
Eigenvalue
3.474
Explained
variation
(rotation
sum
of
squared
Loadings)
69.488
Cronbach’s alpha
.890
AVE
.695
Construct reliability
.919
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El cubre de la cama y el sofá en una casa de la calle meshchanskaya. Sobre Viktor Shklovsky y la percepción de los objetos
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[i2]
Innovación e Investigación en Arquitectura y Territorio. Departamento de Expresión Gráfica y Cartografía.
Arquitectura. Escuela Politécnica Superior. Universidad de Alicante. Nº4 mayo 2016 ISSN: 2341-0515
El cubre de la cama y el sofá en una casa de la
calle meshchanskaya.
Sobre Viktor Shklovsky y la percepción de los objetos.
Palabras clave: Shklovsky, meshchanskaya, sofá, cama,
Barberá Pastor, Carlos
Departamento de Expresión Gráfica y Cartografía, Universidad de Alicante, carlos.barbera@ua.es
Tretya meshchanskaya es el título de la película muda filmada en 1927 y dirigida por el cineasta ruso Abram
Matveyevich Room. La traducción literal, Cama y sofá, alude a dos de los muebles de la pequeña vivienda
de la calle meshchanskaya, mobiliario donde poder dormir y soñar. En el film, las calles, las plazas de la
ciudad de Moscú o el interior de la vivienda, conforman un escenario donde Nikolai, oficial de la construcción, Volodia, oficial de imprenta, y Liudmila, ama de casa, se ganan la vida en la capital Soviética. La película, más que el interés que puede causar la historia en sí, posibilita entablar relaciones interpretables hoy,
desde una opinión, que conforman relaciones entre casa y ciudad propiamente.
Las cortinas que cuelgan frente a las ventanas del interior de la vivienda, el pañuelo que cubre la lámpara,
los paños que hay sobre la mesa, las sábanas sobre la cama, las mantillas sobre el aparador, la cortinilla
frente a la puerta de entrada o el mimbre de la mecedora parecen mostrar el sentido que puede dársele a un
velo. La tela, que por un lado encubre parte de aquello que hay a un lado, por otro permite una mirada distinta al perfil del objeto que reviste. Posibilita mirar e interpretar y dota a la imagen de pormenores que la
caracteriza sin necesidad de definirla con precisión. El uso de los tejidos en el interior de la casa, en la película son empleados como un revestimiento que oculta una pared o una ventana, además son un fondo sobre el que aparecen figuras, semblantes, luces y sombras. El movimiento que se produce en la calle, mediante la luz, se proyecta en la cortina que hay en el salón. Conforman rasgos que no solo entran en la casa
a través del cortinaje sino que en algunos momentos se proyectan sobre las paredes del interior repletas de
telas. La mecedora, que arroja la luz tamizada por el mimbre, muestra a Liudmila escondida, presenciando
al nuevo invitado desde un lado del mueble que permite a su vez ver el rostro de la mujer repleta de luces y
sombras producidas por el mobiliario. Se encuentra a un lado del velo observando quién viene de fuera, con
un aspecto que es transformado a partir de la luz que viene del exterior.
Fig. 1
Fig. 2
La superposición telas en paredes y objetos, como si fueran lienzos, transforman el escenario de manera
que los límites se convierten en fondos sobre los que se proyectan las imágenes que construyen también la
historia de la película. El interior de la casa no solo se conforma como escenografía, donde la tela parece
tener un sentido referido propiamente al cine y por tanto al nuevo medio -ya que es sobre el lienzo de la
pantalla donde se proyecta la imagen-, sino a otros medios de expresión propios del momento con el carácter que tienen para la construcción del mundo. La ciudad, repleta de rótulos, octavillas, pancartas, noticias y
El cubre de la cama y el sofá en una casa de la calle meshchanskaya.
Carlos Barberá Pastor, DOI: 10.14198/i2.2016.4.07
[i2]
Innovación e Investigación en Arquitectura y Territorio. Departamento de Expresión Gráfica y Cartografía.
Arquitectura. Escuela Politécnica Superior. Universidad de Alicante. Nº4 mayo 2016 ISSN: 2341-0515
publicaciones, queda vinculada al interior de la casa invadida también por el papel impreso que adquiere su
protagonismo junto a las telas. Los planos que dibuja Nikolai o las imágenes que son plasmadas en la imprenta en la que trabaja Volodia, justamente se refieren a plasmar sobre papel los planos de un proyecto, la
imagen de un panfleto o el texto de un periódico. Es un medio que adquiere un papel principal por la figuración de la imagen que inunda diversos espacios.
Fig. 3
Fig. 4
Esta manera de interpretar las telas en las que se proyecta una sombra que viene de la calle, relacionado
con los papeles de imprenta o los planos de un proyecto como medio de información para la construcción de
la ciudad, no surge de una manera accidental. Viktor Shklosky, guionista de la película, nos hace ver que
aquello que sucede en el interior de la casa vincula íntimamente el contenido del espacio con los límites que
lo definen, planteando alegóricamente una relación con aquello que sucede en el exterior. A pesar que la
historia que cuenta la película puede ser construida exclusivamente en el escenario referido a la vivienda
por la relación de amor y amistad entre los ocupantes de la casa en la calle meshchanskaya, la imagen de la
ciudad adquiere un papel muy relevante.
La película nos muestra la ciudad de Moscú según la ven Nikolai y Volodia. Es presentada desde puntos de
vista donde el espectador se posiciona como si la viera por primera vez. Ocurre cuando la ciudad es mostrada al espectador, exhibida por Nicolai en el teatro de Moscú en construcción, la llegada en tren de Volodia, o el viaje en avión de éste y Liudmila cuando Nicolai está fuera.
La imagen que ve Liudmila de la ciudad es presentada por Volodia. Éste la invita a un viaje en avión. Las
calles son reconocidas desde arriba, volando por encima de las cubiertas de los edificios de Moscú. El aspecto de admiración de Liudmila revela la sorpresa que causan las imágenes cuando son vistas desde lo
alto, por primera vez.
Fig. 5
Fig. 6
El cubre de la cama y el sofá en una casa de la calle meshchanskaya.
Carlos Barberá Pastor, DOI: 10.14198/i2.2016.4.07
[i2]
Innovación e Investigación en Arquitectura y Territorio. Departamento de Expresión Gráfica y Cartografía.
Arquitectura. Escuela Politécnica Superior. Universidad de Alicante. Nº4 mayo 2016 ISSN: 2341-0515
No obstante, en referencia a las telas que comentábamos al principio, más que tapar nos muestran otra manera de percibir el objeto que envuelven. Las figuras que se proyectan en las cortinas aluden justamente al
cine como medio de expresión entre dos mundos, el de la experiencia del cine y la experiencia vital, o el de
la experiencia del espacio público de la calle y el espacio privado de la casa. La ciudad, justamente, nos hace ver que los acontecimientos en el interior de la casa entre los tres personajes quedan vinculados a las
imágenes que se presencian del exterior. Esto, que no se manifiesta de forma explícita en el argumento, es
uno de los motivos que podrían destacarse del film.
1
Shklosky, guionista de la película, en el libro Teoría de la literatura de los formalistas rusos escribe:
“Si examinamos las leyes generales de la percepción vemos que una vez que las acciones llegan a ser habituales se transforman en automáticas. De modo que todos nuestros hábitos se
refugian en un medio inconsciente y automático. (…) Así la vida desaparece transformándose
en nada. La automatización devora los objetos, los hábitos, los muebles, la mujer y el miedo a
la guerra. “Si la vida compleja de tanta gente se desenvuelve inconscientemente, es como si
esa vida no hubiese existido.” (…) Los objetos percibidos muchas veces comienzan a serlo por
un reconocimiento: el objeto se encuentra delante nuestro, nosotros lo sabemos pero ya no lo
vemos. Por ese motivo no podemos decir nada de él. En arte la liberación del objeto del automatismo perceptivo se logra por diferentes medios; (…) L. Tolstoi, quien, según la opinión de
Mereikovski, parece presentar los objetos tal como los ve; los ve en sí mismos, sin deformarlos.
El procedimiento de singularización en Tolstoi consiste en no llamar al objeto por su nombre
sino en describirlo como si lo viera por primera vez y en tratar cada acontecimiento como si
ocurriera por primera vez; además, en la descripción del objeto no emplea los nombres dados
generalmente a sus partes, sino otras palabras tomadas de la descripción de las partes correspondientes a otros objetos.”
Shklosky, en el argumento de la película nos presenta mediante los velos otra manera de ver la ventana, la
puerta de entrada, la cocina. Sin embargo, la singularización de la que habla no queda tanto referida a la
forma del objeto tapado por el velo sino al modo como pueden ser interpretadas las figuras en un pensamiento. Queda referido a la manera de ver las cosas, que pueden verse distintas a como se presentan. Son
modos que transforman la percepción de los objetos para presentarse como si se vieran por primera vez.
Acorde a establecer un proyecto de vida y a la construcción del escenario donde han de sucederse los
acontecimientos en la casa, la película revela relaciones que se dan en el interior de la vivienda desde acontecimientos ligados a una interpretación de la ciudad. La manera de presentar Moscú y el interior doméstico
de una pequeña vivienda por debajo del nivel de la calle es narrado con la ilusión de ver algo nuevo. Esto le
ocurre sobretodo a Liudmila. Mediante la ilusión de una forma de vida para habitar la casa, la película nos
presenta el deseo y la esperanza de habitar desde una relación con el exterior, con lo público. Nos muestra
una correlación entre casa y ciudad desde la distancia que posibilita la interpretación de un nuevo mundo.
En ese intervalo están las posibilidades de mirar desde “la función de permitir agrupar los objetos y las ac2
ciones heterogéneas y explicar lo desconocido” , de manera que uno pueda descubrir el carácter estético de
un objeto como resultado de nuestra manera de percibir; como dice Shklosky.
Las vistas de la ciudad que nos presenta la película, cuando llega en tren Volodia, o la mirada desde la cubierta del teatro de Moscú en construcción, cuando Nikolai sube a descansar de la jornada de trabajo, o el
recorrido que se sigue a través del tráfico de los coches por la ciudad moscovita, nos exhiben imágenes hechas por unos ojos primerizos. Es una manera de presentarnos la ciudad que nos descubre una mirada de
principiante; no por el ejercicio de la profesión que desarrollan, porque los dos están muy cualificados para
sus actividades, sino por las posibilidades que ofrece una mirada primeriza. No obstante es Liudmila quien
descubre un modo de ver referido a las posibilidades que ofrece el dejarse seducir por aquello que la rodea.
La ciudad y el espacio doméstico de la vivienda son escenografías que el film nos presenta como lugar que
posibilitan nuevos proyectos de vida. Es, el fin de habitar una casa, un compromiso para vincular el exterior
y el interior, la casa y la ciudad, con “el derecho de vincularlo a la poesía”. Esto, la película, lo presenta con
la finalidad de llevar a cabo la vida en una casa a partir de una percepción estética, entendida como un procedimiento particular de percibir los objetos relacionándolos en un ámbito de dejarse seducir.
1
SHKLOSKY, Viktor. “El arte como artificio”. En: Tzvetan Todorov. Teoría de la literatura de los formalistas rusos. 2007.
Mexico: Siglo XXI editores, 1970. p. 55 – 70.
2
Op cit. p. 55.
El cubre de la cama y el sofá en una casa de la calle meshchanskaya.
Carlos Barberá Pastor, DOI: 10.14198/i2.2016.4.07
[i2]
Fig. 7
Innovación e Investigación en Arquitectura y Territorio. Departamento de Expresión Gráfica y Cartografía.
Arquitectura. Escuela Politécnica Superior. Universidad de Alicante. Nº4 mayo 2016 ISSN: 2341-0515
Fig. 8
Los dos lugares conocidos por el ciudadano al vivir y recorrer el espacio de la casa y la ciudad de Moscú, se
presentan desde los puntos de vista desde los cuales miran el guionista y el director de la película, con la
pretensión de hacer de la mirada un medio para descubrir y dejarse sorprender. Alecciona al espectador de
esta posibilidad. No obstante, este planteamiento no es mostrado por Nikolai y Volodia que nos definen las
imágenes primerizas, sino más bien, como hemos dicho, es Liudmila quien lo muestra. La mirada de Liudmila es como el velo de la lámpara que difumina la luz o la cortina de la ventana que permite que sobre ella se
proyecten las sombras. El espectador, que ve por primera vez los planos secuencia de la ciudad de Moscú,
queda identificado con la protagonista. Es ella quien ve por vez primera la ciudad desde el avión. Es quien
experimenta ese tipo de mirada. Es quien descubre una mirada que los dos hombres tienen acostumbrado
en sus vidas. La mujer, que en la película pasa la mayor parte del tiempo en casa, mientras Nikolai y Volodia
se van a trabajar, parece mostrar un velo que manifiesta el deseo por apreciar algo más. Es como si nos
descubriera la necesidad de exigir una mirada primeriza. La ventana, que aparece al principio de la película
con las sombras de la ciudad en las cortinas que cuelgan, al final se presenta con Liudmila enfrentada sobre
esa misma proyección de imágenes que entran en la casa. Ella ansía esa búsqueda de miradas y las dirige,
en ese encuadre, hacia la calle. La decisión de coger el tren, de salir de la casa, como aparece en uno de
los fotogramas cuando se le ve asomándose a la ventana del vagón, es síntoma de esa búsqueda que el
propio Shklosky, guionista del film, nos propone en su escrito El arte como artificio.
Fig. 9
Fig. 10
Carlos Barberá Pastor Carlos Barberá Pastor. Doctor arquitecto. Profesor de Composición Arquitectónica en el
Departamento de Expresión Gráfica y Cartografía de la Universidad de Alicante.
El cubre de la cama y el sofá en una casa de la calle meshchanskaya.
Carlos Barberá Pastor, DOI: 10.14198/i2.2016.4.07
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https://openalex.org/W1967691623
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https://europepmc.org/articles/pmc3415448?pdf=render
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English
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Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines
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PLOS pathogens
| 2,012
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cc-by
| 13,579
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Abstract Funding: This work was supported by grants from the US National Institutes of Health number AI82548 to JBL and EJP AI50668 and AI22662 to JBL and RR02512
to Mark Haskins, University of Pennsylvania. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript. supported by grants from the US National Institutes of Health number AI82548 to JBL and EJP AI50668 and AI22662 to JBL and RR02512
ty of Pennsylvania. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jlok@vet.upenn.edu Competing Interests: The authors have declared that no competing interests exist. * E-mail: jlok@vet.upenn.edu . These authors contributed equally to this work. . These authors contributed equally to this work. resistance arising in populations of nematode parasites of humans
[13,14,15,16]. Screening has and will likely continue to identify
new candidate anthelmintics against nematodes. Increasingly,
however, alternative approaches involving rational design of
agents directed at defined molecular targets are important in
developing new drugs and identifying potential vaccine candidates
in other infectious disease systems and have recently been brought
to bear on parasitic nematodes [17]. Burgeoning descriptive
genomic and transcriptomic resources for parasitic nematodes
notwithstanding [18,19,20,21,22,23], functional characterization
and validation of such molecular targets in these worms has been
hampered by the lack of robust functional genomic tools such as
transgenesis and targeted gene silencing or disruption. For this
reason our laboratory has worked towards a system for transgen-
esis in parasitic nematodes of the genus Strongyloides [24,25,26,27]. Transposon-mediated Chromosomal Integration of
Transgenes in the Parasitic Nematode Strongyloides ratti
and Establishment of Stable Transgenic Lines Hongguang Shao1., Xinshe Li1., Thomas J. Nolan1, Holman C. Massey Jr.1, Edward J. Pearce2,
James B. Lok1* 1 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Department of
Pathology and Immunology, School of Medicine, Washington University, St. Louis, Missouri, United States of America PLoS Pathogens | www.plospathogens.org Abstract Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets
in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system
for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are
expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their
location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal
integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP)
under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In
three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid
encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1
infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-
positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP
expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the
F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations
of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR,
which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST
searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the
basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis. Citation: Shao H, Li X, Nolan TJ, Massey HC Jr, Pearce EJ, et al. (2012) Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode
Strongyloides ratti and Establishment of Stable Transgenic Lines. PLoS Pathog 8(8): e1002871. doi:10.1371/journal.ppat.1002871 Editor: Paul J. Brindley, George Washington University Medical Center, United States of America Received March 9, 2012; Accepted July 6, 2012; Published August 9, 2012 Copyright: 2012 Shao et al. This is an open-access article distributed under the terms of the Creative Commons Attributi
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Shao H, Li X, Nolan TJ, Massey HC Jr, Pearce EJ, et al. (2012) Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode
Strongyloides ratti and Establishment of Stable Transgenic Lines. PLoS Pathog 8(8): e1002871. doi:10.1371/journal.ppat.1002871 August 2012 | Volume 8 | Issue 8 | e1002871 Constructs incorporating regulatory elements of the
piggyBac transposon give reporter gene expression in F2
transgenic larvae of S. ratti has enabled a robust system for generating transgene
expressing S. stercoralis larvae of the F1 generation following gene
transfer [24,25]. In an effort to establish stable transgenic lines, F1
transgenic larvae of S. stercoralis have been reared to infective third-
stages and used to establish patent infections in gerbils [24,25]. Transgene expression has been observed in parasitic females
recovered from the intestines of these animals. Recently we
demonstrated that transgene constructs containing regulatory
sequences from S. stercoralis are expressed at roughly equal
frequencies and in virtually identical anatomical patterns in
Strongyloides ratti [26]. When F1 transgenics of S. stercoralis [24,25]
and S. ratti (unpublished) transformed with conventional plasmid
vectors are subjected to host passage, a substantial proportion of
their F2 progeny harbor transgene sequences, but these are not
expressed in this or subsequent generations of passage. In preliminary experiments [33], transforming S. stercoralis and
S. ratti with pPV356 along with mRNA encoding the piggyBac
transposase resulted in efficient production of F1 transgenics in
both parasites, and, following passage of these through gerbils or
rats, respectively, the first transgene-expressing F2 parasites
observed to date. Given the very low numbers of F2 transgenic
S. stercoralis produced relative to the approximately 50 third-stage
larvae required to establish a patent infection in the gerbil or dog,
we focused the present study on S. ratti. Transgenes with regulatory
elements from S. stercoralis are expressed in virtually identical
patterns and at equal frequencies in S. ratti [26]. Most importantly,
the availability of a well-adapted laboratory host, the rat, makes it
possible to establish patent infections with as few as one or two
infective larvae [26,33]. We assume that like C. elegans, Strongyloides spp. assemble the
majority
of
microinjected
transgenes
into
highly
repetitive
episomal arrays [30,31], and we hypothesize that Strongyloides
spp. actively silence these because of their episomal location, their
highly repetitive character [32] or both. We have addressed both
of
these
scenarios
by
attempting
low-copy
integration
of
transgenes into the chromosomes of S. ratti using the piggyBac
transposon system [33]. As an additional precaution against
epigenetic silencing, we assessed the ability of the gypsy retroviral
insulator sequences from Drosophila [34] to sustain transgene
expression during host passage. Preliminary experiments with this
system yielded the first transgene expressing individuals of the F2
generation in S. stercoralis and S. ratti observed to date [33]. In the
present study we confirmed this finding in S. Constructs incorporating regulatory elements of the
piggyBac transposon give reporter gene expression in F2
transgenic larvae of S. ratti To achieve low-copy integration of transgenes in Strongyloides we
designed constructs that incorporate regulatory elements of the
piggyBac transposon system (Fig. 1). The transgene encoded in both
the donor constructs tested (Fig. 1A, B) included a previously
reported GFP expression cassette in which expression is directed to
the body wall by the promoter for the cellular actin gene Ss-act-2
[24]. In both donor constructs, this reporter transgene was flanked
by the inverted terminal repeats specific for the piggyBac transposon
[35] as well as internal transposon sequences shown to be
necessary for efficient transposition in Drosophila [36]. In construct
pPV356 (Fig. 1B), the reporter transgene is also flanked by the
gypsy retroviral insulator sequences as a further precaution against
epigenetic silencing. It is worth noting that in an effort to define a
minimal functional transposon for studies of gene function in
insects [36], the internal transposon sequences in the piggyBac
encoding vector pXLBacII, from which we derived the piggyBac
elements for our vectors, have been significantly reduced in length
compared to the naturally occurring transposon from Trichoplusia
ni [37]. Although this minimal transposon worked under the
conditions obtaining in this initial study with S. ratti and mobilizes
efficiently in both Trichoplusia [36] and Drosophila [38], it is possible
that its efficiency could be increased in future studies of Strongyloides
and other parasites by optimizing the lengths of internal sequences
in the donor vector. As discussed below, this issue may be
particularly important in attempts to establish stable lines of
dioecious parasitic nematodes with integrated transgenes. Func-
tion of the piggyBac transposase was encoded either in a second
plasmid vector, the helper (pPV402, Fig. 1C), in which the enzyme
was expressed ubiquitously [24] under the promoter for the
ribosomal small subunit gene Ss-rps-21, or in capped and tailed
synthetic mRNA transcribed in vitro under the T7 promoter from
linearized plasmid pPV257 (Fig. 1D). generations of free-living development between parasitic genera-
tions [28]. The free-living females of Strongyloides constitute an
advantageous point of attack for gene transfer because they may
be cultured in vitro and because morphological similarities
between them and hermaphrodites of the free-living nematode
Caenorhabditis elegans have made it relatively straightforward to
adapt the technique of gonadal microinjection, originally devised
for transgenesis in C. elegans [29,30,31], to these parasites [27]. This technique, along with the design of vector constructs
containing both 59 and 39 regulatory sequences from Strongyloides
sp. Author Summary Author Summary Parasitic roundworms sicken and debilitate over one
billion people, most of whom subsist on less than two
US dollars per day. There are no vaccines and few drugs
available to treat and prevent these infections. Basic
research leading to new therapies has been hampered
because we lack methods to study gene function in
parasitic roundworms. One such method is transgenesis, a
process by which gene function is inferred by studying the
effects of transferring native or altered copies of genes into
subject organisms. Our laboratory has developed a system
for transferring synthetic genes into parasitic roundworms
of the genus Strongyloides and for obtaining temporary
expression of these ‘‘transgenes’’. Until now, however, we
have been unable to propagate these transgenic parasites
in the laboratory. This paper describes a new technique
that allows us to establish and maintain self-perpetuating
lines of transgenic parasites for study. This represents a
fundamental advance in the methodology for studying
gene function in parasitic roundworms and should greatly
facilitate the discovery of new therapies. Introduction Parasitic nematodes have an enormous impact on human
welfare, infecting over a billion people and causing debilitating,
disfiguring or blinding disease in hundreds of millions [1,2,3]. More insidious effects of these parasites include complications in
pregnancy and physical and cognitive deficits in children [4,5]. Parasitic nematodes severely degrade the health of domestic
animals, bringing about significant economic losses in developed
agricultural production systems [6,7] and heighten food insecurity
in marginal economies [8]. The need for ongoing research into new agents to prevent or
treat parasitic nematode infection is acute. There are currently no
effective vaccines available for these parasitisms, and chemother-
apy is based on a relatively small arsenal of anthelmintic drugs
[9,10]. Resistance to each of these is widespread among parasites
of livestock [11,12], and suboptimal anthelmintic treatment
responses in human clinical settings may signal genetically based We focus on Strongyloides spp. because unlike most parasitic
nematodes, which display an invariant pattern of development to
infective larvae outside the host, these worms execute one or more August 2012 | Volume 8 | Issue 8 | e1002871 August 2012 | Volume 8 | Issue 8 | e1002871 1 Stable, Integrated Transgenic Lines of S. ratti transgenes, and we established lines of this parasite that stably
transmit and express integrated transgenes in virtually all
progeny. PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 Constructs incorporating regulatory elements of the
piggyBac transposon give reporter gene expression in F2
transgenic larvae of S. ratti C) Helper vector pPV402 in which expression of the piggyBac
transposase gene is driven by Ss-rps-21 promoter and terminated by the Ss-era-1 39 UTR. D) Plasmid pPV257 for in vitro transcription of mRNA
encoding the piggyBac transposase under the T7 promoter. In lieu of helper vector pPV402, this mRNA was capped, tailed and co-injected with donor
vector pPV356 in Experiment 2. doi:10.1371/journal.ppat.1002871.g001 Figure 1. Vector constructs used to establish stable transgenic lines of Strongyloides ratti incorporate elements of the piggyBac
transposon system. A) Donor vector pPV254 incorporating a fluorescent reporter gene in which expression of GFP is driven by the Ss-act-2
promoter and terminated by the Ss era-1 39 UTR. The reporter transgene in pPV254 is flanked by the inverted terminal repeats (ITR) plus internal
sequences common to piggyBac transposable elements. B) Donor vector pPV356, which is like pPV254 in all respects except that the coding
sequence is flanked by the gypsy retroviral insulator sequences from Drosophila. C) Helper vector pPV402 in which expression of the piggyBac
transposase gene is driven by Ss-rps-21 promoter and terminated by the Ss-era-1 39 UTR. D) Plasmid pPV257 for in vitro transcription of mRNA
encoding the piggyBac transposase under the T7 promoter. In lieu of helper vector pPV402, this mRNA was capped, tailed and co-injected with donor
vector pPV356 in Experiment 2. doi:10.1371/journal.ppat.1002871.g001 Experiment 2). This finding is consistent with our hypothesis that
silencing observed in Strongyloides spp. transformed with plasmid
vectors stems from the assembly of the encoded transgenes into
episomal arrays and that this silencing can be circumvented by
integration of the transgenes into the chromosomes of the
parasites. Replicate experiments (Experiments 3 and 4, Table 1),
in which worms were transformed with pPV356 and the helper
plasmid pPV402, yielded markedly higher proportions of F2
parasites expressing the reporter transgene following host passage
than seen when the transposase activity was encoded in capped
RNA. This suggests that in S. ratti, co-transfected helper plasmids
designed for in situ expression of the piggyBac transposase constitute
a more efficient means of providing this activity than in vitro
translated capped RNA. By contrast, co-transfection with capped
RNA encoding the transposase gives efficient piggyBac-mediated
integration of transgenes in Schistosoma mansoni, whereas expression
of the transposase from co-transfected helper plasmids does not
[39]. Co-transformation of S. Constructs incorporating regulatory elements of the
piggyBac transposon give reporter gene expression in F2
transgenic larvae of S. ratti ratti with donor plasmid pPV254,
which lacks the gypsy insulator sequences, and helper plasmid
pPV402 (Table 1, Experiment 5) yielded transgene-expressing
individuals in the F2 generation. This indicates that although the
gypsy insulator sequences effectively resist positional silencing of
transgenes in Drosophila [34], they are not required for sustained
transgene expression in S. ratti. This result further supports the
hypothesis that chromosomal integration of transgenes is the
essential factor provided by this system. rats were screened for GFP expression and hand selected. These
F2 individuals were cultured to mating pairs of free-living adults,
and their progeny (F3) were reared to L3i, selected for expression
of the transgenes, and inoculated into rats. S. ratti eggs and first-
stage larvae in the feces of these rats constituted the F4 generation. This pattern of alternating culture, selection for expression and
host passage was then repeated for subsequent generations of
selection. The results (Fig. 2B), expressed as the percentage of GFP+
individuals in the population screened within each generation,
demonstrate derivation of stable lines with virtually 100%
inheritance and expression of transgenes within 5 generations of
selection. Numbers of individuals available for passage in the F1
and F2 generations were low for all three lines, but could be
amplified owing to the high susceptibility of laboratory rats to S. ratti infection. The decline in the proportion of GFP-positive larvae
in the F5 generation is noteworthy. In our selection scheme
(Fig. 2A), the F5 generation arises from crossing of GFP-positive
free-living males and females in culture. It is possible that some
heterozygous individuals remain in the F5 generation and that the
observed decline in the frequency of GFP-expressing individuals in
the F5 resulted from segregation of homozygous wild-type
individuals among the progeny of crosses between heterozygotes
or between heterozygotes and homozygous transgenic worms. We
are continuing to maintain these lines by serial passage. Line PV2
is currently in the F10 and exhibits GFP fluorescence in 100% of
individuals; PV3 is in the F10 with expression in 98.6% of
individuals, and PV4 is in the F8 with expression in 99.4% of
individuals. While expression rates in PV3 and PV4 may reflect
remaining heterozygosity in these lines, they could also be due to
diminished levels of expression or viability in small proportions of
the worms examined by fluorescence stereomicroscopy. Constructs incorporating regulatory elements of the
piggyBac transposon give reporter gene expression in F2
transgenic larvae of S. ratti We have
cryopreserved transgenic L3i from each line as described [40] (at
280uC instead of in liquid nitrogen) and have thawed viable late-
passage worms with a recovery rate of approximately 10% (data
not shown). All thawed L3i were transgenic, as evidenced by
expression of GFP, indicating
that in the lines observed,
integration of the transgenes did not affect parasite fitness in a
way that would render them more susceptible to damage during
cryopreservation. Constructs incorporating regulatory elements of the
piggyBac transposon give reporter gene expression in F2
transgenic larvae of S. ratti ratti, demonstrated
that the piggyBac system results in chromosomal integration of Transformation of S. ratti with pPV356 in the absence of
transposase-encoding helper constructs gave efficient transmission
and expression of transgenes in F1 progeny, but passage of these
through rats failed to yield any transgene-expressing larvae in the
F2 generation (Table 1, Experiment 1). Transformation of S. ratti
with donor plasmid pPV356 combined with capped RNA
encoding the piggyBac transposase gave efficient transgene trans-
mission and expression in F1 larvae, and it resulted in a small
proportion of F2 individuals expressing the transgene (Table 1, August 2012 | Volume 8 | Issue 8 | e1002871 August 2012 | Volume 8 | Issue 8 | e1002871 2 Figure 1. Vector constructs used to establish stable transgenic lines of Strongyloides ratti incorporate elements of the piggyBac
transposon system. A) Donor vector pPV254 incorporating a fluorescent reporter gene in which expression of GFP is driven by the Ss-act-2
promoter and terminated by the Ss era-1 39 UTR. The reporter transgene in pPV254 is flanked by the inverted terminal repeats (ITR) plus internal
sequences common to piggyBac transposable elements. B) Donor vector pPV356, which is like pPV254 in all respects except that the coding
sequence is flanked by the gypsy retroviral insulator sequences from Drosophila. C) Helper vector pPV402 in which expression of the piggyBac
transposase gene is driven by Ss-rps-21 promoter and terminated by the Ss-era-1 39 UTR. D) Plasmid pPV257 for in vitro transcription of mRNA
encoding the piggyBac transposase under the T7 promoter. In lieu of helper vector pPV402, this mRNA was capped, tailed and co-injected with donor
vector pPV356 in Experiment 2. doi:10.1371/journal.ppat.1002871.g001
Stable, Integrated Transgenic Lines of S. ratti Stable, Integrated Transgenic Lines of S. ratti Figure 1. Vector constructs used to establish stable transgenic lines of Strongyloides ratti incorporate elements of the piggyBac
transposon system. A) Donor vector pPV254 incorporating a fluorescent reporter gene in which expression of GFP is driven by the Ss-act-2
promoter and terminated by the Ss era-1 39 UTR. The reporter transgene in pPV254 is flanked by the inverted terminal repeats (ITR) plus internal
sequences common to piggyBac transposable elements. B) Donor vector pPV356, which is like pPV254 in all respects except that the coding
sequence is flanked by the gypsy retroviral insulator sequences from Drosophila. PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 Expression patterns of a reporter transgene in stable
lines are consistent with those seen in transiently
transformed Strongyloides spp Table 1. Heritable transgene expression and establishment of stable transgene-expressing lines in S. ratti as a function of various pairings of donor and helper plasmids
incorporating elements of the piggyBac transposon. Experiment
Donor
Construct
Helper
No. P0
Injected
No. F1 Larvae GFP
+ (No. Screened)
% F1
Expression
No. F2 Larvae GFP
+ (No. Screened)
% F2
Expression
Stable line
obtained
1
pPV356
None
160
72 (1854)
3.4
0 (1194)
0
No
2
pPV356
Transposase mRNA
195
235 (3716)
6.3
4 (14278)
0.03
No
3
pPV356
pPV402
205
70 (784)
8.9
34 (8511)
0.4
Yes
4
pPV356
pPV402
50
15 (428)
3.5
19 (867)
2.2
Yes
5
pPV254
pPV402
110
45 (1105)
4.1
56 (2282)
2.5
Yes
S Pathogens | www.plospathogens.org Table 1. Heritable transgene expression and establishment of stable transgene-expressing lines in S. ratti as a function of various pairings of donor and helper plasmids
incorporating elements of the piggyBac transposon. Experiment
Donor
Construct
Helper
No. P0
Injected
No. F1 Larvae GFP
+ (No. Screened)
% F1
Expression
No. F2 Larvae GFP
+ (No. Screened)
% F2
Expression
Stable line
obtained
1
pPV356
None
160
72 (1854)
3.4
0 (1194)
0
No
2
pPV356
Transposase mRNA
195
235 (3716)
6.3
4 (14278)
0.03
No
3
pPV356
pPV402
205
70 (784)
8.9
34 (8511)
0.4
Yes
4
pPV356
pPV402
50
15 (428)
3.5
19 (867)
2.2
Yes
5
pPV254
pPV402
110
45 (1105)
4.1
56 (2282)
2.5
Yes The expression pattern of the transcriptional reporter Ss-act-
2prom::gfp::Ss-era-1 39 in parasitic females from a stable integrated
line (PV2) of S. ratti were similar to that reported previously [24]
for F1 larvae of S. stercoralis transformed with a conventional
plasmid vector (pAJ08) and presumably expressing this transcrip-
tional reporter from a multi-copy episomal array. In general,
parasitic females from line PV2 exhibited a zone of fluorescence in
the body wall extending posteriorly from the junction between
pharynx (esophagus) and intestine to a level approximately even
with the posterior margin of the uterus (Fig. 3A, B). Retention of
the body-wall specific pattern of Ss-act-2-regulated GFP expression
in stable S. ratti transformants is consistent with previous findings
that promoters derived from S. stercoralis drive reporter transgene
expression in identical or highly similar anatomical patterns in S. ratti [26]. Moreover, this observation suggests that expression
patterns
of
transgenes
integrated
into
limited
numbers
of
chromosomal sites in Strongyloides spp. Expression patterns of a reporter transgene in stable
lines are consistent with those seen in transiently
transformed Strongyloides spp will be consistent with
patterns resulting from over expression of the same transgenes
from high copy number episomal arrays. The expected body wall specific pattern of the Ss-act-2prom::gfp
reporter was also seen in free-living females (Fig. 3C, D) and free-
living males (Fig. 3E, F). In addition to uniform expression
throughout the body wall, additional loci of GFP expression were
seen in the vulva and rectum of free-living females (Fig. 3D) and in
the cloaca of free-living males (Fig. 3F). No fluorescent signal could
be detected from non-transformed free-living male S. ratti (Fig. 3G,
H). Owing to the failure of non-integrated transgenes to express
beyond the F1 generation, free-living adult Strongyloides expressing
transgenes had not been observed prior to the establishment of
stable integrated lines in S. ratti. Transgene-expressing S. ratti of the F2 generation can
found stable lines with sustained inheritance and
expression of transgenes Having observed the first transgene expression in the F2
generation of S. ratti, we asked whether these F2 individuals could
found stable transgenic lines. We did so using the protocol
illustrated in Figure 2A. Briefly, parental free-living females of S. ratti were transformed by gonadal microinjection with Ss-act-2::gfp
in piggyBac vectors as described previously for plasmid vectors
[24,25]. F1 progeny of microinjected females were reared in
culture and screened for reporter expression by fluorescence
stereomicroscopy. GFP+ progeny were cultured to infective L3
(L3i) and inoculated into rats. F2 progeny arising in feces of these PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 3 Stable, Integrated Transgenic Lines of S. ratti August 2012 | Volume 8 | Issue 8 | e1002871 Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti F2 progeny released in the feces of
these rats are screened for transgene expression and positive larvae reared in culture to free-living males and females and allowed to mate. F3
progeny arising from these crosses are reared to L3i in culture in inoculated into rats. F4 progeny arising in the feces are screened for reporter
transgene expression and used for further alternating rounds of culture and host passage with continued selection on GFP. The timeline indicates the
intervals in days following microinjection of parental worms in which each generation is isolated up to the F5 when transmission and expression are
generally stabilized. B) Frequency of transgene expressing progeny by generation during selection of stable transgenic lines of S. ratti. Data are mean
(6 one standard deviation) percentages of reporter transgene-expressing individuals in each generation of alternating host and culture passage with
selection on GFP (see panel A). Means are derived from three independently established lines (PV2, PV3 and PV4). Sample sizes (n) indicated below
the abscissa are totals of worms examined from all three lines. Statistics: in panel B, 2-way ANOVA, performed using Prism ver. 5.0c (GraphPad
Software, Inc., La Jolla, California, USA), revealed a highly significant effect due to generation (P,0.0001) but no significant effect (P = 0.2) due to
donor plasmid. doi:10.1371/journal.ppat.1002871.g002
Stable, Integrated Transgenic Lines of S. ratti Figure 2. Stable transgenic lines of S. ratti are established by microinje
of host and culture passage, selecting on expression of a fluorescent
stable transgenic lines of S. ratti. Initial gene transfer into parental (P0) free-livin
combination with helper vector or capped transposase encoding mRNA. F1 larva
expressing individuals are reared to infective L3 (L3i) and inoculated into rats t
these rats are screened for transgene expression and positive larvae reared in
progeny arising from these crosses are reared to L3i in culture in inoculated
transgene expression and used for further alternating rounds of culture and host
intervals in days following microinjection of parental worms in which each gene
generally stabilized. B) Frequency of transgene expressing progeny by generatio
(6 one standard deviation) percentages of reporter transgene-expressing individ
selection on GFP (see panel A). Means are derived from three independently es
the abscissa are totals of worms examined from all three lines. Statistics: in p
Software Inc La Jolla California USA) revealed a highly significant effect due Figure 2. Stable transgenic lines of S. Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti We inferred chromosomal integration of the reporter transgene
in S. ratti from a Southern blot of a BsrGI restriction digest of
genomic DNA from each transgenic line which was hybridized
with a gfp-specific probe (Fig. 4A). We detected gfp-specific
sequences in multiple fragments with various molecular weights
in the genomic DNA of each line (Fig. 4B). Based on the presence
of only one BsrGI site in the vector, occurring within the gfp coding
sequence, the smallest predicted transgene-containing restriction
fragments
of
the
genome
would
be
1898 bp
for
worms
transformed with the donor vector pPV356, which contains the
430 bp gypsy insulator sequences or 1468 bp for worms trans-
formed with donor vector pPV254, which does not contain these
sequences (Fig. 4A). The Southern hybridization patterns seen for
PV2, PV3 and PV4 (Fig. 4B) are generally consistent with this
prediction, showing a multiplicity of bands hybridizing with the
gfp-specific probe in the range of approximately 1.8 kb to 7.5 kb. By contrast, this diverse banding pattern is not consistent with the
uniform 8084 and 6758 bp fragments that would be expected to
result from BsrGI digestion of episomal arrays made up of the
donor vectors pPV356 and pPV254, respectively. Chromosomal integration was confirmed by splinkerette-PCR,
which allowed sequencing of the genomic regions bounding unique
transgene insertions in five contigs of the S. ratti genome (Table 2). The current draft genome assembly for S. ratti (http://www.sanger. ac.uk/resources/downloads/helminths/strongyloides-ratti.html) al-
lows assignment of three of these contigs to Chromosome I. Further PLoS Pathogens | www.plospathogens.org PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 August 2012 | Volume 8 | Issue 8 | e1002871 4 Stable, Integrated Transgenic Lines of S. ratti Stable, Integrated Transgenic Lines of S. ratti Figure 2. Stable transgenic lines of S. ratti are established by microinjection of nucleic acid constructs followed alternating rounds
of host and culture passage, selecting on expression of a fluorescent reporter gene product. A) Diagram of major steps in isolation of
stable transgenic lines of S. ratti. Initial gene transfer into parental (P0) free-living females is by gonadal microinjection of donor vectors alone or in
combination with helper vector or capped transposase encoding mRNA. F1 larvae are derived in culture and screened for GFP expression. Transgene
expressing individuals are reared to infective L3 (L3i) and inoculated into rats to establish as parasitic females. Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti ratti are established by microinjection of nucleic acid constructs followed alternating rounds
of host and culture passage, selecting on expression of a fluorescent reporter gene product. A) Diagram of major steps in isolation of
stable transgenic lines of S. ratti. Initial gene transfer into parental (P0) free-living females is by gonadal microinjection of donor vectors alone or in
combination with helper vector or capped transposase encoding mRNA. F1 larvae are derived in culture and screened for GFP expression. Transgene
expressing individuals are reared to infective L3 (L3i) and inoculated into rats to establish as parasitic females. F2 progeny released in the feces of
these rats are screened for transgene expression and positive larvae reared in culture to free-living males and females and allowed to mate. F3
progeny arising from these crosses are reared to L3i in culture in inoculated into rats. F4 progeny arising in the feces are screened for reporter
transgene expression and used for further alternating rounds of culture and host passage with continued selection on GFP. The timeline indicates the
intervals in days following microinjection of parental worms in which each generation is isolated up to the F5 when transmission and expression are
generally stabilized. B) Frequency of transgene expressing progeny by generation during selection of stable transgenic lines of S. ratti. Data are mean
(6 one standard deviation) percentages of reporter transgene-expressing individuals in each generation of alternating host and culture passage with
selection on GFP (see panel A). Means are derived from three independently established lines (PV2, PV3 and PV4). Sample sizes (n) indicated below
the abscissa are totals of worms examined from all three lines. Statistics: in panel B, 2-way ANOVA, performed using Prism ver. 5.0c (GraphPad
Software, Inc., La Jolla, California, USA), revealed a highly significant effect due to generation (P,0.0001) but no significant effect (P = 0.2) due to
donor plasmid. doi:10 1371/journal ppat 1002871 g002 p
doi:10.1371/journal.ppat.1002871.g002 analysis, in which we subjected 2 kb sequences flanking each of the
five identified transposon insertion sites (Table 2) to BlastN/BlastX
searching against the non-redundant nucleotide/protein sequences,
revealed three insertions in coding regions. All insertions were at
TTAA sites (Table 2) as has been found in other studies of the analysis, in which we subjected 2 kb sequences flanking each of the
five identified transposon insertion sites (Table 2) to BlastN/BlastX
searching against the non-redundant nucleotide/protein sequences,
revealed three insertions in coding regions. August 2012 | Volume 8 | Issue 8 | e1002871 Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti All insertions were at
TTAA sites (Table 2) as has been found in other studies of the piggyBac transposon [21,39,40]. Insertion PV2-2 (Table 2) is near the
59 end of a sequence predicted to encode a peptide that is 51%
identical to the hydroxyacylglutathione hydrolase in Loa loa
(Accession XP_003137817). Insertion PV3-1 is within the coding
sequence of a 28S ribosomal RNA gene that has 98% nucleic acid PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 August 2012 | Volume 8 | Issue 8 | e1002871 5 Figure 3. Parasitic and free-living adults from a stable transgenic line of Strongyloides ratti express GFP in the body wall-specific
manner expected for the Ss-act-2prom::gfp transcriptional reporter. Typical patterns of GFP expression in parasitic female, free-living male
and free-living female S. ratti expressing the integrated reporter transgene encoded in pPV356. (A, B) DIC and fluorescence images, respectively, of a
parasitic female S. ratti from integrated line PV2. Note expression predominating in the body wall up to the level of the esophageal/intestinal
boundary (ei). Position of the head is indicated (h). (C, D) DIC and fluorescence images, respectively, of a free-living female S. ratti from integrated
line PV2. Note uniform expression throughout the body wall with additional loci of expression in the vulva (v) and rectum (re). (E, F) DIC and
fluorescence images, respectively, of a free-living male S. ratti from transgenic line PV2. Note uniform expression in the body wall with additional
expression in the cloaca (cl). (G, H) DIC and fluorescence images, respectively, of a non-transformed free-living male S. ratti. The fluorescence image
in panel H was exposed for a period$exposure times in panels B, D and F. Scale bar = 200 mm in all panels. doi:10.1371/journal.ppat.1002871.g003
Stable, Integrated Transgenic Lines of S. ratti Stable, Integrated Transgenic Lines of S. ratti Figure 3. Parasitic and free-living adults from a stable transgenic line of Strongyloides ratti express GFP in the body wall-specific
manner expected for the Ss-act-2prom::gfp transcriptional reporter. Typical patterns of GFP expression in parasitic female, free-living male
and free-living female S. ratti expressing the integrated reporter transgene encoded in pPV356. (A, B) DIC and fluorescence images, respectively, of a
parasitic female S. ratti from integrated line PV2. Note expression predominating in the body wall up to the level of the esophageal/intestinal
boundary (ei). Position of the head is indicated (h). August 2012 | Volume 8 | Issue 8 | e1002871 PLoS Pathogens | www.plospathogens.org Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti Transgene specific sequences are widely distributed in restriction digests of genomic DNA from three stable lines of
transgenic S. ratti. Southern hybridization of genomic DNA (gDNA) of S. ratti from stably transformed lines probed for the gfp coding sequence. A)
Transgene diagram showing position of the probe used for Southern hybridization analysis and the single restriction site for BsrGI, the enzyme used
for restriction digestion of gDNA. B) Southern blot of BsrGI digests of gDNA from pooled free-living adults from three independent integrated lines
(PV2, PV3 and PV4). The positive control lane is a blot of a BsrGI digest of donor plasmid pPV356, and the negative control is a blot of a BsrGI digest of
gDNA from non-transformed S. ratti free-living adults (S.r. gDNA). Note gfp hybridization signals in multiple restriction fragments in gDNA from each
of the three integrated lines. A single hybridizing band of 8.1 kb appears as predicted in the positive control digest of pPV356. No gfp-specific signal
is detected in the negative control digest of gDNA from non-transformed S. ratti. C) Gel analysis of PCR products from genomic DNA templates from
non-transformed control parasites, parasites from each of three stable lines, PV2, PV3 and PV4, with integrated, stably expressed transgenes and F1
progeny of free-living female worms microinjected with donor plasmid pPV356 and helper plasmid pPV402. Also included are PCR products from
control reactions with plasmids pPV356 and pPV254 as templates and from a reaction from which template was omitted. Upper gel image depicts
624 bp amplification products resulting from a forward primer hybridizing to the M13 reverse priming site in the vector and a reverse primer
hybridizing within the transposon sequence. Lower gel image is a loading control showing the expected 554 bp amplification product from reactions
with primers specific for the constitutively expressed cellular actin-encoding gene Sr-act-2. doi:10.1371/journal.ppat.1002871.g004
Stable, Integrated Transgenic Lines of S. ratti Stable, Integrated Transgenic Lines of S. ratti Figure 4. Transgene specific sequences are widely distributed in restriction digests of genomic DNA from three stable lines of
transgenic S. ratti. Southern hybridization of genomic DNA (gDNA) of S. ratti from stably transformed lines probed for the gfp coding sequence. A)
Transgene diagram showing position of the probe used for Southern hybridization analysis and the single restriction site for BsrGI, the enzyme used
for restriction digestion of gDNA. Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti (C, D) DIC and fluorescence images, respectively, of a free-living female S. ratti from integrated
line PV2. Note uniform expression throughout the body wall with additional loci of expression in the vulva (v) and rectum (re). (E, F) DIC and
fluorescence images, respectively, of a free-living male S. ratti from transgenic line PV2. Note uniform expression in the body wall with additional
expression in the cloaca (cl). (G, H) DIC and fluorescence images, respectively, of a non-transformed free-living male S. ratti. The fluorescence image
in panel H was exposed for a period$exposure times in panels B, D and F. Scale bar = 200 mm in all panels. doi:10.1371/journal.ppat.1002871.g003 previously with Salmonella typhi [41], to determine whether the
pattern of piggyBac insertions in S. ratti is consistent with the observed
broad patterns of piggyBac insertions in the genomes of Drosophila
melanogaster [42] and the parasitic trematode S. mansoni [39]. The fact
that three of the five insertions we sequenced were in coding
sequences in the in S. ratti genome is consistent with the piggyBac
transposon’s documented propensity to integrate into the coding
regions of genes in other organisms [42,43]. This observation
underscores the potential of the piggyBac transposon system as a tool
for insertional mutagenesis in unbiased forward genetic investiga-
tions with S. ratti. identity with AB205054 in S. stercoralis, and insertion PV4-1 is within
a gene sequence predicted to encode a peptide with 68% identity to
hypothetical protein T13G4.3 in Caenorhabditis elegans (ADD13552.1)
(XP_001894192.1). Insertions PV2-1 and PV2-3 were in intergenic
regions. Although the wide molecular weight ranges of restriction
fragments of gDNA with gfp-specific sequences in all three lines
(Fig. 4B) are suggestive, the breadth of transgene integrations
throughout the genome of S. ratti cannot be assessed accurately from
the few sequenced integration junctions reported here. Additionally,
splinkerette-PCR, the technique used to sequence and map
integration boundaries, is biased to the restriction sites of the
enzymes used to digest the genomic DNA. In future studies, high-
capacity genome sequencing could be brought to bear, as done Estimates of relative copy number of integrated transgenes
derived by qRT-PCR were similar in the three independent lines PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 August 2012 | Volume 8 | Issue 8 | e1002871 6 Figure 4. PLoS Pathogens | www.plospathogens.org Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti Genomic integration sites for reporter transgenes and their flanking sequences in three stable transgenic lines of
Strongyloides ratti. gration sites for reporter transgenes and their flanking sequences in three stable transgenic lines of Table 2. Genomic integration sites for reporter transgenes and their flanking sequences in three stable transgenic lines of
Strongyloides ratti. with mean relative copy number ranging from 37 to 51 per
genome (Table 3). These means were not significantly different
(P.0.05), indicating that the transformation protocol results in a
consistent number of transgene integrations. To ensure that the
estimates of relative copy number of integrated transgenes did not
reflect sequences in episomal arrays, we conducted PCR on gDNA
from
each stable
line
using
forward
and
reverse
primers
hybridizing within the M13 reverse priming site of the vector
and within the transposon sequence respectively. These primers
did not yield a product from gDNA of the stable integrated lines
(F8 generation), but did from the F1 progeny of free-living female
worms microinjected with donor (pPV356) and helper plasmids
(Fig. 4C). This suggests that donor plasmids are transmitted to the
F1 generation but are lost during passage. We conclude, therefore,
that our estimates of relative transgene copy number reflect
numbers of chromosomal integrations. While random integration
of large numbers of transposons would be desirable in unbiased
studies aimed at gene tagging or disruption, integration of large
numbers of transgene copies in focused studies of gene function is with mean relative copy number ranging from 37 to 51 per
genome (Table 3). These means were not significantly different
(P.0.05), indicating that the transformation protocol results in a
consistent number of transgene integrations. To ensure that the
estimates of relative copy number of integrated transgenes did not
reflect sequences in episomal arrays, we conducted PCR on gDNA
from
each stable
line
using
forward
and
reverse
primers
hybridizing within the M13 reverse priming site of the vector
and within the transposon sequence respectively. These primers
did not yield a product from gDNA of the stable integrated lines
(F8 generation), but did from the F1 progeny of free-living female
worms microinjected with donor (pPV356) and helper plasmids
(Fig. 4C). This suggests that donor plasmids are transmitted to the
F1 generation but are lost during passage. We conclude, therefore,
that our estimates of relative transgene copy number reflect
numbers of chromosomal integrations. bCalculated from three replicate assays.
cDifferences between mean relative copy number for stable lines not significant by one-way ANOVA (P.0.05).
dWildtype S ratti Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti B) Southern blot of BsrGI digests of gDNA from pooled free-living adults from three independent integrated lines
(PV2, PV3 and PV4). The positive control lane is a blot of a BsrGI digest of donor plasmid pPV356, and the negative control is a blot of a BsrGI digest of
gDNA from non-transformed S. ratti free-living adults (S.r. gDNA). Note gfp hybridization signals in multiple restriction fragments in gDNA from each
of the three integrated lines. A single hybridizing band of 8.1 kb appears as predicted in the positive control digest of pPV356. No gfp-specific signal
is detected in the negative control digest of gDNA from non-transformed S. ratti. C) Gel analysis of PCR products from genomic DNA templates from
non-transformed control parasites, parasites from each of three stable lines, PV2, PV3 and PV4, with integrated, stably expressed transgenes and F1
progeny of free-living female worms microinjected with donor plasmid pPV356 and helper plasmid pPV402. Also included are PCR products from
control reactions with plasmids pPV356 and pPV254 as templates and from a reaction from which template was omitted. Upper gel image depicts
624 bp amplification products resulting from a forward primer hybridizing to the M13 reverse priming site in the vector and a reverse primer
hybridizing within the transposon sequence. Lower gel image is a loading control showing the expected 554 bp amplification product from reactions
with primers specific for the constitutively expressed cellular actin-encoding gene Sr-act-2. doi:10.1371/journal.ppat.1002871.g004 PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 7 PLoS Pathogens | www.plospathogens.org Stable, Integrated Transgenic Lines of S. ratti Table 2. Genomic integration sites for reporter transgenes and their flanking sequences in three stable transgenic lines of
Strongyloides ratti. Stable
line
Donor
vector
Insertion
Integration
junction
accession
Contig
Chr.a
Location
59 sequence junction
Target
site
39 sequence junction
PV2
pPV356
PV2-1
JX070935
74278
I
172601
ACTTTAACGTTGTCATGATTTTTTT
TTAA
ATTTCATTATACTATTTTAAAAGTA
‘‘
pPV356
PV2-2
JX070931
74996
I
155901
GGTGGTGGAGATAAAATTAGTTATG
TTAA
TAAAATTGTTAAAGATGGTGATGAA
‘‘
pPV356
PV2-3
JX070932
75336
NDb
3211
TTCGATATCTCTTGTTACTTTTCAA
TTAA
AAAAAAAAATATTTTTTAAGGCAAA
PV3
pPV356
PV3-1
JX070933
75034
NDb
3303
GTATCCAAAGTGCTTGTTATATTAC
TTAA
AAAGTTAATGGAAGCATAATAAATT
PV4
pPV254
PV4-1
JX070934
75488
I
603672
ATAAGAACACGTCCTAAGTGAAATC
TTAA
TGCTGTTCGAGGATGAAAATTTTCA
Contig identities, insertion locations within contigs and, where possible, chromosome numbers are derived from the draft S.ratti genome sequence. aChromosome. bNot determined. doi:10.1371/journal.ppat.1002871.t002 Table 2. Genomic integration sites for reporter transgenes and their flanking sequences in three stable transgenic lines of
Strongyloides ratti. Table 2. Wildtype S. ratti.
doi:10.1371/journal.ppat.1002871.t003 ratti.
ournal.ppat.1002871.t003 Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti While random integration
of large numbers of transposons would be desirable in unbiased
studies aimed at gene tagging or disruption, integration of large
numbers of transgene copies in focused studies of gene function is less than desirable given the risk of non-physiologic effects of over
expression and non-specific effects resulting from insertional
mutagenesis. It is noteworthy in this regard that no unusual or
unexpected phenotypes have been evident among worms from the
three stable lines in the present study. A technique for single-copy
integration of transgenes in C. elegans based on the Mos-1
transposon system has been developed recently [44] and could
provide a strategy for achieving similar results in Strongyloides spp. in the future. Note that a stable, GFP-expressing line was established using
the donor plasmid pPV254, which lacks the gypsy insulator
sequences, (Fig. 1; Tables 1 and 2). Furthermore, the proportion of
GFP expressing individuals among all larvae screened did not vary
significantly as a function of donor construct (Fig. 2B). Together,
these observations indicate that under conditions pertaining in the
present study, where integrations appeared to occur widely
throughout the genome, with mean relative copy numbers ranging
from 37–51 per genome, the gypsy insulator sequences are not
required for stable expression of transgenes in integrated lines of S. Table 3. Analysis of relative transgene copy number via qRT-PCR for stable transgenic lines of S. ratti transformed with the
piggyBac transposon system. Stable line
Donor
construct
Assay
replicate
Ct e (Mean ± SD)a
Ct t (Mean ± SD)a
DCt
DDCt
22DDCt
Relative copy
number (Mean ± SD)b,c
PV2
pPV356
1
19.9560.26
21.4560.04
1.49
25.62
49
2
19.5360.05
21.3660.09
1.83
25.72
53
5162
3
19.8660.16
21.1060.24
1.24
25.66
51
PV3
pPV356
1
20.4960.12
22.3160.10
1.82
25.29
39
2
20.0860.15
22.2360.07
2.20
25.35
41
3766
3
20.1960.25
22.1860.10
1.99
24.92
30
PV4
pPV254
1
21.0260.19
22.4460.11
1.42
25.69
52
2
20.5060.25
22.2860.07
1.78
25.77
55
4869
3
20.5260.13
22.2160.06
1.69
25.21
37
Controld
none
1
23.9760.18
31.0860.15
7.11
0
1
2
23.9160.11
31.4660.30
7.55
0
1
1
3
24.8860.20
31.7960.20
6.91
0
1
aCalculated from three technical replicates within the assay. bCalculated from three replicate assays. cDifferences between mean relative copy number for stable lines not significant by one-way ANOVA (P.0.05). dWildtype S. ratti. Stable, Integrated Transgenic Lines of S. ratti Stable, Integrated Transgenic Lines of S. ratti parthenogenetic females are clonal [56] and so matings in
subsequent free-living generations are effective selfings of the
parasitic
female
parent,
further
hastening
the
selection
of
homozytotic transgenic parasites. These advantages would not
be available in other parasitic nematode taxa of interest, which are
dioecious. Nevertheless,
if
sufficient
numbers
of
transgenic
progeny from the F1 and subsequent generations, comprising
both male and female larvae, and a sufficiently well adapted host-
parasite system in which to carry out line selection were available,
these conditions could combine to enable the establishment of
stable transgenic lines of dioecious parasitic nematodes, albeit with
a more lengthy and labor intensive process than in Strongyloides spp. The ability to transfer genes into Strongyloides spp. and related
genera by gonadal microinjection of free-living females [27,57]
also constitutes a major advantage in undertaking transgenesis that
will be unavailable in the majority of parasitic nematode species
that cannot undertake full generations of free-living development. However, larval stages of animal parasitic nematodes that arise in
the extrinsic environment or in arthropod vectors can be cultured
transiently and may be susceptible to gene transfer by other routes. This potential was recently demonstrated emphatically in the
heritable transformation of Brugia malayi by chemically mediated
gene transfer [58]. Gene transfer in this study was achieved by
incubating developing third-stage larvae with co-precipitates of the
DNA vector with calcium phosphate. This method succeeded with
larvae that were inoculated along with the co-precipitate into the
peritoneal cavities of susceptible gerbils but not with larvae
cultured in vitro with co-precipitate, even in a culture system that
promoted
L3–L4 molting. This
finding may indicate that
completion of a molt cycle with subsequent remodeling of the
nascent cuticle, which occurs in the host, but may not occur
normally in cultured B. malayi L3, is necessary for sufficient uptake
of the DNA-CaP04 co-precipitate. In any case, this method of
chemically mediated gene transfer into an accessible larval stage
represents one that, in theory, could be adaptable to transposon
mediated integration of transgenes into a broad range of parasitic
nematodes of medical and veterinary importance. ratti. However, the gypsy sequences were originally characterized
as resisting positional silencing effects in Drosophila, which vary
significantly based on the site of transgene integration [34]. Although positional silencing of transgenes has not been docu-
mented in S. Ethics statement Rats and gerbils were used for parasite strain maintenance and
host passage of transgenic S. ratti in this study. These procedures
were approved by the Institutional Animal Care and Use
Committee (IACUC) of the University of Pennsylvania (Protocol
No. 803511). This protocol, as well as routine husbandry care of
the animals, was in strict accordance with the Guide for the Care and
Use of Laboratory Animals of the National Institutes of Health. With regard to the use of a transposon-based approach to
transgene integration per se, improvements in the efficiency of
transposition will enable such techniques as transposition induced
gene knockouts [42], exon and enhancer traps [53], and excision-
based site directed mutagenesis [54] to further the genetic analysis
of these species. The use of transposon integration sites as genetic
markers [55] could aid in the assembly of chromosome scaffolds
for the ongoing genome sequencing projects for these worms. PLoS Pathogens | www.plospathogens.org Stable, Integrated Transgenic Lines of S. ratti ratti, it is possible that, as we attempt to achieve
integrations at lower copy number or to adapt methods for single
copy transposon-mediated transgenesis [44] to this parasite, such
positional effects will manifest themselves and be a significant
factor in stable transgene expression. In such cases it may be
prudent to include the gypsy sequences as data from the present
study also indicate that they do not adversely affect the efficiency
of integration. In summary, we have developed a transposon-based system for
integration of transgenes into the chromosomes of Strongyloides spp. and for the establishment of stable transgenic lines. Strongyloides ratti
lends itself particularly well to establishment of stable integrated
lines owing to its efficiency of infection in laboratory rats. Three
such integrated lines have been established in S. ratti, and
boundary sequences of multiple unique transgene integration sites
have been mapped. The possibility of introducing heritable, stably
expressed transgenes into a parasitic nematode is an advance that
offers important new opportunities to study gene function in these
organisms. Perpetuation of stable transgenic lines will greatly
facilitate functional approaches involving expression transgenes
with dominant interfering or activating mutations as was recently
done with very limited samples of transiently transformed S. stercoralis [45]. Transposon mediated transgenesis will also be a useful adjunct
to other methods of genetic analysis such as RNAi. The majority
of animal parasitic nematodes exhibit very limited sensitivity to
exogenously administered RNAi [46]. RNAi targets in Haemonchus
contortus that are located on cell surfaces in contact with the worm’s
external environment show greater RNAi sensitivity than internal
targets [47], suggesting that this, and perhaps other parasitic
species, are deficient in mechanisms that spread administered
dsRNA from cell to cell. Endogenously expressing interfering
RNAs from transgenes in stably transformed worms could be a
means of surmounting barriers to uptake and transport of
administered dsRNA. This approach has shown promise in
transiently transformed schistosomes [48,49,50]. Furthermore, by
stable heterologous expression of dsRNA transporters such as C. elegans SID-2 [51] or of Argonaut proteins such as RDE-4, it may
be possible to complement specific deficiencies in RNAi processing
suggested by recent transcriptomic studies in parasitic nematodes
[33,52]. Transgene sequences in stable lines generated by the
piggyBac transposon system are integrated in TTAA sites
in the genome of S. ratti doi:10.1371/journal.ppat.1002871.t003
PLoS Pathogens | www.plospathogens.org
8
August 2012 | Volume 8 | Issue 8 | e1002871 ve transgene copy number via qRT-PCR for stable transgenic lines of S. ratti transformed with the August 2012 | Volume 8 | Issue 8 | e1002871 8 August 2012 | Volume 8 | Issue 8 | e1002871 Worm strains, animal infection and maintenance of
transgenic lines The ED321 strain of S. ratti was maintained in rats and gerbils
and cultured as described [59]. Free-living adult S. ratti were
isolated via the Baermann funnel technique from charcoal
coprocultures maintained at 22uC for 48 hours. The worms were
washed twice with sterile deionized water to reduce carryover of
fecal bacteria and plated on Nematode Growth Medium (NGM
agar) plates with lawns of Escherichia coli HB101. Agar plate
cultures of S. ratti were incubated at 22uC unless otherwise noted. Rats were infected with S. ratti by subcutaneous injection of
infective larvae (L3i). Naı¨ve rats are highly susceptible with S. ratti,
frequently developing patent infection after inoculation of only one
or two infective larvae [60,61]. This makes them the host of choice
for early generations of passage during isolation of stable lines, While the present finding constitutes a substantial advance in
transgenesis for Strongyloides and related genera, its impact would
be greater if the technology could be adapted to other parasitic
nematodes of medical or agricultural importance. We envision two
potential hurdles, the lack of parthenogenetic parasitic females and
challenges in gene transfer, that would need to be surmounted in
order to accomplish this. The property of parthenogenesis in
parasitic females of Strongyloides spp. [28] represents an advantage
in deriving transgenic lines by host passage in that it makes it
possible to obtain large numbers of progeny from parasitic females
without the presence of males. Moreover, progeny from individual August 2012 | Volume 8 | Issue 8 | e1002871 9 Stable, Integrated Transgenic Lines of S. ratti when very few transgenic L3i are available. Rats, however, mount
a vigorous protective immune response to S. ratti infection, and
eliminate their infections about one month following inoculation
[62]. Gerbils are also susceptible to infection with S. ratti but
require 50–100 L3i to establish a patent infection. However,
gerbils do not expel their infections as rapidly as rats and so allow
maintenance for 6 months or more in individual animals [63]. This makes them the hosts of choice for maintaining established
stable transgenic lines, where numbers of L3i are not limiting. Therefore, once established by 3 or 4 generations of alternating
passage through free-living culture, selection and rat infection,
stable transgenic lines of S. ratti were maintained by serial passage
in gerbils thereafter. Genomic Southern blotting Genomic DNA was extracted from control and transformed S. ratti from the F8 generation of each stable line using the Gentra
Puregene Tissue Kit (QIAGEN). Genomic DNA and donor
plasmid pPV356 were digested with BsrGI. Each donor vector,
pPV254 and pPV356, contains only one BsrGI site. BsrGI was
selected because the location of its single cut site within the gfp
coding sequence would result in two fragment ends that hybridize
with the probe for each integration event, thus increasing the
intensity of the signal in our Southern blot. Restriction
fragments
were
separated
by
electrophoresis
through 1% agarose, transferred to nylon membranes and fixed
there by cross linking with UV light using a UV Stratalinker 1800
(STRATAGENE). A probe homologous to a 723 bp segment of
the gfp coding sequence was generated by PCR using primers:
GFP-metF (59 ATGAGTAAAGGAGAACTTTTC 39) and GFP-
702R (59 ATCGCCAATTGGAGTATTTTGT 39). The PCR
product was purified by agarose gel electrophoresis followed by gel
extraction with the QIAquik kit (Qiagen). The probe was labeled
with digoxigenin–dUTP using DIG High Prime DNA labeling
and detection starter kit II (Roche). The DIG-labeled gfp gene
probe was hybridized to Southern blots at 42uC overnight with
gentle agitation. Membranes were washed at high stringency, the
probe detected immunologically with CSPD and the resulting blot
imaged on X-ray film (Kodak). g
g
y
p
pPV402 (Fig. 1C; GenBank accession number, JX013634), the
piggyBac transposase helper vector, was made by excising the
piggyBac transposase coding sequence from pPV257 (see below)
with the restriction enzymes AgeI and AvrII and cloning them into
the pAJ50 vector [24] (Addgene Plasmid #14918) from which the
gfp coding region had been removed with the same restriction
enzymes. This yielded pPV402, in which the piggyBac transposase
is expressed under the control of the Ss-rps-21 promoter, which
drives expression of transgenes in all tissues including the germline
of microinjected P0 female worms and in F1 transformants [24]. To prepare in vitro transcribed synthetic mRNA for microin-
jection in lieu of a helper plasmid, pPV257 (Fig. 1D; GenBank
accession number JX017375) was made by first amplifying the pPV402 (Fig. Plasmid constructs We created donor vector pPV254 (Fig. 1A; GenBank accession
number, JX013636) by excising the previously described reporter
cassette, Ss-act-2p::gfp::Ss-era-1 39 UTR, from pAJ08 [24] (Addgene
Plasmid #14912) with HindIII and EagI (Unless otherwise noted,
all restriction and other DNA processing enzymes used in this
study were obtained from New England Biolabs) and ligating with
T4 DNA Ligase into the piggyBac donor vector pXL-BacII [38]
containing inverted terminal repeats (ITRs) of the piggyBac
transposon and essential internal sequences surrounding a multi-
enzyme cloning site (MCS), which was cut with the same
restriction enzymes. As a precaution against epigenetic silencing, the gypsy retroviral
insulator sequences, which retard positional silencing effects in
Drosophila [34], were introduced at the 59 and 39 ends of the
expression cassette in donor vector pPV254 to create the new
donor vector pPV356 (Fig. 1B; GenBank accession number,
JX013635). Tandem 430 bp gypsy insulators were excised from
plasmid pCa4B2G, a kind gift from Norbert Perrimon, Harvard
University School of Medicine, by digestion with Sac I and Not I. The fragment containing the gypsy insulators was isolated by gel
purification and blunt ended by Pfu turbo DNA Polymerase
(Stratagene). Plasmid pPV254 was then digested with Hind III and
Xba I, and the larger (3970 bp) fragment, which contained the
piggyBac ITRs but lacked the expression cassette, was isolated and
blunt ended as described above. These two fragments were then
ligated to yield a piggyBac plasmid with two gypsy insulators. Subsequently, a multi-cloning site (MCS) (GGGCCCACTAGTC-
GGCCGGAATTCCTAGGACCGGTACCCTGCAGAAGCT)
was cloned into the plasmid between the gypsy insulators to
create a multi-purpose insulated piggyBac vector. Finally, the
expression cassette, Ss-act-2p::gfp::Ss-era-1 39UTR from pAJ08
was cloned into the MCS by digestion with Xba I and Hind III
and ligation with T4 DNA Ligase to yield pPV356. Genomic Southern blotting 1C; GenBank accession number, JX013634), the
piggyBac transposase helper vector, was made by excising the
piggyBac transposase coding sequence from pPV257 (see below)
with the restriction enzymes AgeI and AvrII and cloning them into
the pAJ50 vector [24] (Addgene Plasmid #14918) from which the
gfp coding region had been removed with the same restriction
enzymes. This yielded pPV402, in which the piggyBac transposase
is expressed under the control of the Ss-rps-21 promoter, which
drives expression of transgenes in all tissues including the germline
of microinjected P0 female worms and in F1 transformants [24]. Microinjection of Strongyloides ratti Microinjection of Strongyloides ratti
Constructs were microinjected into one arm of the syncytial
gonad of free-living S. ratti females as described for C. elegans [31]. Microinjected worms were recovered on NGM ager plates with
lawns of Escherichia coli HB101 and cultured at 22uC with free-
living males. At 48 h following injection, parental (P0) females and
their broods were observed, and both total and GFP-positive F1
eggs and larvae were counted. Over the ensuing week, GFP
positive third stage larvae (L3i) of the F1 generation were reared,
manually selected and stored in BU buffer [64] for animal
inoculation. Basic steps of this procedure and their timing are
illustrated in Fig. 2A. Frequencies of GFP expression in worms
screened from each of three transgenic lines, including one
transformed with donor vector pPV254 and two transformed with
pPV356, were compared statistically as detailed in the caption to
Fig. 2. The criterion for significance was P#0.05. Worm strains, animal infection and maintenance of
transgenic lines Cohorts of L3i from each stable line were also
cryopreserved as described [40] and the viability of thawed
parasites verified by observation of patency following gerbil
inoculation. piggyBac transposase coding sequence from pBSII-IE1-orf (http://
piggybac.bio.nd.edu/) with the PCR primers HelpKpnIAgeIF
(CCTGCAGCCCGGGTACCGGTATATAATAAAATGGG)
and HelpAvrIIEcoRIR (GTGGCGGCCGCTGAATTCCTAG-
GGGATCCAAATTC) using the Pfu Turbo DNA polymerase
(Stratagene). The PCR amplicon was cloned into the pCR-Blunt
II-TOPO vector (Invitrogen) to yield pPV257. This construct
was linearized with AvrII and used for in vitro transcription
from the T7 promoter of the parental pCR-Blunt II-TOPO
plasmid using the mMessage mMachine T7 Ultra kit (Ambion,
Austin, TX, USA), which includes capping and poly-A tailing
reactions, according to the manufacture’s instructions. PLoS Pathogens | www.plospathogens.org Acknowledgments The authors gratefully acknowledge help from the following colleagues:
Mark Viney, University of Bristol, for providing starting material and
advice on optimizing in vitro culture techniques for S. ratti, Malcolm Fraser,
University of Notre Dame and Paul Brindley, George Washington
University, for providing plasmids pXL-BAC II and pBSII-IE1-orf
containing elements of the piggyBac transposon and for advice on expressing
these, Norbert Perrimon, Harvard University, for the providing plasmid
pCa4B2G containing the gypsy insulator sequences and Alejandro
Sanchez-Flores, Wellcome Trust Sanger Institute, for consulting on S. ratti
genome informatics. Estimation of relative copy numbers of integrated
transgenes Screening for transgenic larvae based on GFP fluorescence was
carried out with a Olympus SZX12 stereomicroscope equipped
with coaxial epifluorescence. Fine-scale examination of transgene
expressing worms was carried out with an Olympus BX60
compound microscope with Nomarski Differential Interference
Contrast (DIC) optics and epifluorescence (Olympus America Inc.,
Center Valley, Pennsylvania, USA). Images from the Olympus
BX60 were captured with a Spot RT Color digital camera and
processed using either the Spot Advanced image analysis software
package (Diagnostic Instruments, Inc., Sterling Heights, Michigan,
USA) or Adobe Photoshop 7.0. Image-processing algorithms such
as contrast and brightness adjustments were always applied in
linear fashion across the entire image. g
Relative numbers of transgene copies integrated into each of the
stable lines were estimated by real-time PCR using gDNA as
template as described [66]. Relative copy number was expressed
as the mean of Ct (22DDCt) determined from three independent
amplifications. Transgene DNA was quantified using primers
specific for the coding sequence of gfp: gfp-F (59ACCCTT-
GTTAATAGAATCGAG39) and gfp-R (59TCAATGTTGTGT-
CTAATTTTGAAG39). The gene aap-1, which encodes the
ortholog
of
the
phosphoinositide
3-kinase
(PI3K)
p50/p55
adaptor/regulatory subunit, exists as a single copy in S. ratti
genome and so was selected as a reference gene for relative copy
number determination. Primers aap-1F (59TACCAGAAGAT-
GATGTAGATGC39)
and
aap1R
(59AGTTTATTGACTT-
TAGTTGTCAATG39) were designed based on the sequence of
S ratti aap-1. The size of amplicons from the aap-1- and gfp-specific
primers were identical at 210 bp. Real-time PCR amplification
was performed with a 7500 Fast Real-time PCR system using the
SYBR Green Master Mix (Applied Biosystems, Foster City, CA,
USA). 10 ng of genomic DNA template were added in a total
volume of 20 ml for each reaction. Each sample was set up in
triplicate to give three technical replicates of the reaction. Reaction conditions were: start at 50uC for 2 min, initial
denaturation at 95uC for 10 min, followed by 40 cycles of 95uC
for 15 sec, 56uC for 1 min. The dissociation curve was run at
56uC for 60 min. Three independent amplifications (denoted
‘‘measuments’’ in Table 3) were performed on gDNA from each
line with three technical replicates per amplification. As stipulated
previously [66], quality control criteria for reliable results were a 3. Hotez PJ, Fenwick A, Savioli L, Molyneux DH (2009) Rescuing the bottom
billion through control of neglected tropical diseases. Lancet 373: 1570–
1575. 4. Brooker S (2010) Estimating the global distribution and disease burden of
intestinal nematode infections: adding up the numbers–a review. Int J Parasitol
40: 1137–1144. Author Contributions Conceived and designed the experiments: HS XL TJN HCM EJP JBL. Performed the experiments: HS XL TJN. Analyzed the data: HS XL TJN
HCM EJP JBL. Contributed reagents/materials/analysis tools: HS XL
TJN HCM. Wrote the paper: HS XL TJN HCM EJP JBL. Conceived and designed the experiments: HS XL TJN HCM EJP JBL. Performed the experiments: HS XL TJN. Analyzed the data: HS XL TJN
HCM EJP JBL. Contributed reagents/materials/analysis tools: HS XL
TJN HCM. Wrote the paper: HS XL TJN HCM EJP JBL. Stable, Integrated Transgenic Lines of S. ratti Nested
PCR was carried out with Splnk-2 paired with 39PB2 or 59PB2
and a 1:20 dilution of spPCR products as template. Amplification
with Phusion Polymerase was carried out using the same thermal
cycling conditions detailed for primary spPCR. Primary and
nested
PCR
products
were
analyzed
by
1%
agarose
gel
electrophoresis. After treatment using Shrimp Alkaline Phospha-
tase (United States Biological) and Exonuclease I, nested PCR
products were sequenced using primers, 39PB SEQ and 59PB
SEQ, targeting the 39 and 59 ends of the transgene coding
sequence, respectively. Ct,25 for an amplicon and a standard deviation ,0.3 for
technical replicates in an amplification. Relative copy number determinations for the integrated
transgenes could be subject to inteference from transgene
sequences in extrachromosomal arrays formed by the donor
vector. To assess this eventuality, we conducted diagnostic PCR
reactions with primers designed to detect vector sequences,
presumed to be in extrachromosomal arrays, persisting in
worms from the three stable transgenic lines. We preformed
PCR using a pair of donor vector-specific primers for diagnostic
PCR: M13 reverse, hybridizing with the M13 reverse priming
site in the vector, and 59PB2 (see splikerette PCR protocol
above) hybridizing within the piggyBac transposon sequences of
both vectors. These primers amplify an identical 624 bp product
from both donor vectors used in the study. The primers SrAct-
2F (59-TGGAGATGAGGCCCAATCC-39) and SrAct-2R(59-
GTGATGAAGATGAAGCAGCTGTG-39) were designed to
amplify a 554 bp fragment of endogenous gene Sr-act-2, which
was used as loading control. The DNA templates in the amount
of 5 ng were used in the PCR reaction, which was carried out
with Phusion Polymerase. Thermal cycling conditions were
98uC for 1 min followed by 25 cycles of 98uC for 20 sec, 57uC
for 15 sec and 72uC for 40 sec, with a extension at 72uC for
10 min. 1. Chan MS, Medley GF, Jamison D, Bundy DA (1994) The evaluation of
potential global morbidity attributable to intestinal nematode infections.
Parasitology 109: 373–387. 2. Hotez P, Molyneux DH, Fenwick A, Ottesen EA, Sachs SE, et al. (2006)
Incorporating a rapid-impact package for neglected tropical diseases with
programs for HIV/AIDS, tuberculosis, and malaria. PLoS Med 3: 0001–0009. Stable, Integrated Transgenic Lines of S. ratti Stable, Integrated Transgenic Lines of S. ratti to annealed splinkerette oligonucleotides (SPLNK-GATC-TOP
and SPLNK-BOT in protocol S1) with GATC sticky ends for 8 h
at 16uC in a total volume of 30 ml. Following ligation of gDNA
restriction fragments to splinkerette oligos, those containing
transgene integrations were identified, and integration boundaries
recovered by primary and nested spPCR using appropriate
combinations of primers Splnk-1 and Splnk-2, which target the
ligated splinkerette oligos, and 39PB1 and 59PB1, which target the
39 and 59 ends of the transgene coding sequence, respectively. For
primary spPCR, Splnk-1 was paired with either 39PB1 or 59PB1,
and amplification was carried out with Phusion Polymerase and
approximately 20 ng of splinkerette-ligated genomic DNA as
template. Thermal cycling conditions were 98uC for 1 min
followed by 30 cycles of 98uC for 20 sec, 55uC for 15 sec and
72uC for 2 min, with a final extension at 72uC for 10 min. Nested
PCR was carried out with Splnk-2 paired with 39PB2 or 59PB2
and a 1:20 dilution of spPCR products as template. Amplification
with Phusion Polymerase was carried out using the same thermal
cycling conditions detailed for primary spPCR. Primary and
nested
PCR
products
were
analyzed
by
1%
agarose
gel
electrophoresis. After treatment using Shrimp Alkaline Phospha-
tase (United States Biological) and Exonuclease I, nested PCR
products were sequenced using primers, 39PB SEQ and 59PB
SEQ, targeting the 39 and 59 ends of the transgene coding
sequence, respectively. to annealed splinkerette oligonucleotides (SPLNK-GATC-TOP
and SPLNK-BOT in protocol S1) with GATC sticky ends for 8 h
at 16uC in a total volume of 30 ml. Following ligation of gDNA
restriction fragments to splinkerette oligos, those containing
transgene integrations were identified, and integration boundaries
recovered by primary and nested spPCR using appropriate
combinations of primers Splnk-1 and Splnk-2, which target the
ligated splinkerette oligos, and 39PB1 and 59PB1, which target the
39 and 59 ends of the transgene coding sequence, respectively. For
primary spPCR, Splnk-1 was paired with either 39PB1 or 59PB1,
and amplification was carried out with Phusion Polymerase and
approximately 20 ng of splinkerette-ligated genomic DNA as
template. Thermal cycling conditions were 98uC for 1 min
followed by 30 cycles of 98uC for 20 sec, 55uC for 15 sec and
72uC for 2 min, with a final extension at 72uC for 10 min. Splinkerette-PCR Chromosomal integrations of transgenes were further confirmed
and mapped by splinkerette-PCR (spPCR) using a modification of
Splinkerette Protocol S1 [65]. Genomic DNA was extracted from
free-living adult worms of the transgenic lines using the Gentra
Puregene Tissue Kit (QIAGEN). Purified DNA (,100 ng) was
digested with BstYI, BamHI or Bgl II for 2 h in a total volume of
15 ml for each reaction. Digested DNA was ligated with T4 Ligase To prepare in vitro transcribed synthetic mRNA for microin-
jection in lieu of a helper plasmid, pPV257 (Fig. 1D; GenBank
accession number JX017375) was made by first amplifying the August 2012 | Volume 8 | Issue 8 | e1002871 10 3. Hotez PJ, Fenwick A, Savioli L, Molyneux DH (2009) Rescuing the bottom
billion through control of neglected tropical diseases. Lancet 373: 1570–
1575.
4. Brooker S (2010) Estimating the global distribution and disease burden of
intestinal nematode infections: adding up the numbers–a review. Int J Parasitol
40: 1137–1144. Stable, Integrated Transgenic Lines of S. ratti Kalinna BH, Brindley PJ (2007) Manipulating the manipulators: advances in
parasitic helminth transgenesis and RNAi. Trends Parasitol 23: 197–204. 18. Ghedin E, Wang S, Spiro D, Caler E, Zhao Q, et al. (2007) Draft genome of the
filarial nematode parasite Brugia malayi. Science 317: 1756–1760. 19. Mitreva M, Jasmer DP, Zarlenga DS, Wang Z, Abubucker S, et al. (2011) The
draft genome of the parasitic nematode Trichinella spiralis. Nat Genet 43: 228–235. 50. Duvoisin R, Ayuk MA, Rinaldi G, Suttiprapa S, Mann VH, et al. (2012) Human
U6 promoter drives stronger shRNA activity than its schistosome orthologue in
Schistosoma mansoni and human fibrosarcoma cells. Transgenic Res 21: 511–521. 20. Ma YF, Zhang Y, Kim K, Weiss LM (2004) Identification and characterisation
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Incorporating a rapid-impact package for neglected tropical diseases with
programs for HIV/AIDS, tuberculosis, and malaria. PLoS Med 3: 0001–0009. PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 11 Stable, Integrated Transgenic Lines of S. ratti Lok JB (2009) Transgenesis in parasitic nematodes: building a better array. Trends Parasitol 25: 345–347. 33. Lok JB (2011) Nucleic acid transfection and transgenesis in parasitic nematodes. Parasitology 139: 574–588. 64. Hawdon JM, Schad GA (1991) Long term storage of hookworm infective larvae
in buffered saline solution maintains larval responsiveness to host signals. J Helm
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number and zygosity in transgenic plants. Plant Cell Rep 23: 263–271. 35. Fraser MJ, Ciszczon T, Elick T, Bauser C (1996) Precise excision of TTAA-
specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the PLoS Pathogens | www.plospathogens.org August 2012 | Volume 8 | Issue 8 | e1002871 August 2012 | Volume 8 | Issue 8 | e1002871 PLoS Pathogens | www.plospathogens.org 12
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English
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Measurement of the forward-backward asymmetry of electron and muon pair-production in <math xmlns="http://www.w3.org/1998/Math/MathML">
<mi>p</mi>
<mi>p</mi>
</math> collisions at <math xmlns="http://www.w3.org/1998/Math/MathML">
<msqrt>
<mi>s</mi>
</msqrt>
</math> = 7 TeV with the ATLAS detector
|
OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information)
| 2,015
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cc-by
| 26,166
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Published for SISSA by
Springer Received: March 13, 2015
Revised: July 17, 2015
Accepted: August 10, 2015
Published: September 9, 2015 Received: March 13, 2015
Revised: July 17, 2015
Accepted: August 10, 2015
Published: September 9, 2015 Measurement of the forward-backward asymmetry of
electron and muon pair-production in pp collisions at
√s = 7 TeV with the ATLAS detector JHEP09(2015)049 Open Access, Copyright CERN,
for the benefit of the ATLAS Collaboration.
Article funded by SCOAP3. The ATLAS collaboration E-mail: atlas.publications@cern.ch Abstract: This paper presents measurements from the ATLAS experiment of the forward-
backward asymmetry in the reaction pp →Z/γ∗→l+l−, with l being electrons or muons,
and the extraction of the effective weak mixing angle. The results are based on the full
set of data collected in 2011 in pp collisions at the LHC at √s = 7 TeV, corresponding
to an integrated luminosity of 4.8 fb−1. The measured asymmetry values are found to
be in agreement with the corresponding Standard Model predictions. The combination
of the muon and electron channels yields a value of the effective weak mixing angle of
sin2 θlept
eff
= 0.2308±0.0005(stat.)±0.0006(syst.)±0.0009(PDF), where the first uncertainty
corresponds to data statistics, the second to systematic effects and the third to knowledge
of the parton density functions. This result agrees with the current world average from the
Particle Data Group fit. Keywords: Hadron-Hadron Scattering Keywords: Hadron-Hadron Scattering ArXiv ePrint: 1503.03709 ArXiv ePrint: 1503.03709 doi:10.1007/JHEP09(2015)049 doi:10.1007/JHEP09(2015)049 Open Access, Copyright CERN,
for the benefit of the ATLAS Collaboration. Contents
1
Introduction
1
2
The ATLAS detector
3
3
Signal and background modelling
4
4
Event reconstruction and selection
5
4.1
Electron reconstruction
5
4.2
Muon reconstruction
6
4.3
Event selection and background estimation
7
5
Measurement of AFB
8
5.1
Correcting for dilution
14
6
Measurement of sin2 θ lept
eff
14
6.1
Impact of PDFs on the sin2 θ lept
eff
measurement
17
6.2
Results for sin2 θ lept
eff
17
6.3
Determination of Aµ
18
7
Conclusions
21
The ATLAS collaboration
26 JHEP09(2015)049 7
Conclusions The ATLAS collaboration 1
Introduction The vector and axial-vector couplings in the neutral current annihilation process q¯q →
Z/γ∗→ℓ+ℓ−lead to a forward-backward asymmetry AFB in the polar angle distribution
of the final state lepton ℓ−with respect to the quark direction in the rest frame of the
dilepton system. This paper presents measurements of the forward-backward asymmetry
in electron and muon pairs from Z/γ∗boson decays and the extraction of the weak mixing
angle by the ATLAS experiment. The results are based on the full set of pp collision data
collected in 2011 at the LHC at a centre-of-mass energy of √s = 7 TeV, corresponding to
an integrated luminosity of 4.8 fb−1. The differential cross section for the annihilation process can be written at leading
order as
d
4
2 3
dσ
d(cos θ) = 4πα2
3ˆs
3
8A(1 + cos2 θ) + B cos θ
,
(1.1) (1.1) where α is the fine-structure constant,
√
ˆs is the centre-of-mass energy of the quark and
anti-quark, and θ is the angle between the lepton and the quark in the rest frame of the where α is the fine-structure constant,
√
ˆs is the centre-of-mass energy of the quark and
anti-quark, and θ is the angle between the lepton and the quark in the rest frame of the – 1 – dilepton system. The coefficients A and B are functions of
√
ˆs and of the electroweak
vector and axial-vector couplings. In the case that the dilepton system has non-vanishing
transverse momentum, pT, the four-momentum of the incoming (anti-)quark is not known,
as it is no longer collinear with the incoming beams. The impact of this effect on the
asymmetry measurement is minimized by choosing a particular rest frame of the dilepton
system, the Collins-Soper (CS) frame [1], in which the angle between the lepton and the
quark, θ∗
CS, is calculated. The sign of cos θ∗
CS is defined with respect to the direction of the
quark, which is, however, ambiguous in pp collisions. It is therefore chosen by measuring the
longitudinal boost of the final-state dilepton system in the laboratory frame, and assuming
that this is in the same direction as that of the quark in the initial state. This assumption
leads to a fraction of events with wrongly assigned quark direction, which causes a dilution
of the observed asymmetry. ¯gf
V = √ρf
T 3
f −2Qf sin2 θeff
, with sin2 θeff= κf sin2 θW , 1
Introduction The probability of correct quark direction assignment increases
with the boost of the dilepton system, thus reducing the dilution for dileptons produced at
large rapidities. With this assumption, cos θ∗
CS can be written as a function of the lepton
momenta in the laboratory frame, JHEP09(2015)049 cos θ∗
CS = pz,ℓℓ
|pz,ℓℓ|
2(p+
1 p−
2 −p−
1 p+
2 )
mℓℓ
q
m2
ℓℓ+ p2
T,ℓℓ
(1.2) (1.2) with p±
i =
1
√
2(Ei ± pz,i), where E is the energy and pz the longitudinal momentum of the lepton (i = 1) and anti-
lepton (i = 2). The variables pz,ℓℓ, mℓℓ, and pT,ℓℓdenote the longitudinal momentum,
invariant mass and transverse momentum of the dilepton system, respectively. The first
factor in eq. 1.2 defines the sign of cos θ∗
CS according to the longitudinal direction of flight
of the dilepton system, as discussed above. The events with cos θ∗
CS ≥0 are classified as
forward (F), while those having cos θ∗
CS < 0 are classified as backward (B). The asymmetry
AFB is then defined as AFB = σF −σB
σF + σB
,
(1.3) (1.3) where σF and σB are the cross sections for the respective forward and backward configura-
tions. At leading order, the second term in eq. 1.1, B cos θ, describes the asymmetry AFB. This analysis measures AFB as a function of the invariant mass of the dilepton system. The results, which are presented in section 5, include the detector-level values, as well as
the corrections needed to take into account detector effects and dilution. Several Standard Model parameters can be extracted from the dependence of the
AFB values on the invariant dilepton mass. One of these is the electroweak mixing angle
sin2 θW, which is defined at tree level as 1 −m2
W /m2
Z. Depending on the renormalisation
scheme, higher-order loop corrections may modify this relation. This analysis extracts the
effective leptonic weak mixing angle sin2 θ lept
eff
[2, 3] at the mZ scale from the detector-level
AFB values. 1
Introduction The value of AFB at the peak of the Z/γ∗resonance (mℓℓ= mZ), A0,ℓ
FB, can be written
as a function of the asymmetry parameters Aℓand Aq, A0,ℓ
FB = 3
4AqAℓ,
(1.4) (1.4) with ℓ(q) denoting the leptons (quarks) in the final (initial) state. The parameters Aℓand
Aq are directly related to the electroweak vector and axial-vector couplings, as described
in section 6.3. The most precise measurements of the electron and muon asymmetry
parameters were performed by SLD [3], yielding Ae = 0.15138 ± 0.00216 and Aµ = 0.142 ±
0.015. The precision of the Aµ measurement is dominated by the statistical uncertainty,
thus making it an interesting parameter to measure with the large number of Z →µµ events
produced at the LHC. The Aµ result from this analysis is presented in section 6.3. The
determination of Aµ in the LEP/SLD results is entirely based on asymmetry measurements
in electron and muon final states without any assumptions on the involved Af. In contrast,
the determination of Aµ presented here uses the Standard Model prediction of Aq. 1
Introduction The effective weak mixing angle is related to the electroweak vector coupling ¯gf
V via ¯gf
V = √ρf
T 3
f −2Qf sin2 θeff
, with sin2 θeff= κf sin2 θW , – 2 – where the electroweak radiative corrections to the tree-level couplings are absorbed into
the fermion-dependent factors κf and ρf, T 3
f is the third component of the weak isospin
and Qf the electric charge of the fermion f. Using this definition of the effective weak
mixing angle, the coupling retains its tree-level form multiplied by the additional factor
√ρf. The relationship between the leptonic and quark sin2 θeffcan be approximated as
a flavour-dependent shift in the leptonic sin2 θeff[3]. Although the sin2 θeffvalue from b-
quarks differs the most from the one from leptons, only a few percent of the events in this
analysis come from initial-state b-quarks. In particular, the effect of the quark sin2 θeffon
the measured AFB is an order of magnitude smaller than the effect of the leptonic sin2 θeff. This analysis therefore measures the leptonic sin2 θeff, denoted by sin2 θ lept
eff
in the following. Its value is extracted from the measured AFB as a function of the invariant mass of the
dilepton system by comparing it to MC predictions produced with varying values of the
weak mixing angle. Details are given in section 6. l JHEP09(2015)049 The most precise measurement of sin2 θ lept
eff
comes from the combination of results from
the LEP and SLD experiments [3]. Those studies yield an average leptonic sin2 θ lept
eff
=
0.23153 ± 0.00016. The two most precise single measurements are extracted from the
forward-backward asymmetry in b-quark final states, A0,b
FB, at LEP (sin2 θ lept
eff
= 0.23221 ±
0.00029) and from the leptonic left-right polarization asymmetry, ALR, at SLD (sin2 θ lept
eff
=
0.23098 ± 0.00026). These two values differ by approximately three standard deviations. More recently, the CDF [4] and D0 [5] experiments at the Tevatron and the CMS [6, 7]
experiment at the LHC have also measured sin2 θ lept
eff
. The CDF (D0) measurement was
performed using Z →µµ (Z →ee) events from p¯p collisions, and the CMS measurements
were performed using Z →µµ events from pp collisions. These results are compared to
those from this analysis in section 6. 1ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in
the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre
of the LHC ring, and the y-axis points upward. Cylindrical coordinates (r,φ) are used in the transverse
plane, φ being the azimuthal angle around the beam pipe. The pseudorapidity is defined in terms of the
polar angle θ as η = −ln tan(θ/2). 2
The ATLAS detector The ATLAS detector [8] is a general-purpose detector installed at the LHC [9] at CERN. The detector subsystem closest to the interaction point, the inner detector (ID), provides – 3 – precise position and momentum measurements of charged particle trajectories. It covers
the pseudorapidity range1 |η| < 2.5 and provides full azimuthal coverage. The ID consists of
three subdetectors arranged in a coaxial geometry around the beam axis: the silicon pixel
detector, the semiconductor microstrip detector and the straw-tube transition-radiation
tracker. A solenoidal magnet generates a 2 T magnetic field in which the ID is immersed. Electromagnetic calorimetry in the region |η| < 3.2 is based on a high-granularity,
lead/liquid-argon (LAr) sampling technology. Hadronic calorimetry uses a scintillator-
tile/steel detector in the region |η| < 1.7 and a copper/LAr detector in the region 1.5 <
|η| < 3.2. The most forward region of the detector (3.1 < |η| < 4.9) is equipped with
a forward calorimeter, measuring both the electromagnetic and hadronic energies using
copper/LAr and tungsten/LAr modules. JHEP09(2015)049 A large muon spectrometer (MS) constitutes the outermost part of the detector. It
consists of three large air-core superconducting toroidal magnet systems (each with eight
coils): one barrel providing a field of about 0.5 T and two endcaps each providing a field
of about 1 T. The deflection of the muon trajectories in the magnetic field is measured in
three layers of precision drift tube chambers for |η| < 2. In higher |η| regions (2.0 < |η| <
2.7), two layers of drift tube chambers are used in combination with one layer of cathode
strip chambers in the innermost endcap wheels of the MS. Three layers of resistive plate
chambers in the barrel (|η| < 1.05) and three layers of thin gap chambers in the endcaps
(1.05 < |η| < 2.4) provide the muon trigger and also measure the muon trajectory in the
non-bending plane. A three-level trigger system is used to select events in real time. A hardware-based
Level-1 trigger uses a subset of detector information to reduce the event rate to a design
value of at most 75 kHz. The rate of accepted events is then reduced to about 300 Hz by
two software-based trigger levels, Level-2 and the Event Filter. 3
Signal and background modelling Details are given in section 6. The cross
section is calculated at next-to-next-to-leading order (NNLO) in the strong coupling using
PHOZPR [16] with MSTW2008 NNLO PDFs. The ratio of this cross section to the leading-
order (LO) cross section is the mℓℓ-dependent K-factor, applied to the generated signal
for all plots shown in the following. However, the main observable described here (AFB) is
not affected by this LO-to-NNLO rescaling. The impact of higher-order corrections in αs
and αem on AFB and on sin2 θ lept
eff
is assessed using the HORACEv3.1 [17], MCFMv6.6 [18]
and POWHEGv1 [19] generators, as described in section 5. The POWHEG simulation is
combined with PYTHIA 6.4 for showering and hadronization. JHEP09(2015)049 Background contributions containing prompt isolated electron or muon pairs are es-
timated using Monte Carlo simulation normalized using the best available cross sec-
tion prediction at (N)NLO. The background from Z/γ∗→ττ is also generated us-
ing PYTHIAv6.4. Diboson (WW, WZ, and ZZ) samples are generated with HER-
WIGv6.510 [20, 21] using the MRSTMCal PDFs [22]. Pair-production of top quarks is
generated with MC@NLOv4.01 [23, 24] using the CTQE6L1 PDFs [25], combined with
HERWIG for showering and hadronization. The contributions from multi-jet and W+jets background events containing non-
isolated leptons from heavy-flavour decays and hadrons misidentified as leptons are es-
timated using data-driven techniques, as described in section 4.3. Since the contribution
from W+jets is found to be a small fraction of the multi-jet background over the whole
invariant mass range, the term ‘multi-jet background’ is used in the following to denote
the sum of these contributions. 4
Event reconstruction and selection The analysis uses pp data collected in 2011, corresponding to an integrated luminosity of
4.8 fb−1 for the electron channels and 4.6 fb−1 for the muon channel. All events analysed
were acquired under good operating conditions of the ATLAS detector. Events in the
electron channels passed the single electron trigger, with an electron ET > 20 or 22 GeV
(depending on the instantaneous luminosity). Events in the muon channel passed the single
muon trigger with a muon pT threshold of 18 GeV. The presence of a reconstructed collision
vertex with at least three tracks with pT > 400 MeV is required. For the muon channel,
there is an additional requirement that the longitudinal position of this vertex be within
200 mm of the nominal interaction point. 3
Signal and background modelling Monte Carlo (MC) simulated event samples used to model signal and background pro-
cesses are generated and passed through the ATLAS detector simulation [10], based on
the GEANT4 toolkit [11]. Simulated events acquire weights such that the resulting dis-
tributions match the ones observed in the data for the following variables: the average
number of interactions per bunch crossing, the z coordinate of the interaction vertex, the
lepton energy/momentum scale and resolution, and the trigger, identification and recon-
struction efficiencies. The Z/γ∗production is detected by the emission of charged lepton pairs, ee or µµ. The contribution from Z/γ∗→ττ followed by τ decays to electrons or muons is consid-
ered as background and subtracted from the signal. Signal samples are generated with – 4 – PYTHIAv6.4 [12] with the MSTW2008LO [13] parton distribution functions (PDFs) and
a value of sin2 θ lept
eff
= 0.232 for the effective weak mixing angle. Final-state radiation from
QED is taken into account using PHOTOS [14] in the exponentiated mode with multi-
photon emission. For the sin2 θ lept
eff
measurement, where sensitivity to PDFs is expected to
be significant, additional PDF sets are also used, including one specifically prepared for this
analysis, based on the ATLAS-epWZ12 PDFs [15]. Details are given in section 6. The cross
section is calculated at next-to-next-to-leading order (NNLO) in the strong coupling using
PHOZPR [16] with MSTW2008 NNLO PDFs. The ratio of this cross section to the leading-
order (LO) cross section is the mℓℓ-dependent K-factor, applied to the generated signal
for all plots shown in the following. However, the main observable described here (AFB) is
not affected by this LO-to-NNLO rescaling. The impact of higher-order corrections in αs
and αem on AFB and on sin2 θ lept
eff
is assessed using the HORACEv3.1 [17], MCFMv6.6 [18]
and POWHEGv1 [19] generators, as described in section 5. The POWHEG simulation is
combined with PYTHIA 6.4 for showering and hadronization. PYTHIAv6.4 [12] with the MSTW2008LO [13] parton distribution functions (PDFs) and
a value of sin2 θ lept
eff
= 0.232 for the effective weak mixing angle. Final-state radiation from
QED is taken into account using PHOTOS [14] in the exponentiated mode with multi-
photon emission. For the sin2 θ lept
eff
measurement, where sensitivity to PDFs is expected to
be significant, additional PDF sets are also used, including one specifically prepared for this
analysis, based on the ATLAS-epWZ12 PDFs [15]. 4.1
Electron reconstruction This analysis uses electrons in two distinct regions of the detector:
the central
region (|η| < 2.47) where tracking information is available, and the forward region – 5 – (2.5 < |η| < 4.9), where the electron reconstruction relies only on information from the
calorimeter. The inclusion of electrons in the forward region allows the reconstruction of
events where the Z/γ∗candidates are emitted at large rapidity, thus reducing the effect of
dilution due to the imperfect knowledge of the direction of the initial state quark. For both the central and forward electrons, the reconstruction begins with identify-
ing energy deposits in the calorimeters consistent with electromagnetic showers. Electron
candidates in the central region are matched to a track reconstructed in the ID. A trans-
verse energy requirement, ET > 25 GeV, is applied to both the central and forward candi-
dates. Electron candidates in transition regions between the barrel and endcap calorimeters
(1.37 < |η| < 1.52) and between the endcap and forward calorimeters (3.16 < |η| < 3.35)
are excluded from this analysis. JHEP09(2015)049 The central candidates must satisfy either ‘medium’ or ‘tight’ identification criteria,
based on shower shape and track quality variables [26] optimized for the 2011 data [27]. Forward electron candidates must satisfy similar medium quality criteria optimized specif-
ically for forward electrons. When combining one central and one forward electron there
is an additional requirement that the central electron be isolated. It is implemented by
requiring that the transverse energy deposition in the calorimeter within a cone defined
by ∆R =
q
(∆φ)2 + (∆η)2 = 0.2 around the electron candidate be less than 5 GeV, ex-
cluding the electron candidate itself. The reconstruction efficiencies in simulated events
are corrected to match the measured efficiencies [27]. Selected events consist of either two
medium candidates in the central region (central-central, referred to as CC) or one tight
central electron candidate and one medium forward electron candidate (central-forward,
referred to as CF). In the CC electron channel, the electrons are required to have opposite
charges. This requirement is not applicable in the CF electron channel, since the forward
electron has no charge information. The effect of charge misidentification is found to be
negligible in both the CC and CF electron channels [27]. 4.3
Event selection and background estimation Events must contain two oppositely charged leptons in the muon and CC electron channels
or one central electron and one forward electron in the CF electron channel. In the muon
and CC electron channels, dilepton pairs with invariant masses up to 1000 GeV are used. In the CF electron channel, the AFB measurement is performed only for dilepton masses
up to 250 GeV, because the background dominates at larger masses, leading to sizeable
systematic uncertainties. Contributions of different background sources are estimated using either simulation or
data-driven techniques. For dibosons (ZZ, WZ, WW), Z/γ∗→ττ and t¯t, contributions
are estimated using simulation. The dominant background for the muon and CC electron
channels, across the whole invariant mass range, is that due to t¯t events. Background from
Z/γ∗→ττ production (followed by τ →ℓν) populates the low end of the dilepton invariant
mass distribution. For the electron channels, the multi-jet background is estimated using a
combination of data-driven techniques. In the CC electron channel, the reverse identifica-
tion method [30] is used for dilepton invariant masses below 125 GeV, while the fake-factor
method [31], is employed for higher invariant masses. An overlap region is defined between
110 and 200 GeV, where the estimates from both methods are compared and a scale factor
for the reverse identification estimate is determined using the fake-factor result. Since the
CF electron channel only extends to a dilepton invariant mass of 250 GeV, only the reverse
identification method is used. In the muon channel, the multi-jet background is estimated
from data in a control region defined by inverting the muon isolation cut. The numbers
of events in the control and signal regions observed in MC simulation are then used to
transfer the data distribution from the control region to the signal region. JHEP09(2015)049 Figures 1 and 2 show the mℓℓand cos θ∗
CS distributions of events in the three channels. The total numbers of selected events are 1.2×106, 0.35×106 and 1.7×106 for the CC elec-
tron, CF electron and muon channels respectively. In the region close to the Z peak, the
background contamination is estimated to be less than 1% for the muon and CC electron
channels, and about 5% for the CF electron channel. The background contributions in the
muon and CC electron channels increase to about 5% and 16% in the low- and high-mass
regions, respectively. 4.2
Muon reconstruction Muons identified as ‘combined muons’ by the reconstruction and identification algo-
rithms [28, 29] are used in this analysis. They are reconstructed by associating and
combining two independently reconstructed tracks, one in the ID and one in the MS. Combined muons are required to have transverse momentum pT > 20 GeV, and must lie
within |η| < 2.4. The ID tracks associated with the muons must satisfy quality require-
ments on the number of hits recorded by each subdetector [28]. To reject muons from
cosmic rays, the longitudinal coordinate of the point of closest approach of the track to
the beamline is required to be within 10 mm of the collision vertex (see section 4.3). Re-
jection of multi-jet background is improved by requiring the muons to be isolated. The
isolation parameter is the relative momentum isolation, defined as the sum of the pT of all
other tracks within a cone of ∆R = 0.2 around the muon track, divided by the muon pT:
P ptrack
T
/pµ
T < 0.1. The kinematic variables of the muons are measured by the ID, in order
to minimize the impact of residual misalignments between the ID and the MS. This choice
also reduces the impact of muon bremsstrahlung in the calorimeter on the measurement. Charge misidentification for muons is very low, with negligible effect on this analysis. – 6 – 4.3
Event selection and background estimation The CF electron channel has a background contamination of about
30% in the low-mass region. Agreement between data and simulation is observed within
the uncertainties over the whole invariant mass range and also in the cos θ∗
CS distributions. These uncertainties contain both the statistical and systematic components and include
the effects of multiple pp collisions occurring in the same or in neighbouring bunch crossing
(pileup), energy/momentum scale and resolution, trigger efficiency, misalignment of the in-
ner detectors, data-driven background estimates, and PDFs. Details are given in section 5. Figure 2(c) highlights the cos θ∗
CS distribution for the CF electron channel to better
illustrate the reduced impact of dilution: the forward-backward asymmetry is large enough
to be observed directly from the plot. Some differences between data and simulation are
observed in the lowest and highest bins in cos θ∗
CS. As a cross check, the analysis was
repeated excluding the bins in question and the impact on the AFB and sin2 θ lept
eff
results
was found to be negligible. – 7 – [GeV]
ee
CC
m
70
2
10
2
10
×
2
3
10
Events / GeV
1
−
10
1
10
2
10
3
10
4
10
5
10
-1
= 7 TeV, 4.8 fb
s
Data 2011
ee
→
*
γ
Z/
Other backgrounds
Multijets
ATLAS
CC electron
> 25 GeV
T
p
| < 2.47
η
|
(a)
[GeV]
ee
CF
m
70 80 90
2
10
2
10
×
2
Events / GeV
2
10
3
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4
10
5
10
-1
= 7 TeV, 4.8 fb
s
Data 2011
ee
→
*
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Z/
Other backgrounds
Multijets
ATLAS
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
(b)
[GeV]
µ
µ
m
70
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3
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Events / GeV
1
−
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1
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6
10
ATLAS
-1
= 7 TeV, 4.6 fb
s
muons
> 20 GeV
T
p
| < 2.4
η
|
Data 2011
µ
µ
→
*γ
Z/
Other backgrounds
Multijet
(c)
Figure 1. Dilepton invariant mass distributions obtained from the event selections described in the
ext, for the (a) CC electron, (b) CF electron and (c) muon channels. 4.3
Event selection and background estimation [GeV]
ee
CF
m
70 80 90
2
10
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2
Events / GeV
2
10
3
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4
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5
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-1
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s
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*
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Z/
Other backgrounds
Multijets
ATLAS
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
(b) [GeV]
ee
CC
m
70
2
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3
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Events / GeV
1
−
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1
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5
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-1
= 7 TeV, 4.8 fb
s
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ee
→
*
γ
Z/
Other backgrounds
Multijets
ATLAS
CC electron
> 25 GeV
T
p
| < 2.47
η
|
( )
[GeV]
ee
CF
m
70 80 90
2
10
2
10
×
2
Events / GeV
2
10
3
10
4
10
5
10
-1
= 7 TeV, 4.8 fb
s
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ee
→
*
γ
Z/
Other backgrounds
Multijets
ATLAS
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
( ) [GeV]
ee
CC
m
70
2
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2
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3
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Events / GeV
1
−
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1
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5
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-1
= 7 TeV, 4.8 fb
s
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ee
→
*
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Z/
Other backgrounds
Multijets
ATLAS
CC electron
> 25 GeV
T
p
| < 2.47
η
| JHEP09(2015)049 [GeV]
ee
m
(a)
(b)
[GeV]
µ
µ
m
70
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2
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3
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Events / GeV
1
−
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1
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10
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-1
= 7 TeV, 4.6 fb
s
muons
> 20 GeV
T
p
| < 2.4
η
|
Data 2011
µ
µ
→
*γ
Z/
Other backgrounds
Multijet
(c) (b) (a)
(b)
[GeV]
µ
µ
m
70
2
10
2
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3
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Events / GeV
1
−
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1
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6
10
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-1
= 7 TeV, 4.6 fb
s
muons
> 20 GeV
T
p
| < 2.4
η
|
Data 2011
µ
µ
→
*γ
Z/
Other backgrounds
Multijet
(c) (c) Figure 1. 4.3
Event selection and background estimation Dilepton invariant mass distributions obtained from the event selections described in the
text, for the (a) CC electron, (b) CF electron and (c) muon channels. Data are shown by open circles
and the total expectation is shown as a line with a band representing the total uncertainty (statistical
and systematic added in quadrature). The data-driven estimate for the multi-jet background and
the simulation-based estimates for all other backgrounds are shown by the shaded areas. 4.3
Event selection and background estimation Data are shown by open circles
and the total expectation is shown as a line with a band representing the total uncertainty (statistical
and systematic added in quadrature). The data-driven estimate for the multi-jet background and
he simulation-based estimates for all other backgrounds are shown by the shaded areas. 5
Measurement of AFB For each invariant mass bin, the AFB value is obtained from the corresponding cos θ∗
CS
distribution by measuring the numbers of forward and backward events: For each invariant mass bin, the AFB value is obtained from the corresponding cos θ∗
CS
distribution by measuring the numbers of forward and backward events: AFB =
Ncos θ∗
CS≥0 −Ncos θ∗
CS<0
Ncos θ∗
CS≥0 + Ncos θ∗
CS<0
. Ncos θ∗
CS≥0 + Ncos θ∗
CS<0
. For comparison, expected AFB values are calculated from both the PYTHIA and POWHEG
samples described in section 3. Background contributions are subtracted from the number
of forward and backward events measured at detector-level. Some background contri- – 8 – CS
*
θ
cos
1
−
0.5
−
0
0.5
1
Events / 0.1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
10
-1
= 7 TeV, 4.8 fb
s
Data 2011
ee
→
*
γ
Z/
Other backgrounds
Multijets
ATLAS
CC electron
> 25 GeV
T
p
| < 2.47
η
|
(a)
CS
*
θ
cos
1
−
0.5
−
0
0.5
1
Events / 0.1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
10
ATLAS
-1
= 7 TeV, L = 4.6 fb
s
muons
> 20 GeV
T
p
| < 2.4
η
|
Data 2011
μ
μ
→
*γ
Z/
Other backgrounds
Multijet
(b)
CS
*
θ
cos
1
−
0.5
−
0
0.5
1
Events / 0.1
0
10
20
30
40
50
60
70
3
10
×
-1
= 7 TeV, 4.8 fb
s
Data 2011
ee
→
*γ
Z/
Other backgrounds
Multijets
ATLAS
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
(c)
CS
*
θ
cos
1
−
0.5
−
0
0.5
1
Events / 0.1
2
10
3
10
4
10
5
10
6
10
7
10
-1
= 7 TeV, 4.8 fb
s
Data 2011
ee
→
*γ
Z/
Other backgrounds
Multijets
ATLAS
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
(d)
Figure 2. Distributions of the cosine of the polar angle in the Collins-Soper frame (cos θ∗
CS)
obtained from the event selections described in the text, for the (a) CC electron and (b) muon
channels. 5
Measurement of AFB The corresponding distribution for the CF electron channel is shown using both (c) a
linear and (d) a logarithmic scale. Data are shown by open circles and the total expectation is
shown as a line with a band representing the total uncertainty (statistical and systematic added
in quadrature). The data-driven estimate for the multi-jet background and the simulation-based
estimates for all other backgrounds are shown by the shaded areas. CS
*
θ
cos
1
−
0.5
−
0
0.5
1
Events / 0.1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
10
ATLAS
-1
= 7 TeV, L = 4.6 fb
s
muons
> 20 GeV
T
p
| < 2.4
η
|
Data 2011
μ
μ
→
*γ
Z/
Other backgrounds
Multijet
(b) CS
*
θ
cos
1
−
0.5
−
0
0.5
1
Events / 0.1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
10
-1
= 7 TeV, 4.8 fb
s
Data 2011
ee
→
*
γ
Z/
Other backgrounds
Multijets
ATLAS
CC electron
> 25 GeV
T
p
| < 2.47
η
| JHEP09(2015)049 (a) (b) CS
*
θ
cos
1
−
0.5
−
0
0.5
1
Events / 0.1
2
10
3
10
4
10
5
10
6
10
7
10
-1
= 7 TeV, 4.8 fb
s
Data 2011
ee
→
*γ
Z/
Other backgrounds
Multijets
ATLAS
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
(d) CS
*
θ
cos
1
−
0.5
−
0
0.5
1
Events / 0.1
0
10
20
30
40
50
60
70
3
10
×
-1
= 7 TeV, 4.8 fb
s
Data 2011
ee
→
*γ
Z/
Other backgrounds
Multijets
ATLAS
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
(c) Events / 0.1 CS
*
θ
cos
1
−
0.5
−
0
0.5
1
2
10
3
10
4
10
5
10
g
Multijets
|
C
η
|
| < 4.9
F
η
2.5 < |
(d) (c) (d) Figure 2. Distributions of the cosine of the polar angle in the Collins-Soper frame (cos θ∗
CS)
obtained from the event selections described in the text, for the (a) CC electron and (b) muon
channels. 2Numerical values of all results are available in HepData [32]. 5
Measurement of AFB The corresponding distribution for the CF electron channel is shown using both (c) a
linear and (d) a logarithmic scale. Data are shown by open circles and the total expectation is
shown as a line with a band representing the total uncertainty (statistical and systematic added
in quadrature). The data-driven estimate for the multi-jet background and the simulation-based
estimates for all other backgrounds are shown by the shaded areas. butions, such as multi-jet events, display no asymmetry and hence dilute the measured
asymmetry. Other background contributions, such as t¯t, display an asymmetry. The
detector-level asymmetry values after background subtraction (Ameas
FB ) in the electron and
muon channels are shown in figure 3 as a function of the invariant mass of the lepton
pair.2 Good agreement between data and simulation is observed. Figure 4 shows the same
information in a narrower mass range around the Z pole. – 9 – meas
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CC electron
> 25 GeV
T
p
| < 2.47
η
|
[GeV]
ee
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(a)
FB
meas
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CF electron
| < 2.47
C
η
> 25 GeV, |
T
p
| < 4.9
F
η
2.5 < |
[GeV]
ee
m
70 80 90
2
10
2
10
×
2
σ
/
∆
2
−1
−
012
(b)
FB
meas
A
0.6
−
0.4
−
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−
0
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0.8
1
1.2
Data
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(c)
Figure 3. Detector-level forward-backward asymmetry (Ameas
FB ) values as a function of the dilepton
invariant mass for the (a) CC electron, (b) CF electron and (c) muon channels, after background
subtraction. 5
Measurement of AFB Detector-level forward-backward asymmetry (Ameas
FB ) values as a function of
t
f
th
( ) CC
l
t
(b) CF
l
t
d ( )
h
l
ft FB
meas
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(c) FB
meas
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(c) (c) Figure 3. Detector-level forward-backward asymmetry (Ameas
FB ) values as a function of the dilepton
invariant mass for the (a) CC electron, (b) CF electron and (c) muon channels, after background
subtraction. For the data, the black inner error bars represent the statistical component and
the lighter outer error bars the total error (statistical and systematic added in quadrature). The
boxed shaded regions for the MC expectations represent only the statistical uncertainty; theoretical
uncertainties for MC are included in the systematic uncertainties on the data. The lower panel of
each plot shows the pull value (∆/σ) for each mass bin, where ∆is the difference between data and
simulation and σ is the sum in quadrature of the data and simulation uncertainties. The asymmetry values Ameas
FB
are unfolded from detector level to particle level (Aobs
FB),
to allow comparisons with theoretical predictions. The unfolding procedure corrects for
effects collectively referred to as ‘mass-bin migration’ (MBM) as described below. The asymmetry values Ameas
FB
are unfolded from detector level to particle level (Aobs
FB),
to allow comparisons with theoretical predictions. The unfolding procedure corrects for
effects collectively referred to as ‘mass-bin migration’ (MBM) as described below. 5
Measurement of AFB For the data, the black inner error bars represent the statistical component and
the lighter outer error bars the total error (statistical and systematic added in quadrature). The
boxed shaded regions for the MC expectations represent only the statistical uncertainty; theoretical
uncertainties for MC are included in the systematic uncertainties on the data. The lower panel of
each plot shows the pull value (∆/σ) for each mass bin, where ∆is the difference between data and
simulation and σ is the sum in quadrature of the data and simulation uncertainties. meas
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CC electron
> 25 GeV
T
p
| < 2.47
η
|
[GeV]
ee
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(a) FB
meas
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CF electron
| < 2.47
C
η
> 25 GeV, |
T
p
| < 4.9
F
η
2.5 < |
[GeV]
ee
m
70 80 90
2
10
2
10
×
2
σ
/
∆
2
−1
−
012
(b) JHEP09(2015)049 (b) (a) (a)
(b)
FB
meas
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(c)
3. 5
Measurement of AFB • Detector effects: the finite resolution of the detector, as well as lepton reconstruction
efficiencies, deform the measured Z/γ∗line shape and the dependence of the asym-
metry values on the dilepton mass with respect to what one would measure with an
ideal apparatus covering the same kinematic range. – 10 – FB
meas
A
0.3
−
0.2
−
0.1
−
0
0.1
0.2
0.3
Data
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CC electron
> 25 GeV
T
p
| < 2.47
η
|
[GeV]
ee
m
80
85
90
95
100
105
110
σ
/
∆
2
−1
−
012
(a)
FB
meas
A
0.3
−
0.2
−
0.1
−
0
0.1
0.2
0.3
Data
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CF electron
| < 2.47
C
η
> 25 GeV, |
T
p
| < 4.9
F
η
2.5 < |
[GeV]
ee
m
80
85
90
95
100
105
110
σ
/
∆
2
−1
−
012
(b)
FB
meas
A
0.3
−
0.2
−
0.1
−
0
0.1
0.2
0.3
Data
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
80
85
90
95
100
105
110
σ
/
∆
2
−1
−
012
(c)
Figure 4. Detector-level forward-backward asymmetry (Ameas
FB ) values as a function of the dilepton
invariant mass for the (a) CC electron, (b) CF electron and (c) muon channels in a narrow region
around the Z pole, after background subtraction. For the data, the black inner error bars represent
the statistical component and the lighter outer error bars the total error (statistical and systematic
added in quadrature). The boxed shaded regions for the MC expectations represent only the
statistical uncertainty; theoretical uncertainties for MC are included in the systematic uncertainties
on the data. The lower panel of each plot shows the pull values (∆/σ, as defined for figure 3). 5
Measurement of AFB The boxed shaded regions for the MC expectations represent only t
cal uncertainty; theoretical uncertainties for MC are included in the systematic uncertaint ( )
(
FB
meas
A
0.3
−
0.2
−
0.1
−
0
0.1
0.2
0.3
Data
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
80
85
90
95
100
105
110
σ
/
∆
2
−1
−
012
(c) FB
meas
A
0.3
−
0.2
−
0.1
−
0
0.1
0.2
0.3
Data
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
80
85
90
95
100
105
110
σ
/
∆
2
−1
−
012
(c) (c) Figure 4. Detector-level forward-backward asymmetry (Ameas
FB ) values as a function of the dilepton
invariant mass for the (a) CC electron, (b) CF electron and (c) muon channels in a narrow region
around the Z pole, after background subtraction. For the data, the black inner error bars represent
the statistical component and the lighter outer error bars the total error (statistical and systematic
added in quadrature). The boxed shaded regions for the MC expectations represent only the
statistical uncertainty; theoretical uncertainties for MC are included in the systematic uncertainties
on the data. The lower panel of each plot shows the pull values (∆/σ, as defined for figure 3). • QED radiative corrections: radiative corrections [33], or real photon emission in the
final-state (FSR), deform the shape of the dilepton invariant mass distribution. This
deformation is particularly pronounced below the Z peak. The events are moved
from the Z peak (i.e. expected AFB positive and small) towards smaller values of
invariant mass, significantly reducing the magnitude of the observed AFB in the region
66 GeV< mℓℓ< mZ. In the high-mass region (mℓℓ> mZ), the deformation due to
radiative corrections is still present, but is reduced in magnitude. To account for these
corrections, dileptons are unfolded to the pre-FSR state, referred to as ‘Born level’. • QED radiative corrections: radiative corrections [33], or real photon emission in the
final-state (FSR), deform the shape of the dilepton invariant mass distribution. This
deformation is particularly pronounced below the Z peak. 5
Measurement of AFB FB
meas
A
0.3
−
0.2
−
0.1
−
0
0.1
0.2
0.3
Data
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CC electron
> 25 GeV
T
p
| < 2.47
η
|
[GeV]
ee
m
80
85
90
95
100
105
110
σ
/
∆
2
−1
−
012 FB
meas
A
0.3
−
0.2
−
0.1
−
0
0.1
0.2
0.3
Data
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CF electron
| < 2.47
C
η
> 25 GeV, |
T
p
| < 4.9
F
η
2.5 < |
[GeV]
ee
m
80
85
90
95
100
105
110
σ
/
∆
2
−1
−
012
(b) ATLAS JHEP09(2015)049 (a) (b) (a)
(b)
FB
meas
A
0.3
−
0.2
−
0.1
−
0
0.1
0.2
0.3
Data
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
80
85
90
95
100
105
110
σ
/
∆
2
−1
−
012
(c)
4. Detector-level forward-backward asymmetry (Ameas
FB ) values as a function of the dilept
nt mass for the (a) CC electron, (b) CF electron and (c) muon channels in a narrow reg
the Z pole, after background subtraction. For the data, the black inner error bars represe
istical component and the lighter outer error bars the total error (statistical and systema
in quadrature). 5
Measurement of AFB The events are moved
from the Z peak (i.e. expected AFB positive and small) towards smaller values of
invariant mass, significantly reducing the magnitude of the observed AFB in the region
66 GeV< mℓℓ< mZ. In the high-mass region (mℓℓ> mZ), the deformation due to
radiative corrections is still present, but is reduced in magnitude. To account for these
corrections, dileptons are unfolded to the pre-FSR state, referred to as ‘Born level’. – 11 – The unfolding procedure is carried out using an iterative Bayesian unfolding
method [34], as implemented in the RooUnfold toolkit [35]. The response matrices are
built from the PYTHIA signal sample and the number of iterations (ten) is chosen in such
a way as to optimize the result of closure tests on simulated samples. Additional checks are
performed to ensure that the use of the PYTHIA LO generator for the unfolding does not
bias the result. Since FSR is a significant correction, an alternative real-photon emission
generator is investigated, using a simulated sample generated with SHERPA [36], which
uses a module called PHOTONS++ [37] for higher-order QED corrections. The impact of
NLO electroweak (EWK) corrections on the response matrix are estimated by reweighting
the PYTHIA simulation to the prediction from the HORACE MC event generator and
redoing the unfolding. In order to estimate NLO QCD effects on the Aobs
FB values, a test
is performed using a simulated POWHEG sample as pseudo-data and unfolding the asym-
metry values using the PYTHIA-derived response matrices. These studies all show that
any biases are much smaller than the present statistical uncertainties of the measurement. JHEP09(2015)049 The systematic uncertainties on Aobs
FB have contributions from the sources discussed in
the following. • Unfolding uncertainty: estimated using a partially data-driven method. A set of
weights is derived as a function of mℓℓand cos θ∗
CS to reweight the Ameas
FB
values from
simulation to the one observed in data. These weights are applied to the generator-
level asymmetry values. The response matrix used in the unfolding is applied to
the resulting values to fold and subsequently unfold them. Particular care is taken
to make the matrices used for the folding and the unfolding statistically indepen-
dent. The generator-level AFB dependence on mℓℓis compared before and after the
fold-unfold operation, and the difference is taken as an estimate of the uncertainty
introduced by the unfolding. 5
Measurement of AFB • Uncertainty due to finite size of the simulated event samples: the statistical uncer-
tainty on the response matrices is propagated through the unfolding procedure. • Uncertainty due to finite size of the simulated event samples: the statistical uncer-
tainty on the response matrices is propagated through the unfolding procedure. • Multi-jet background modelling: in the CC electron channel, the difference between
the two background estimation methods described in section 4.3 is taken as the sys-
tematic uncertainty and is found to be negligible with respect to other uncertainties. In the CF electron channel, this uncertainty is estimated by comparing templates
based on different electron isolation requirements. For the muon channel, the impact
of this background (and its uncertainty) is negligible. • Other experimental systematic uncertainties: these include the impact of pileup and
of detector alignment, as well as energy/momentum scale and resolution, and trigger
and reconstruction efficiencies. The associated systematic uncertainties are estimated
following the prescriptions in refs. [26, 28, 38]. The uncertainties related to energy
scaling and resolution are among the largest contributions to the total error in all
three channels, since they result in both a shifting and a broadening of the invariant
mass peak, causing events to migrate between mass bins. – 12 – CC electrons
Uncertainty
66–70 GeV
70–250 GeV
250–1000 GeV
Unfolding
∼1×10−2
(2–5)×10−3
∼4×10−4
Energy scale/resolution
∼7×10−3
(0.5–2)×10−3
∼2×10−2
MC statistics
∼5×10−3
(0.1–1)×10−3
(3–20)×10−3
PDF
∼2×10−3
(1–8)×10−4
(0.7–3)×10−3
Other
∼1×10−3
(0.1–2)×10−3
(5–9)×10−3
CF electrons
Uncertainty
66–70 GeV
70–250 GeV
250–1000 GeV
Unfolding
∼2×10−2
(0.5–2)×10−2
—
Energy scale/resolution
∼1×10−2
(0.5–7)×10−2
—
MC statistics
∼1×10−2
(1–7)×10−3
—
Background
∼3×10−2
(0.5–1)×10−2
—
PDF
∼4×10−3
(2–6)×10−4
—
Other
∼1×10−3
(1–5)×10−4
—
Muons
Uncertainty
66–70 GeV
70–250 GeV
250–1000 GeV
Unfolding
∼1×10−2
(1–4)×10−3
∼5×10−4
Energy scale/resolution
∼8×10−3
(3–6)×10−3
∼5×10−3
MC statistics
∼5×10−3
(0.1–1)×10−3
(2–30)×10−3
PDF
∼2×10−3
(1–8)×10−4
(0.3–3)×10−3
Other
∼1×10−3
(0.5–1)×10−3
(3–10)×10−3
. Absolute systematic uncertainties on the Aobs
FB values, after unfolding for mass-bi
l
h
l JHEP09(2015)049 Table 1. Absolute systematic uncertainties on the Aobs
FB values, after unfolding for mass-bin migra-
tion. Approximate values in three invariant mass intervals are given. Table 1. Absolute systematic uncertainties on the Aobs
FB values, after unfolding for mass-bin migra-
tion. Approximate values in three invariant mass intervals are given. 5.1
Correcting for dilution A similar unfolding procedure is used to further correct the Aobs
FB values to remove dilution
effects, which occur when the wrong choice is made for the direction of the quark. The
unfolding for dilution and the extrapolation from the detector acceptance to the full phase
space are performed using the PYTHIA signal sample, where the description of the initial
state allows a straightforward definition of the polar angle of the lepton with respect to the
quark. The fully corrected asymmetry values for dileptons at the Born level Acor
FB are shown
in figure 6. The magnitude of the correction is larger than in the previous unfolding step. In addition, the contribution from the PDFs becomes the dominant systematic uncertainty. Good agreement is observed in general between the measured and predicted values. The
muon channel measurement exhibits a discrepancy with respect to the PYTHIA prediction
for masses above the Z boson mass, where the measured asymmetry is consistently larger
than the prediction. This effect could not be explained by the analysis procedure and
might be a feature of the simulation. JHEP09(2015)049 5
Measurement of AFB • PDF uncertainties: the CT10 PDF set [39], which provides a reliable uncertainty
estimate and is widely used in ATLAS, is also used here to estimate the PDF un-
certainty. Its eigenvectors are used and the result quoted at 68% confidence level. For each error set, the MC signal sample is reweighted, the response matrices are
recalculated and the unfolding is repeated. This contribution is found to be small
when unfolding only mass-bin migration effects. • PDF uncertainties: the CT10 PDF set [39], which provides a reliable uncertainty
estimate and is widely used in ATLAS, is also used here to estimate the PDF un-
certainty. Its eigenvectors are used and the result quoted at 68% confidence level. For each error set, the MC signal sample is reweighted, the response matrices are
recalculated and the unfolding is repeated. This contribution is found to be small
when unfolding only mass-bin migration effects. The magnitudes of the systematic uncertainties on the Aobs
FB values are summarized in
table 1, for three invariant mass regions. Figure 5 shows the Aobs
FB values obtained from
leptons unfolded to Born level for all three channels. Expectations from PYTHIA and
POWHEG are in good agreement with the measured values, as illustrated in the pull
distribution at the bottom of each plot. – 13 – 6
Measurement of sin2 θ lept
eff The extraction of the effective weak mixing angle (sin2 θ lept
eff
) from the detector-level asym-
metry values (Ameas
FB ) is presented here. Within the region of interest (0.218 ≤sin2 θ lept
eff
≤0.236) 17 MC simulated samples
were generated with varying values of sin2 θ lept
eff
. The generator used for the templates is
PYTHIA, which allows the value of sin2 θ lept
eff
to be tuned without changing mZ. Within
the range of the sin2 θ lept
eff
variations, the Z boson line shape remains unchanged in the
generated samples. From the generator-level information in the samples, weights are cal-
culated, in bins of mℓℓand cos θ∗
CS, to transform the Ameas
FB
values to the ones expected
(Arew
FB ) for a different value of sin2 θ lept
eff
. The reweighting technique is validated on simu-
lated samples. For each channel, the Arew
FB values obtained from the reweighted datasets
are compared to those obtained from the data, using a χ2 test over the mass range 70–
250 GeV, taking statistical and systematic uncertainties into account. The mass range has
been optimized for the maximum sensitivity and stability of the measurement. A parabola
is fitted to the resulting distributions of χ2 values for each channel independently. The
minimum of the parabola yields the sin2 θ lept
eff
result. The χ2/ndf value at the minimum
is 22.4/16 for the CC electron channel, 21.9/16 for the CF electron channel and 22.6/16
for the muon channel. The fit results are found to be stable with respect to the invariant
mass range over which the template comparisons are performed, as well as with respect to
the sin2 θ lept
eff
range over which the χ2 is minimized. As discussed in section 5, the use of a LO generator and a specific implementation
of the real photon emission in the final state does not bias the unfolded Ameas
FB
values. In
order to assess the impact of these potential sources of systematic effects on the templates
used to extract the sin2 θ lept
eff
value, additional tests are performed. Effects related to the
modelling of the real photon emission are tested with SHERPA, and are found to be neg-
ligible. 6
Measurement of sin2 θ lept
eff obs
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, MBM unfolding
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
[GeV]
ee
m
70 80 90
2
10
2
10
×
2
σ
/
∆
2
−1
−
012
(b) obs
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, MBM unfolding
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CC electron
> 25 GeV
T
p
| < 2.47
η
|
[GeV]
ee
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
( ) JHEP09(2015)049 (b) (a) obs
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, MBM unfolding
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(c) (c) Figure 5. Forward-backward asymmetry (Aobs
FB) values as a function of the dilepton invariant mass
for the (a) CC electron, (b) CF electron and (c) muon channels. Leptons are unfolded to Born level
to account for mass bin migration, and the results are compared to truth-level MC information. For
the data, the black inner error bars represent the statistical component and the lighter outer error
bars the total error (statistical and systematic added in quadrature). The boxed shaded regions
for the MC expectations represent only the statistical uncertainty; theoretical uncertainties are
included in the systematic uncertainties on the data. The lower panel of each plot shows the pull
values (∆/σ, as defined for figure 3). sin2 θ lept
eff
measurement by comparing LO and NLO predictions of the AFB vs. mass distri-
butions calculated with MCFM [18]. Differences are propagated through the extraction of
sin2 θ lept
eff
and the resulting variation of the weak mixing angle is treated as an additional
systematic error. 6
Measurement of sin2 θ lept
eff The effects of NLO QCD corrections are investigated further in the context of the – 14 – obs
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, MBM unfolding
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CC electron
> 25 GeV
T
p
| < 2.47
η
|
[GeV]
ee
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(a)
obs
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, MBM unfolding
ee
→
*γ
PYTHIA, Z/
ee
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CF electron
> 25 GeV
T
p
| < 2.47
C
η
|
| < 4.9
F
η
2.5 < |
[GeV]
ee
m
70 80 90
2
10
2
10
×
2
σ
/
∆
2
−1
−
012
(b)
obs
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, MBM unfolding
µ
µ
→
*γ
PYTHIA, Z/
µ
µ
→
*γ
POWHEG, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
> 20 GeV
T
p
| < 2.4
η
|
[GeV]
µ
µ
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(c)
Figure 5. Forward-backward asymmetry (Aobs
FB) values as a function of the dilepton invariant mass
for the (a) CC electron, (b) CF electron and (c) muon channels. Leptons are unfolded to Born level
to account for mass bin migration, and the results are compared to truth-level MC information. For
the data, the black inner error bars represent the statistical component and the lighter outer error
bars the total error (statistical and systematic added in quadrature). The boxed shaded regions
for the MC expectations represent only the statistical uncertainty; theoretical uncertainties are
included in the systematic uncertainties on the data. The lower panel of each plot shows the pull
values (∆/σ, as defined for figure 3). 6
Measurement of sin2 θ lept
eff The effects of NLO EWK corrections are found to be small compared to
the rest of the uncertainties, but are accounted for as an additional systematic uncertainty
on the final result. The systematic uncertainties already described for the Ameas
FB
values are also esti-
mated for the sin2 θ lept
eff
measurement. As the background is small in all channels of the – 15 – cor
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, full unfolding
ee
→
*γ
PYTHIA, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CC electron
[GeV]
ee
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(a)
cor
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, full unfolding
ee
→
*γ
PYTHIA, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CF electron
[GeV]
ee
m
70 80 90
2
10
2
10
×
2
σ
/
∆
2
−1
−
012
(b)
cor
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, full unfolding
µ
µ
→
*γ
PYTHIA, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
[GeV]
µ
µ
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(c)
Figure 6. Forward-backward asymmetry (Acor
FB) values as a function of the dilepton invariant mass
for the (a) CC electron, (b) CF electron and (c) muon channels. Leptons are unfolded to Born
level to account for mass bin migration and dilution effects are corrected. The measurement is
extrapolated to the full phase space, and the results are compared to truth-level MC information. For the data, the black inner error bars represent the statistical component and the lighter outer
error bars the total error (statistical and systematic added in quadrature). The boxed shaded
regions for the MC expectations represent only the statistical uncertainty; theoretical uncertainties
are included in the data systematic uncertainties. The lower panel of each plot shows the pull values
(∆/σ, as defined for figure 3). 6.2
Results for sin2 θ lept
eff The measured values of the weak mixing angle obtained with ATLAS-epWZ12 LO PDF are
shown in table 2. Of the three channels, the CF electron channel has the lowest statistical
uncertainty, despite having the fewest selected events, as detailed in section 4.3. This is
because the Ameas
FB
values in this channel are less affected by dilution due to the larger
average rapidity of the dilepton system compared to the other two channels. Details of the
main sources of systematic uncertainty on the result in each channel are given in table 3. The sin2 θ lept
eff
measurements from all three channels are combined. Given the total
covariance matrix C, the weights assigned to the three measurements for the combina-
tion are calculated using wi = P
k C−1
ik / P
jk C−1
jk , with the indices i, j and k representing
the three measurements (CC electron, CF electron, muon). The total error is given by
σ2 = (W TC−1)−1W, with W T = (1, 1, 1). Uncertainties due to energy/momentum scale
and resolution are treated as completely uncorrelated across channels. Since the energy
scale and resolution for the CF electron channel is dominated by the forward calorimeter
performance, this is a good approximation for the correlation of the CC and CF elec-
tron channels. The systematic uncertainties due to the MC statistical uncertainty are
also treated as fully uncorrelated. Theory-related systematic uncertainties, such as those
associated with PDFs and higher-order EWK and QCD corrections, are treated as fully
correlated across channels. All the remaining uncertainties are treated as fully correlated The measured values of the weak mixing angle obtained with ATLAS-epWZ12 LO PDF are
shown in table 2. Of the three channels, the CF electron channel has the lowest statistical
uncertainty, despite having the fewest selected events, as detailed in section 4.3. This is
because the Ameas
FB
values in this channel are less affected by dilution due to the larger
average rapidity of the dilepton system compared to the other two channels. Details of the
main sources of systematic uncertainty on the result in each channel are given in table 3. The sin2 θ lept
eff
measurements from all three channels are combined. 6
Measurement of sin2 θ lept
eff cor
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, full unfolding
ee
→
*γ
PYTHIA, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CC electron
[GeV]
ee
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(a) cor
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, full unfolding
ee
→
*γ
PYTHIA, Z/
ATLAS
-1
= 7TeV, 4.8fb
s
CF electron
[GeV]
ee
m
70 80 90
2
10
2
10
×
2
σ
/
∆
2
−1
−
012
(b) JHEP09(2015)049 (a) (b) cor
FB
A
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
1.2
Data, full unfolding
µ
µ
→
*γ
PYTHIA, Z/
ATLAS
-1
= 7TeV, 4.6 fb
s
muon
[GeV]
µ
µ
m
70
2
10
2
10
×
2
3
10
σ
/
∆
2
−1
−
012
(c) (c) Figure 6. Forward-backward asymmetry (Acor
FB) values as a function of the dilepton invariant mass
for the (a) CC electron, (b) CF electron and (c) muon channels. Leptons are unfolded to Born
level to account for mass bin migration and dilution effects are corrected. The measurement is
extrapolated to the full phase space, and the results are compared to truth-level MC information. For the data, the black inner error bars represent the statistical component and the lighter outer
error bars the total error (statistical and systematic added in quadrature). The boxed shaded
regions for the MC expectations represent only the statistical uncertainty; theoretical uncertainties
are included in the data systematic uncertainties. The lower panel of each plot shows the pull values
(∆/σ, as defined for figure 3). sin2 θ lept
eff
extraction, a simple, but slightly conservative, approach is used to obtain its
uncertainty. The sin2 θ lept
eff
measurement is repeated without the subtraction of the back-
ground, and the result is compared to the baseline measurement, which has the background
subtracted. The uncertainty on sin2 θ lept
eff
from the background is taken to be 10% of the
observed difference, to take into account the uncertainties on the cross sections of the
background components known with least precision [40]. – 16 – 6.1
Impact of PDFs on the sin2 θ lept
eff
measurement This measurement is sensitive to the PDFs describing the flavour composition of the initial
state, since the AFB values depend on the flavour and charge of the initial partons. In
addition, the uv and dv valence quark distributions have an impact on the measurement
due to differences in the weak couplings, while dilution effects introduce a dependence on
the sea-to-valence quark ratio within the accessible Bjorken-x range. The ATLAS measurements of inclusive W and Z boson production [41] are sensitive
to the same effects and indicate that some of the existing PDF sets may not provide a good
description of the data. JHEP09(2015)049 For this measurement of sin2 θ lept
eff
a LO version of the ATLAS-epWZ NNLO and
NLO fits was prepared. In the following, the PDFs extracted from these fits are called
ATLAS-epWZ12 LO PDFs. These fits include inclusive W +, W −and Z production data
at √s = 7 TeV [41], together with the combined HERA data on inclusive neutral- and
charged-current interactions from e+p and e−p scattering [42]. The settings for the fit
are kept the same as those for the NLO and NNLO fits as much as possible. The main
differences between the LO fit and the higher-order fits are that the gluon PDF distribution
has a simple parameterization which requires it to be positive definite at all scales, and
that the value of the strong coupling constant αs(mZ) is set to be higher than the one for
the NLO or NNLO fits. A value of αs(mZ) of 0.130 is chosen with a scanning procedure,
such that it yields the best level of agreement (χ2) between data and the fit result. This
value is consistent with that used by other LO PDF sets. These PDFs are available in
LHgrid formats [43]. 6.2
Results for sin2 θ lept
eff Given the total
covariance matrix C, the weights assigned to the three measurements for the combina-
tion are calculated using wi = P
k C−1
ik / P
jk C−1
jk , with the indices i, j and k representing
the three measurements (CC electron, CF electron, muon). The total error is given by
σ2 = (W TC−1)−1W, with W T = (1, 1, 1). Uncertainties due to energy/momentum scale
and resolution are treated as completely uncorrelated across channels. Since the energy
scale and resolution for the CF electron channel is dominated by the forward calorimeter
performance, this is a good approximation for the correlation of the CC and CF elec-
tron channels. The systematic uncertainties due to the MC statistical uncertainty are
also treated as fully uncorrelated. Theory-related systematic uncertainties, such as those
associated with PDFs and higher-order EWK and QCD corrections, are treated as fully
correlated across channels. All the remaining uncertainties are treated as fully correlated – 17 – across the channels to which they are applicable. The central value of the combination
is found to be stable when varying the magnitudes of the main correlated uncertainties. Following this procedure, the combined (CC+CF) electron result, as well as the electron-
muon combination, is shown in table 2. The systematic uncertainty on the combined result
is dominated by the PDF uncertainty (±0.0009), which is calculated using the variations
provided in the ATLAS-epWZ12 LO PDF eigenvalue set, similar to the calculation de-
scribed in section 5. The contributions to the total systematic uncertainties on the results
are included in table 3. The results obtained with the ATLAS-epWZ12 LO PDF set have been compared
to those obtained using other leading order PDF sets. The PDF sets ATLAS-epWZ12
LO and HERAPDF1.5LO [44] yield very similar results, the ATLAS-epWZ12 LO result
being larger by 1 × 10−4 (with a PDF uncertainty of 9 × 10−4). The MSTW2008LO set
produces a downward shift in the resulting sin2 θ lept
eff
which is significant (∆sin2 θ lept
eff
=
−2 × 10−3). However, MSTW sets are known to give a poor description of the ATLAS
W and Z data [41]. The CT10 PDF set (which is NLO) was also tested, and yields a
sin2 θ lept
eff
value which is smaller than, but compatible with, the results obtained with the
ATLAS-epWZ12 LO PDF (∆sin2 θ lept
eff
= −8 × 10−4). 6.2
Results for sin2 θ lept
eff The PDF analysis of the ATLAS
data also suggests an increased strange-quark sea density (¯s/ ¯d ∼1) compared to other
PDF sets [15]. In order to probe the sensitivity of this measurement to the enhanced
strange-quark density, a dedicated PDF set was prepared with a suppressed strange sea
corresponding to uncertainty, which indicates a low sensitivity of the measurement to this
effect. This is due to the fact that the sea composition only affects the measurement
through the dilution, which, to first approximation, does not change the position of the
minimum of the χ2 in the template fits. JHEP09(2015)049 As a cross-check of the measured weak mixing angle and the unfolding procedure,
sin2 θ lept
eff
is extracted from the unfolded particle-level asymmetries. Similarly to the pro-
cedure described in the beginning of section 6, samples with various values of sin2 θ lept
eff
,
generated using MCFM v6.8 [18] at LO in perturbative QCD and interfaced to APPLGRID
v1.4.69 [45] using the ATLAS-epWZ12 LO PDF set, are compared to the measured particle-
level asymmetries using a χ2 test. The correlations between the systematic uncertainties
are included in the χ2 calculation. A parabolic fit to the χ2 distribution yields a value of
sin2 θ lept
eff
in good agreement with the detector-level extraction. There are several other measurements of sin2 θ lept
eff
at the mZ scale. The results from
this analysis, as well as the results from the other collider experiments, are summarized in
table 4 and shown in figure 7. These include measurements from LEP, SLC, the Tevatron,
and the LHC, as well as the results of the PDG global fit [46]. Differences with respect
to the combined LEP/SLC measurements are also displayed. The combined result of
this analysis agrees within 0.1 σ with the most precise leptonic asymmetry measurement,
and within 1.2 σ of the sin2 θ lept
eff
value extracted from the LEP A0,b
FB measurement. The
combined result is in agreement with the PDG global fit at the level of 0.6 σ. 6.3
Determination of Aµ As described in section 1, the forward-backward asymmetry around the Z pole for the muon
channel, A0,µ
FB, can be expressed in terms of the muon and quark asymmetry parameters Aµ – 18 – sin2 θ lept
eff
CC electron
0.2302 ± 0.0009(stat.) ± 0.0008(syst.) ± 0.0010(PDF) = 0.2302 ± 0.0016
CF electron
0.2312 ± 0.0007(stat.) ± 0.0008(syst.) ± 0.0010(PDF) = 0.2312 ± 0.0014
Muon
0.2307 ± 0.0009(stat.) ± 0.0008(syst.) ± 0.0009(PDF) = 0.2307 ± 0.0015
El. combined
0.2308 ± 0.0006(stat.) ± 0.0007(syst.) ± 0.0010(PDF) = 0.2308 ± 0.0013
Combined
0.2308 ± 0.0005(stat.) ± 0.0006(syst.) ± 0.0009(PDF) = 0.2308 ± 0.0012 Table 2. The sin2 θ lept
eff
measurement results in each of the three studied channels: electron central-
central, electron central-forward and muon. Results of the statistical combination of both electron
channels and all three channels are shown as well. JHEP09(2015)049 CC electrons
CF electrons
Muons
Combined
Uncertainty source
[10−4]
[10−4]
[10−4]
[10−4]
PDF
10
10
9
9
MC statistics
5
2
5
2
Electron energy scale
4
6
—
3
Electron energy resolution
4
5
—
2
Muon energy scale
—
—
5
2
Higher-order corrections
3
1
3
2
Other sources
1
1
2
2 Table 3. Contributions to the systematic uncertainties on the sin2 θ lept
eff
values extracted from the
three analysis channels and on the combined result. Null entries (denoted by “—”) correspond to
uncertainties that do not apply to a specific channel. Higher-order corrections include NLO QCD
and NLO EWK contributions. Other sources include the effect of pileup, background uncertainties,
lepton trigger/reconstruction/identification efficiency uncertainties, muon momentum resolution
and effects of detector misalignment. and Aq, cf. eq. 1.4 and ref. [3]. Both are functions of the vector and axial-vector couplings
of the quark/muon, Aq/µ =
2gq/µ
V gq/µ
A
(gq/µ
V )2 + (gq/µ
A )2 =
2gq/µ
V /gq/µ
A
1 + (gq/µ
V /gq/µ
A )2 . (6.1) (6.1) The asymmetry parameters are related to the flavour-dependent weak mixing angle
sin2 θ q/µ
eff
by
/
/
/ The asymmetry parameters are related to the flavour-dependent weak mixing angle
sin2 θ q/µ
eff
by gq/µ
V /gq/µ
A
= 1 −4|Qq/µ| sin2 θ q/µ
eff
,
(6.2) (6.2) where Qq/µ is the quark (q) or muon (µ) charge. 6.3
Determination of Aµ The measurement of the forward-backward
asymmetry can be interpreted as a determination of Aµ when assuming sin2 θ q
eff= sin2 θ µ
eff
= sin2 θ lept
eff
, which is valid within an uncertainty of 1.5 × 10−4 [3]. Given the value of where Qq/µ is the quark (q) or muon (µ) charge. The measurement of the forward-backward
asymmetry can be interpreted as a determination of Aµ when assuming sin2 θ q
eff= sin2 θ µ
eff
= sin2 θ lept
eff
, which is valid within an uncertainty of 1.5 × 10−4 [3]. Given the value of – 19 – ∆/σ
∆/σ
sin2 θ lept
eff
(w.r.t. LEP+SLC)
(w.r.t. ATLAS)
CMS [6]
0.2287 ± 0.0032
−0.9
−0.6
D0 [5]
0.23146 ± 0.00047
−0.1
0.5
CDF [4]
0.2315 ± 0.0010
−0.03
0.4
LEP, A0,b
FB [3]
0.23221 ± 0.00029
—
1.2
LEP, A0,l
FB [3]
0.23099 ± 0.00053
—
−0.1
SLC, ALR [3]
0.23098 ± 0.00026
—
−0.1
LEP+SLC [3]
0.23153 ± 0.00016
—
0.6
PDG global fit [46]
0.23146 ± 0.00012
−0.4
0.6
ATLAS
0.2308 ± 0.0012
−0.6
— JHEP09(2015)049 Table 4. Comparison of the results of this analysis with other published results for sin2 θ lept
eff
. The comparison includes the most precise measurements from LEP and SLC, and the results from
the leptonic sin2 θ lept
eff
measurements from the hadron collider experiments CMS, D0, and CDF. Also shown are the values of sin2 θ lept
eff
from the LEP+SLC global combination (which includes
all sin2 θ lept
eff
measurements performed at the two colliders) and from the PDG global fit. Each
∆/σ column shows the difference between the result and the quoted reference value, divided by the
quadratic sum of the associated uncertainties. Table 4. Comparison of the results of this analysis with other published results for sin2 θ lept
eff
. The comparison includes the most precise measurements from LEP and SLC, and the results from
the leptonic sin2 θ lept
eff
measurements from the hadron collider experiments CMS, D0, and CDF. Also shown are the values of sin2 θ lept
eff
from the LEP+SLC global combination (which includes
all sin2 θ lept
eff
measurements performed at the two colliders) and from the PDG global fit. Each
∆/σ column shows the difference between the result and the quoted reference value, divided by the
quadratic sum of the associated uncertainties. 6.3
Determination of Aµ lept
eff
θ
2
sin
0.225
0.23
0.235
PDG Fit
LEP+SLC
LR
SLD, A
0,l
FB
LEP, A
0,b
FB
LEP, A
CDF
D0
CMS
ATLAS combined
µ
ATLAS,
ATLAS, e CF
ATLAS, e CC
ATLAS
-1
= 7 TeV, 4.8 fb
s
Figure
7. Comparison of the results of this analysis with other published results for
sin2 θ lept
eff
. This includes the most precise measurements from LEP and SLC, and the leptonic
sin2 θ lept
eff
measurements from the hadron collider experiments CMS [6], D0 [5], and CDF [4]. Also
shown are the values of sin2 θ lept
eff
from the LEP+SLC global combination [3] (which includes all
sin2 θ lept
eff
measurements performed at the two colliders) and from the PDG global fit [46]. The
vertical dotted line shows the central value of the ATLAS combined measurement reported here,
while the vertical dashed line represents that of the current PDG global fit [46]. lept
eff
θ
2
sin
0.225
0.23
0.235
PDG Fit
LEP+SLC
LR
SLD, A
0,l
FB
LEP, A
0,b
FB
LEP, A
CDF
D0
CMS
ATLAS combined
µ
ATLAS,
ATLAS, e CF
ATLAS, e CC
ATLAS
-1
= 7 TeV, 4.8 fb
s Figure
7. Comparison of the results of this analysis with other published results for
sin2 θ lept
eff
. This includes the most precise measurements from LEP and SLC, and the leptonic
sin2 θ lept
eff
measurements from the hadron collider experiments CMS [6], D0 [5], and CDF [4]. Also
shown are the values of sin2 θ lept
eff
from the LEP+SLC global combination [3] (which includes all
sin2 θ lept
eff
measurements performed at the two colliders) and from the PDG global fit [46]. The
vertical dotted line shows the central value of the ATLAS combined measurement reported here,
while the vertical dashed line represents that of the current PDG global fit [46]. Figure
7. Comparison of the results of this analysis with other published results for
sin2 θ lept
eff
. This includes the most precise measurements from LEP and SLC, and the leptonic
sin2 θ lept
eff
measurements from the hadron collider experiments CMS [6], D0 [5], and CDF [4]. Also
shown are the values of sin2 θ lept
eff
from the LEP+SLC global combination [3] (which includes all
sin2 θ lept
eff
measurements performed at the two colliders) and from the PDG global fit [46]. Table 4. Comparison of the results of this analysis with other published results for sin2 θ lept
eff
.
The comparison includes the most precise measurements from LEP and SLC, and the results from
the leptonic sin2 θ lept
eff
measurements from the hadron collider experiments CMS, D0, and CDF.
Also shown are the values of sin2 θ lept
eff
from the LEP+SLC global combination (which includes
all sin2 θ lept
eff
measurements performed at the two colliders) and from the PDG global fit. Each
∆/σ column shows the difference between the result and the quoted reference value, divided by the
quadratic sum of the associated uncertainties. 6.3
Determination of Aµ The
vertical dotted line shows the central value of the ATLAS combined measurement reported here,
while the vertical dashed line represents that of the current PDG global fit [46]. Figure
7. Comparison of the results of this analysis with other published results for
sin2 θ lept
eff
. This includes the most precise measurements from LEP and SLC, and the leptonic
sin2 θ lept
eff
measurements from the hadron collider experiments CMS [6], D0 [5], and CDF [4]. Also
shown are the values of sin2 θ lept
eff
from the LEP+SLC global combination [3] (which includes all
sin2 θ lept
eff
measurements performed at the two colliders) and from the PDG global fit [46]. The
vertical dotted line shows the central value of the ATLAS combined measurement reported here,
while the vertical dashed line represents that of the current PDG global fit [46]. – 20 – sin2 θ lept
eff
, Aµ is small compared to Aq and the uncertainty on sin2 θ µ
eff(∼1.5 × 10−3) plays
a more important role in eq. 1.4 than the error introduced by the assumption about sin2 θ q
eff
mentioned above. With the present precision, it is therefore possible to use the value of
sin2 θ µ
effobtained from the muon asymmetry measurement to derive Aµ =
2(1 −4 sin2 θ lept
eff
)
1 + (1 −4 sin2 θ lept
eff
)2 . (6.3) (6.3) The uncertainties on Aµ are propagated from the uncertainties on sin2 θ lept
eff
. The result is
Aµ = 0.153 ± 0.007(stat.) ± 0.006(syst.) ± 0.007(PDF) = 0.153 ± 0.012(tot.), uncertainties on Aµ are propagated from the uncertainties on sin2 θ lept
eff
. The result is The uncertainties on Aµ are propagated from the uncertainties on sin2 θ lept
eff
. The result is
Aµ = 0.153 ± 0.007(stat.) ± 0.006(syst.) ± 0.007(PDF) = 0.153 ± 0.012(tot.), Aµ = 0.153 ± 0.007(stat.) ± 0.006(syst.) ± 0.007(PDF) = 0.153 ± 0.012(tot.), Aµ = 0.153 ± 0.007(stat.) ± 0.006(syst.) ± 0.007(PDF) = 0.153 JHEP09(2015)049 which is of similar precision and in good agreement with the measurement from e+e−
collisions of 0.142 ± 0.015 [3]. 6.3
Determination of Aµ It is worth stressing, however, that the determination of
Aµ in the LEP/SLD results is based entirely on asymmetry measurements in the different
lepton final states without any assumptions on other Af, whereas the determination of Aµ
presented here uses the Standard Model prediction of Aq. which is of similar precision and in good agreement with the measurement from e+e−
collisions of 0.142 ± 0.015 [3]. It is worth stressing, however, that the determination of
Aµ in the LEP/SLD results is based entirely on asymmetry measurements in the different
lepton final states without any assumptions on other Af, whereas the determination of Aµ
presented here uses the Standard Model prediction of Aq. Acknowledgments We thank CERN for the very successful operation of the LHC, as well as the support staff
from our institutions without whom ATLAS could not be operated efficiently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Aus-
tralia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and
FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST
and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR,
Czech Republic; DNRF, DNSRC and Lundbeck Foundation, Denmark; EPLANET, ERC
and NSRF, European Union; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia;
BMBF, DFG, HGF, MPG and AvH Foundation, Germany; GSRT and NSRF, Greece;
RGC, Hong Kong SAR, China; ISF, MINERVA, GIF, I-CORE and Benoziyo Center, Is-
rael; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Nether-
lands; BRF and RCN, Norway; MNiSW and NCN, Poland; GRICES and FCT, Portugal;
MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MSTD,
Serbia; MSSR, Slovakia; ARRS and MIZˇS, Slovenia; DST/NRF, South Africa; MINECO,
Spain; SRC and Wallenberg Foundation, Sweden; SER, SNSF and Cantons of Bern and
Geneva, Switzerland; NSC, Taiwan; TAEK, Turkey; STFC, the Royal Society and Lever-
hulme Trust, United Kingdom; DOE and NSF, United States of America. JHEP09(2015)049 The crucial computing support from all WLCG partners is acknowledged gratefully,
in particular from CERN and the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF
(Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF
(Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (U.K.) and BNL
(U.S.A.) and in the Tier-2 facilities worldwide. Open Access. This article is distributed under the terms of the Creative Commons
Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in
any medium, provided the original author(s) and source are credited. 7
Conclusions The forward-backward asymmetry in electron and muon pairs from Z/γ∗decays is
measured using the 7 TeV pp LHC collision data recorded with the ATLAS detector in
2011 corresponding to an integrated luminosity of 4.8 fb−1. The data are analysed over a
range of dilepton invariant masses from 66 GeV to 1000 GeV in the central-central electron
and muon channels, and up to 250 GeV in the central-forward electron channel. The latter
includes events where one electron is reconstructed in the forward pseudorapidity range
(2.5 < |η| < 4.9). The forward-backward asymmetry is measured separately for the three channels as a
function of the dilepton invariant mass and unfolded for detector effects and final-state
radiation. Additionally, a leading-order interpretation which accounts for the effects of
dilution and full detector acceptance is presented. The resulting AFB values are found to
be in agreement with the corresponding Standard Model predictions. The detector level asymmetry values are used to extract the value of the leptonic
effective weak mixing angle, sin2 θ lept
eff
, separately for the three data samples using a
χ2 minimization method. The results are in good agreement with each other and with
measurements at e+e−colliders, at the Tevatron and by CMS at the LHC. Results from the electron and muon final states are combined, yielding
sin2 θ lept
eff
= 0.2308 ± 0.0005(stat.)±0.0006(syst.)±0.0009(PDF)=0.2308 ± 0.0012(tot.). Results from the electron and muon final states are combined, yielding sin2 θ lept
eff
= 0.2308 ± 0.0005(stat.)±0.0006(syst.)±0.0009(PDF)=0.2308 ± 0.0012(tot.) The dominant uncertainty comes from knowledge of the PDFs. The result from the muon channel, when converted to the asymmetry parameter
Aµ, yields Aµ = 0.153 ± 0.007(stat.) ± 0.006(syst.) ± 0.007(PDF) = 0.153 ± 0.012(tot.),
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M. Wright53, M. Wu55, S.L. Wu174, X. Wu49, Y. Wu88, E. Wulf35, T.R. Wyatt83,
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S. Yacoob146b,am, M. Yamada65, H. Yamaguchi156, Y. Yamaguchi156, A. Yamamoto65,
K. Yamamoto63, S. Yamamoto156, T. Yamamura156, T. Yamanaka156, K. Yamauchi102,
Y. Yamazaki66, Z. Yan22, H. Yang33e, H. Yang174, U.K. Yang83, Y. Yang110, S. Yanush92,
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A.L. The ATLAS collaboration Zhemchugov64, J. Zhong119, B. Zhou88, L. Zhou35, N. Zhou164,
C.G. Zhu33d, H. Zhu33a, J. Zhu88, Y. Zhu33b, X. Zhuang33a, A. Zibell175, D. Zieminska60,
N.I. Zimine64, C. Zimmermann82, R. Zimmermann21, S. Zimmermann21,
S. Zimmermann48, Z. Zinonos54, M. Ziolkowski142, G. Zobernig174, A. Zoccoli20a,20b,
M. zur Nedden16, G. Zurzolo103a,103b, V. Zutshi107, L. Zwalinski30 .S. Weber17, S.W. Weber175, J.S. Webster31, A.R. Weidberg119, P. Weigell100, JHEP09(2015)049 ,
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zur Nedden16, G. Zurzolo103a,103b, V. Zutshi107, L. Zwalinski30 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany NY, United States of America 3 Department of Physics, University of Alberta, Edmonton AB, Canada y
y
4
(a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University
Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara; (d)
Turkish Atomic Energy Authority, Ankara, Turkey 4
(a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi U
Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara; of Physics, Ankara University, Ankara; (b) Departme Department of Physics, Ankara University, Ankara;
Department of Physics, Gazi University,
Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara; (d) Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara; (d) Turkish Atomic Energy Authority, Ankara, Turkey 5 LAPP, CNRS/IN2P3 and Universit´e de Savoie, Annecy-le-Vieux, France LAPP, CNRS/IN2P3 and Universit´e de Savoie, An 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States of America Physics Division, Argonne National Laboratory, Argo 7 Department of Physics, University of Arizona, Tucson AZ, United States of America 8 Department of Physics, The University of Texas at Arlington, Arlington TX, United States of
America 9 Physics Department, University of Athens, Athens, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece
11 11 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 11 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan
12 12 Institut de F´ısica d’Altes Energies and Departament de F´ısica de la Universitat Aut`onoma de
Barcelona, Barcelona, Spain Barcelona, Barcelona, Spain 13
(a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, 13
(a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences,
University of Belgrade, Belgrade, Serbia University of Belgrade, Belgrade, Serbia 14 Department for Physics and Technology, University of Bergen, Bergen, Norwa 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley
CA, United States of America 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berke
CA, United States of America 16 Department of Physics, Humboldt University, Berlin, Germany 16 Department of Physics, Humboldt University, Berlin, Germany 17 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics,
University of Bern, Bern, Switzerland – 36 – 18 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 19
(a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus
University, Istanbul; (c) Department of Physics Engineering, Gaziantep University, Gaziantep,
Turkey 20
(a) INFN Sezione di Bologna; (b) Dipartimento di Fisica e Astronomia, Universit`a di Bologna,
Bologna, Italy 21 Physikalisches Institut, University of Bonn, Bonn, Germany 22 Department of Physics, Boston University, Boston MA, United States of America 23 Department of Physics, Brandeis University, Waltham MA, United States of America
( )
( ) 24
(a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits
Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao
Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao
Paulo, Brazil JHEP09(2015)049 Physics Department, Brookhaven National Laboratory, Upton NY, United States of America
26
(a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for
Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj
Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara,
Timisoara, Romania 28 Cavendish Laboratory, University of Cambridge, Cambridge, United Kin 29 Department of Physics, Carleton University, Ottawa ON, Canada 30 CERN, Geneva, Switzerland 31 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 32
(a) Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago; (b) Departamento
de F´ısica, Universidad T´ecnica Federico Santa Mar´ıa, Valpara´ıso, Chile 33
(a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of
Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics,
Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics
Department, Shanghai Jiao Tong University, Shanghai, China 33
(a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of
( ) Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics,
Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics
Department, Shanghai Jiao Tong University, Shanghai, China
34 Modern Physics, University of Science and Technology of China, Anhui; ( ) Department of Physics,
Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Department, Shanghai Jiao Tong University, Shanghai, China 34 Laboratoire de Physique Corpusculaire, Clermont Universit´e and Universit´e Blaise Pascal and
CNRS/IN2P3, Clermont-Ferrand, France 35 Nevis Laboratory, Columbia University, Irvington NY, United States of America Institute, University of Copenhagen, Kobenhavn, D 36 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark
( ) 37
(a) INFN Gruppo Collegato di Cosenza, Labora
Fisica, Universit`a della Calabria, Rende, Italy 37
(a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di
Fisica Universit`a della Calabria Rende Italy (a) INFN Gruppo Collegato di Cosenza, Laboratori N Fisica, Universit`a della Calabria, Rende, Italy y
38
(a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science,
Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 38
(a) AGH University of Science and Technology, Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland
39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow,
Poland 39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow,
Poland 40 Physics Department, Southern Methodist University, Dallas TX, United States of America Physics Department, Southern Methodist University, Dallas TX, United States of America
41 Physics Department, University of Texas at Dallas, Richardson TX, United States of America
42 DESY H
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47 INFN Laboratori Nazionali di Frascati, Frascati, Italy 46 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom
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(a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit`a di Genova, Genova, Italy 50
(a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit 50
(a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit`a di Genova, Genova, Italy
51
(a) E. Barcelona, Barcelona, Spain Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) 51
(a) E. Andronikashvili Institute of Physics, Iv. Barcelona, Barcelona, Spain Javakhishvili Tbilisi State University, Tbilisi; (b)
High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia – 37 – 52 II Physikalisches Institut, Justus-Liebig-Universit¨at Giessen, Giessen, Germany 53 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 54 II Physikalisches Institut, Georg-August-Universit¨at, G¨ottingen, Germany 55 Laboratoire de Physique Subatomique et de Cosmologie, Universit´e Grenoble-Alpes, CNRS/IN2P3,
Grenoble, France 56 Department of Physics, Hampton University, Hampton VA, United States of America 57 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge MA, United States
of America (a) Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b)
( ) 58
(a) Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b)
Physikalisches Institut Ruprecht Karls Universit¨at Heidelberg Heidelberg; (c) ZITI Institut 58
(a) Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b)
Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (c) ZITI Institut f
technische Informatik, Ruprecht-Karls-Universit¨at Heidelberg, Mannheim, Germany 58
(a) Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b)
Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (c) ZITI Institut f¨ur y
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ZITI Institut fur
technische Informatik, Ruprecht-Karls-Universit¨at Heidelberg, Mannheim, Germany technische Informatik, Ruprecht-Karls-Universit¨at Heidelberg, Mannheim, Germany aculty of Applied Information Science, Hiroshima Ins JHEP09(2015)049 60 Department of Physics, Indiana University, Bloomington IN, United States of Americ 61 Institut f¨ur Astro- und Teilchenphysik, Leopold-Franzens-Universit¨at, Innsbruck, Austria 62 University of Iowa, Iowa City IA, United States of America y
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63 Department of Physics and Astronomy, Iowa State University, Ames IA, United States of America 63 Department of Physics and Astronomy, Iowa State University, Ames IA, United States of Americ 63 Department of Physics and Astronomy, Iowa State University, Ames IA, Department of Physics and Astronomy, Iowa State U 64 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 64 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 65 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 65 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan duate School of Science, Kobe University, Kobe, Jap 67 Faculty of Science, Kyoto University, Kyoto, Japan 68 Kyoto University of Education, Kyoto, Japan 69 Department of Physics, Kyushu University, Fukuoka, Japan Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 70 Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argenti stituto de F´ısica La Plata, Universidad Nacional de L 70 Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONI 71 Physics Department, Lancaster University, Lancaster, United Kingdom 72
(a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Universit`a del Salento, Lecce,
Italy 72
(a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Universit`a del Salento, Lecce,
Italy 73 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom liver Lodge Laboratory, University of Liverpool, Live 74 Department of Physics, Joˇzef Stefan Institute and University of Ljubljana, Ljubljana, Slovenia 75 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 76 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 77 Department of Physics and Astronomy, University College London, London, United Kingdom 78 Louisiana Tech University, Ruston LA, United States of America 79 Laboratoire de Physique Nucl´eaire et de Hautes Energies, UPMC and Universit´e Paris-Diderot and
CNRS/IN2P3, Paris, France 80 Fysiska institutionen, Lunds universitet, Lund, Sweden 81 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 81 Departamento de Fisica Teorica C-15, Universidad Auton Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 82 Institut f¨ur Physik, Universit¨at Mainz, Mainz, Germany 83 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 84 CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France epartment of Physics, University of Massachusetts, A 86 Department of Physics, McGill University, Montreal QC, Canada 86 Department of Physics, McGill University, Montreal QC, Canada partment of Physics, McGill University, Montreal QC 87 School of Physics, University of Melbourne, Victoria, Australia 88 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America 88 Department of Physics, The University of Michigan, Ann Arbor 89 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States
of America 90
(a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Universit`a di Milano, Milano, Italy 90
(a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Universit`a di Milano, Milano, Italy
91 91 B.I. Barcelona, Barcelona, Spain Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of
Belarus 91 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of
Belarus 92 National Scientific and Educational Centre for Particle and High Energy Physics, Minsk, Republi
of Belarus 92 National Scientific and Educational Centre for Particle and High Energy Physics, Minsk, Republic
of Belarus 93 Department of Physics, Massachusetts Institute of Technology, Cambridge MA, United States of
America – 38 – 94 Group of Particle Physics, University of Montreal, Montreal QC, Canada 95 P.N. Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 96 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 97 National Research Nuclear University MEPhI, Moscow, Russia 98 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow,
Russia 99 Fakult¨at f¨ur Physik, Ludwig-Maximilians-Universit¨at M¨unchen, M¨unchen, Germany 100 Max-Planck-Institut f¨ur Physik (Werner-Heisenberg-Institut), M¨unchen, Germany 101 Nagasaki Institute of Applied Science, Nagasaki, Japan 102 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan
( )
(b) Graduate School of Science and Kobayashi Maskawa Institute, Nagoya University, Nagoya, Japan
103
(a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 103
(a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 104 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States
of America JHEP09(2015)049 105 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University
Nijmegen/Nikhef, Nijmegen, Netherlands 105 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University
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Netherlands 107 Department of Physics, Northern Illinois University, DeKalb IL, United States of America udker Institute of Nuclear Physics, SB RAS, Novosib 109 Department of Physics, New York University, New York NY, United States of Am 110 Ohio State University, Columbus OH, United States of America 111 Faculty of Science, Okayama University, Okayama, Japan 112 Homer L. Barcelona, Barcelona, Spain Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK,
United States of America 113 Department of Physics, Oklahoma State University, Stillwater OK, United States of America 114 Palack´y University, RCPTM, Olomouc, Czech Republic 115 Center for High Energy Physics, University of Oregon, Eugene OR, United States of America
116 Center for High Energy Physics, University of Orego 116 LAL, Universit´e Paris-Sud and CNRS/IN2P3, Orsay, France 117 Graduate School of Science, Osaka University, Osaka, Japan 118 Department of Physics, University of Oslo, Oslo, Norway 119 Department of Physics, Oxford University, Oxford, United Kingdom epartment of Physics, Oxford University, Oxford, Un 120
(a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 121 Department of Physics, University of Pennsylvania, Philadelphia PA 121 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America
122 Petersburg Nuclear Physics Institute Gatchina Russia Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America
122 Petersburg Nuclear Physics Institute, Gatchina, Russia 122 Petersburg Nuclear Physics Institute, Gatchina, Russia etersburg Nuclear Physics Institute, Gatchina, Russia 123
(a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 123
(a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Barcelona, Barcelona, Spain Petersburg State Polytechnical University, St. Barcelona, Barcelona, Spain Fermi, Universit`a di Pisa, Pisa, Italy
124 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of
America 124 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of
America 125
(a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa; (b) Faculdade
de Ciˆencias, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra,
Coimbra; (d) Centro de F´ısica Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de
Fisica, Universidade do Minho, Braga; (f) Departamento de Fisica Teorica y del Cosmos and
CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de
Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 126 Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 127 Czech Technical University in Prague, Praha, Czech Republic 128 Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 129 State Research Center Institute for High Energy Physics, Protvino, Russia 130 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United K cs Department, Rutherford Appleton Laboratory, Di ticle Physics Department, Rutherford Appleton Labo 131 Physics Department, University of Regina, Regina SK, Canada 132 Ritsumeikan University, Kusatsu, Shiga, Japan 133
(a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy
4
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(b) 134
(a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Universit`a di Roma Tor
Vergata, Roma, Italy – 39 – 135
(a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Universit`a Roma Tre,
Roma, Italy 136
(a) Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies -
Universit´e Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques
Nucleaires, Rabat; (c) Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech;
(d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des
sciences, Universit´e Mohammed V-Agdal, Rabat, Morocco 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay
(Commissariat `a l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France 138 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA,
United States of America JHEP09(2015)049 JHEP09(2015)049 140 Department of Physics and Astronomy, University of Sheffield, Sheffield, United 141 Department of Physics, Shinshu University, Nagano, Japan 142 Fachbereich Physik, Universit¨at Siegen, Siegen, Germany epartment of Physics, Simon Fraser University, Burn 143 Department of Physics, Simon Fraser University, Burnaby BC, Canada 144 SLAC National Accelerator Laboratory, Stanford CA, United States of America 145
(a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)
Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of
Sciences, Kosice, Slovak Republic 145
(a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)
Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Acade Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of
Sciences, Kosice, Slovak Republic Sciences, Kosice, Slovak Republic 146
(a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics,
University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand,
Johannesburg, South Africa 147
(a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Swede 148 Physics Department, Royal Institute of Technology, Stockholm, Sweden 149 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY,
United States of America 150 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom
151 151 School of Physics, University of Sydney, Sydney, Australia 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 153 Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel
154 154 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv,
Israel 155 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greec 156 International Center for Elementary Particle Physics and Department of Physics, The University of
Tokyo, Tokyo, Japan 157 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 158 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 159 Department of Physics, University of Toronto, Toronto ON, Canada 160
(a) TRIUMF, Vancouver BC; (b) Department of Physics and Astronomy, York University, Toronto
ON, Canada 161 Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan
162 Department of Physics and Astronomy, Tufts University, Medford MA, United States of America 162 Department of Physics and Astronomy, Tufts University, Medford MA, United 163 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia
164 164 Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States
America 165
(a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento
di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy 166 Department of Physics, University of Illinois, Urbana IL, United States of America 167 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 168 Instituto de F´ısica Corpuscular (IFIC) and Departamento de F´ısica At´omica, Molecular y Nuclear
and Departamento de Ingenier´ıa Electr´onica and Instituto de Microelectr´onica de Barcelona
(IMB-CNM), University of Valencia and CSIC, Valencia, Spain – 40 – 169 Department of Physics, University of British Columbia, Vancouver BC, Canada 170 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 171 Department of Physics, University of Warwick, Coventry, United Kingdom 172 Waseda University, Tokyo, Japan y,
y ,
p
173 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 73 Department of Particle Physics, The Weizmann I 174 Department of Physics, University of Wisconsin, Madison WI, United States of America 175 Fakult¨at f¨ur Physik und Astronomie, Julius-Maximilians-Universit¨at, W¨urzburg, Germany 176 Fachbereich C Physik, Bergische Universit¨at Wuppertal, Wuppertal, Germany 77 Department of Physics, Yale University, New Hav 177 Department of Physics, Yale University, New Haven CT, United States of America
178 178 Yerevan Physics Institute, Yerevan, Armenia 178 Yerevan Physics Institute, Yerevan, Armenia 179 Centre de Calcul de l’Institut National de Physique Nucl´eaire et de Physique des Particules
(IN2P3), Villeurbanne, France JHEP09(2015)049 a Also at Department of Physics, King’s College London, London, United Kingdom b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan c Also at Novosibirsk State University, Novosibirsk, Russia d Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom
e Also at TRIUMF Vancouver BC Canada d Also at Particle Physics Department, Rutherford Appleton Laboratory, Did d Also at Particle Physics Department, Rutherfor Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom
e Also at TRIUMF, Vancouver BC, Canada e Also at TRIUMF, Vancouver BC, Canada e Also at TRIUMF, Vancouver BC, Canada f Also at Department of Physics, California State f Also at Department of Physics, California State University, Fresno CA, United States of America
g Also at Department of Physics, University of Fribourg, Fribourg, Switzerland g Also at Department of Physics, University of Fribourg, Fribourg, Switzerland g Also at Department of Physics, University of Frib h Also at Tomsk State University, Tomsk, Russia Also at CPPM, Aix-Marseille Universit´e and CNR Also at CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France i Also at CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France j Also at Universit`a di Napoli Parthenope, Napoli, Italy k Also at Institute of Particle Physics (IPP), Canada l Also at Department of Physics, St. am Also at Discipline of Physics, University of KwaZulu-Natal, Durban, South Africa
∗Deceased al Also at Department of Physics, The University of Michigan, Ann Arbor MI, United States of
America am Also at Discipline of Physics, University of KwaZulu-Natal, Durban, South Africa
∗D
d ak Also at Institut f¨ur Experimentalphysik, Universit¨at Hamburg, Hamburg, Germany aj Also at Department of Physics, Nanjing University, Jiangsu, China Barcelona, Barcelona, Spain Petersburg,
Russia m Also at Chinese University of Hong Kong, China m Also at Chinese University of Hong Kong, China n Also at Louisiana Tech University, Ruston LA, United States of America n Also at Louisiana Tech University, Ruston LA, United States of America Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain o Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barce p Also at Department of Physics, The University of Texas at Austin, Austin TX, United States of
America q Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia r Also at CERN, Geneva, Switzerland s Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan t Also at Manhattan College, New York NY, United States of America t Also at Manhattan College, New York NY, United States of America u Also at Institute of Physics, Academia Sinica, Taipei, Taiwan u Also at Institute of Physics, Academia Sinica, Taipei, Taiwan v Also at LAL, Universit´e Paris-Sud and CNRS/IN2P3, Orsay, France w Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan x Also at Laboratoire de Physique Nucl´eaire et de Hautes Energies, UPMC and Universit´e
Paris-Diderot and CNRS/IN2P3, Paris, France x Also at Laboratoire de Physique Nucl´eaire et de Hautes Energies, UPMC and Universit´e x Also at Laboratoire de Physique Nucl´eaire et de Hautes Energies, UPMC and Universit´e Paris-Diderot and CNRS/IN2P3, Paris, France /
y Also at School of Physical Sciences, National Institute of Science Education and Research,
Bhubaneswar, India y Also at School of Physical Sciences, National Institute of Science Education and Research,
Bhubaneswar, India z Also at Dipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy z Also at Dipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy aa Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia
b aa Also at Moscow Institute of Physics and Technology State University, Dolg ab Also at section de Physique, Universit´e de Gen`eve, Geneva, Switzerland ab Also at section de Physique, Universit´e de Gen`eve, Geneva, Switzerland ac Also at International School for Advanced Studies (SISSA), Trieste, Italy ac Also at International School for Advanced Studies (SISSA), Trieste, Italy ad Also at Department of Physics and Astronomy, University of South Carolina, Columbia SC, United
States of America ae Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China af Also at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia ag Also at National Research Nuclear University MEPhI, Moscow, Russia ah Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest,
Hungary – 41 – ai Also at Department of Physics, Oxford University, Oxford, United Kingdom ai Also at Department of Physics, Oxford University, Oxford, United Kingdom aj Also at Department of Physics, Nanjing University, Jiangsu, China JHEP09(2015)049 – 42 –
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English
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A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part II, modeling
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PLOS computational biology/PLoS computational biology
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RESEARCH ARTICLE Editor: Mark Alber, University of California
Riverside, UNITED STATES Editor: Mark Alber, University of California
Riverside, UNITED STATES Riverside, UNITED STATES
Received: January 18, 2017
Accepted: May 4, 2017
Published: July 14, 2017
Copyright: © 2017 Bottier 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. Received: January 18, 2017
Accepted: May 4, 2017
Published: July 14, 2017 Copyright: © 2017 Bottier 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. A new index for characterizing micro-bead
motion in a flow induced by ciliary beating:
Part II, modeling Mathieu Bottier1,2,3, Marta Peña Ferna´ndez1,2,3, Gabriel Pelle1,2,3, Daniel Isabey1,2,3,
Bruno Louis1,2,3, James B. Grotberg4, Marcel Filoche1,2,3,5* 1 Eq. 13, Institut Mondor de Recherche Biome´dicale, Inserm U955, Cre´teil, France, 2 Universite´ Paris-Est,
Faculte´ de me´decine, Cre´teil, France, 3 CNRS ERL 7240, Cre´teil, France, 4 Department of Biomedical
Engineering, University of Michigan, Ann Arbor, MI, USA, 5 Laboratoire de Physique de la Matière
Condense´e, Ecole Polytechnique, CNRS, Universite´ Paris Saclay, 91128 Palaiseau Cedex, France a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * marcel.filoche@polytechnique.edu * marcel.filoche@polytechnique.edu Abstract Mucociliary clearance is one of the major lines of defense of the human respiratory system. The mucus layer coating the airways is constantly moved along and out of the lung by the
activity of motile cilia, expelling at the same time particles trapped in it. The efficiency of the
cilia motion can experimentally be assessed by measuring the velocity of micro-beads trav-
eling through the fluid surrounding the cilia. Here we present a mathematical model of the
fluid flow and of the micro-beads motion. The coordinated movement of the ciliated edge is
represented as a continuous envelope imposing a periodic moving velocity boundary condi-
tion on the surrounding fluid. Vanishing velocity and vanishing shear stress boundary condi-
tions are applied to the fluid at a finite distance above the ciliated edge. The flow field is
expanded in powers of the amplitude of the individual cilium movement. It is found that the
continuous component of the horizontal velocity at the ciliated edge generates a 2D fluid
velocity field with a parabolic profile in the vertical direction, in agreement with the experi-
mental measurements. Conversely, we show than this model can be used to extract micro-
scopic properties of the cilia motion by extrapolating the micro-bead velocity measurement
at the ciliated edge. Finally, we derive from these measurements a scalar index providing a
direct assessment of the cilia beating efficiency. This index can easily be measured in
patients without any modification of the current clinical procedures. OPEN ACCESS Citation: Bottier M, Peña Ferna´ndez M, Pelle G,
Isabey D, Louis B, Grotberg JB, et al. (2017) A new
index for characterizing micro-bead motion in a
flow induced by ciliary beating: Part II, modeling. PLoS Comput Biol 13(7): e1005552. https://doi. org/10.1371/journal.pcbi.1005552 Author summary Mucociliary clearance is the first line of defense mechanisms of the human airways. The
mucus transporting debris, particles, microorganisms and pollutants is carried away by
the coordinated motion of cilia beating at the surface of the airway epithelium. We present
here a mathematical and numerical model aiming at defining a global index for assessing
the efficiency of this beating. Numerical simulations show that the bead velocity parallel
to the wall varies according a parabolic profile with the distance to the wall. The velocity Funding: This work has been supported by a grant
from Fondation pour la Recherche Me´dicale
(https://www.frm.org/, DI being the PI): FRM
programme Bio-Inge´nierie pour la Sante´ 2014,
DBS 20140930771. The PhD Scholarship of MB
has been provided by CNRS INSIS 2013 (http://
www.cnrs.fr/). Some authors of this article are
participants in BEAT-PCD (COST Action 1407). The 1 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. extrapolated at the wall is demonstrated to be a measurement of the momentum transfer
between cilia and the surrounding fluid. This model allows us to interpret experimental
measurements performed in a companion article and to propose a universal index charac-
terizing the beating efficiency, which can be extracted in the current clinical setting. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. Characterizing micro-bead motion in a ciliary beating induced flow: Modeling This work intends to build a mathematical model of the experiments described in a com-
panion paper [11], and to derive from this model an efficient and reliable method to assess the
cilia beating efficiency in a clinical setting. In the experiments, ciliated cell clusters issued from
nasal brushing are immersed in a cell survival medium devoid of mucus, pushing forward the
medium as they beat. One has to stress here that the absence of mucus is an important ingredi-
ent of the experimental setting and the developed mathematical model. Polystyrene micro-
beads are then used as massless tracers to visualize and quantify the fluid velocity field around
the cilia. The theoretical and numerical work presented here therefore aim at a quantitative
modeling of the ciliary beating, then of the fluid flow generated by it. In the literature, two main types of ciliary beating models can be found: discrete-cilia models
and volume-force models [12]. In discrete-cilia model, each cilium is modeled as a discrete
body and its shape is parametrized along its stroke period [1, 13, 14]. Discrete-cilia models are
themselves divided into two types: in the prescribed beating models, the cilium motion is
imposed as an input to the simulation [15]; in the couple-internal mechanics/fluid-structure
interaction models [16, 17], cilia motions originate from the coupling between the internal
structure of cilia and the external viscous forces. In contrast, in volume-force models cilia are
modeled through a phenomenological continuous force distribution, varying in space and
time as the cilia beat [18–20]. In this second type of model, the envelope modeling approach
accounts for the formation of metachronal waves above the cilia layer [21]. The cilia tips are
seen from the fluid as a “wavy wall”, hereby ignoring the details of the sub-layer dynamics
[22, 23] (see Fig 1, right). Many studies have addressed experimentally [24–26] and numerically [27–31] the effect of
fluid visco-elasticity on transport and locomotion. We want to stress here that in our model,
no finite layer of viscous mucus sits on top of the cilia, since they are surrounded by a semi-
infinite layer of watery fluid. In the following, we first compute the wave envelope boundary condition from the cilia
motions, based on the work of Ross [22]. Materials and methods We present here a two-dimensional model of cilia, fluid, and micro-beads motion. We then derive the non linear equations for a fluid
flow periodic in the direction of the metachronal wave. These equations are expanded in ε,
which is the ratio of the cilium amplitude to the fluid layer thickness, then solved using a Fou-
rier decomposition. The steady contribution to the flow field in the vertical direction above the
cilia is shown to exhibit a parabolic profile, to a very good approximation. We finally show
that measuring microbead velocities as a function of the distance to the ciliated edge enables
us to compute a scalar index which accounts for the transfer of momentum between the cilia
and the fluid, and therefore assesses the cilia beating efficiency. Introduction Mucociliary clearance is one of the major defense mechanisms of the respiratory airway sys-
tem. The mucus layer coating the epithelial surface of the airways filters the inhaled air by trap-
ping potentially harmful material (fungi, bacteria and other particles) [1–4]. This mucus layer
is continuously carried away and out of the airways by the activity of motile cilia. Neighboring
cilia beat in an organized manner with a small phase lag, their tips creating an undulating sur-
face on top of the cilia layer which deforms in a wave-like fashion called the metachronal wave
[5–7]. The beat pattern of an individual cilium displays a two-stroke effective-recovery motion
[8]. During the effective stroke, cilia beat forwards and engage with the mucous layer, propel-
ling it forward. In contrast, during the recovery stroke, they return to their initial position in
the underlying periciliary fluid, minimizing thereby the drag on the mucus in the opposite
direction (Fig 1, left). This asymmetry in the beat pattern is responsible for a net fluid flow in
the direction of the effective stroke. In the airways, each mature ciliated cell may be covered
with up to 200 cilia, with a surface density around 5–8 cilia/μm2 [6, 9]. A cilium, approximately
6 μm long and of diameter around 0.2 μm, beats 12 to 15 times per second, resulting in a veloc-
ity of the mucus layer of several mm/min [10]. Fig 1. Schematic representation of the stroke of an individual cilium and the envelope model. (Left) Simplification of the stroke cycle as an ellipse. (Right) Representation of the envelope model covering the cilia layer and the propagation of the metachronal wave. (Inspired by Velez-Cordero et al. [23])
https://doi org/10 1371/journal pcbi 1005552 g001 Fig 1. Schematic representation of the stroke of an individual cilium and the envelope model. (Left) Simplification of the stroke cycle as an ellipse. (Right) Representation of the envelope model covering the cilia layer and the propagation of the metachronal wave. (Inspired by Velez-Cordero et al. [23])
https://doi.org/10.1371/journal.pcbi.1005552.g001 Fig 1. Schematic representation of the stroke of an individual cilium and the envelope model. (Left) Simplification of the stroke cycle as an ellipse. (Right) Representation of the envelope model covering the cilia layer and the propagation of the metachronal wave. (Inspired by Velez-Cordero et al. [23])
https://doi org/10 1371/journal pcbi 1005552 g001 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 2 / 21 From the individual cilium motion to the metachronal wave Each individual cilium is assumed to undergo a periodic motion in which its tip follows an
elliptic trajectory, see Fig 1, left. Taking the limit of a continuous cilia distribution, the cilia
array is simplified as an undulating surface that covers the cilia layer, ignoring the details of
the sub-layer dynamics (Fig 1, right) [22, 23]. The x axis is chosen parallel to the ciliated edge,
each ciliary tip being located around y = 0 on average (the ‘’ notation stands for dimensioned
quantities, and will be removed once we switch to dimensionless quantities). The tip of a cil-
ium located at the horizontal coordinate ξ is assumed to follow a periodic elliptic trajectory
centered in (ξ, 0) during each elementary beat (Fig 2). At time t, the tip coordinates ðX
w; Y
wÞ 3 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 The motion equations of each tip are thus rewritten in a dimensionless form: Xwðx; tÞ
¼ x ε cosðkx þ tÞ
Ywðx; tÞ
¼ bε sinðkx þ tÞ
ð4Þ
( ð4Þ In the following, we assume that the ciliary beating amplitude is much smaller than the film
thickness, i.e. ε 1. In the limit of continuously distributed cilia along the x axis, the envelope
of their tip motions forms a continuous boundary that generates a forcing on the fluid layer
above the cilia. Starting from Eq 4, we now express the position of a particle of this envelope
(or wall) in the Eulerian frame of reference (x, y, t). A tip located at position x at time t corre-
sponds to a cilium centered in (ξ, 0) such that: x ¼ x ε cosðkx þ tÞ
ð5Þ ð5Þ This equation shows that (x −ξ) is of order ε. Therefore the vertical location of the cilia wall
yw(x, t), which is also the y-coordinate of the corresponding cilium, Yw(ξ, t), can be developed
around ξ = x using a Taylor expansion: ywðx; tÞ ¼ Ywðx; tÞ ¼ Ywðx; tÞ þ x x
ð
Þ @Yw
@x
x¼x
þ ðx xÞ
2
2
@2Yw
@x2
x¼x
þ
ð6Þ ð6Þ Since both Yw and (ξ −x) are first order in ε (Eqs 4 and 5), the expansion to the second order
in ε of yw is Since both Yw and (ξ −x) are first order in ε (Eqs 4 and 5), the expansion to the second order
in ε of yw is ywðx; tÞ ¼ εb sinðkx þ tÞ þ ε2bk cos 2ðkx þ tÞ
ð7Þ ð7Þ Now that we have determined the location of the ciliated wall at time t, we can derive its veloc-
ity. The horizontal component of the wall velocity is obtained as the time derivative of the
Lagrangian velocity of the tip at location x at time t, while its vertical component is calculated
from the time derivative of the vertical coordinate of the wall at position x and time t: uwðx; tÞ
¼
@Xw
@t
x
¼ εsinðkx þ tÞ
vwðx; tÞ
¼ @yw
@t ¼ εb cosðkx þ tÞ ε2bk sinð2ðkx þ tÞÞ
ð8Þ
8
>
>
>
<
>
>
>
: ð8Þ The value of ξ to insert in the above horizontal velocity is given by Eq 5. Characterizing micro-bead motion in a ciliary beating induced flow: Modeling Fig 2. Schematic elliptic motion of an individual ciliary tip. https://doi.org/10.1371/journal.pcbi.1005552.g002 Fig 2. Schematic elliptic motion of an individual ciliary tip. Fig 2. Schematic elliptic motion of an individual ciliary tip. Fig 2. Schematic elliptic motion of an individual ciliary tip. are
X
w
¼ x a cosðotÞ
Y
w
¼ ba sinðotÞ
ð1Þ
(
Fig 2. Schematic elliptic motion of an individual ciliary tip. https://doi.org/10.1371/journal.pcbi.1005552.g002 https://doi.org/10.1371/journal.pcbi.1005552.g002 https://doi.org/10.1371/journal.pcbi.1005552.g002 are X
w
¼ x a cosðotÞ
Y
w
¼ ba sinðotÞ
ð1Þ
( ð1Þ where β is the ellipse eccentricity, 2a is its major axis in the x direction, and 2βa its minor axis
in the y direction. For β > 0, the tip orbits clockwise, while for β < 0, the tip orbits
counterclockwise. To reproduce the backwards traveling metachronal wave of wavelength λ, we introduce in
the periodic motion of each cilium a phase shift 2px
l
which linearly depends on the cilium
position: X
w
¼ x a cos
ot þ 2px
l
Y
w
¼ ba sin
ot þ 2px
l
ð2Þ
8
>
>
>
<
>
>
>
: ð2Þ The corresponding wave frequency is f = 2πω and its speed is c = f λ. By setting the space and
time units respectively to be h and ω−1, dimensionless parameters ε and k, and variables
(Xw, Yw) are introduced: The corresponding wave frequency is f = 2πω and its speed is c = f λ. By setting the space and
time units respectively to be h and ω−1, dimensionless parameters ε and k, and variables
(Xw, Yw) are introduced: ε ¼ a
h ; k ¼ 2ph
l
; Xw ¼ X
w
h ; Yw ¼ Y
w
h ; x ¼ x
h ; y ¼ y
h ; x ¼ x
h ; t ¼ ot
ð3Þ ð3Þ putational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017
4 / 21 OS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017
4 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 4 / 21 In summary, at location x in the Eulerian frame, the second order expansion in ε of the vertical PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling The motion equations of each tip are thus rewritten in a dimensionless form: This
contribution is proportional to k and will be the origin of the steady horizontal motion of the
fluid above the cilia. We can observe therefore that all velocity terms at the ciliated wall are oscillatory, except one
steady contribution to the horizontal velocity at the second order in ε that appears in uw,2. This
contribution is proportional to k and will be the origin of the steady horizontal motion of the
fluid above the cilia. Characterizing micro-bead motion in a ciliary beating induced flow: Modeling position yw and of the velocity vector (uw, vw) of the ciliated wall are: position yw and of the velocity vector (uw, vw) of the ciliated wall are: ywðx; tÞ ¼ εb siny þ ε2bk
2
1 þ cos 2y
ð
Þ
ð
Þ
ð10Þ ð10Þ uwðx; tÞ ¼ ε siny þ ε2k
2
1 þ cos 2y
ð
Þ
ð
Þ
ð11Þ uwðx; tÞ ¼ ε siny þ ε2k
2
1 þ cos 2y
ð
Þ
ð
Þ ð11Þ vwðx; tÞ ¼ εb cosy ε2bk sinð2yÞ
ð12Þ ð12Þ where θ = kx + t is the local phase of the metachronal wave. We now introduce the first and
second orders of the ε-expansion of the wall velocity, called ~U w;1 ¼ ðuw;1; vw;1Þ and
~U w;2 ¼ ðuw;2; vw;2Þ, respectively, defined such that: where θ = kx + t is the local phase of the metachronal wave. We now introduce the first a
second orders of the ε-expansion of the wall velocity, called ~U w;1 ¼ ðuw;1; vw;1Þ and
~U w;2 ¼ ðuw;2; vw;2Þ, respectively, defined such that: ~U w ¼ ε ~U w;1 þ ε2
2
~U w;2
ð13Þ ð13Þ In summary, the first and second orders in ε of the location and velocities of the cilia wall are: yw;1ðyÞ
¼ b siny ¼ b
2i eiy b
2i e iy
yw;2ðyÞ
¼ bk 1 þ cos 2y
ð
Þ
ð
Þ ¼ bk þ bk
2 e2iy þ bk
2 e 2iy
uw;1ðyÞ
¼ siny ¼ 1
2i eiy 1
2i e iy
uw;2ðyÞ
¼ k 1 þ cos 2y
ð
Þ
ð
Þ ¼ k þ k
2 e2iy þ k
2 e 2iy
vw;1ðyÞ
¼ b cosy ¼ b
2 eiy þ b
2 e iy
vw;2ðyÞ
¼ 2bk sinð2yÞ ¼ ibk e2iy ibk e 2iy
ð14Þ
8
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
<
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
: ð14Þ We can observe therefore that all velocity terms at the ciliated wall are oscillatory, except one
steady contribution to the horizontal velocity at the second order in ε that appears in uw,2. The motion equations of each tip are thus rewritten in a dimensionless form: Expanding the hori-
zontal component of the wall velocity in Taylor series to the second order in ε yields uwðx; tÞ
¼
@Xw
@t
x¼x
þ ðx xÞ @uw
@x
x¼x
þ . . . ¼
ε sinðkx þ tÞ þ ½ε cosðkx þ tÞ½εk cosðkx þ tÞ
¼
ε sinðkx þ tÞ þ ε2k cos 2ðkx þ tÞ
ð9Þ ð9Þ In summary, at location x in the Eulerian frame, the second order expansion in ε of the vertical In summary, at location x in the Eulerian frame, the second order expansion in ε of the vertical PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 5 / 21 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling frequency are h = 50 μm and f = 10 Hz. In water (ρ = 1 g.cm−3 and μ = 1 cP), the above expres-
sion leads to a Womersley number α 0.4, which means α2 of the order of 0.1. Boundary conditions. Boundary conditions are periodic in the horizontal direction with
a period equal to the wavelength of the metachronal wave (corresponding here to λ/h = 2π/k): ~U
2p
k ; y; t
¼ ~Uð0; y; tÞ
ð16Þ ð16Þ At the upper side of the domain (y = 1), one assumes vanishing vertical velocity and vanishing
shear stress due to the presence of stagnant fluid above the channel: At the upper side of the domain (y = 1), one assumes vanishing vertical velocity and vanishing
shear stress due to the presence of stagnant fluid above the channel: vðx; 1; tÞ ¼ 0
and
@u
@y
ðx;1;tÞ
¼ 0
ð17Þ vðx; 1; tÞ ¼ 0
and
@u
@y
ðx;1;tÞ
¼ 0
ð17Þ ð17Þ Next to the cilia envelope (y = yw), we introduce a Robin-type boundary condition, which Next to the cilia envelope (y = yw), we introduce a Robin-type boundary condition, which
involves both the velocity and its normal derivative (Eq 18). The factor in front of the normal
derivative, called ϕ, accounts for the partial momentum transfer between the wall and the fluid
due to the non continuous coverage of the cilia. ϕ = 0 corresponds to a no slip boundary condi-
tion, while ϕ ! +1 corresponds to a vanishing shear stress at the wall (perfect sliding). This
condition is analogous to that of a fluid flow next to a porous wall, where the presence of pores
reduces the transfer of momentum between the wall and the fluid [32, 33]: ~U @~U
@y
! ðx;yw;tÞ
¼ ~U wðx; tÞ
ð18Þ ð18Þ If expressed in dimensional quantities, the parameter ϕ becomes a length ϕ = hϕ. For a porous
wall, this length is proportional to the square root of the permeability. In our case, it is inter-
preted as a characteristic sliding length and will be related to the cilia surface density. preted as a characteristic sliding length and will be related to the cilia surface density. Computing the flow field We now compute the oscillatory flow field of a fluid of density ρ and viscosity μ in the channel
comprised between y = yw(x, t) and y = h in the vertical direction. Due to the periodic nature
of the forcing from the wall, we will consider periodic solution in the x direction. Given the
normalization chosen in Eq 3, the normalization factors for velocity and pressure are hω and
μω, respectively. Hence, the dimensionless Navier-Stokes equation reads a2
@~U
@t þ ð~U r
! Þ ~U
! ¼ r
! p þ D~U ;
ð15Þ ð15Þ where ~U ¼ ðu; vÞ is the dimensionless velocity field, p is the dimensionless pressure, and α is
the Womersley number defined by a2 ¼ rh2o
m . In the limit α ! 0, one recovers the classical
stationary Stokes equation. In our case, typical values for the channel thickness and beating PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 6 / 21 org/10.1371/journal.pcbi.1005552
July 14, 2017
7 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling Inserting Eqs 19 and 20 into Eq 18 finally gives the first two orders of the ε-expansion of the
boundary condition at the ciliated wall: Inserting Eqs 19 and 20 into Eq 18 finally gives the first two orders of the ε-expansion of the
boundary condition at the ciliated wall: ~U 1ð0Þ @~U 1
@y
0
¼ ~U w;1
ð21Þ ð21Þ ~U 2ð0Þ @~U 2
@y
0
¼ ~U w;2 2yw;1
@~U 1
@y
0
@2~U 1
@y2
0
! ð22Þ ð22Þ Expanding the flow field ~U in powers of ε, ~U ¼ ε ~U 1 þ ε2
2 ~U 2, the boundary conditions
can be expressed at all orders in ε. Since yw is first order in ε (see Eq 10), the boundary condi-
tion can be expanded around y = 0 both for the velocity ~U and its normal derivative @~U=@y: ~UðywÞ
¼
~Uð0Þ þ yw
@~U
@y
y¼0
þ Oðε3Þ
¼
ε~U 1ð0Þ þ ε2
2
~U 2ð0Þ þ εyw;1 þ ε2
2 yw;2
ε @~U 1
@y
0
þ ε2
2
@~U 2
@y
0
"
#
þ Oðε3Þ
¼
ε ~U 1ð0Þ þ ε2
2
~U 2ð0Þ þ 2yw;1
@~U 1
@y
"
#
þ Oðε3Þ
ð19Þ ¼
ε~U 1ð0Þ þ ε2
2
~U 2ð0Þ þ εyw;1 þ ε2
2 yw;2
ε @~U 1
@y
0
þ ε2
2
@~U 2
@y
0
"
#
þ Oðε3Þ
¼
ε ~U 1ð0Þ þ ε2
2
~U 2ð0Þ þ 2yw;1
@~U 1
@y
"
#
þ Oðε3Þ
ð19Þ @~U
@y
yw
¼
@~U
@y
0
þ yw
@2~U
@y2
0
þ Oðε3Þ
¼
ε @~U 1
@y
0
þ ε2
2
@~U 2
@y
0
þ εyw;1 þ ε2
2 yw;2
ε @2~U 1
@y2
0
þ ε2
2
@2~U 2
@y2
0
"
#
þ Oðε3Þ
¼
ε @~U 1
@y
0
þ ε2
2
@~U 2
@y
0
þ 2yw;1
@2~U 1
@y2
0
"
#
þ Oðε3Þ
ð20Þ ð20Þ PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017
7 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017
7 / 21 7 / 21 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling two orders in ε translate into 4 equations at the wall boundary: two orders in ε translate into 4 equations at the wall boundary: equations at the wall boundary: c1;yjðx;0Þ c1;yy ¼ siny
ð30Þ c1;yjðx;0Þ c1;yy ¼ siny
ð30Þ
j
ð
Þ ð30Þ c1;yjðx;0Þ c1;yy ¼ siny
ð30Þ
c1;xjðx;0Þ c1;xy ¼ b cosy
ð31Þ c1;xjðx;0Þ c1;xy ¼ b cosy
ð31Þ ð31Þ c2;yjðx;0Þ c2;yy ¼ kð1 þ cosð2yÞÞ 2b siny ðc1;yy c1;yyyÞ
ð32Þ
c2;xjðx;0Þ c2;xy ¼ 2bk sinð2yÞ 2b siny ðc1;xy c1;xyyÞ
ð33Þ ð32Þ c2;xjðx;0Þ c2;xy ¼ 2bk sinð2yÞ 2b siny ðc1;xy c1;xyyÞ
ð33Þ ð33Þ The stream function can thus be computed in first approximation as if the domain were a
i h
h
l
i d b
0
d
1 The stream function can thus be computed in first approximation as if the domain were a
straight channel comprised between y = 0 and y = 1. The stream function can thus be computed in first approximation as if the domain were a
straight channel comprised between y = 0 and y = 1. traight channel comprised between y = 0 and y = 1 First order of the stream function. ψ1(x, y, t) is a periodic function of period 2π/k along x
that solves the following system: First order of the stream function. ψ1(x, y, t) is a periodic function of period 2π/k along x
that solves the following system: D2c1 a2Dc1;t ¼ 0
c1;y c1;yy ¼ siny
at y ¼ 0
c1;x c1;xy ¼ b cosy
at y ¼ 0
c1;yy ¼ 0
at y ¼ 1
c1;x ¼ 0
at y ¼ 1
ð34Þ
8
>
>
>
>
>
>
>
<
>
>
>
>
>
>
>
: ð34Þ We expand ψ1 in Fourier series along the x direction, each term corresponding to a wave trav-
eling forward or backward at dimensionless speed 1. The stream function The stream function Since the flow is two-dimensional and incompressible, a natural formulation of the problem is
obtained by introducing the stream function ψ such that: u ¼ @c
@y
and
v ¼ @c
@x
ð23Þ ð23Þ Such a solution automatically fulfills the continuity equation divð~UÞ ¼ 0. The dimensionless
Navier-Stokes equation now reads: Such a solution automatically fulfills the continuity equation divð~UÞ ¼ 0. The dimensionless
Navier-Stokes equation now reads: a2ðcyt þ cycyx cxcyyÞ
¼ px þ cyxx þ cyyy
a2ð cxt þ cycxx þ cxcxyÞ
¼ py cxxx cxyy
ð24Þ
( ð24Þ We derive a Partial Differential Equation (PDE) in ψ only by taking the y-derivative of the first
equation and subtracting it to it the x-derivative of the second equation: We derive a Partial Differential Equation (PDE) in ψ only by taking the y-derivative of the first
equation and subtracting it to it the x-derivative of the second equation: a2fcxxt þ cyyt þ cycyyx cxcyyy þ cycxxx cxcxxyg ¼ cyyyy þ 2cxxcyy þ cxxxx
ð25Þ ð25Þ This equation can finally be rewritten in the following compact form: This equation can finally be rewritten in the following compact form: a2fDct þ cyDcx cxDcyg ¼ D2c
ð26Þ ð26Þ Asymptotic expansion of the stream function. We now expand the stream function to
the second order in ε, as it was done for the flow field: c ¼ εc1 þ ε2
2 c2
ð27Þ ð27Þ The zero order term ψ0 vanishes since the fluid is set into motion only by the cilia beating
(ε > 0). The two orders in ε are solved in sequence. The first order of Eq 26 is: a2Dc1;t ¼ D2c1
ð28Þ ð28Þ while the second order is: while the second order is: while the second order is: a2fDc2;t þ 2c1;yDc1;x 2c1;xDc1;yg ¼ D2c2
ð29Þ ð29Þ The boundary condition at the lower wall (the cilia envelope) couples the first and second
orders ψ1 and ψ2 through the Robin condition of Eq 18. The two velocity components and the PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 8 / 21 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling and B 1 ¼ B1. Therefore, ψ1 can be expressed as: c1ðx; y; tÞ ¼ ½A1e ky þ B1e g1y eiy þ c:c: ;
ð40Þ ð40Þ where c.c. stands for complex conjugate. The flow field at first order in ε is then: u1 ¼ c1;y ¼ 2 Ref½kA1e ky þ g1B1e g1y eiyg
v1 ¼ c1;x ¼ 2k Imf½A1e ky þ B1e g1y eiyg
ð41Þ
( ð41Þ A1 and B1 are determined from the two boundary conditions at y = 0 in Eq (34): kA1 g1B1 ðk2A1 þ g2
1B1Þ
¼ 1
2i
ik½A1 þ B1 ð kA1 g1B1Þ
¼ b
2
ð42Þ
8
>
>
<
>
>
: ð42Þ which finally gives: which finally gives: A1 ¼
i
2ð1 þ k Þ
bg1
k 1
g1 k
0
B
@
1
C
A
and
B1 ¼
i
2ð1 þ g1 Þ
1 b
g1 k
ð43Þ ð43Þ Second order of the stream function: The steady contribution. ψ2(x, y, t), the second
order in ε of the stream function, is a periodic function of period 2π/k along x that solves the
following system: D2c2 a2Dc2;t ¼ 2a2fc1;yDc1;x c1;xDc1;yg
c2;y c2;yy ¼ kð1 þ cosð2yÞÞ 2b siny c1;yy þ 2b siny c1;yyy
at y ¼ 0
c2;x c2;xy ¼ 2bk sinð2yÞ 2b siny c1;xy þ 2b siny c1;xyy
at y ¼ 0
ð44Þ
8
>
>
<
>
>
: ð44Þ Since the first order ψ1 of the stream function contains only terms in eiθ and e−iθ, as seen Since the first order ψ1 of the stream function contains only terms in eiθ and e−iθ, as seen in the
previous section, a rapid analysis of the above boundary conditions shows that the second
order ψ2 will only contain terms either constant or proportional to e2iθ and e−2iθ. We are inter-
ested here only in the constant term, which corresponds to the first non vanishing contribu-
tion to the steady flow field in the ε-expansion. From Eq 29, we know that the steady part of
ψ2, here called cðsÞ
2 , solves the following equation D2cðsÞ
2 ¼ 2a2 fc1;yDc1;x c1;xDc1;yg
ðsÞ
ð45Þ ð45Þ where the superscript ‘(s)’ on the right hand side stands for the steady part. c1ðx; y; tÞ ¼
X
þ1
n¼ 1
anðyÞ einðkxþtÞ
ð35Þ ð35Þ Due to the linearity of the PDE in Eq 34, each term of this Fourier series satisfies the same
equation, which implies: ða0000
n 2k2n2a00
n þ k4n4anÞ ina2ða00
n k2n2anÞ ¼ 0 :
ð36Þ ð36Þ this linear ODE is solved using its characteristic equation: this linear ODE is solved using its characteristic equation: d4 ð2k2n2 þ ina2Þd2 þ k2n2ðk2n2 þ ina2Þ ¼ 0
ð37Þ ð37Þ whose 4 solutions are: d ¼ kjnj
and
d ¼ gn
with gn ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
k2n2 þ ina2
p
ð38Þ ð38Þ The coefficients of the Fourier decomposition of ψ1 are determined by the boundary condi-
tions. In real experiments carried out on fluid flow next to ciliated edges [11], the value of k is
much larger than 1 (which means that the channel width along y is much larger than the meta-
chronal wavelength). This enables us to translate the boundary condition at y = 1 (the last two
of Eq 34) into the cancellation of the coefficients attached to the roots with positive real part in
Eq 37. Finally, since the only non homogeneous terms appearing in these boundary conditions
are in eiθ and e−iθ (sin θ and cos θ), we only retain the coefficients corresponding to n = 1 and
n = −1, which leads to the following expression for ψ1: c1ðx; y; tÞ ¼ ½A1e ky þ B1e g1y eiy þ ½A 1e ky þ B 1e g 1y e iy
ð39Þ ð39Þ Since g 1 ¼ g1, imposing that ψ1 is real valued implies that the coefficients also satisfy A 1 ¼ A1 9 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling vanishes and can be removed from the equation satisfied by cðsÞ
2 , leaving only the first paren-
thesis: cðsÞ
2;yyyy ¼ 2a2ðc1;yc1;yx c1;xc1;yyÞ
ðsÞ
y
ð47Þ ð47Þ cðsÞ
2;yyyy ¼ 2a2ðc1;yc1;yx c1;xc1;yyÞ
ðsÞ
y Integrating along y yields: cðsÞ
2;yyy ¼ 2a2ðc1;yc1;yx c1;xc1;yyÞ
ðsÞ þ 2A2
ð48Þ ð48Þ where 2A2 is an integration constant. Note that ε2/2 × 2A2 = ε2 A2 can actually be interpreted
as the steady component of the pressure gradient pðsÞ
x which appears in the right hand side of
the Navier-Stokes equation (Eq 15). The model therefore predicts a constant pressure gradient
along x. From the previous section, we know that ψ1 can be written as: c1ðx; y; tÞ ¼ a1ðyÞ eiy þ c:c:
with
a1ðyÞ ¼ A1e ky þ B1e g1y
ð49Þ ð49Þ Inserting this expression of ψ1 into the right hand side parenthesis of Eq 48 leaves: ðc1;yc1;yx c1;xc1;yyÞ
ðsÞ ¼ ikja0
1j
2 ika1a@
1 þ c:c: ¼ 2k Imða1a@
1Þ
ð50Þ ð50Þ (The first term of the above right hand side being imaginary, its contribution vanishes when
adding its complex conjugate). Eq 48 and the first boundary condition of Eq 44 thus provide
the following simpler system for cðsÞ
2 (the second boundary condition deals with x-derivatives
that vanish in the steady flow due to the translation invariance of the problem): cðsÞ
2;yyy ¼ 4a2k Imða1a00
1Þ þ 2A2
cðsÞ
2;yðx; 0Þ cðsÞ
2;yyðx; 0Þ ¼ k þ C
ð51Þ
8
<
: ð51Þ where C stands for the constant contribution of −2β sin θ (ψ1, yy −ϕ ψ1, yyy)|y = 0. More precisely, the
bracketed term can be rewritten as: c1;yDc1;x c1;xDc1;y
¼
c1;yðc1;xxx þ c1;xyyÞ c1;xðc1;xxy þ c1;yyyÞ
¼
ðc1;yc1;xyy c1;xc1;yyyÞ þ ðc1;yc1;xxx c1;xc1;xxyÞ
¼
ðc1;yc1;xy c1;xc1;yyÞy þ ðc1;yc1;xx c1;xc1;xyÞx
ð46Þ ð46Þ Let us examine the x-dependency of the parentheses appearing in the last line. All products
contain terms that are either constant or oscillating in x. Therefore the contribution of the sec-
ond parenthesis to the steady stream function can only be constant. Its x-derivative then PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 10 / 21 371/journal.pcbi.1005552
July 14, 2017
11 / 21 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling This expression is now inserted into the first equation of Eq 51, which gives after 2 integra-
tions: cðsÞ
2;y ¼ A2y2 þ B2y þ C2 4a4k Re
A1B1
e ðkþg1Þy
ðk þ g1Þ
2
! þ jB1j
2 e ðg1þg1Þy
ðg1 þ g1Þ
2
"
#
;
ð57Þ ð57Þ where B2 and C2 are integration constants. We observe here that the steady contribution to the
horizontal fluid velocity appears as the sum of one parabolic and two exponential profiles. The
parabolic profile depends on integration constants A2, B2, and C2 that are determined using
the boundaries conditions. Velocity and shear stress vanish at y = 1, so that the fluid in the
flowing layer matches the stagnant fluid above. The boundary conditions take the following
form: cðsÞ
2;yð1Þ
¼ 0 ¼ A2 þ B2 þ C2 þ G1
cðsÞ
2;yyð1Þ
¼ 0 ¼ 2A2 þ B2 þ G2
cðsÞ
2;yð0Þ cðsÞ
2;yyð0Þ
¼ k þ C ¼ C2 B2 þ G3 þ k þ C
ð58Þ
8
>
>
>
<
>
>
>
: ð58Þ where G1, G2, and G3 are defined such that: where G1, G2, and G3 are defined such that: where G1, G2, and G3 are defined such that: G1
4a4k
¼
Re
A1B1
e ðkþg1Þ
ðk þ g1Þ
2
! jB1j
2
e ðg1þg1Þ
ðg1 þ g1Þ
2
G2
4a4k
¼
Re
A1B1
e ðkþg1Þ
ðk þ g1Þ
þ jB1j
2 e ðg1þg1Þ
ðg1 þ g1Þ
G3 þ k þ C
4a4k
¼
Re
A1B1
ðk þ g1Þ
2
! This constant
C is determined using the expression of ψ1 in Eq 49: c1;yy c1;yyy ¼ ða00
1 a000
1 Þ eiy þ c:c:
ð52Þ ð52Þ Therefore the constant contribution C can be expressed from the constants A1 and B1 com-
puted in the previous section: C ¼ 2b Imða00
1ð0Þ a000
1 ð0ÞÞ ¼ 2b Imðk2A1 þ g2
1B1 þ k3 A1 þ g3
1 B1Þ
ð53Þ ð53Þ Using the values of A1 and B1 obtained in the previous section gives immediately: C ¼ 2b Im ik2
2
bg1
k 1
g1 k
0
B
@
1
C
A þ ig2
1
2
1 b
g1 k
2
64
3
75 ¼ bk þ bð1 bÞ Reðg1Þ
ð54Þ ð54Þ We now turn to Im(a1a00
1) in order to determine cðsÞ
2 . Using Eq 40, this term can be rewritten: Imða1a00
1Þ
¼
ImfðA1e ky þ B1e g1yÞðk2 A1e ky þ g2
1 B1e g1 yÞg
¼
Imfg2
1A1 B1e ðkþg1Þy þ k2 A1B1e ðkþg1Þy þ g2
1B1 B1e ðg1þg1Þyg
ð55Þ ð55Þ Using the relation g2
1 ¼ k2 ia2 allows us to simplify the above expression to: Imða1a00
1Þ
¼
Imf ia2A1 B1e ðkþg1Þy ia2jB1j
2e ðg1þg1Þyg
¼
a2½ReðA1 B1e ðkþg1ÞyÞ þ jB1j
2e ðg1þg1Þy
ð56Þ Imða1a00
1Þ
¼
Imf ia2A1 B1e ðkþg1Þy ia2jB1j
2e ðg1þg1Þyg
¼
a2½ReðA1 B1e ðkþg1ÞyÞ þ jB1j
2e ðg1þg1Þy
ð56Þ Imða1a00
1Þ
¼
Imf ia2A1 B1e ðkþg1Þy ia2jB1j
2e ðg1þg1Þyg
¼
a2½ReðA1 B1e ðkþg1ÞyÞ þ jB1j
2e ðg1þg1Þy
ð56Þ ð56Þ PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 11 / 21 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling and the coefficients of the parabolic profile are: and the coefficients of the parabolic profile are: A2 ¼ G3
1 þ 2
;
B2 ¼
2G3
1 þ 2
;
C2 ¼ G3
1 þ 2
ð61Þ ð61Þ This leads to the following steady flow: uðyÞ ¼ ε2
2
G3
1 þ 2 y2 þ
2G3
1 þ 2 y G3
1 þ 2
¼ ε2
2
G3
1 þ 2 ðy 1Þ
2
ð62Þ ð62Þ One recovers here a parabolic profile with vanishing velocity and vanishing shear stress at
y = 1. Moreover, from Eq 59, the term proportional to 4α4k in G3 can be neglected, leading to: G3 k C ¼ k bk bð1 bÞ Reðg1Þ ðb2 2b 1Þk
ð63Þ ð63Þ The dimensionless extrapolated velocity at the ciliated wall in this case is then: uð0Þ ¼ ε2
2
1 þ 2b b2
1 þ 2
k ¼ ε2
2
2 ðb 1Þ
2
1 þ 2
k
ð64Þ ð64Þ One can see here that the extrapolation of the flow velocity (hence of the microbead) at the cili-
ated wall depends on β, ϕ, and k. It has a maximum value, ε2k/(1 + 2ϕ), which is achieved for
β = 1, i.e., for a circular motion of the cilia tips. The case β = 0, together with a finite value of
ε = a/h corresponds to the limit of the flat horizontal motion of the cilia tips. The dimension-
less fluid velocity at the cilia wall in this situation is: uð0Þ ¼
ε2k
2ð1 þ 2 Þ
ð65Þ ð65Þ One would think that the limit case β = 0 would lead to a vanishing steady velocity of the fluid,
since all cilia undergo the same oscillatory motion, with a left/right symmetry. What we see
here is that the metachronal wave (antiplectic, i.e. going backwards for k > 0) breaks this sym-
metry and leads to a positive steady contribution to the fluid velocity. When k = 0 (no wave),
one recovers a pure oscillatory motion of the fluid, with no net fluid motion. Finally, the limit β ! +1 corresponds to a flat vertical motion of the cilia tips. Re
A1B1
ðk þ g1Þ
jB1j
2
ðg1 þ g1Þ
2
jB1j
2
ðg1 þ g1Þ
ð59Þ
8
>
>
>
>
>
>
>
>
>
>
<
>
>
>
>
>
>
>
>
>
>
: ð59Þ The coefficients of the steady parabolic profile are finally: The coefficients of the steady parabolic profile are finally: The coefficients of the steady parabolic profile are finally: A2
¼ G1 ð1 þ ÞG2 G3
1 þ 2
B2
¼ 2G1 þ G2 þ 2G3
1 þ 2
C2
¼ 2 G1 þ G2 G3
1 þ 2
ð60Þ
8
>
>
>
>
>
>
>
<
>
>
>
>
>
>
>
: ð60Þ In summary, the velocity field of the fluid flow can be expanded in powers of ε. The first non
vanishing order in ε for the oscillatory part of the fluid flow is of order 1 while at the second
order, a steady contribution appears. This steady flow is the one responsible for the micro-
bead motion observed in the experiments. It has essentially a parabolic profile whose coeffi-
cients are directly determined from the values of the quantities k, α, β, and ϕ, which are in turn
deduced from the fundamental parameters of the cilia motion and the surrounding fluid (fre-
quency, amplitude, density, viscosity). In the experiments, fitting the measured micro-bead
velocities with a parabolic profile will allow us to extract quantities that are not easily measur-
able, such as the cilium amplitude a or the transfer parameter ϕ. The steady velocity profile. In experiments carried out on real samples, the values of k
are larger than 10 (in most cases much larger), and α2 is about 0.1. We can therefore safely
assume that e−k 1 and α 1. In this situation, G1 and G2 are negligible compared to G3, PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 12 / 21 Modeling the micro-bead motion Once the oscillatory velocity field ~U ¼ ðu; vÞ is computed, the trajectories and the crossing
times of micro-beads in this field are calculated by solving their equation of motion: m d~U
b
dt ¼ ~F drag ;
ð67Þ ð67Þ where m is the individual mass of the micro-bead, ~U
b its velocity, and ~F drag is the drag force
applied on the bead by the fluid flow. Assuming a spherical shape for the bead and small speed
differences between the bead and the fluid, the drag force takes the Stokes expression ~F drag ¼ 6pRmð~U
b ~U Þ ;
ð68Þ ð68Þ where R stands for the bead diameter. Using the previously defined space and time units h and
ω−1, the adimensioned version of Eq 67 reads: d~U b
dt ¼
~U ~U b
Stk
with
Stk ¼ mo
6pRm
ð69Þ ð69Þ Stk is the bead Stokes number, which characterizes the effective inertia of the bead in the fluid
flow. For spherical particles of radius R and density ρb, this number also reads: Stk ¼
4
3 pR3rbo
6pRm
¼ 2
9
R2rbo
m
ð70Þ ð70Þ The micro-beads are about 4.5 μm diameter, and made out of polystyrene of density ρb of
order 1 g.cm−3. At 10 Hz in water, the corresponding Stokes number is about 10−4, which
means that the micro-beads can be considered as massless tracers. Their velocity can be
assumed to be permanently equal to the fluid velocity at the same location. For each micro-bead entering the simulation window at x = 0 and a given altitude y0, the
effective speed is computed as: Veffðy0Þ ¼
L
tðy0Þ ¼
L
H
T y0dt ¼
L
I
T y0
ds
k~UðsÞk
;
ð71Þ ð71Þ where τ(y0) is the crossing time of the micro-bead entering at (0, y0), and T y0 is the trajectory
followed by this micro-bead. During each elementary step of this trajectory, the infinitesimal
duration is dt ¼ ds=k~UðsÞk, ~U being the fluid velocity at curvilinear abscissa s of the trajectory. The effective speed Veff corresponds to the quantity measured in our experiments. where τ(y0) is the crossing time of the micro-bead entering at (0, y0), and T y0 is the trajectory
followed by this micro-bead. Characterizing micro-bead motion in a ciliary beating induced flow: Modeling Modeling the micro-bead motion During each elementary step of this trajectory, the infinitesimal
duration is dt ¼ ds=k~UðsÞk, ~U being the fluid velocity at curvilinear abscissa s of the trajectory. The effective speed Veff corresponds to the quantity measured in our experiments. Since
ε = a/h is the adimensioned horizontal beating amplitude, the limit has to be taken with
ε ! 0, βε remaining constant as the adimensioned vertical amplitude b/h. The horizontal
component of the steady velocity is then: uð0Þ ¼ lim
b!þ1
ε2
2
1 þ 2b b2
1 þ 2
k ¼ b2k
2h2ð1 þ 2 Þ
ð66Þ ð66Þ Interestingly, the metachronal wave also breaks the left/right symmetry in this case, but in the
opposite direction. In summary, the existence of an antiplectic metachronal wave triggers a net
fluid motion along Ox, in the positive direction for smaller values of β and in the opposite
direction for larger values of β, the critical value βc being obtained when (βc −1)2 = 2 (accord-
ing to Eq 64), i.e., for bc ¼ 1 þ
ffiffiffi
2
p
2:41. In realistic situations, the trajectories of cilia tips
are very flat, so that one is always in the case of small values of β: the fluid steady motion occurs
always in the direction opposite to the metachronal wave propagation. PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 13 / 21 Simulation of micro-bead motions The fluid velocity field is solved using the Fourier transform decomposition exposed earlier. This field is periodic both in space and time. Fig 3A and 3B displays the horizontal and vertical
components of the fluid velocity, respectively, while Fig 3D presents the micro-bead trajecto-
ries in this fluid, simulated by numerically solving Eq 69. One can observe that the beads follow
slightly wavy trajectories when close to the cilia wall, but that these trajectories become almost
perfect straight lines when moving away from the wall farther than a cilia length. This means
that the transit time of a micro-bead across the simulation window is dominated by the steady
part of the flow field. 14 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 15 / 21 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling Fig 4. Parabolic velocity profile. (A) Contribution of the different order of the micro-beads velocity The blue dashed line represent the effective velocity
(in μm.s-1). Green dashed line represent the contribution of the first order of the fluid velocity. The red dashed line represented the contribution of the
second order. Black points are the contribution of the parabolic term of the second order. Magenta points are the contribution of the exponential function. Ciliary beating parameters are the following: CBF = 10.0 Hz, CBA = 8.0 μm, λ = 10 μm, ϕ = 0 and h = 100 μm. (B) Examples of parabolic fitting on
microbead velocity measurements on 3 different ciliated edges (see companion paper [11]). htt
//d i
/10 1371/j
l
bi 1005552 004 Fig 4. Parabolic velocity profile. (A) Contribution of the different order of the micro-beads velocity The blue dashed line represent the effective velocity
(in μm.s-1). Green dashed line represent the contribution of the first order of the fluid velocity. The red dashed line represented the contribution of the
second order. Black points are the contribution of the parabolic term of the second order. Magenta points are the contribution of the exponential function. Ciliary beating parameters are the following: CBF = 10.0 Hz, CBA = 8.0 μm, λ = 10 μm, ϕ = 0 and h = 100 μm. (B) Examples of parabolic fitting on
microbead velocity measurements on 3 different ciliated edges (see companion paper [11]). Fig 4. Parabolic velocity profile. (A) Contribution of the different order of the micro-beads velocity The blue dashed line represent the effective velocity
(in μm.s-1). Green dashed line represent the contribution of the first order of the fluid velocity. The red dashed line represented the contribution of the
second order. Black points are the contribution of the parabolic term of the second order. Magenta points are the contribution of the exponential function. Ciliary beating parameters are the following: CBF = 10.0 Hz, CBA = 8.0 μm, λ = 10 μm, ϕ = 0 and h = 100 μm. (B) Examples of parabolic fitting on
microbead velocity measurements on 3 different ciliated edges (see companion paper [11]). https://doi.org/10.1371/journal.pcbi.1005552.g004 https://doi.org/10.1371/journal.pcbi.1005552.g004 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling Fig 3. Numerical simulation of the velocity field and micro-bead trajectories. (A) Color view of the horizontal velocity field. The dashed square
represents the zoomed-in area represented in Fig 3C. The beating parameters are: CBF = 10 Hz, CBA = 8 μm, λ = 10 μm, Φ = 0, h = 50 μm). (B) Color
view of the vertical velocity field. The dashed square represents the zoomed-in area represented in Fig 3C. (C) Zoomed in horizontal (top frame) and
vertical (bottom frame) velocity field. (D) Particle trajectories for several insertion points. The particles enter the fluid flow on the left side of the window
and travel to the right. htt
//d i
/10 1371/j
l
bi 1005552 003 Fig 3. Numerical simulation of the velocity field and micro-bead trajectories. (A) Color view of the horizontal velocity field. The dashed square
represents the zoomed-in area represented in Fig 3C. The beating parameters are: CBF = 10 Hz, CBA = 8 μm, λ = 10 μm, Φ = 0, h = 50 μm). (B) Color
view of the vertical velocity field. The dashed square represents the zoomed-in area represented in Fig 3C. (C) Zoomed in horizontal (top frame) and
vertical (bottom frame) velocity field. (D) Particle trajectories for several insertion points. The particles enter the fluid flow on the left side of the window
and travel to the right. https://doi.org/10.1371/journal.pcbi.1005552.g003 Fig 4A displays the various contributions to the flow profile in the y-direction for typical
values of the input parameters CBF, CBA, λ, ϕ, and h. One observes that the total bead velocity
(blue line) is essentially dominated by the steady parabolic contribution (black line) deter-
mined by the coefficients A2, B2, and C2 in Eq 57. The oscillatory part coming from the first
order in ε (green line) brings a significant contribution only very close to the ciliated edge, and
the exponential contribution in the steady part is negligible everywhere. Consequently, mea-
suring the bead velocity as function of the distance to the ciliated edge essentially amounts to
measuring the steady parabolic profile of the flow. Fie 4B shows the excellent agreement
between the model and the actual measurements performed on different ciliated edges (here 3
samples from 3 different subjects are presented). Discussion In human airways, the coordinated motion of the cilia covering the epithelium induces a com-
plex displacement of a double layer consisting in a bottom periciliary layer (PCL) and a top
mucus layer. Both layers have similar thicknesses, about 5 to 10 μm, but very different viscosi-
ties. The situation presented in this paper is however very different from the in vivo condition. It reproduces in fact the ex vivo experiments performed on a few ciliated cells obtained from
nasal brushing. In these experiments, clusters formed of a few ciliated cells are immersed in
water, and observed between two microscope slides by high speed video-microscopy. The cili-
ated edges are therefore lying horizontally in the simulation plane (Ox, Oy), which is perpen-
dicular to the optical axis Oz of the microscope. The first and foremost difference lies in the fact that the fluid surrounding the cilia is now
essentially water, i.e., a homogeneous Newtonian fluid. Secondly, unlike the in vivo situation
where mucus is propelled only by the forward trajectory of the cilia tips, here both forward
and backward parts of the stroke cycle contribute to the fluid motion. Consequently, as
expressed through the mathematical model developed here, the oscillatory motion of the cilia
induces at first order in the amplitude a an oscillatory motion of the fluid, and at second order
a net steady component that pushes the fluid in the direction of the cilia beating. The net
motion of the fluid originates from the phase shift between neighboring cilia tips, which breaks
the left/right symmetry. More precisely, it creates an unbalance on the envelope between the
respective densities of tips moving forward and those moving backwards. This unbalance
appears in the second term of Eq 9. One can also note that the direction of the net fluid motion
does not depend on the sense of rotation of the tips (clockwise or counterclockwise), but only
the direction of the metachronal wave: the fluid is propelled in the direction opposite to the
metachronal wave. Characterizing micro-bead motion in a ciliary beating induced flow: Modeling is only a way to assess of the ability of the ciliated edge to transfer momentum into the sur-
rounding fluid, thus defining a usable clinical index. Finally, the extrapolated velocity at the wall can be compared to the one predicted from
Eq 64: U0 ¼ ho uð0Þ ¼ ho a2
2h2
1 þ 2b b2
1 þ 2
2ph
l ¼ pa2o
l
1 þ 2b b2
1 þ 2
h
0
B
@
1
C
A
ð74Þ ð74Þ The cilium beating amplitude a (CBF), the the cilium beating frequency ω/2π (CBF), and the
metachronal wavelength λ are measured directly by microscopic measurements on the cilia,
while h is extracted from the parabolic fitting of the microbead velocity profile. In the approxi-
mation of a flat beating (β = 0), this set of measurements therefore provides a way to assess ϕ,
the equivalent sliding length introduced in the boundary condition of Eq 18. The ciliary beating efficiency index The horizontal component of the steady pressure gradient pðsÞ
x (see Eq 48), applies a force on
the fluid directed negatively along Ox. This force is exactly compensated by the force exerted
by the cilia on the fluid, proportional to the dimensionless shear stress @u/@y (see S1 File for
the detailed force balance between the cilia and the fluid). The dimensionless steady force
applied by the cilia to a volume fluid of length 2π/k (the dimensionless wavelength) in the Ox
direction and dimensionless thickness H/h in the Oz direction thus reads: FðsÞ
w ¼ H
h
2p
k
@uðsÞ
@y
ðy¼0Þ
¼ lH
h2
@uðsÞ
@y
ðy¼0Þ
ð72Þ ð72Þ Using the parabolic velocity profile u(s)(y) = u(0)(y −1)2 found in Eq 62, and putting back
dimensional quantities finally leads to the expression of the local shear stress τw applied to the
fluid by the cilia: Using the parabolic velocity profile u(s)(y) = u(0)(y −1)2 found in Eq 62, and putting back
dimensional quantities finally leads to the expression of the local shear stress τw applied to the
fluid by the cilia: tw ¼ ðmoh2ÞFðsÞ
w
lH
¼ moh2
lH
lH
h2 2uð0Þ ¼ 2mU0
h
ð73Þ ð73Þ where U0 is the velocity extrapolated at y = 0 from the measurements of microbead velocities
above the cilia. This shear stress characterizes the momentum transfer between cilia and the
surrounding fluid. Since h is also directly measured by fitting the microbead velocity profile
with a parabolic profile, it means that τw can be directly deduced using this microbead tracking
technique. Consequently, we propose this shear stress as an index for assessing the efficiency
of the ciliary beating. One has to stress that we do not intend to reproduce in this model the in
vivo condition. The shear stress measured using this micro-bead velocity technique is not
assumed to be identical to the shear stress experienced by mucus in the pulmonary airways. It PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 16 / 21 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling independent and additive steady and oscillatory contributions to the flow field, respectively. Regarding micro-bead velocity far from the ciliated edge, the system then behaves as an equiv-
alent stationary system with an effective wall velocity Uw = ε2k/2. At a distance larger than a fraction of the metachronal wavelength from the ciliated edge
(the wavelength λ is of order 10–20 μm in all experiments, see [11]), the oscillatory behavior of
the flow field vanishes and only the net steady horizontal flow remains. The microbead track-
ing technique therefore probes this steady part of the flow field, which exhibits a parabolic pro-
file along y. The steady contribution to the dimensionless horizontal wall velocity is ε2k/2 (see
Eq 11). In the case of flat beating (β = 0) and no slip boundary condition at the cilia wall
(ϕ = 0), this value is exactly the steady fluid velocity at the wall expressed in Eq 65. As a conse-
quence, it also corresponds to the extrapolated micro-bead velocity at the wall. Our model
shows that the synchronized elliptic motion of the cilia generates a correction to this velocity
by a factor (1 + 2β −β2) (Eq 64). In addition, an imperfect momentum transfer between the
cilia and the fluid (ϕ > 0) reduces this velocity. Introducing a partial slip at the cilia wall, through a Robin type boundary condition with an
effective sliding length ϕ = hϕ (Eq 18), reduces the fluid velocity by a factor (1 + 2ϕ), a result
that is also obtained in a purely steady model for a parabolic profile. As shown in the compan-
ion paper [11], this effective sliding length is directly correlated to the average density of cilia. This is very consistent with the use of this type of boundary condition in fluid flow next to
porous flow, the pore openings corresponding to the empty space between cilia. In clinical
application of this model, it is expected that ϕ will not be a fitting parameter but predicted by
the mere measurement of the cilia density. Discussion This property due to the antiplectic nature of the wave might have an inter-
esting consequence: Although the forcing mechanism on the fluid is very different in the pres-
ence of mucus (the tips of the cilia enter the mucus layer and push it forward), the fluid
covering the cilia would still be pushed in the same direction if the mucus viscosity was signifi-
cantly lowered or if the mucus would disappear. In the ex vivo experiments, the Womersley number α is close to 0.4 while the dimensionless
wave vector k = 2πh/λ is at least about 10 and most of the time much larger than that (h is com-
prised between 30 μm and 140 μm in the experiments, see [11]). This means that the contribu-
tion of the convective acceleration to momentum conservation is very small in all cases, and
that in first approximation, the system can be understood using a linear Stokes equation point
of view. The steady and oscillatory contributions to the wall velocity decouple and induce PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 17 / 21 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling In the model, the ciliated edge is supposed to be flat. This assumption is generally valid in
real experiments (see [11]). Cases in which cell clusters appear to have a rough, curved, or dis-
rupted ciliated edge are removed from the measurement procedure. In the model, the ciliated edge is supposed to be flat. This assumption is generally valid in
real experiments (see [11]). Cases in which cell clusters appear to have a rough, curved, or dis-
rupted ciliated edge are removed from the measurement procedure. An important assumption lies in the fact that the fluid surrounding the cilia is stagnant at a
distance h above the edge. The stagnation of the fluid above the cilia is observed experimen-
tally, and originates from the external environment of the measured cell cluster. Various expla-
nations can account for it: it can be due to the presence of other cell clusters at a few hundred
microns distance which create a complex fluid flow in the entire system, or by the friction of
the fluid on the upper and lower slides, or the appearance of boundary layers. Determining
this value would require modeling the full 3D geometry of the system whose geometry is not
easily accessible by microscopic observation. The distance h therefore is treated as the only fit-
ting parameter of the model (the sliding length ϕ being calculated from the measured cilia
density). Finally, the bead velocities are found in the model to be almost always parallel to the cilia
wall. In real experiments, a vertical component (i.e., in the direction perpendicular to the
wall) might appear, although much smaller than the horizontal component. This is in partic-
ular the case when the ciliated edge is not strictly flat, or when the observation window is
such that the ciliated edge is on one side of the window and the bead passing on the other
side of the same window. The micro-beads are in this situation not set into motion only by
the considered ciliated edge, and external influence originating from outside the observation
window interfere, contributing to create a more complex fluid flow pattern. Such situations
should be discarded from the analysis, either through eye examination or by an automatized
procedure. Model limitations The model proposed in this article relies on several assumptions, hence has a few limitations
that we examine now. First, the motion of each individual cilium tip is assumed to be elliptic. Although this is a
generally accepted hypothesis, microscopic imaging of the actual motion of the cilium shows a
more complicated trajectory [34]. In particular, the path followed by the tip on the backward
trajectory seems to be closer to the forward path than for an elliptic motion. However, we have
seen that the net steady motion of the fluid is essentially generated by the metachronal wave. This discrepancy of the trajectory should be accounted for through a change of the parameter
β representing the ellipse eccentricity. One also has to stress that, since our model reproduces
an experimental setup in which cilia are surrounded only by water, one might expect a differ-
ent motion from the one achieved in in vivo situations where cilia are beating in a periciliary
liquid while their tips are entering the bottom of the mucus layer. The model also assumes an exact synchronization of the individual cilia motions, generat-
ing a metachronal wave of constant velocity. This implies that the phase shift between two cilia
is a linear function of the distance separating them along the direction of the metachronal
wave propagation. Ex vivo experiments carried out in [11] show that this hypothesis is satisfac-
torily verified at the observational scale of the experiment, i.e., an edge of a cluster containing a
few ciliated cells. A deviation from the linear phase shift would disrupt the translational peri-
odicity of the system and alter our solution based on a Fourier decomposition along the hori-
zontal axis. The fluid velocity at the cilia wall computed in our model can thus be considered
as an “ideal velocity”, reached for a perfect metachronal wave. Any measured discrepancy with
respect to this ideal velocity could be therefore be attributed either to an imperfect momentum
transfer (through the effective slip length ϕ), or to a perturbed metachronal wave. PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 18 / 21 Author Contributions Conceptualization: Mathieu Bottier, Marta Peña Ferna´ndez, James B. Grotberg, Marcel
Filoche. Conceptualization: Mathieu Bottier, Marta Peña Ferna´ndez, James B. Grotberg, Marcel
Filoche. Formal analysis: Mathieu Bottier, Marta Peña Ferna´ndez, Bruno Louis, James B. Grotberg,
Marcel Filoche. Funding acquisition: Daniel Isabey, Bruno Louis. Investigation: Mathieu Bottier, Marta Peña Ferna´ndez, Gabriel Pelle, Daniel Isabey, Bruno
Louis, James B. Grotberg, Marcel Filoche. Methodology: Mathieu Bottier, Gabriel Pelle, James B. Grotberg, Marcel Filoche. Project administration: Bruno Louis, Marcel Filoche. Software: Mathieu Bottier, Marta Peña Ferna´ndez, Gabriel Pelle, Bruno Louis, James B. Grot-
berg, Marcel Filoche. Supervision: Daniel Isabey, Bruno Louis, James B. Grotberg, Marcel Filoche. Validation: Mathieu Bottier, Gabriel Pelle, Bruno Louis, James B. Grotberg, Marcel Filoche. Visualization: Mathieu Bottier, Marta Peña Ferna´ndez, Gabriel Pelle, Bruno Louis, James B. Grotberg, Marcel Filoche. Writing – original draft: Mathieu Bottier, Bruno Louis, James B. Grotberg, Marcel Filoche. Writing – review & editing: Mathieu Bottier, Gabriel Pelle, Daniel Isabey, Bruno Louis, James
B. Grotberg, Marcel Filoche. Supporting information S1 File. Force balance on a fluid volume. Derivation of shear stress exerted by the cilia
motion onto the surrounding fluid. (PDF) Characterizing micro-bead motion in a ciliary beating induced flow: Modeling References 1. Fulford GR, Blake JR. Muco-ciliary Transport in the Lung. J Theor Biol. 1986; 121:381–402. https://doi. org/10.1016/S0022-5193(86)80098-4 PMID: 3796001
2. Wanner A, Salathe M, O’Riordan TG. Mucociliary clearance in the airways. Am J Respir Crit Care Med. 1996; 154:1868–1902. https://doi.org/10.1164/ajrccm.154.6.8970383 PMID: 8970383
3. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian
airways. J Clin Invest. 2002; 109:571–577. https://doi.org/10.1172/JCI15217 PMID: 11877463
4. Fahy JV, Dickey BF. Airway mucus function and dysfunction. N Engl J Med. 2010; 363(23):2233–47. https://doi.org/10.1056/NEJMra0910061 PMID: 21121836
5. Sanderson MJ, Sleigh MA. Ciliary activity of cultured rabbit tracheal epithelium: beat pattern and meta-
chrony. J Cell Sci. 1981; 47:331–47. PMID: 7263784
6. Sleigh MA, Blake JR, Liron N. The propulsion of mucus by cilia. Am Rev Respir Dis. 1988; 137
(13):726–41. https://doi.org/10.1164/ajrccm/137.3.726 PMID: 3278666
7. Gheber L, Priel Z. On metachronism in ciliary systems: a model describing the dependence of the meta
chronal wave properties on the intrinsic ciliary parameters. Cell Motil Cytoskeleton. 1990; 16(3):
167–81. https://doi.org/10.1002/cm.970160304 PMID: 2364445
8. Gheber L, Priel Z. Extraction of cilium beat parameters by the combined application of photoelectric
measurements and computer simulation. Biophys J. 1997; 72:449–462. https://doi.org/10.1016/S0006-
3495(97)78686-7 PMID: 8994632 1. Fulford GR, Blake JR. Muco-ciliary Transport in the Lung. J Theor Biol. 1986; 121:381–402. https://doi. org/10.1016/S0022-5193(86)80098-4 PMID: 3796001 2. Wanner A, Salathe M, O’Riordan TG. Mucociliary clearance in the airways. Am J Respir Crit Care Med. 1996; 154:1868–1902. https://doi.org/10.1164/ajrccm.154.6.8970383 PMID: 8970383 3. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian
airways. J Clin Invest. 2002; 109:571–577. https://doi.org/10.1172/JCI15217 PMID: 11877463 4. Fahy JV, Dickey BF. Airway mucus function and dysfunction. N Engl J Med. 2010; 363(23):2233–47. https://doi.org/10.1056/NEJMra0910061 PMID: 21121836 5. Sanderson MJ, Sleigh MA. Ciliary activity of cultured rabbit tracheal epithelium: beat pattern and meta-
chrony. J Cell Sci. 1981; 47:331–47. PMID: 7263784 6. Sleigh MA, Blake JR, Liron N. The propulsion of mucus by cilia. Am Rev Respir Dis. 1988; 137
(13):726–41. https://doi.org/10.1164/ajrccm/137.3.726 PMID: 3278666 7. Gheber L, Priel Z. On metachronism in ciliary systems: a model describing the dependence of the meta-
chronal wave properties on the intrinsic ciliary parameters. Cell Motil Cytoskeleton. 1990; 16(3):
167–81. https://doi.org/10.1002/cm.970160304 PMID: 2364445 8. Gheber L, Priel Z. Extraction of cilium beat parameters by the combined application of photoelectric
measurements and computer simulation. Biophys J. 1997; 72:449–462. Conclusion We have developed a mathematical model of micro-bead velocity in a fluid set into motion by
the periodic beating of a ciliated edge. The cilia wall is represented as a continuous envelope
whose motion is calculated from the coordinated movement of all cilia. The boundary condi-
tion imposed by the effective wall induces a motion of the fluid above the wall. This motion
has two components: one oscillatory, at the spatial and temporal periodicity of the metachro-
nal wave and the cilia beating, respectively, and one steady, oriented along the direction of the
wall. The oscillatory component vanishes at a distance of the order of the metachronal wave-
length. The steady component extends on a much longer distance and exhibits a parabolic pro-
file in the direction perpendicular to the wall. The parameters governing this profile are the
fluid viscosity, the ciliary beating frequency (CBF), the ciliary beating amplitude (CBA), the
metachronal wavelength (λ), the cilia density (through the sliding length ϕ), and the distance
from the wall to the region of stagnant fluid (h). Polystyrene micro-beads immersed in the
fluid act as massless tracers, allowing the measurement of the local fluid velocity, hence the
measurement of the velocity profile. All aforementioned parameters can be determined
through local measurement by high speed video-microscopy, except for the distance h which
remains the only fitting parameter of the model extracted from the velocity profile. The veloc-
ity extrapolated from the profile at the wall is found to be linearly related to the shear stress
exerted by the cilia on the fluid. This shear stress is proposed as a new index for assessing the
efficiency of the ciliary beating. One has to stress that this index can be measured from nasal
brushing in the clinical setting without any modification of the current clinical procedures. Preliminary tests (see [11]) have already shown that this index has the potential to be a power-
ful screening test, able to distinguish patients suffering from various alterations of the cilia
beating such Primary Ciliary Dyskinesia (PCD). 19 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 Characterizing micro-bead motion in a ciliary beating induced flow: Modeling 9. Hill DB, Swaminathan V, Estes A, Cribb J, O’Brien ET, Davis CW, et al. Force Generation and Dynam-
ics of Individual Cilia under External Loading. Biophys J. 2010; 98:57–66. https://doi.org/10.1016/j.bpj. 2009.09.048 PMID: 20085719 10. Salathe M. Regulation of mammalian ciliary beating. Annu Rev Physiol. 2007; 69:401–22. https://doi. org/10.1146/annurev.physiol.69.040705.141253 PMID: 16945069 11. Bottier M, Blanchon S, Pelle G, Bequignon E, Isabey D, Coste A, et al. A New Index for Characterizing
Micro-bead Motion in a Flow Induced by Ciliary Beating: part I, Experimental Analysis. PLoS Comput
Biol. 2017. 12. Smith DJ, Gaffney EA, Blake JR. Modelling mucociliary clearance. Resp Physiol Neurobi. 2008; 163:
178–188. https://doi.org/10.1016/j.resp.2008.03.006 13. Blake J. A model for the micro-structure in ciliated organisms. J Fluid Mech. 1972; 55. https://doi.org/10. 1017/S0022112072001612 14. Lauga E, Powers TR. The hydrodynamics of swimming microorganisms. Rep Prog Phys. 2009; 72(9):
096601. https://doi.org/10.1088/0034-4885/72/9/096601 15. Lee WL, Jayathilake PG, Tan Z, Le DV, Lee HP, Khoo BC. Muco-ciliary transport: Effect of mucus vis-
cosity, cilia beat frequency and cilia density. Comput Fluid. 2011; 49(1):214–221. https://doi.org/10. 1016/j.compfluid.2011.05.016 16. Mitran SM. Metachronal wave formation in a model of pulmonary cilia. Comput Struct. 2007; 85:
763–774. https://doi.org/10.1016/j.compstruc.2007.01.015 PMID: 19169426 17. Yang X, Dillon RH, Fauci LJ. An Integrative Computational Model of Multiciliary Beating. Bull Math Biol. 2008; 70(4):1192–1215. https://doi.org/10.1007/s11538-008-9296-3 PMID: 18236120 18. Keller SR. Fluid mechanical investigations of ciliary propulsion. California Institute of Technology; 1975. 19. Blake JR, Winet H. On the mechanics of muco-ciliary transport. Biorheology. 1980; 17:125–34. PMID:
7407342 20. Smith DJ, Gaffney EA, Blake JR. Discrete Cilia Modelling with Singularity Distributions: Application to
the Embryonic Node and the Airway Surface Liquid. Bull Math Biol. 2007; 69(5):1477–1510. https://doi. org/10.1007/s11538-006-9172-y PMID: 17473955 21. Childress S. Mechanics of Swimming and Flying. Cambridge University Press; 1981. Cambridge
Books 22. Ross SM, Corrsin S. Results of an analytical model of mucociliary pumping. J Appl Physiol. 1974;
34(3):333–40. 23. Velez-Cordero JR, Lauga E. Waving transport and propulsion in a generalized Newtonian fluid. J Non-
Newton Fluid. 2013; 199:37–50. https://doi.org/10.1016/j.jnnfm.2013.05.006 24. Shen XN, Arratia PE. Undulatory Swimming in Viscoelastic Fluids. Phys Rev Lett. 2011; 106:208101. https://doi.org/10.1103/PhysRevLett.106.208101 PMID: 21668264 25. Liu B, Powers TR, Breuer KS. Force-free swimming of a model helical flagellum in viscoelastic fluids. Proc
Natl Acad Sci USA. 2011; 108:19516–19520. https://doi.org/10.1073/pnas.1113082108 PMID: 22106263 26. Dasgupta M, Liu B, Fu HC, Berhanu M, Breuer KS, Powers TR, et al. References https://doi.org/10.1016/S0006-
3495(97)78686-7 PMID: 8994632 20 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
July 14, 2017 Speed of a swimming sheet in
Newtonian and viscoelastic fluids. Phys Rev E. 2013; 87:013015. https://doi.org/10.1103/PhysRevE. 87.013015 27. Lauga E. Propulsion in a viscoelastic fluid. Phys Fluids. 2007; 19. https://doi.org/10.1063/1.2751388 28. Fu H, Wolgemuth W, Powers TR. Swimming speeds of filaments in nonlinearly viscoelastic fluids. Phys
Fluids. 2009; 21. https://doi.org/10.1063/1.3086320 29. Teran J, Fauci L, Shelley M. Viscoelastic Fluid Response Can Increase the Speed and Efficiency of a
Free Swimmer. Phys Rev Lett. 2010; 104:038101. https://doi.org/10.1103/PhysRevLett.104.038101
PMID: 20366685 30. Zhu L, Do-Quang M, Lauga E, Brandt L. Locomotion by tangential deformation in a polymeric fluid. Phys Rev E. 2011; 83:011901. https://doi.org/10.1103/PhysRevE.83.011901 31. Zhu L, Lauga E, Brandt L. Self-propulsion in viscoelastic fluids: Pushers vs. pullers. Phys Fluids. 2012;
24(5). https://doi.org/10.1063/1.4718446 32. Beavers GS, Joseph DD. Boundary conditions at a naturally permeable wall. J Fluid Mech. 1967;
30(1):197–207. https://doi.org/10.1017/S0022112067001375 33. Verma VS, Tripathee SM. A planar model for muco-ciliary transport in the human lung: effects of mucus
visco-elasticity, cilia beating and porosity. IJMMPS. 2013; 1(1). 34. Chilvers MA, O’Callaghan C. Analysis of ciliary beat pattern and beat frequency using digital high speed
imaging: comparison with the photomultiplier and photodiode methods. Thorax. 2000; 55:314–317. https://doi.org/10.1136/thorax.55.4.314 PMID: 10722772 21 / 21 PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1005552
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https://figshare.com/articles/journal_contribution/Supplementary_Figure_6_from_MAPK_Reliance_via_Acquired_CDK4_6_Inhibitor_Resistance_in_Cancer/22469687/1/files/39921176.pdf
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Supplementary Figure 5 from MAPK Reliance via Acquired CDK4/6 Inhibitor Resistance in Cancer
| null | 2,023
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cc-by
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Supplementary Figure 6: Acquired CDK4/6i resistance leads to dependence on MAPK signaling. A. LAPC4 cells were treated 24h with 0.5 or 1µM MEK
inhibitor (U0126) or CTRL and immunoblotted for p-ERK1/2, demonstrating loss of the hyperphosphorylation of ERK1/2 observed in the LNCaP PDR models when
MEK is inhibited, confirming that the MAPK pathway is activated in the PDR models. MEK inhibition does not affect RB hyperphosphorylation (upper band tRB). B.
LAPC4 PDR cells are sensitized to MEK inhibition, as analyzed via Trypan blue exclusion after 13 days treatment with 0.5 or 1µM U0126 or CTRL, whereas the
parental cells are less responsive, indicating that the PDR cells have become more reliant on the MAPK activation. 0
0.2
0.4
0.6
0.8
1
1.2
LAPC4
L4-PDR1
Rel. cell viability
Cell viability
CTRL
0.5uM U0126
1uM U0126
tRB =
LaminB -
A
*
pERK1/2 -
Vinculin -
0.5µM PD - + - - - - -
µM U0126 - - 0.5 1 - 0.5 1
LAPC4
L4-PDR1
B
Supplementary Figure 6: Acquired CDK4/6i resistance leads to dependence on MAPK signaling. A. LAPC4 cells were treated 24h with 0.5 or 1
inhibitor (U0126) or CTRL and immunoblotted for p-ERK1/2, demonstrating loss of the hyperphosphorylation of ERK1/2 observed in the LNCaP PDR 0
0.2
0.4
0.6
0.8
1
1.2
LAPC4
L4-PDR1
Rel. cell viability
Cell viability
CTRL
0.5uM U0126
1uM U0126
tRB =
LaminB -
A
*
pERK1/2 -
Vinculin -
0.5µM PD - + - - - - -
µM U0126 - - 0.5 1 - 0.5 1
LAPC4
L4-PDR1
B
Supplementary Figure 6: Acquired CDK4/6i resistance leads to dependence on MAPK signaling. A. LAPC4 cells were treated 24h with 0.5 or 1
inhibitor (U0126) or CTRL and immunoblotted for p-ERK1/2, demonstrating loss of the hyperphosphorylation of ERK1/2 observed in the LNCaP PDR 0
0.2
0.4
0.6
0.8
1
1.2
LAPC4
L4-PDR1
Rel. cell viability
Cell viability
CTRL
0.5uM U0126
1uM U0126
tRB =
LaminB -
A
*
pERK1/2 -
Vinculin -
0.5µM PD - + - - - - -
µM U0126 - - 0.5 1 - 0.5 1
LAPC4
L4-PDR1
B 0
0.2
0.4
0.6
0.8
1
1.2
LAPC4
L4-PDR1
Rel. cell viability
Cell viability
CTRL
0.5uM U0126
1uM U0126
tRB =
LaminB -
A
*
pERK1/2 -
Vinculin -
0.5µM PD - + - - - - -
µM U0126 - - 0.5 1 - 0.5 1
LAPC4
L4-PDR1
B tRB =
LaminB -
A
pERK1/2 -
Vinculin -
0.5µM PD - + - - - - -
µM U0126 - - 0.5 1 - 0.5 1
LAPC4
L4-PDR1 0
0.2
0.4
0.6
0.8
1
1.2
LAPC4
L4-PDR1
Rel. cell viability
Cell viability
CTRL
0.5uM U0126
1uM U0126
*
B B A Supplementary Figure 6: Acquired CDK4/6i resistance leads to dependence on MAPK signaling. A. LAPC4 cells were treated 24h with 0.5 or 1µM MEK
inhibitor (U0126) or CTRL and immunoblotted for p-ERK1/2, demonstrating loss of the hyperphosphorylation of ERK1/2 observed in the LNCaP PDR models when
MEK is inhibited, confirming that the MAPK pathway is activated in the PDR models. MEK inhibition does not affect RB hyperphosphorylation (upper band tRB). B. LAPC4 PDR cells are sensitized to MEK inhibition, as analyzed via Trypan blue exclusion after 13 days treatment with 0.5 or 1µM U0126 or CTRL, whereas the
parental cells are less responsive, indicating that the PDR cells have become more reliant on the MAPK activation.
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https://cancerci.biomedcentral.com/counter/pdf/10.1186/s12935-023-03027-0
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Spatially resolved visualization of reprogrammed metabolism in hepatocellular carcinoma by mass spectrometry imaging
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Cancer cell international
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Cancer Cell International
Ma et al. Cancer Cell International
(2023) 23:177
https://doi.org/10.1186/s12935-023-03027-0
Open Access
RESEARCH
Spatially resolved visualization
of reprogrammed metabolism
in hepatocellular carcinoma by mass
spectrometry imaging
Bangzhen Ma1, Yang Zhang2, Jiwei Ma2, Xinguo Chen1,2, Chenglong Sun3,4*
and Chengkun Qin1*
Abstract
Background Metabolic reprogramming refers to tumor-associated metabolic alterations during tumorigenesis
and has been regarded as one of the most important features of cancer. Profiling the altered metabolites and lipids
in hepatocellular carcinoma with spatial signature will not only enhance our understanding of tumor metabolic
reprogramming, but also offer potential metabolic liabilities that might be exploited for hepatocellular carcinoma
therapy.
Methods We perform matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) analysis
on both hepatocellular carcinoma xenograft mouse model and hepatocellular carcinoma patients. Discriminatory
metabolites that altered during the development of hepatocellular carcinoma are screened and imaged in xenograft
mouse model and are further validated in 21 hepatocellular carcinoma patients.
Results We discover stepwise metabolic alterations and progressively increasing metabolic heterogeneity during
the growth of hepatocellular carcinoma. Arginine and its metabolites spermine and spermidine, choline and
phosphatidylcholine metabolism, and fatty acids were found to be significantly reprogrammed in hepatocellular
carcinoma tissues.
Conclusions The spatially resolved profiling of the metabolites and lipids in highly heterogeneous hepatocellular
carcinoma tissue will contribute to obtaining precise metabolic information for the understanding of tumor
metabolic reprogramming.
Keywords Hepatocellular carcinoma, MALDI-MS, Spatially resolved imaging, Metabolites, lipids
*Correspondence:
Chenglong Sun
chenglongsun1989@163.com
Chengkun Qin
qinchengkun_slyy@163.com
1
Shandong Provincial Hospital, Shandong University, Jinan 250021, China
2
Shandong Provincial Hospital Affiliated to Shandong First Medical
University, Jinan 250021, China
3
School of Pharmaceutical Sciences, Qilu University of Technology
(Shandong Academy of Sciences), Jinan 250014, China
4
Key Laboratory for Applied Technology of Sophisticated Analytical
Instruments of Shandong Province, Shandong Analysis and Test Center,
Qilu University of Technology (Shandong Academy of Sciences),
Jinan 250014, China
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
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in this article, unless otherwise stated in a credit line to the data.
Ma et al. Cancer Cell International
(2023) 23:177
Background
Hepatocellular carcinoma is one of the most common
malignant tumors worldwide, ranking sixth in incidence
among malignant tumors and third in cause of death
from tumors [1]. Reprogrammed metabolism is regarded
as an important feature of tumors, which helps to elucidate the mechanism of its occurrence and provides
potential targets for clinical treatment [2–4]. Disturbed
energy metabolism in cancer cells can alter many biologically relevant metabolic pathways, such as cell proliferation and regulation, and as a common feature of
all cancer cells, altered metabolism has always been an
important direction of cancer research [5, 6].
Performing metabolomic studies on tumor tissues can
not only characterize the metabolic signature of tumors,
but also help to identify potential metabolic markers
associated with tumor initiation, progression, and metastasis [7]. Recently, researchers have successfully carried
out liquid chromatography-mass spectrometry (LC-MS)
based metabolomic analysis on hepatocellular carcinoma
tissues and have made significant progress in profiling
the metabolic signatures of hepatocellular carcinoma
[8, 9]. For example, Xu’s group performed LC-MS based
nontargeted tissue metabolomics analysis on fifty pairs
of hepatocellular carcinoma samples and matched normal tissues, and found that the glycolysis, gluconeogenesis, and β-oxidation were upregulated and tricarboxylic
acid cycle and Δ-12 desaturase were downregulated in
hepatocellular carcinoma [10]. Ferrarini et al. explored
the metabolomic characteristics of tumor and nontumor
tissues from 40 hepatocellular carcinoma patients using
LC-MS and gas chromatography (GC)-MS platforms. A
total of 18 metabolites in tricarboxylic acid (TCA) cycle,
glycolysis, purines, and lipid metabolism pathways were
screened as key molecules related to the development of
hepatocellular carcinoma [8]. Liu et al. discovered that
the levels of DL-3-phenyllactic acid, L-tryptophan, glycocholic acid and 1-methylnicotinamide in hepatocellular carcinoma tissues were significantly higher than those
in healthy controls by performing LC-MS based untargeted metabolomic profiling [11]. However, it should
be noted that hepatocellular carcinoma tumor tissue is
Page 2 of 10
highly heterogeneous, which means that the distributions
of metabolites in hepatocellular carcinoma tissue is also
heterogeneous. Traditional metabolomics studies carried out based on LC-MS technique requires experimental processes such as tissue homogenization, metabolite
extraction, and chromatographic separation, in which,
unfortunately, information on the spatial distributions of
metabolites in heterogeneous tumor tissues is completely
destroyed.
Mass spectrometry imaging (MSI) allows in situ analysis of metabolites in tissue sections without disrupting
their spatial distribution characteristics [12–18]. Matrixassisted laser desorption/ionization-mass spectrometry
imaging (MALDI-MSI) and desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) are currently the two most commonly used mass spectrometry
imaging techniques for the visualization of metabolites in
biological tissues [19–24]. By performing MSI analysis on
highly heterogeneous tumor tissues, the researchers successfully screened tumor in situ diagnostic markers and
identified potential therapeutic targets through metabolic pathway analysis, paving way for deeper understanding of tumor molecular characteristics and better
tumor therapy [25–31].
In this study, we conduct MALDI-MSI analysis on the
tumor tissues of hepatocellular carcinoma xenograft
mouse model and hepatocellular carcinoma patients.
Discriminatory metabolites that altered during the
growth and progression of hepatocellular carcinoma
were first screened and imaged in xenograft mouse
model and then were further validated in postoperative
human hepatocellular carcinoma tissues. The design of
this work is illustrated in Fig. 1.
Methods
Chemicals and reagents
1,5-Diaminonaphthalene (1,5-DAN) was purchased
from Shanghai Aladdin Biochemical Technology Co.,
Ltd (Shanghai, China). HPLC-grade acetonitrile (ACN)
and methanol (MeOH) were afforded by Merck (Darmstadt, Germany). O.C.T. embedding agent was provided
by Sakura Finetek Japan Co., Ltd. (Tokyo, Japan). The
Fig. 1 Schematic illustrations for spatially resolved visualization of reprogrammed metabolism in hepatocellular carcinoma by mass spectrometry
imaging
Ma et al. Cancer Cell International
(2023) 23:177
indium tin oxide (ITO)-coated slides were purchased
from Bruker (Daltonics, Billerica, MA). The SUPERFROST PLUS slides were provided by Thermo Fisher
Scientific (Waltham, MA, USA). Experimental water was
provided by Wahaha Co., Ltd. (Hangzhou, China).
Cell culture
A human hepatocellular carcinoma cell line (HepG2
cell) was purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The cell culture medium was
MEM with 10% fetal bovine serum (FBS), streptomycin
(100 µg/mL), and penicillin (100 µg/mL). Cells were cultured at 37 °C, 5% CO2 atmosphere.
Establishment of in vivo experimental animal models
The human hepatocellular carcinoma cell line HepG2
was stored in liquid nitrogen until use. The healthy male
BALB/c nude mice (4 weeks old) were purchased from
Spelford Biotechnology Co., Ltd (Beijing, China) and
housed in the animal house of the Provincial Hospital of
Shandong University. All animal husbandry and experiments were performed in accordance with the policy
guidelines established by the Institutional Animal Husbandry and Use Committee of Shandong University Provincial Hospital. The human hepatocellular carcinoma
cell line HepG2 was cultured in cell culture flasks, and
after routine recovery, HepG2 cells were centrifuged,
washed twice with PBS, and resuspended in PBS. 100
µL of cell suspension, with a total cell count of approximately 1 × 106, was injected into the subcutaneous space
of the foreleg to establish an ectopic transplantation
tumor model.
Nude mice were randomly divided into three groups
of three mice each when the subcutaneous tumor volume grew to 100 mm3. The nude mice were housed in
the SPF environment of the Animal Experiment Center
of the Provincial Hospital of Shandong University, and
the subcutaneous tumor sampling of one group of nude
mice was per-formed every 7 days. The anesthetic drug
aphrodisiac, 280 mg/kg, was administered intraperitoneally once before sampling, and all mice were anesthetized and their tumor specimens were taken. All mice
were anesthetized and their tumor specimens were taken.
After sampling, the mice were killed by inhalation of carbon dioxide. The nude mice were disposed of according
to laboratory regulations. The subcutaneous tumors were
stored in a -80 refrigerator for freezing and then analyzed
by MALDI mass spectrometry. The animal experiment
was conducted at Laboratory Animal Research Center of
Shandong Provincial Hospital with approval by the Animal Care and Use Committee of Shandong Provincial
Hospital (No. 2022-040).
Page 3 of 10
Collection of human hepatocellular carcinoma tissue
sample
This study was performed according to the Declaration
of Helsinki and Good Clinical Practice and approved by
the Ethnical Committee of Shandong Provincial Hospital
(No. 2022-069). This study included hepatocellular carcinoma tissue from 21 patients who underwent surgery.
The post-operative hepatocellular carcinoma tissues were
immediately placed in dry ice and then transferred to a
-80 °C refrigerator.
Preparation of tissue section
Human and mouse hepatocellular carcinoma tissues were
embedded in Tissue-Tek O.C.T. agent, and then were cut
into continuous twelve-micron tissue sections using a
cryostat microtome (CryoStar NX50 NOVPD, Thermo,
Bremen, Germany). For each hepatocellular carcinoma
tissue, one of the hepatocellular carcinoma frozen tissue sections was subjected to H&E staining, and two of
the hepatocellular carcinoma frozen tissue sections were
subjected to MALDI mass spectrometry imaging analysis
in positive and negative ion modes, respectively.
Matrix coating
1,5-DAN was selected as the MALDI matrix for hepatocellular carcinoma analysis. 1,5-DAN (2.5 mg/mL) in
ACN/H2O (70:30, v/v) was sprayed onto the tissue sections by a HTX TM-Sprayer™ (HTX Technologies, Carrboro, USA). The flow rate of MALDI matrix solution was
set to 75 µL/min. Nozzle nitrogen pressure was set to 10
psi, and nozzle temperature was set to 55 °C. A total of 12
sprays of matrix solution were performed on the surface
of hepatocellular carcinoma tissue sections. The nozzle
travel speed was set to 80 cm/min. The space between the
two spray tracks was set to 0.3 cm. The nozzle-to-slice
distance was set to 4 cm.
MALDI-MS imaging and data analysis
RapifleX MALDI Tissuetyper™ TOF/TOF MS (Bruker
Daltonics, Billerica, MA) was used for MALDI-MS imaging of hepatocellular carcinoma tissue sections. MALDIMSI analysis experiments were performed in positive and
negative ion mode in the range of m/z 70-1000. The spatial resolution for the MALDI-MS imaging experiments
was set to 100 μm. LasAtten was optimized according the
ion intensities and resolutions. The MS images of metabolites were constructed using SCiLS Lab 2018b software
(GmbH, Bremen, Germany). The ion intensities and in
situ data analysis were also calculated using SCiLS Lab
2018b software.
Ma et al. Cancer Cell International
(2023) 23:177
Results
Visual characterization of the global metabolic profile of
hepatocellular carcinoma during growth
We first establish xenograft model in nude mice with
human hepatocellular carcinoma HepG2 cells. A total
of nine nude mouse were used to place xenografts in
this study. Tumor tissues from three nude mouse were
taken on the 7th, 14th and 21st days post transplantation
(Fig. 2A). The tumor tissue sections at different growth
processes were prepared for H&E staining. Figure 2B
shows the typical H&E stain images of hepatocellular carcinoma tissue sections at different growth stages. Then,
we performed MALDI-MS imaging analysis on two adjacent tissue sections of H&E-stained sections in positive
and negative ion detection modes, respectively.
After MALDI-MS imaging experiment, we carried
out data-driven segmentation analysis on hepatocellular
carcinoma tissue sections according to the region-specific MALDI-MS fingerprints. In the data-driven segmentation analysis, different tumor regions with similar
MALDI-MS fingerprints are grouped and assigned specific colors, meaning that tissue regions assigned the
Fig. 2 Imaging of the global metabolic profile of transplanted hepatocellular carcinoma tumors after 7, 14 and 21 days of growth. (A) Collection
of tumor tissues at different growth stages. (B) H&E stain imaged of hepatocellular carcinoma tissue sections. (C) Data-driven tissue segmentation
analysis. (D) Principal component analysis (PCA) for transplanted tumors
Page 4 of 10
same color have very close metabolic characteristics.
Figure 2 C shows the data-driven segmentation result of
transplanted tumors after 7, 14 and 21 days of growth.
The results suggest that not only the tumor volume
increases gradually during the growth of hepatocellular
carcinoma, but also its metabolism is found to be significantly altered. In both positive and negative ion modes,
7-day transplanted tumor tissue sections were given
more of cool blue color, while 21-day transplanted tumor
were given more of a hot red color. In addition, we found
that 21-day transplanted tumor tissue sections exhibited a variety of different colors, suggesting that tumors
exhibit significant metabolic heterogeneity at this stage of
growth. Moreover, we extracted MALDI-MS data from
distinct segmentation regions and per-formed unsupervised principal component analysis. The results showed
that the metabolic characteristics of distinct segmentation regions were different and showed stepwise alterations with the growth of tumors (Fig. 2D).
Screening and imaging of altered metabolites during the
growth of hepatocellular carcinoma
Arginine metabolism pathway is altered in hepatocellular
carcinoma growth
Probabilistic latent semantic analysis (PLSA) model
was further used for multivariate statistical analysis
and screening of differentially expressed metabolites in
distinct hepatocellular carcinoma tissues. PLSA score
plots show a clear separation among 7-day, 14-day, and
21-day transplanted tumor tissue, indicating significant
metabolite alteration during the growth of hepatocellular carcinoma (Fig. 3A). On this basis, we compressed all
metabolites into two fundamental components to characterize the main trends of altered metabolites. Component
1 represents metabolites that are significantly upregulated during the growth of hepatocellular carcinoma,
while Component 2 indicates a class of metabolites that
are downregulated during the growth of hepatocellular
carcinoma in nude mice (Fig. 3B).
By screening discriminatory metabolites among different transplanted tumors, we found that the expression of
arginine ([M + H]+, m/z 175.1) increased gradually during
the growth of hepatocellular carcinoma (Fig. 3C). Arginine can be further metabolized to spermine and spermidine under the catalysis of ornithine decarboxylase
(ODC), spermidine synthase (SRM), and spermine synthase (SMS). Here, the MALDI-MSI data suggest that the
levels of spermine ([M + H]+, m/z 203.2) and spermidine
([M + H]+, m/z 146.1) also exhibited upregulated expressions during the growth of hepatocellular carcinoma
(Fig. 3D and E).
Ma et al. Cancer Cell International
(2023) 23:177
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258.1) expression also increases gradually during the
growth of hepatocellular carcinoma (Fig. 4C).
The expressions of fatty acids are altered during the
growth of hepatocellular carcinoma
Here, we also discovered that fatty acid (FA) undergoes
significant metabolic reprogramming during hepatocellular carcinoma progression. However, it should
be noticed that polyunsaturated FAs showed different
expression trends from monounsaturated and saturated
FAs. Polyunsaturated FAs, such as FA-20:4 ([M-H]−, m/z
303.2) and FA-20:3 ([M-H]−, m/z 305.2), exhibited gradually up-regulated expression in 7-day, 14-day, and 21-day
transplanted tumor tissues (Fig. 5A and B). The level of
FA-20:2 ([M-H]−, m/z 307.2) did not differ significantly
in 7-day, 14-day, and 21-day transplanted tumor tissues
(Fig. 5C). As for FA-20:1 ([M-H]−, m/z 309.2), its expression showed a gradual decrease with tumor progression
(Fig. 5D).
Other altered metabolites in hepatocellular carcinoma
Fig. 3 (A) Data-driven PLSA analysis for transplanted hepatocellular carcinoma tumors after 7, 14 and 21 days of growth. (B) Two main PLSA components that distinguish different hepatocellular carcinoma tissues. (C-E)
MS images and levels of arginine, spermine, and spermidine in different
transplanted tumors. Comp, component
Cholines and phosphatidylcholines are altered during the
growth of hepatocellular carcinoma
Choline is an important component of phosphatidylcholines (PC), and it also regulates cellular lipid metabolism and transmethylation metabolism. In this study,
we discovered that choline ([M]+, m/z 104.1) exhibited
an upregulated expression in the growth of hepatocellular carcinoma (Fig. 4A). Choline can combine with
phosphoric acid to form phosphocholine, which can further generate PC under the catalysis of choline kinase
(CHKA) and diacylglycerol cholinephosphotransferase (CPT). Coincidentally, the level of phosphocholine
([M + H]+, m/z 184.1) was found to be also upregulated
with the growth of hepatocellular carcinoma (Fig. 4B).
In addition, many PCs such as PC-32:0 ([M + H]+, m/z
734.6) and PC-34:2 ([M + H]+, m/z 758.6) also show a
gradually increasing expression trend (Fig. 4D and E).
However, it should be noted that compared to choline and phosphocholine, PCs increase less with tumor
growth. Glycerolphosphocholin (GPC) is an important
product of phosphatidylcholine metabolism, and our
MALDI-MSI results indicate that GPC ([M + H]+, m/z
In addition, we screened out some other metabolites that
are altered during the growth of transplanted tumors.
Malic acid is an important intermediate metabolite in the
tricarboxylic acid (TCA) cycle, and it was found that the
expression of malic acid exhibited a continuous increase
with tumor growth (Fig. S1). Glutamate can enter the
TCA cycle to provide energy for cell growth. Here, we
discovered that glutamate also showed an upregulated
expression in the growth of hepatocellular carcinoma
(Fig. S2). Phosphorylation is essential for aerobic oxidation and anaerobic glycolysis of glucose in cells, and the
MALDI-MSI result suggest that the level of glucosephosphate is significantly increased in 21-day transplanted tumor than that in 7-day and 14-day transplanted
tumors (Fig. S3).
Verification of discriminatory metabolites screened
from xenograft mouse model on human postoperative
hepatocellular carcinoma tissue
By performing MALDI-MSI based spatial metabolomics
analysis on hepatocellular carcinoma xenograft mouse
model, we successfully screened for metabolites associated with hepatocellular carcinoma progression. Further,
we performed MALDI-MS imaging analysis on 21 postoperative human hepatocellular carcinoma tissues. The
postoperative hepatocellular carcinoma tissue samples
contained both tumor tissue and adjacent non-tumor tissue. Figure 6B and L demonstrate the MALDI-MS images
of arginine, spermine, spermidine, choline, phosphocholine, glycerophosphocholine, PC-32:0, PC-34:2, FA-20:4,
FA-20:3, and FA-20:2 in representative human hepatocellular carcinoma tissue sections. Arginine, as well as
spermine and spermidine, increased gradually during the
Ma et al. Cancer Cell International
(2023) 23:177
Page 6 of 10
Fig. 4 (A) MS images and levels of choline in different transplanted tumors. (B) MS images and levels of phosphocholine in different transplanted tumors.
(C) MS images and levels of glycerophosphocholine (GPC) in different transplanted tumors. (D) MS images and levels of PC-32:0 in different transplanted
tumors. (E) MS images and levels of PC-34:2 in different transplanted tumors
growth of hepatocellular carcinoma in xenograft mouse
model. In human hepatocellular carcinoma tissue, the
expression of arginine, spermine, and spermidine were
found to be significantly higher in tumor regions than in
non-tumor regions (Fig. 6B and D). The expressions of
choline and phosphatidylcholines are altered during the
growth of hepatocellular carcinoma in xenograft mouse
model. The MALDI-MSI results also suggest that the
metabolism of choline and phosphatidylcholines were
reprogrammed in tumor tissue compared to surrounding
Ma et al. Cancer Cell International
(2023) 23:177
Page 7 of 10
Fig. 5 MS images and levels of fatty acids including FA-20:4 (A), FA-20:3 (B), FA-20:2 (C), and FA-20:1 (D) in different transplanted tumors
non-tumor tissue. However, choline and GPC increased
with tumor growth in xenograft mouse model, whereas
in human hepatocellular carcinoma tissue, the expression
of choline was not significantly upregulated in tumor tissue, and the expression of GPC was downregulated in
tumor tissues (Fig. 6E and G). Similar to the xenograft
mouse model, the levels of phosphocholine and PC-32:0
were significantly higher in tumor tissue than in paired
non-tumor tissue (Fig. 6F and H). There was no significant difference in the expression of PC-34:2 in tumor
and surrounding non-tumor tissue (Fig. 6I). In xenograft
mouse model, we found that some FAs showed abnormal expression during tumor progression. Polyunsaturated FAs represented by FA-20:4 and FA-20:3 showed
progressively higher expression during the growth of
transplanted tumors, while FA-20:1 showed little change
and FA-20:1 expression gradually decreased. In human
hepatocellular carcinoma tissue, we discovered that
polyunsaturated FAs, monounsaturated FAs, and saturated FAs all exhibited significantly higher expressions in
tumor tissue than surrounding non-tumor tissue (Fig. 6J
and L, and Fig. S4).
Discussion
Tumor cells need to absorb energy and nutrient compounds from the surrounding environment to maintain
their growth and progression. In addition, there is growing evidence showed that tumor cells can also reprogram
Ma et al. Cancer Cell International
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Fig. 6 (A) H&E stain images of human postoperative hepatocellular carcinoma tissue sections. (B-L) MS images of arginine, spermine, spermidine, choline, phosphocholine, glycerophosphocholine, PC-32:0, PC-34:2, FA-20:4, FA-20:3, and FA-20:2 in human postoperative hepatocellular carcinoma tissues.
NS, No significant differences
their metabolic networks to increase energy supply and
biomolecule synthesis, and the altered metabolism is
recognized as an important feature of tumors. Exploring how reprogrammed tumor metabolic networks affect
tumor growth is an important prerequisite for discovering potential metabolic targets to better cancer treatment [32, 33]. However, it should be noted that tumor
tissues are highly heterogeneous and the spatial distribution characteristics of metabolites in different microregions of tumor tissues may be completely different.
Mass spectrometry imaging can direct analyze metabolites in tissue sections, visualize the spatial distributions
and relative contents of different metabolites in distinct
micro-regions of tumors, thus provide technical support
to accurately and spatiotemporally characterize the metabolic features of tumors during their development. In this
study, we carried out MALDI-MS imaging analysis on
both hepatocellular carcinoma xenograft mouse model
and hepatocellular carcinoma patients. Discriminatory
metabolites that altered during the growth and progression of hepatocellular carcinoma were first screened and
imaged in xenograft mouse model and then were further validated in postoperative human hepatocellular
carcinoma tissues. Overall, the metabolic profile of the
transplanted tumor shows stepwise alterations during
its growth. In addition, we also found that the metabolic
heterogeneity of the tumor increases as it grows. Metabolite-driven segmentation analysis indicate that the transplanted tumor exhibited slight heterogeneity after 7 days
of growth, but the metabolic heterogeneity significantly
increased after 21 days of growth (Fig. 2C).
However, there are some limitations in the visualization
of metabolites in biological tissues using MALDI-MSI.
For example, when performing MALDI-MSI analysis,
we must use matrix, and the presence of matrix-related
ions is an important factor restricting the imaging of low
molecular weight metabolites. The use of multiple MSI
techniques is a good means to improve the coverage of
metabolite visualization [34]. In addition, the resolution
of the MALDI-MSI used in this study is 100 μm. Whereas
most of the cells in the heterogeneous tumor microenvironment have a diameter of about 10 μm, which makes it
difficult to visualize and analyze metabolites at the singlecell level.
Polyamines are a class of metabolites containing two
or more amino groups, mainly including spermine, spermidine and putrescine. Polyamines play important roles
in DNA synthesis and replication, regulation of gene
Ma et al. Cancer Cell International
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transcription and translation, cell proliferation and apoptosis, and maintenance of cell membrane stability [35].
Previous studies have shown that disorders in the metabolism of polyamines and polyamine synthases are closely
related to the development of tumors, and targeting the
altered polyamine metabolism is a rational approach for
tumor treatment. In this study, we found that polyamines
including spermine and spermidine were significantly
upregulated in human hepatocellular carcinoma tissues,
and exhibited a gradually increase during the growth
of hepatocellular carcinoma in xenograft mouse model
(Figs. 3 and 6). This is consistent with previous reports of
significantly elevated levels of polyamines in other cancers such as esophageal and gastric cancers [22, 36].
Choline and phosphatidylcholine showed significant
alterations in both hepatocellular carcinoma xenograft
mouse model and human hepatocellular carcinoma tissue. Choline is an important precursor molecule for
the synthesis of phosphatidylcholine, which in turn is
an important class of phospholipids in cells. Abnormal
phospholipid metabolism is closely related to the occurrence, progression, and metastasis of many tumors [37,
38]. In fact, we found that phospholipids with different
carbon chain composition can exhibit different changing trends in hepatocellular carcinoma. Some phospholipids such as PC-32:0 showed significant upregulation
in hepatocellular carcinoma tissues, while others such as
PC-34:2 showed little difference in tumor and adjacent
non-tumor tissues (Figs. 4 and 6). We speculate that this
may be due to the different biological roles played by different phospholipids in tumor progression, however, this
speculation needs further validation.
Fatty acids are important components of cell membranes and play an essential role in cellular signaling
and energy metabolism. In the present study, we found
that most of the fatty acids were significantly upregulated in tumor tissue, except for individual fatty acids
such as FA-20:2 and FA-20:1, which showed little change
or downregulation during the growth of transplanted
tumors. This echoes the biological role of fatty acids and
the previous studies [39, 40]. During the rapid growth
and proliferation of tumor cells, fatty acids are needed for
β-oxidation to provide energy and also indispensable for
the synthesis of lipids.
In summary, we built hepatocellular carcinoma xenograft model and carried out MALDI-MS imaging analysis
on tumor tissues at different growth stages. Combined
with multivariate statistical analysis, we successfully
screened discriminatory metabolites that significantly
altered during the growth of transplanted tumor. Furthermore, we performed MALDI-MSI analysis on 21
human postoperative hepatocellular carcinoma tissues
to validate the discriminatory metabolites that screened
on xenograft model. We discovered that arginine and its
Page 9 of 10
metabolites, spermine and spermidine, were significantly
upregulated in tumor tissue of hepatocellular carcinoma,
and their ex-pressions showed a continuous increase
with tumor growth. The metabolism of choline and phosphatidylcholine are altered during the growth of hepatocellular carcinoma. We also found that the levels of
most fatty acids in tumor tissues are significantly higher
than those in non-tumor tissues. These findings not only
enhance our understanding of hepatocellular carcinoma
metabolism, but also provide potential metabolic targets
for better cancer treatment.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12935-023-03027-0.
Supplementary Material 1: Fig. S1–Fig. S4.
Authors’ contributions
ZBM, YZ, CLS, and CKQ performed the investigation and designed the research
content. ZBM, JWM, and XGC collected postoperative samples. ZBM and CLS
carried out the experiment and performed the data analysis. ZBM, CLS, and
CKQ wrote and revised the manuscript. All authors have read and approved
the final manuscript.
Funding
This research was funded by the Shandong Province Science and Technology
Development Plan (No.003130113) and Shandong First Medical University
(Shandong Academy of Medical Sciences) Youth Science Foundation
Incubation Grant Program (No.202201-051), and the Science, Education
and Industry Integration Innovation Pilot Project from Qilu University of
Technology (2022PY001).
Data Availability
All data generated and analyzed in this study are available by reasonable
request of the corresponding author.
Declarations
Ethics approval and consent to participate
The experiments were approved by the Ethnical Committee of Shandong
Provincial Hospital (No. 2022-069), and the participants gave informed written
consent.
Consent for publication
Informed consent for publication was collected from all participants.
Competing interests
The authors declare no competing interests.
Received: 19 June 2023 / Accepted: 10 August 2023
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Differentiation in native as well as introduced ranges: germination reflects mean and variance in cover of surrounding vegetation
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Differentiation in native as well as introduced ranges:
germination reflects mean and variance in cover of
surrounding vegetation Tina Heger1,2,4,*, Gabriele Nikles1 and Brooke S. Jacobs3,5 Tina Heger
, Gabriele Nikles and Brooke S. Jacobs
1Technical University of Munich, Restoration Ecology, Emil-Ramann-Str. 6, 85354 Freising, Germany
2Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), Altensteinstr. 34, 14195 Berlin, Germany
3Department of Plant Sciences, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA
4Present address: Biodiversity Research/Systematic Botany, University of Potsdam, Maulbeerallee 2a, 14469 Potsdam, Germany
5Present address: California Department of Fish and Wildlife, Water Branch, 1416 9th Street, Sacramento, CA 95811, USA Received: 26 January 2017 Editorial decision: 22 January 2018 Accepted: 2 February 2018 Published: 3 February 2018
Associate Editor: Kristina Hufford
Citation: Heger T, Nikles G, Jacobs BS. 2018. Differentiation in native as well as introduced ranges: germination reflects mean and
variance in cover of surrounding vegetation. AoB PLANTS 10: ply009; doi: 10.1093/aobpla/ply009 Abstract. Germination, a crucial phase in the life cycle of a plant, can be significantly influenced by competition and
facilitation. The aim of this study was to test whether differences in cover of surrounding vegetation can lead to popula-
tion differentiation in germination behaviour of an annual grassland species, and if so, whether such a differentiation can
be found in the native as well as in the introduced range. We used maternal progeny of Erodium cicutarium previously
propagated under uniform conditions that had been collected in multiple populations in the native and two introduced
ranges, in populations representing extremes in terms of mean and variability of the cover of surrounding vegetation. In
the first experiment, we tested the effect of germination temperature and mean cover at the source site on germination,
and found interlinked effects of these factors. In seeds from one of the introduced ranges (California), we found indica-
tion for a 2-fold dormancy, hindering germination at high temperatures even if physical dormancy was broken and water
was available. This behaviour was less strong in high cover populations, indicating cross-generational facilitating effects
of dense vegetation. In the second experiment, we tested whether spatial variation in cover of surrounding vegetation
has an effect on the proportion of dormant seeds. Contrary to our expectations, we found that across source regions, high
variance in cover was associated with higher proportions of seeds germinating directly after storage. In all three regions,
germination seemed to match the local environment in terms of climate and vegetation cover. Research Article Research Article Differentiation in native as well as introduced ranges:
germination reflects mean and variance in cover of
surrounding vegetation We suggest that this is
due to a combined effect of introduction of preadapted genotypes and local evolutionary processes. Keywords: Bet-hedging; competition; eco-evolutionary experience; facilitation; genetic adaptation; physical and
physiological dormancy; preadaptation. AoB PLANTS https://academic.oup.com/aobpla
© The Author(s) 2018
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
© The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. *Corresponding author’s e-mail address: t.heger@wzw.tum.de Introduction high percentage of germination can help to effectively
exploit transient resources (Grime et al. 1981). If neigh-
bouring plants are present already during the phase of
germination, this can have negative effects on plant fit-
ness (Novoplansky 2009; Novoa and González 2014). It high percentage of germination can help to effectively
exploit transient resources (Grime et al. 1981). If neigh-
bouring plants are present already during the phase of
germination, this can have negative effects on plant fit-
ness (Novoplansky 2009; Novoa and González 2014). It Germination is a crucial step in the life cycle of plants,
and this holds especially true for annuals in situations
where resources are limited. Fast germination and a *Corresponding author’s e-mail address: t.heger@wzw.tum.de 1 © The Author(s) 2018 AoB PLANTS https://academic.oup.com/aobpla Heger et al. – Differentiation in germination reflects vegetation cover has been shown that percentage germination can be
substantially lower if other species are present (Oliveira
et al. 2014). Consequently, early germination is advan-
tageous in situations involving interspecific competition
among seedlings (Burke and Grime 1996). Continuous
exposure to a high level of interspecific competition,
as found in grassland communities with high cover of
grasses and herbs, may thus foster directional selection
for early germination (Orrock and Christopher 2010). expectation would be that low variability in competi-
tion and thus good predictability will lead to directional
selection of morphological traits. This may then render
it advantageous to wait for a reliable cue instead of
germinating rapidly as soon as good conditions for ger-
mination are given (cf. Cohen 1967). As far as we know,
studies testing whether high or low variability in levels
of competition leads to delayed germination are not
available yet. It is well-known that different populations of the
same species can vary in their germination behaviour
(e.g. Fenner and Thompson 2005). Variation in require-
ments for germination, dormancy breaking mecha-
nisms, germination rates and germination speed within
species has been observed in many species. They can be
due to genetic differentiation as well as maternal effects
(Baskin and Baskin 1998, pp. 181 and 187). Population
differentiation with respect to germination responses
to gradients in temperature can occur within the native
(Schwaegerle and Bazzaz 1987; Postma et al. 2016) as
well as introduced ranges (Beckmann et al. 2011). Introduction It has
been hypothesized that rapid adaptive changes in ger-
mination strategies may contribute to the success of
globally distributed invaders (Hierro et al. 2009). To our
knowledge, it has not been explored, however, whether
population differentiation in germination can also be
driven by differing levels of cover of the surrounding
vegetation, and whether such an evolutionary response
(if present) can be rapid enough to also occur within an
introduced range. We approached these research questions with two
germination experiments using seeds from the native
and two introduced ranges of the annual grassland
species Erodium cicutarium. This species is native to
Europe, has been transported to many regions of the
world and is now considered an invasive species, e.g.,
in California (USA) and in Chile (Figueroa et al. 2004;
Brooks and Berry 2006). Competition has been shown
to have transgenerational effects on trait expression
in this species (Heger et al. 2014; Heger 2016), and the
populations are differentiated in response to environ-
mental heterogeneity also in the introduced range
(Baythavong and Stanton 2010; Baythavong 2011). In
the first experiment, we specifically wanted to know if
competition at a source site, estimated as mean cover
of surrounding vegetation, has an effect on the ger-
mination ability of the populations. We expected that
seeds from high cover sites would germinate earlier
than those from sites with lower cover (Hypothesis
1). Further, we wanted to test whether the source
populations differ in their germination response to
temperature, and if so, whether these differences
can be related to the region the populations occur in
(native range: Germany; introduced range: Chile and
California) or mean vegetation cover at the source
site. We expected that populations from hot climates
would differ in germination percentage and speed
under high temperatures from populations from tem-
perate climate, but that these differences would be
less pronounced if cover at the source site had been
high (Hypothesis 2). With the second experiment, we
additionally wanted to know whether across regions,
seeds from populations with highly variable vegeta-
tion cover have higher proportions of dormant seeds
than those from population with low variability in
cover (Hypothesis 3). Factors that can lead to population differentiation are
not only the average environmental conditions experi-
enced by the plants, but also the spatial and temporal
variability in environmental conditions. Introduction In environ-
ments where unfavourable conditions occur irregularly,
a bet-hedging strategy can evolve (Baskin and Baskin
1998, p. 567). Plants using a bet-hedging strategy pro-
duce seeds that differ in their germination timing, while
showing no heritable differences. Bet-hedging strate-
gies seem to be ubiquitous (Simons 2011), and can
also result from different kinds of environmental vari-
ation (Tielboerger et al. 2012). Hierro et al. (2009), for
instance, found delayed germination in pappus seeds
of Centaurea solstitialis populations that experience
greater inter-annual variation in winter precipitation. Because competition can have negative effects on
growth and survival, spatially varying levels of compe-
tition may promote a bet-hedging strategy. In environ-
ments where it is not predictable whether offspring
will face high competition or not, and where there-
fore adaptive trait differentiation does not happen,
it may be advantageous to ‘spread the risk’ and pro-
duce some seeds with longer dormancy. An alternative AoB PLANTS https://academic.oup.com/aobpla Experiment 1: does vegetation cover at source
site influence germination ability and response to
temperature? For two closely related species in the genus
Erodium, however, it has been shown that changes in
temperature can induce germination (Rice 1985). In an
experiment testing germination ability after dry storage,
an increase in germination rate during the duration of
the experiment (i.e. 2 years) has been observed (Van
Assche and Vandelook 2006). The seed coat imperme-
ability in E. cicutarium thus can be removed by mech-
anical scarification, by dry storage or by temperatures
fluctuating between cool and hot. When either of these
factors renders the seed coat permeable, seeds ger-
minate as soon as conditions are favourable (Rice 1987;
Van Assche and Vandelook 2006). In a comparison of
the seed bank persistence of 70 dicotyledons in Britain,
however, E. cicutarium was one of the species with the
most persistent seed bank. In the fifth year after sowing,
still 4 % of the sown seeds germinated (Roberts 1986). Baskin and Baskin (1998, p. 376) report the optimum
temperature for germination as 15 °C/20 °C (night/day). The species is native to Europe and invasive in many
parts of the world. For this study, we collected maternal
seed families in the native range (Bavaria, Germany) and
in two introduced ranges (California, USA, and Chile). Pollen analysis indicates that E. cicutarium has been
introduced to California prior to the first settlements of
Spanish missionaries, and that it was already well estab-
lished in the 1750s (Mensing and Byrne 1998). In Chile,
the species has most probably been introduced more re-
cently (i.e. during the 19th century). i
We randomly selected two brown, filled seeds from each
field collected maternal family and propagated them in
the glasshouse under uniform conditions. Upon flowering,
individuals were allowed to produce self-fertilized seeds. After 4 months, we harvested all ripe seeds. Seeds from col-
lection sites in Germany and California were propagated at
Gewächshauslaborzentrum Dürnast, Technical University
of Munich, those collected in Chile were propagated at the
glasshouse facilities of the University of California-Davis. All
seeds were stored in paper envelopes at room tempera-
ture and under dry conditions after harvesting until the be-
ginning of the experiment. Experimental setup. In the native range, a germin-
ation peak in autumn can be observed in E. cicutarium
(Poschlod et al. 2003). Experiment 1: does vegetation cover at source
site influence germination ability and response to
temperature? conditions (Hegi 1964). It can be found in open vegeta-
tion such as sand dunes and nutrient-poor grasslands,
but also in dense lawns and ruderal vegetation. The pink
flowers are mostly self-pollinated. The long seedpod is
shaped like the bill of a stork. When fruits are ripe, dry
conditions stimulate a spring mechanism powered by
the awns curling up in a spiral, and up to five achenes
are dispersed ballistically. If humidity changes, the spi-
ralled awns wind and unwind. This movement can cause
self-burial of the achenes in the soil (Evangelista et al. 2011). Seed sources. We chose seed collection sites differing in
vegetation cover [see Supporting Information—Table
S1] and made sure that the sites had not been subject
to recent changes in land use. At each collection site, we
randomly selected mature seeds from up to 20 individu-
als and stored them in paper envelopes. In Germany and
California, percentage of cover of the surrounding vege-
tation was estimated in three 50 × 50 cm plots per col-
lection site, each centred on an E. cicutarium plant (cf. Heger 2016). In Chile, percentage of vegetation cover
was estimated in 15 cm radiuses surrounding each sam-
pled individual. We used these data to calculate means
and variances of vegetation cover per population. With
an ANOVA (function ‘aov’ in R version 3.1.2; R Core Team
2014) we tested whether seed sources classified as ‘high
cover’ differed systematically in their mean daily tem-
peratures from those classified as ‘low cover’, but neither
the main effect of cover at source site nor its interaction
with source region were significant.i One seed is contained in each achene, so each fruit
may hold up to five seeds. A single plant can produce
up to 9900 seeds (Blackshaw and Harker 1998), but
this maximum is reached only under optimum condi-
tions, and usually seed production is much lower (own
observations). In the fresh fruit, physiological and phys-
ical dormancy both hinder germination; later, physical
dormancy prevails. Seeds have a hard seed coat, and
scarification can break physical dormancy (Baskin and
Baskin 1998, p. 376). No study is available on whether
mechanical scarification, e.g., by abrasion from soil par-
ticles happens under natural conditions (Baskin and
Baskin [2000] report this lack of evidence for other spe-
cies, too). Study species: E. cicutarium, redstem filaree Study species: E. cicutarium, redstem filaree Erodium cicutarium is a winter annual to biennial herb
in the Geraniaceae family. It occurs on various soil types
from soils rich in lime to lime deficient, and prefers dry 2 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2018 Heger et al. – Differentiation in germination reflects vegetation cover AoB PLANTS https://academic.oup.com/aobpla Experiment 1: does vegetation cover at source
site influence germination ability and response to
temperature? In the summer dry regions in
California and Chile, germination usually also starts after
the first rains in autumn (Rice 1985 for two close rela-
tives in California). We retrieved daily temperature max-
ima and minima during autumn (i.e. September through
November for Germany and California, and April through
June for Chile) from climate stations as close as possible
to the sampling sites (mappedplanet.de; data from IPCC,
1960 to 1990). We calculated means for every region and
used these to programme a climate chamber (Mueve TK
252; day/night cycle of 12 h without light). Programmed
temperature treatments were 13.0 °C/4.6 °C for German
autumn, 24.2 °C/8.4 °C for Californian autumn and
16.2 °C/6.6 °C for Chilean autumn. The species is native to Europe and invasive in many
parts of the world. For this study, we collected maternal
seed families in the native range (Bavaria, Germany) and
in two introduced ranges (California, USA, and Chile). Pollen analysis indicates that E. cicutarium has been
introduced to California prior to the first settlements of
Spanish missionaries, and that it was already well estab-
lished in the 1750s (Mensing and Byrne 1998). In Chile,
the species has most probably been introduced more re-
cently (i.e. during the 19th century). 3 © The Author(s) 2018 AoB PLANTS https://academic.oup.com/aobpla Heger et al. – Differentiation in germination reflects vegetation cover probability for a seed to not germinate (i.e. to ‘survive’)
during the upcoming time interval. From each of the three source regions, we selected
those five collection sites with the highest and those
five sites with the lowest mean cover of vegetation sur-
rounding E. cicutarium [see Supporting Information—
Table S1]. Ten maternal seed families were randomly
selected per population. Within each of the six groups of
vegetation cover at source site (two levels: high and low)
crossed with source region (three levels), 360 seeds were
drawn at random across all selected populations and
maternal families. We selected only brown, filled seeds. The seeds were pooled per group. For every combination
of cover × source region × germination temperature, we
loaded six petri dishes with 20 seeds each. We spread the
seeds homogeneously on filter paper within each petri
dish. In the later analyses, the data for the 120 seeds
were accumulated; petri dish was included as random
factor. Experiment 1: does vegetation cover at source
site influence germination ability and response to
temperature? Prior to this, all seeds were physically scarified by
placing 10 seeds at a time between two small sheets of
sandpaper (Klingspor PL 31 grain size 150). Using light
pressure, the top sheet was moved 15 s back and forth
and 15 s from left to right. To statistically compare germination among treat-
ment groups, and to assess the significance of the three
covariates germination temperature, seed source region
and vegetation cover at source site we used Cox models,
following the procedure proposed in McNair et al. (2012). Before building the models, we checked the proportional
hazards assumption for each covariate based Kaplan–
Meier survivor functions (function ‘survfit’ in package
‘survival’; Therneau 2015), to make sure the data meet
the requirements for applying these kinds of models. Covariates were then checked for multicolinearity using
the variance inflation factor (function ‘vif’ in package
‘HH’; Heiberg 2016). Next, all covariates meeting these
requirements were transformed into dummy variables,
each named after the factor level which is re-coded as
‘1’ [see Supporting Information—Table S3]. Cox mod-
els were built starting with one covariate (re-coded as
dummy) plus the frailty term using the function ‘coxph’
from the package ‘survival’. We then used a forward
selection procedure based on the Akaike Information
Criterion (function ‘AIC’; R Core Team 2014) to build the
final model. To make sure all seeds had access to approximately
the same amount of water during the experiment and to
prevent moulding, we prepared solutions of polyethyl-
ene glycol 6000 (PEG-6000) and created a water poten-
tial within the optimum range for E. cicutarium according
to Blackshaw (1992) [see Supporting Information—
Table S2]. We pipetted 4 mL of this solution into each
petri dish, covered it with parafilm and placed it at a ran-
dom position into the preheated germination chamber. Due to space limitations we had to run one temperature
treatment after the other. Every other day, we recorded
and removed germinated seeds. A seed was classified
as germinated if the radicle was visible. After 2 weeks,
we terminated the experiment. Seeds were regarded as
viable but dormant if they were still brown and filled. Experiment 2: do more seeds from populations
with highly variable cover of surrounding
vegetation stay dormant? The aim of this treatment was to find
out whether for some seeds, neither dry cool storage nor
storage under hot fluctuating temperatures had termi-
nated the impermeability of the seed coat. We watered
the seeds and placed the racks back into the chamber
using the same optimum conditions as described in the
previous paragraph. We surveyed again every 2 to 4 days
for 35 days. Phase 3—germination after scarification. As a next
step, we scarified the remaining seeds by cutting the tip
with a razor blade. The aim of this treatment was to find
out whether for some seeds, neither dry cool storage nor
storage under hot fluctuating temperatures had termi-
nated the impermeability of the seed coat. We watered
the seeds and placed the racks back into the chamber
using the same optimum conditions as described in the
previous paragraph. We surveyed again every 2 to 4 days
for 35 days. the wool without prior scarification. We made sure that
every maternal family was represented in each rack so
that every rack was a replicate (for some families this
was not possible because not enough seeds were avail-
able [see Supporting Information—Table S4]). Racks
were loosely covered with parafilm. the wool without prior scarification. We made sure that
every maternal family was represented in each rack so
that every rack was a replicate (for some families this
was not possible because not enough seeds were avail-
able [see Supporting Information—Table S4]). Racks
were loosely covered with parafilm. Phase 1—germination after dry storage. To test for
germination directly after the period of dry storage,
we placed racks containing the non-scarified seeds
in a germination chamber set to day/night cycles of
12 h (always dark) with temperatures at 41 °C/21 °C. Similar temperatures have been observed under field
conditions in California in August (Rice 1985). Under
these conditions, we expected those seeds to ger-
minate for which the impermeability of the seed coat
had been broken by the previous storage, and which
are not restrained by high temperature. We recorded
germination every other day and removed germinated
seeds. When germination had slowed, we switched
to a weekly census. At every census we randomly
exchanged the position of the racks in the chamber,
and watered seeds with distilled water if necessary. Moulded seeds were cleaned with distilled water and
placed back in the tube containing fresh cotton wool. Experiment 2: do more seeds from populations
with highly variable cover of surrounding
vegetation stay dormant? If this was not possible because seeds were decayed
they were removed and the loss was recorded. When
germination had stopped, seeds were still stored in the
chamber with the same temperature cycles. Statistical analysis. For the analysis of data from
Experiment 2, we wanted to know if seed sources dif-
fer concerning which mechanisms break their physical
dormancy. We therefore did not focus on the germin-
ation percentage over time observed in the different
seed sources as in the previous analysis for Experiment
1. Instead, we assigned each seed to a category de-
pending on whether it germinated during one of the
three experimental phases or not. Many seeds moulded
during the experiment, which made us establish an
additional category for these events. Our response vari-
able thus had the five categories ‘dry storage’, ‘dry-
hot’, ‘scarification’, ‘moulded’ and ‘no germination’. Some seeds were lost during the experiment and were
excluded from the data set. To test for the effects of seed source region (factor
with the three levels Germany, California and Chile), vari-
ance of cover at the source site (continuous variable) and
their interaction on this categorical response variable,
we performed a multinomial logistic regression using
the function ‘multinom’ in the package ‘nnet’ (Venables
and Ripley 2002). We ordered the levels of the response
variable to make ‘dry storage’ the baseline category. We
calculated P-values based on Wald ratios using the func-
tion ‘pchisq’. Overall significance of the effect variables
was checked using the function ‘Anova’ from the pack-
age ‘car’ (Fox and Weisberg 2011). After 148 days (21 weeks), we removed the racks from
the chambers and stored them in paper bags at room
temperature for 5 months. Brown, filled seeds were
regarded as viable and left in the tubes, the others were
removed and recorded as dead. To check whether after
this further period of dry storage more seeds would ger-
minate we placed the racks again into the chamber for
17 days, using the same temperature cycle. Phase 2—germination after dry-hot treatment. Next,
we wanted to check whether for the remaining seeds,
exposure to dry and hot conditions with fluctuating
temperature would break dormancy as suggested in
Rice (1985). We stopped watering the tubes, removed
the parafilm, and changed the temperature cycle to
45 °C/25 °C. We left the seeds under these conditions
for 110 days. Experiment 2: do more seeds from populations
with highly variable cover of surrounding
vegetation stay dormant? After this period we surveyed the seeds
once more, watered the tubes containing seeds classi-
fied as viable with distilled water and placed the racks
into the chamber set to the optimum germination con-
ditions according to Blackshaw (1992), i.e. a 12 h cycle
of 21 °C/4 °C. We checked every 2 to 4 days for germin-
ation. After 20 days, we surveyed again and removed
non-viable seeds. Experiment 2: do more seeds from populations
with highly variable cover of surrounding
vegetation stay dormant? Seed sources. For Experiment 2, we again used seeds
originating from the three regions Germany, California
and Chile. For information on seed collection methods
for German and Chilean origins see Experiment 1. The
Californian seed families were collected at 24 sites,
using the same protocol for sampling and vegetation
surveying as described above for the Chilean sources. All
families were propagated at the glasshouse facilities of
the University of California-Davis under uniform condi-
tions using the protocol described above. Statistical analysis. For analysing data of Experiment
1 we used a time-to-event analysis as suggested by
McNair et al. (2012). In time-to-event analyses, the
fate of single seeds is followed instead of looking at
cumulative germination or specific qualities of the ger-
mination process of a cohort of seeds (e.g. percentage
germinated seeds in a petri dish). To characterize and
visualize the temporal pattern of germination, we com-
puted life table estimates of survivor functions for each
of the 18 groups of seeds defined by the combination of
treatment factors (3 × 3 × 2 for the factors germination
temperature, seed source region and vegetation cover
at source site). This was done using the ‘lifetab’ func-
tion from the ‘KMsurve’ package (Klein et al. 2012) in R
version 3.1.2 (R Core Team 2014). In the case of germin-
ation data, the survival function helps to estimate the Experimental setup. For each source site, the variance
of percentage cover was calculated from the single
cover estimates. We selected the two populations with
the highest and those two with the lowest variance in
percentage cover [see Supporting Information—Table
S4]. Per population, we randomly selected up to 10
maternal families (118 families in total), and up to eight
brown and filled seeds from every family. We equipped
eight racks usually used for molecular genetic analy-
ses (e.g. PCR) each with 118 Eppendorf tubes (1.5 mL). Every tube was filled with cotton wool, and tubes were
watered with distilled water. We assigned individual
seeds randomly to tubes and placed them on top of 4 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2018 Heger et al. – Differentiation in germination reflects vegetation cover Phase 3—germination after scarification. As a next
step, we scarified the remaining seeds by cutting the tip
with a razor blade. Effects of temperature and mean vegetation
cover at source site on germination The results of the final Cox model (Table 1) together with
the plotted life table estimates of survivor functions
(Fig. 1) show that in the first experiment, the germin-
ation temperature had a significant influence on ger-
mination of all seeds. Temperatures simulating German
autumn slowed the germination process compared to
the other two temperatures. Under temperatures simu-
lating Chilean autumn, all groups of seeds showed simi-
lar germination behaviour, and nearly all of the seeds 5 © The Author(s) 2018 AoB PLANTS https://academic.oup.com/aobpla Heger et al. – Differentiation in germination reflects vegetation cover Figure 1. Life table estimates of survivor functions for Experiment 1 for germination temperatures simulating German (A), Californian (B) and
Chilean (C) autumn. The probability of not germinating is plotted against time. Colours are coding the seed source regions (black: Germany;
red: California; orange: Chile). Solid lines summarize data for seeds from high cover source sites; dashed lines those from low cover sites. For
every group, data from 120 seeds have been accumulated. Figure 1. Life table estimates of survivor functions for Experiment 1 for germination temperatures simulating German (A), Californian (B) and
Chilean (C) autumn. The probability of not germinating is plotted against time. Colours are coding the seed source regions (black: Germany;
red: California; orange: Chile). Solid lines summarize data for seeds from high cover source sites; dashed lines those from low cover sites. For
every group, data from 120 seeds have been accumulated. Figure 1. Life table estimates of survivor functions for Experiment 1 for germination temperatures simulating German (A), Californian (B) and
Chilean (C) autumn. The probability of not germinating is plotted against time. Colours are coding the seed source regions (black: Germany;
red: California; orange: Chile). Solid lines summarize data for seeds from high cover source sites; dashed lines those from low cover sites. For
every group, data from 120 seeds have been accumulated. Table 1. Summary table of the final Cox model for the data from Experiment 1. SE denotes the standard error. Covariates are dummy variables
derived from the original categorical variables germination temperature (levels German, Californian, Chilean autumn), seed source region
(Germany, California, Chile) and cover at source site (high, low). The dummy variables are coded as 1 if the factor level is the one given in the
name of the covariate (e.g. Effects of temperature and mean vegetation
cover at source site on germination 1B suggest that under these
conditions, seeds from German sources germinated
earlier and to a higher percentage than seeds from the
other regions. According to the results of the final Cox
model (Table 1), Californian seeds, in particular, had a
comparably low chance of germinating under these
conditions at any given point in time. This is indicated
by the low value of exp(coef) for the interaction of ‘tem-
perature California’ and ‘source region California’ (cf. McNair et al. 2012). However, Californian seeds origi-
nating from a population with high cover of surround-
ing vegetation had, under Californian temperatures
and compared to seeds from low cover sites, a signifi-
cantly higher probability for germination at any given
time during the experiment (high value of exp(coef) in
Table 1). The coefficient was negative, opposed to this,
for the significant interaction of temperatures simulat-
ing Californian autumn and high cover at source site,
indicating a low probability of germination for this
treatment combination (Table 1).iil Effects of temperature and mean vegetation
cover at source site on germination ‘Temperature California’ compares germination of seeds under Californian temperature to those under the other
two temperatures [see Supporting Information—Table S3]). Significant results (P < 0.05) are highlighted with bold font. Table 1. Summary table of the final Cox model for the data from Experiment 1. SE denotes the standard error. Covariates are dummy variables
derived from the original categorical variables germination temperature (levels German, Californian, Chilean autumn), seed source region
(Germany, California, Chile) and cover at source site (high, low). The dummy variables are coded as 1 if the factor level is the one given in the
name of the covariate (e.g. ‘Temperature California’ compares germination of seeds under Californian temperature to those under the other
two temperatures [see Supporting Information—Table S3]). Significant results (P < 0.05) are highlighted with bold font. Table 1. Summary table of the final Cox model for the data from Experiment 1. SE denotes the standard error. Covariates are dummy variables
derived from the original categorical variables germination temperature (levels German, Californian, Chilean autumn), seed source region
(Germany, California, Chile) and cover at source site (high, low). The dummy variables are coded as 1 if the factor level is the one given in the
name of the covariate (e.g. ‘Temperature California’ compares germination of seeds under Californian temperature to those under the other
two temperatures [see Supporting Information—Table S3]). Significant results (P < 0.05) are highlighted with bold font. Covariate
Coefficient
exp(coef)
SE of coef
Chisq
P
Temperature California
1.427
4.167
0.2717
27.59
<0.001
Source region California
−0.021
0.980
0.2947
0.00
0.940
Cover at source high
0.197
1.217
0.2387
0.68
0.410
Temperature California: source region California
−3.064
0.047
0.4963
38.12
<0.001
Temperature California: cover at source high
−1.788
0.167
0.3948
20.51
<0.001
Source region California: cover at source high
−0.242
0.785
0.4157
0.34
0.560
Temperature California: source region California: cover at source high
2.346
10.442
0.7083
10.97
<0.001
Frailty (petri dish)
690.29
<0.001 6 © The Author(s) 2018 6 Heger et al. – Differentiation in germination reflects vegetation cover lower percentage under Californian temperatures than
German seeds from low cover sites. in this treatment had germinated by the end of the
experiment. Germination patterns under temperatures
simulating Californian autumn were significantly differ-
ent from those under the other two regimes (Table 1;
Fig. 1B). The graphs in Fig. Significant effects of variation in cover at source
site on germination The multinomial logistic regression revealed highly signifi-
cant effects of source region, variance of cover of surround-
ing vegetation at source site and the interaction of both (all
P < 0.001). Whether seeds germinated after storage, after
dry-hot treatment or after scarification was significantly
influenced by the region the seeds originated from, the de-
gree of spatial variation in the cover of surrounding vegeta-
tion at the source site and their interaction. Seeds originating from German populations showed
the highest overall proportions of germination (Fig. 2). Those German seeds that germinated did so directly
after storage; neither the dry-hot treatment nor scari-
fication with a razor blade caused additional germin-
ation events. Compared to seeds from Germany, seeds
from California and Chile had a much higher probability
to germinate after dry-hot treatment or scarification (cf. high positive coefficients for these categories in Table 2). Across source regions, a more heterogeneous cover at
the source site was connected to a higher probability
for seeds to germinate after dry storage instead of after
dry-hot treatment (high negative coefficient in Table 2). This was true especially for seeds from Chile. Looking at
the interaction between origin California and variance in
cover, the pattern was reversed: for every unit of increase
in variance of cover at the source site, the probability to
germinate after dry-hot treatment (as compared to ger-
mination after dry storage) increased significantly (log
odds increased by 18.9; Table 2). The final Cox model did not reveal a significant influ-
ence of vegetation cover at the source sites on germin-
ation in general. Nevertheless, the survivor functions
indicate some differences among these seed sources. Chilean seeds from high cover source sites germinated
earlier and to a higher percentage than those from
the Chilean low cover source sites; especially under
temperatures of German autumn. German seeds from
higher cover source sites germinated later and to a Figure 2. Cumulated number of seeds that germinated during one of the three phases of the experiment, or that moulded or did not germin-
ate, given separately for (A) German, (B) Californian and (C) Chilean seed sources. Each bar represents one source population (cf. Supporting
Information—Table S4). The x-axis gives the variance of cover estimated at the source sites (note: for practical reasons variance is graphed
as levels and axes do not give continuous values). Figure 2. Significant effects of variation in cover at source
site on germination Cumulated number of seeds that germinated during one of the three phases
ate, given separately for (A) German, (B) Californian and (C) Chilean seed sources. Each
Information—Table S4). The x-axis gives the variance of cover estimated at the source
as levels and axes do not give continuous values). Figure 2. Cumulated number of seeds that germinated during one of the three phases of the experiment, or that moulded or did not germin-
ate, given separately for (A) German, (B) Californian and (C) Chilean seed sources. Each bar represents one source population (cf. Supporting
Information—Table S4). The x-axis gives the variance of cover estimated at the source sites (note: for practical reasons variance is graphed
as levels and axes do not give continuous values). 7 © The Author(s) 2018 AoB PLANTS https://academic.oup.com/aobpla Heger et al. – Differentiation in germination reflects vegetation cover For all seed sources, seeds that germinated after dry
storage did so during the first 36 days, afterwards (even
after further storage for 5 months) no additional ger-
mination occurred. In this experiment, a relatively high
proportion of seeds moulded (13.6 %). A chi-square test
(function ‘chisq.test’) revealed a significant difference in
proportions of moulded seeds across regions (P = 0.006),
with German seeds showing the lowest percentage of
moulding (8.4 %). For seeds from California and Chile,
the probability to mould was even higher than the prob-
ability to germinate directly after storage (Table 2). temperatures. Although the seeds had been scarified,
and although water was available, many seeds originat-
ing from California and Chile did not germinate. Baskin and Baskin (1998, pp. 305 and 307) report
rapid germination of E. cicutarium seeds following rain
in the field, and identify scarification as the main dor-
mancy breaking mechanism (p. 376). For seeds originat-
ing from Germany, this seems to be true, but not for the
other two source areas. Seeds originating from California
(and probably those from Chile) instead seem to have an
enduring combinatory dormancy: in addition to physical
dormancy, which is due to the hard seed coat and can
be broken by scarification, physiological dormancy is
hindering germination even after dry storage and even
if the seed coat is permeable and water is available. For
winter annuals with relatively weak physiological dor-
mancy, it has been observed that high summer tem-
peratures gradually break dormancy. Interlinked effects of temperature, source region
and mean vegetation cover at source site The results from Experiment 1 show that mean cover
of surrounding vegetation at the source site is linked to
differences in germination, but only in interaction with
temperature and source region. We found early ger-
mination in populations from high cover at source site
(see Hypothesis 1) for Chilean seeds grown under tem-
peratures simulating German autumn, but not for the
other sources or temperatures. High cover of surround-
ing vegetation thus does not seem to impose a clear
enough selective pressure to lead to early germination
across regions and temperatures. Temperature overall had a strong influence on ger-
mination. In the intermediate temperature regime cre-
ated to simulate Chilean autumn, all populations were
similar in terms of germination velocity and percent-
age of germinated seeds. However, in the temperature
regime with the highest temperatures (i.e. the one simu-
lating Californian autumn), we observed pronounced dif-
ferences in germination. For seeds from the cool German
climate, high temperatures induced early germination. Seeds originating from California (and according to
the survivor functions also those from Chile) showed
reduced and retarded germination under these high Table 2. Results of a multinomial regression with reference category ‘spontaneous’. Coefficients are given in bold where P-values calculated
from Wald ratios1 indicated significance (P < 0.05). The coefficients give an estimate for the log odds for each category in comparison to the
baseline, i.e. ‘spontaneous’. For instance, a one-unit increase in variance in cover is associated with a decrease in the log odds of germinating
after dry-hot treatment versus spontaneously of 21.36. 1R code used to calculate P-values: pchisq(summary.MNL$Wald.ratios^2, 1, low = F). Table 2. Results of a multinomial regression with reference category ‘spontaneous’. Coefficients are given in bold where P-values calculated
from Wald ratios1 indicated significance (P < 0.05). The coefficients give an estimate for the log odds for each category in comparison to the
baseline, i.e. ‘spontaneous’. For instance, a one-unit increase in variance in cover is associated with a decrease in the log odds of germinating
after dry-hot treatment versus spontaneously of 21.36. 1R code used to calculate P-values: pchisq(summary.MNL$Wald.ratios^2, 1, low = F). Significant effects of variation in cover at source
site on germination If such seeds are
exposed to high temperatures, they first gain the ability
to germinate at low temperatures (e.g. 6 °C/15 °C), and
only with additional loss of dormancy are they able to
also germinate at higher temperatures (e.g. 15 °C/25 °C;
Baskin and Baskin 1998, p. 58). A combination of phys-
ical and physiological dormancy has been reported for
Geranium carolinianum (Baskin and Baskin 1998, p. 123)
and G. robertianum (Vandelook and Van Assche 2010)
which, along with E. cicutarium, are members of the
Geraniaceae. Based on experiments done with seeds
collected along roadsides in Belgium, Van Assche and
Vandelook (2006) propose that both forms of dormancy
also exist in seeds of E. cicutarium, and that both at
the same time can be broken by dry storage leading to
high germination percentages at low temperatures in
autumn. It is very interesting that in our study, seeds
originating from California remained dormant in high
fractions, although they had been stored for 12 months,
exactly like the German seeds (only Chilean seeds had
been stored for a longer time period). For the Californian
seeds, this period of dry storage did not break the physio-
logical dormancy. Table 2. Results of a multinomial regression with reference category ‘spontaneous’. Coefficients are given in bold where P-values calculated
from Wald ratios1 indicated significance (P < 0.05). The coefficients give an estimate for the log odds for each category in comparison to the
baseline, i.e. ‘spontaneous’. For instance, a one-unit increase in variance in cover is associated with a decrease in the log odds of germinating
after dry-hot treatment versus spontaneously of 21.36. 1R code used to calculate P-values: pchisq(summary.MNL$Wald.ratios^2, 1, low = F). AoB PLANTS https://academic.oup.com/aobpla Higher variability in vegetation cover at the
source site is connected to higher probability for
germination directly after storage Jimenez et al. (2016) suggest, based on low germin-
ation percentages and high coefficients of variation in
seed size in Chile, that E. cicutarium is characterized by
a diversified bet-hedging strategy. Based on our data,
it is not possible to test this suggestion for the species
in general. The relatively high proportions of seeds that
did not germinate after physical dormancy was broken
(see Fig. 2), however, may be seen as indication for the
existence of a bet-hedging strategy in this species. In
any case, according to the results of Experiment 2, there
is no indication that high spatial variability in vegetation
cover induces delayed germination in a high propor-
tion of seeds as would be expected for a bet-hedging
strategy. Instead, across source regions, high variance
in vegetation cover at the source site is associated with
higher probability for germination after dry storage, and
thus lower numbers of dormant seeds. A potential ex-
planation for this observation is that if the future level
of competition is unpredictable, it may be advantageous
to germinate as soon as current temperature and water
availability allow for it, because high levels of competi-
tion could occur in which case fast germination could be
advantageous. In cases where future levels of competi-
tion (and facilitation) are more predictable, on the other
hand, it may be advantageous to wait for environmental
cues like dry-hot scarification, to assure a better timing
of the germination event. Because potential negative
effects of competitors here occur regularly, they could
have triggered adaptive variation in morphological or
other traits, making early germination less important. Heger et al. (2014) observed lower vegetative biomass
production and slower growth rates, and at the same
time lower abortion rates of developing seed in plants
originating from populations with high vegetation cover. Altered germination strategies in the introduced
range have been observed in other species (Beckmann
et al. 2011). However, based on our results, it is not clear
whether E. cicutarium dormancy strategies evolved in
the new range, or whether they have been carried over
from a different part of the native range. To be better
able to decide on this question, the experiment needs to
be repeated with seeds from populations in the native
Mediterranean region, especially because the likely re-
gion of origin of the Californian and Chilean populations
is Spain. Interlinked effects of temperature, source region
and mean vegetation cover at source site (Intercept)
Source region
California
Source region
Chile
Variance of cover
at source site
Source region California
× variance of cover
Source region Chile
× variance of cover
Dry-hot
−27.6428
29.0628
29.2283
−21.3641
18.8987
−38.3071
Scarification
−19.7016
19.0287
18.7088
−81.8308
71.9138
−151.2599
Moulded
−1.4749
2.5771
2.1557
−8.7310
6.3293
17.5775
No germination
−0.2848
2.3961
2.6281
−9.8595
11.1567
5.8745 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2018 8 Heger et al. – Differentiation in germination reflects vegetation cover under the cooler temperature regimes. The warmer
temperatures seem to favour germination in these
sources. In German sites, warm temperatures are not
necessarily connected to drought, and there probably
was no selective pressure inducing a forced dormancy
under high temperatures as in California and Chile. In
the temperate climate, a strict binding of germination to
autumn temperatures may not be necessary. Kudoh et al. (2007) observed suppressed germination
under higher temperature regimes in invasive popula-
tions of Cardamine hirsuta. Temperature-dependent dor-
mancy in this species seems to be mediated by abscisic
acid (ABA). Native populations did not consistently show
this behaviour; patterns of temperature effects on ger-
mination in the native populations were quite variable. Kudoh et al. (2007) suggest that suppressed germin-
ation at high temperature is a mechanism to assure
germination in autumn, and thus to reinforce a winter
annual life cycle. This suggestion seems reasonable also
for our study system. Availability of water resulting from
early rainfall events may not be a good enough predictor
for good growing conditions in Mediterranean climates,
because short periods of rain may still be followed by
hot and dry periods. Only the combination of available
water and cooler temperatures may be a reliable cue for
a grassland annual in California and Chile assuring high
survival rates of germinated seedlings. Further research
is needed to clarify the mechanisms underlying the
observed suppressed germination in these populations. AoB PLANTS https://academic.oup.com/aobpla Higher variability in vegetation cover at the
source site is connected to higher probability for
germination directly after storage Seeds originating from high cover sources in California
(and according to the life table estimates, also those
from Chile) germinated to a higher percentage and
also faster than those from low cover sources in these
regions. The high cover of surrounding vegetation may
have had a facilitative effect, attenuating the stressful
climatic conditions in California and Chile. This observa-
tion is in line with Hypothesis 2. As low germination per-
centages can be connected to the cost of lower overall
abundances, selection may have favoured higher fixed
rates of germination in populations where such facili-
tative effects can be expected. The faster germination
may have helped to lessen the negative effects of high
cover of neighbours in later life stages. German seed sources grown under the hot Californian
temperatures showed a pattern very different from
those of the other two source regions. Here, seeds from
high cover sources germinated later than those from
low cover sources, and a slightly higher proportion of
seeds stayed dormant. Overall, German seed sources
germinated earlier under Californian temperatures than Looking at seeds that originated in California only,
though, this result is reversed: the more variable the
vegetation cover, the less seeds germinate directly
after dry storage. A possible interpretation is that in
California, where we found delayed germination also
in response to high temperatures, unpredictability of
future cover of neighbours represents an additional 9 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2018 Heger et al. – Differentiation in germination reflects vegetation cover stressor also inducing a delay in germination. Follow-up
experiments testing for the interaction of high cover
and high variability in cover are needed to further clarify
the mechanisms causing these observations. Since our
experimental phases did not simulate the natural se-
quence of temperatures in either range, it would be
interesting to repeat this experiment in the field, ideally
with a higher number of seeds to be better able to mirror
the high number of seeds produced by this species. that in all three ranges, populations are differentiated in
terms of their germination behaviour as a consequence
of differences among populations in cover of surround-
ing vegetation. It seems unlikely that this pattern is due
to the introduction of several preadapted genotypes fol-
lowed by sorting processes. It is much more probable
that such a behaviour has evolved in each population
in each of the ranges independently. Higher variability in vegetation cover at the
source site is connected to higher probability for
germination directly after storage Parallel evolution
in the native and introduced range has been observed
before (Maron et al. 2004), but we found no study indi-
cating that such a pattern can arise in response to cover
of surrounding vegetation. Germination behaviour and population
differentiation match climatic conditions
and vegetation cover in native as well as
introduced ranges In both experiments, seeds from German origins
tended to germinate earlier and to higher proportions
than Californian and Chilean seeds. This pattern seems
to contradict findings in other species that invasive pop-
ulations have higher germination rates (Beckmann et al. 2011). Our experiment, though, was not designed to
allow for direct comparisons of native and invasive pop-
ulations. Native regions have not been replicated; more-
over, it is very likely that the populations in California
and Chile originate from native populations in Spain and
not Germany. Experiment 2 showed that German seeds are able to
germinate after 12 months of storage at room tempera-
ture, whereas seeds originating from Chile and California
showed significantly higher probabilities to germinate
after dry-hot scarification compared to after storage. Experiment 1 revealed a 2-fold dormancy in seeds from
California and probably Chile, allowing the seeds to stay
dormant, even if scarified, until lower temperatures are
reached. Taken together, these results suggest that ger-
mination behaviour in E. cicutarium varies among these
three regions in accordance with the respective climatic
conditions. In the Mediterranean regions Chile and
California, overall higher levels of dormancy, the occur-
rence of enduring physiological dormancy in addition
to physical dormancy and the responsiveness to dry-
hot scarification may all serve to fine-tune germination
events to the dry hot summer and rainy winter seasons. Conclusions Our study indicates that the cover of surrounding vege-
tation experienced in a population can influence ger-
mination behaviour across generations. Climate and
vegetation as well as other factors present in the ecosys-
tems may influence population differentiation and ger-
mination behaviour. Our results, however, indicate that
in the study systems, differing levels of vegetation cover
have driven population differentiation in germination
behaviour. This evolutionary response was rapid enough
to also occur within the introduced range. Because we
had harvested the seeds used in the experiments from
plants grown under uniform, glasshouse conditions, we
suppose that maternal effects do not contribute signifi-
cantly to the patterns we found. It is likely that native Mediterranean populations
show similar germination behaviour, and that already
the first plants introduced to California profited from
this preadaptedness. Alternatively, rapid adaptation
of germination behaviour in the new range may have
occurred. This has been found for C. solstitialis (Hierro
et al. 2009) as well as for Achillea millefolium, Hieracium
pilosella and Hypericum perforatum (Beckmann et al. 2011). Leiblein-Wild and Tackenberg (2014) found for
Ambrosia artemisiifolia that populations in the intro-
duced range matched environmental conditions; this
was not found, though, when investigating germination
rate and speed (Leiblein-Wild et al. 2014). Whether pre-
adaptation or evolution in the novel range have led to
the observed patterns in E. cicutarium has to be clarified
in future studies, e.g., by including populations from dif-
ferent parts of the native range. For spatial variation in vegetation cover, we found
effects that were independent of source region of the
seeds, with higher variability generally being connected
to higher fractions of seed germinating after dry storage. Mean cover of surrounding vegetation, though, seems
to affect the germination behaviour of populations dif-
ferently depending on the source region. In seeds from
Mediterranean climates, especially from California, we
observed delayed germination under hot temperatures
even though we had scarified the seeds and water was
available, which we interpret as indication for an add-
itional physiological dormancy in these populations. The While the effect of mean cover of surrounding vegeta-
tion on germination behaviour was not consistent across
regions, we found an effect of variability of cover that
was independent of seed source region. This indicates 10 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2018 Heger et al. – Differentiation in germination reflects vegetation cover and earlier versions of the manuscripts. J. Literature Cited Baskin CC, Baskin JM. 1998. Seeds. Ecology, biogeography, and evo-
lution of dormancy and germination. San Diego, CA: Academic
Press. T.H. received funding by Deutsche Forschungsgemein-
schaft (DFG) (grant numbers HE 5893/3-1 and HE 5893/3-
2). B.S.J. was supported by a NSF International Research
Fellowship Award (grant #0853094). This work was sup-
ported by the German Research Foundation (DFG) and
the Technical University of Munich (TUM) in the frame-
work of the Open Access Publishing Programme. Baskin JM, Baskin CC. 2000. Evolutionary considerations of claims
for physical dormancy-break by microbial action and abrasion
by soil particles. Seed Science Research 10:409–413. by soil particles. Seed Science Research 10:409–413. Baythavong BS. 2011. Linking the spatial scale of environmental
variation and the evolution of phenotypic plasticity: selection
favors adaptive plasticity in fine-grained environments. The
American Naturalist 178:75–87. Baythavong BS, Stanton ML. 2010. Characterizing selection on
phenotypic plasticity in response to natural environmental het-
erogeneity. Evolution 64:2904–2920. Supporting Information The following additional information is available in the
online version of this article— Table S1. Gives information on the collection sites for
Experiment 1. Table S2. Characterizes the three temperatures treat-
ments applied in Experiment 1. Table S2. Characterizes the three temperatures treat-
ments applied in Experiment 1. Table S2. Characterizes the three temperatures treat-
ments applied in Experiment 1. Table S3. Explains the dummy variables used for the
statistical analysis of Experiment 1. Table S3. Explains the dummy variables used for the
statistical analysis of Experiment 1. Table S4. Gives information of the collections sites for
Experiment 2. Conclusions Kollmann
commented on the experimental design and the draft
manuscript, and J. Joshi and two anonymous reviewers
made further helpful suggestions to improve the text. Permissions for seed collection and field work in nature
reserves in Germany were provided by Regierung von
Niederbayern (NSG Sandharlander Heide, 6 June 2011
and 13 August 2012) and Regierung von Oberbayern
(NSG Panzerwiese, 9 June 2011). delay in germination was less strong in populations that
had experienced high cover of surrounding vegetation. We suggest that high vegetation cover ameliorates heat
and drought, thus making the need for delayed germin-
ation less urgent. Under these conditions of potentially
strong selective pressures caused by heat and drought,
the facilitative effects of neighbours may be more im-
portant than competitive effects during later life stages. p
p
g
g
The strong differences in dormancy behaviour we
found among the populations in the native range and
the introduced ranges, with different cues inducing ger-
mination and an additional physiological dormancy only
in Californian (and maybe also Chilean) populations,
may be due either to the introduction of genotypes prea-
dapted to the Mediterranean climates in the introduced
range, or to evolutionary changes in the new range. We
believe the first option to be more likely. The differenti-
ation we observed in accordance with mean and vari-
ance in vegetation cover, opposed to this, we interpret
as fine-tuned adjustments to the local environments,
which are more likely to have evolved locally in each of
the populations. Conflict of Interest None declared. Brooks ML, Berry KH. 2006. Dominance and environmental corre-
lates of alien annual plants in the Mojave Desert, USA. Journal
of Arid Environments 67:100–124. Contributions by the Authors B.S.J. and T.H. collected the seeds and conceived and
designed the experiments; G.N. conducted the experi-
ments; T.H. performed the statistical analyses in the pre-
sented version and wrote the text; B.S.J. and G.N. edited
the text. Beckmann M, Bruelheide H, Erfmeier A. 2011. Germination
responses of three grassland species differ between native and
invasive origins. Ecological Research 26:763–771. Blackshaw RE. 1992. Soil temperature, soil moisture, and seed bur-
ial depth effects on Redstem Filaree (Erodium cicutarium) emer-
gence. Weed Science 40:204–207. Blackshaw RE, Harker KN. 1998. Redstem Filaree (Erodium cicu-
tarium) development and productivity under noncompetitive
conditions. Weed Technology 12:590–594. AoB PLANTS https://academic.oup.com/aobpla Acknowledgements Burke MJW, Grime JP. 1996. An experimental study of plant com-
munity invasibility. Ecology 77:776–790. V. Moos conducted Phase 1 of Experiment 2 as a stu-
dent’s project, and I. Kapps provided help during both
experiments. K. Rice provided helpful comments regard-
ing the experimental design, interpretation of results Cohen D. 1967. Optimizing reproduction in a randomly varying
environment when a correlation may exist between the condi-
tions at the time a choice has to be made and the subsequent
outcome. Journal of Theoretical Biology 16:1–14. 11 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2018 Heger et al. – Differentiation in germination reflects vegetation cover Evangelista D, Hotton S, Dumais J. 2011. The mechanics of explo-
sive dispersal and self-burial in the seeds of the filaree, Erodium
cicutarium (Geraniaceae). The Journal of Experimental Biology
214:521–529. McNair JN, Sunkara A, Frobish D. 2012. How to analyse seed
germination data using statistical time-to-event analysis:
non-parametric and semi-parametric methods. Seed Science
Research 22:77–95. Mensing S, Byrne R. 1998. Pre-mission invasion of Erodium cicutar-
ium in California. Journal of Biogeography 25:757–762. Fenner M, Thompson K. 2005. The ecology of seeds. Cambridge, UK;
New York, NY: Cambridge University Press. Figueroa JA, Castro SA, Marquet PA, Jaksic FM. 2004. Exotic plant
invasions to the Mediterranean region of Chile: causes, history
and impacts. Revista Chilena De Historia Natural 77:465–483. Novoa A, González L. 2014. Impacts of Carpobrotus edulis (L.)
N.E.br. on the germination, establishment and survival of native
plants: a clue for assessing its competitive strength. PLoS One
9:e107557. Fox J, Weisberg S. 2011. An {R} companion to applied regression. Thousand Oaks, CA: Sage. Novoplansky A. 2009. Picking battles wisely: plant behaviour under
competition. Plant, Cell & Environment 32:726–741. Grime JP, Mason G, Curtis AV, Rodman J, Band SR. 1981. A compara-
tive study of germination characteristics in a local flora. Journal
of Ecology 69:1017–1059. Oliveira G, Clemente A, Nunes A, Correia O. 2014. Suitability and
limitations of native species for seed mixtures to re-vegetate
degraded areas. Applied Vegetation Science 17:726–736. Heger T. 2016. Light availability experienced in the field affects abil-
ity of following generations to respond to shading in an annual
grassland plant. Journal of Ecology 104:1432–1440. Orrock JL, Christopher CC. 2010. Density of intraspecific competi-
tors determines the occurrence and benefits of accelerated ger-
mination. American Journal of Botany 97:694–699. Heger T, Jacobs BS, Latimer AM, Kollmann J, Rice KJ. 2014. Acknowledgements Does
experience with competition matter? Effects of source com-
petitive environment on mean and plastic trait expression in
Erodium cicutarium. Perspectives in Plant Ecology Evolution and
Systematics 16:236–246. Poschlod P, Kleyer M, Jackel A-K, Dannemann A, Tackenberg
O. 2003. BIOPOP - a database of plant traits and inter-
net application for nature conservation. Folia Geobotanica
38:263–271. Hegi G. 1964. Illustrierte Flora von Mittel-Europa. Mit besonderer
Berücksichtigung von Deutschland, Österreich und der Schweiz. Zum Gebrauch in Schulen und zum Selbstunterricht. Jena,
Germany: Weissdorn-Verlag. Postma FM, Lundemo S, Ågren J. 2016. Seed dormancy cycling and
mortality differ between two locally adapted populations of
Arabidopsis thaliana. Annals of Botany 117:249–256. R Core Team. 2014. R: a language and environment for statis-
tical computing. Vienna, Austria: R Foundation for Statistical
Computing. http://www.R-project.org/. (30 October 2015) Heiberg RM. 2016. Statistical analysis and data display: Heiberger
and Holland. R package version 3.1-31. http://CRAN.R-project. org/package=HH. (23 June 2016) Computing. http://www.R-project.org/. (30 October 2015) Rice KJ. 1985. Responses of Erodium to varying microsites: the role
of germination cueing. Ecology 66:1651–1657. Hierro JL, Eren Ö, Khetsuriani L, Diaconu A, Török K, Montesinos D,
Andonian K, Kikodze D, Janoian L, Villarreal D, Estanga-Mollica
ME, Callaway RM. 2009. Germination responses of an invasive
species in native and non-native ranges. Oikos 118:529–538. Rice KJ. 1987. Evidence for the retention of genetic variation in ero-
dium seed dormancy by variable rainfall. Oecologia 72:589–596. Roberts HA. 1986. Seed persistence in soil and seasonal emergence
in plant species from different habitats. Journal of Applied
Ecology 23:639–656. Jimenez MA, Gaxiola A, Armesto JJ, Gonzalez-Browne C, Meserve
PL, Kelt DA, Gutierrez JR, Jaksic FM. 2016. Bet-hedging strategies
of native and exotic annuals promote coexistence in semiarid
Chile. Journal of Arid Environments 126:62–67. Schwaegerle KE, Bazzaz FA. 1987. Differentiation among nine popu-
lations of Phlox: response to environmental gradients. Ecology
68:54–64. Klein JP, Moeschenberger ML, Yan J. 2012. KMsurv. R package version
0.1-5. Original by Klein, Moeschberger and modifications by Jun Yan
(2012). http://CRAN.R-project.org/package=KMsurv (24 June 2016). Simons AM. 2011. Modes of response to environmental change and
the elusive empirical evidence for bet hedging. Proceedings of
the Royal Society B-Biological Sciences 278:1601–1609. Kudoh H, Nakayama M, Lihová J, Marhold K. 2007. Does invasion
involve alternation of germination requirements? A compara-
tive study between native and introduced strains of an annual
Brassicaceae, Cardamine hirsuta. Ecological Research 22:869–875. Therneau T. 2015. A package for survival analysis in S. version 2.38. Acknowledgements Therneau T. 2015. A package for survival analysis in S. version 2.38. http://CRAN.R-project.org/package=survival (23 June 2016). http://CRAN.R-project.org/package=survival (23 June 201 Tielboerger K, Petru M, Lampei C. 2012. Bet-hedging germination in
annual plants: a sound empirical test of the theoretical founda-
tions. Oikos 121:1860–1868. Leiblein-Wild MC, Kaviani R, Tackenberg O. 2014. Germination and
seedling frost tolerance differ between the native and invasive
range in common ragweed. Oecologia 174:739–750. Van Assche JA, Vandelook FEA. 2006. Germination ecology of eleven
species of Geraniaceae and Malvaceae, with special reference to
the effects of drying seeds. Seed Science Research 16:283–290. Leiblein-Wild MC, Tackenberg O. 2014. Phenotypic variation
of 38 European Ambrosia artemisiifolia populations meas-
ured in a common garden experiment. Biological Invasions
16:2003–2015. Vandelook F, Van Assche JA. 2010. A combined physical and physio-
logical dormancy controls seasonal seedling emergence of
Geranium robertianum. Plant Biology 12:765–771. Maron JL, Vila M, Bommarco R, Elmendorf S, Beardsley P. 2004. Rapid evolution of an invasive plant. Ecological Monographs
74:261–280. Venables WN, Ripley BD. 2002. Modern applied statistics with S, 4th
edn. New York, NY: Springer. 12 © The Author(s) 2018 AoB PLANTS https://academic.oup.com/aobpla
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The system of lessons and lessons of mathematics in elementary grades
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TECHNIQUE AND IT. JOURNAL 2023 TECHNIQUE AND IT. JOURNAL 2023 MATYAQUBOVA FOTIMA URINBAYEVNA
URGANCH DAVLAT UNIVERSITETI TALABASI Annotatsiya: Boshlang‘ich sinflarda matematika darsi, uning xususiyatlari,
darsni tshkil qilish tizimi, darsga qo‘yilgan talablar to‘g‘risidagi bilimlarni
mustahkamlash. Kalit so‘zlar: Dars, dars tizimi, dars mazmuni, dars tarkibi, dars tiplari,
matеmatika darslari xususiyatlari, matеmatika darsi turlari: murakkab dars, yangi
matеrialni o‘rganish darsi, mustahkamlash darsi, umumlashtiruvchi dars, nazorat
qilish darslari. Аннотация: Закрепление знаний об уроке математики в начальных
классах, его особенностях, системе организации урока, требованиях к
проведению урока. Ключевые слова: Yрок, система урока, содержание урока, содержание
урока, виды урока, особенности урока математики, виды урока математики:
комплексный урок, урок для изучения нового материала, урок-закрепление,
урок-обобщающий, контрольный урок. Abstract: Strengthening knowledge about the mathematics lesson in
primary grades, its features, lesson organization system, requirements for the
lesson. Key words: Lesson, lesson system, lesson content, lesson content, lesson
types, features of mathematics lessons, types of mathematics lesson: complex
lesson, lesson for learning new material, reinforcement lesson, summarizing
lesson, control lessons. The lesson is a historical, complex form of organizing the teaching of 1 TECHNIQUE AND IT. JOURNAL 2023 mathematics at school, proven by many years of experience and meeting the basic
requirements of our time. The acquisition of mathematical knowledge by students
depends not only on the choice of the correct method in teaching, but also on the
form of organization of the educational process. A lesson is an educational work organized by a fixed number of students
under the guidance of a teacher, according to a specific schedule, according to the
program. During the lesson, students learn the theoretical information from
mathematics, counting skills, problem solving, various measurements, that is, all
educational work is performed in the classroom. The uniqueness of the
mathematics lesson, first of all, follows from the features of this academic subject. One of its features is that, along with arithmetic material, elements of algebra and
geometry are also studied. Another peculiar side of the elementary course of
mathematics is the joint consideration of theoretical problems. That is why
practical skills and abilities are instilled in each lesson with the introduction of new
knowledge. Usually, several didactic goals are realized in the lesson: the passage of new
material; strengthening the subject; strengthening knowledge; generalization,
systematization of knowledge; creating reliable learning and skills, etc. Another
feature of mathematics lessons is the abstractness of the teaching material. Therefore, it also depends on visual aids, careful selection of active teaching
methods, student activity, and the skill level of class students. 5. Testing knowledge and skills. 5. Testing knowledge and skills. 5. Testing knowledge and skills. Most of the methodological literature is based on the division of lessons
according to their didactic purpose. Below are the types of math lessons: 1. A lesson in mastering new knowledge. In them, students get acquainted
with new concepts, calculation methods, solutions to new types of problems, new
properties of figures, numbers; 2. A lesson to consolidate knowledge, skills and abilities. They work
examples and tasks related to consolidating the knowledge, skills and abilities
gained in the previous lesson; 3. Repetition - lessons-generalizations. Examples of this include repetition
and debriefing at the end of a particular section, or at the beginning and end of a
school year; 4. Lesson control of knowledge, skills and abilities. 4. Lesson control of knowledge, skills and abilities. 5. A mixed or complex lesson in which there are several didactic goals, and
all of them are important. 5. A mixed or complex lesson in which there are several didactic goals, and
all of them are important. In conclusion, we can say that one of the important requirements of our time
is the activation of the knowledge and creative activity of students. Each lesson
should focus on thinking and creativity. MATYAQUBOVA FOTIMA URINBAYEVNA
URGANCH DAVLAT UNIVERSITETI TALABASI Another requirement
for modern mathematics classes is the development of independent thinking and
creative activity of students. The student develops such mental operations as
analysis, synthesis, comparison, generalization, conclusion. The system of
mathematics lessons in elementary grades - in each lesson, students work with
several concepts. Each of them can be mastered at different stages of this lesson. The understanding of each concept is carried out by repetition, recall of another 2 TECHNIQUE AND IT. JOURNAL 2023 concept, and this concept serves to explain the following concepts. In the learning process, each training material is presented in expanded form,
this training material is the foundation for understanding the materials taught after
it. If we consider the process of mastering another concept, then it is formed as a
result of the interconnected teaching of several lessons. Thus, the formation of
mathematical concepts is formed not in one lesson, but in the process of passing a
series of interrelated lessons. We call such classes a shared class system. Therefore, the teacher must arrange the lessons in a logical sequence that reveals
the content of the subject. The biggest requirement in the structure of the lesson system is to take into
account the educational purpose of the lesson, taking into account the
methodological and general pedagogical aspects of the principles of teaching. A
well-thought-out system of classes on a topic depends on the correct distribution of
study time by topic. It should be aimed at developing students' independence by
considering specific examples, drawing specific conclusions from them and
drawing general conclusions from them. After this knowledge is formed and fixed
in the lesson system, examples and tasks should be solved. After that, it is
necessary to develop the skills through exercises, as well as to ensure the
continuous integration and generalization of the acquired knowledge. When
determining the content of the topic of the program, distributing the material of the
topic for class hours, that is, obtaining knowledge, the following main stages are
considered. 1. Preparation of new material for training. 2. Perception of new educational material and creation of new knowledge. 3. Consolidation of knowledge and the formation of skills through various
exercises. 4. Repetition, generalization and systematization of knowledge. 3 TECHNIQUE AND IT. JOURNAL 2023 References: 1. Dzhumaev M.E. Workshop on the methodology of teaching mathematics in
elementary grades. (for O O'Y) Tashkent. "Teacher" 2004 1. Dzhumaev M.E. Workshop on the methodology of teaching mathematics in
elementary grades. (for O O'Y) Tashkent. "Teacher" 2004
2. Dzhumaev M.E. Methodology for organizing laboratory classes in mathematics
in elementary grades. Tashkent. "Generation of the new century" 2006. 20 b / t. 3. Tadzhieva Z.G., Abdullaeva B.S., Dzhumaev M.E., Sidelnikova R.I., Sadykova
A.V. Methods of teaching mathematics.-T. "Turon-Iqbal" 2011. 336s. 2. Dzhumaev M.E. Methodology for organizing laboratory classes in mathematics
in elementary grades. Tashkent. "Generation of the new century" 2006. 20 b / t. 3. Tadzhieva Z.G., Abdullaeva B.S., Dzhumaev M.E., Sidelnikova R.I., Sadykova
A.V. Methods of teaching mathematics.-T. "Turon-Iqbal" 2011. 336s.
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The unique social sense of puerperium: Increased empathy and Schadenfreude in parents of newborns
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The unique social sense of
puerperium: Increased empathy
and Schadenfreude in parents of
newborns Ana-María Gómez-Carvajal1,2,14, Hernando Santamaría-García3,4,14, Adolfo M. García 5,6,7,
Mario Valderrama9, Jhony Mejia9, Jose Santamaría-García1, Mateo Bernal1, Jaime Silva10,
Agustín Ibáñez 5,6,11,12,13 & Sandra Baez1,14* Pregnancy and puerperium are typified by marked biobehavioral changes. These changes, which are
traceable in both mothers and fathers, play an important role in parenthood and may modulate social
cognition abilities. However, the latter effects remain notably unexplored in parents of newborns (PNs). To bridge this gap, we assessed empathy and social emotions (envy and Schadenfreude) in 55 PNs
and 60 controls (childless healthy participants without a romantic relationship or sexual intercourse in
the previous 48 hours). We used facial electromyography to detect physiological signatures of social
emotion processing. Results revealed higher levels of affective empathy and Schadenfreude in PNs,
the latter pattern being accompanied by increased activity of the corrugator suppercilii region. These
effects were not explained by potential confounding variables (educational level, executive functioning,
depression, stress levels, hours of sleep). Our novel findings suggest that PNs might show social
cognition changes crucial for parental bonding and newborn care. In the course of adult development, the transition to parenthood defines a new stage marked by unique biobehav-
ioral changes1,2. In particular, puerperium is a special and variable period in the life of all who become parents,
comprising between 6 and 8 weeks after childbirth3. The evolutionary pressure for survival forces parents to pro-
vide care and well-being to their offspring4, leading to new demands on the familial context and major changes
in social relationships2. These changes can modulate specific social cognition domains. Social cognition refers to
“the set of mental operations underlying social interactions”5, which includes processes implicated in perceiving,
interpreting, and generating responses to the intentions and behaviors of others5,6. Among these, empathy plays a
key role throughout puerperium, as it proves crucial to acknowledge and address the infant’s needs in the absence
of verbal communication7. Surprisingly, however, social cognition abilities in parents of newborns (PNs) have not
been previously studied. p
y
Neurobiological and endocrine adaptations during pregnancy and postpartum include an increase in hor-
mones (e.g., prolactin, oxytocin (OXY), progesterone) and the expression of specific hormonal neuroreceptors8,9,
which prepare the body for childbirth, nursing, and upbringing10. Indeed, some authors propose that pregnancy 1Universidad de los Andes, Bogotá, Colombia. 2Neuroscience Research Group NEUROS, School of Medicine and
Health Sciences, Universidad del Rosario, Bogotá, Colombia. 3Memory and cognition Center, Intellectus. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 www.nature.com/scientificreports/ In fact, increased maternal sensitivity to mother-baby
interactions during postpartum8,15 is associated with amplified protective mechanisms for warning and defense1
and hypervigilance towards signals of threat and potential harm8. These changes influence how PNs process emo-
tional information in the environment and how they shape their behaviors accordingly8,10. y
p
g y
Baby stimuli activate the “parenting” brain circuits, which share cortico-limbic circuits that regulate other
forms of social attachment and behavior, and they are more active during early post-partum7. Also, parental sen-
sitivity is critically related to modulations of executive functions, including attentional control, working memory,
and flexible task-switching15, emotion regulation, reward/motivation system, and parental thoughts15. Parents
experience a dynamic change in their thoughts and behavior and this intense and chronic mental focus activates
motivational-reward pathways and empathy15. p
y
p
y
Taken together, these findings suggest that PNs might experience major adaptations in their social cogni-
tion abilities. Although no experimental study has examined the issue, these changes might prove particularly
conspicuous in particular affective domains, such as empathy and social emotions22. Essential for human inter-
action23,24, these two domains underpin the welfare and interactive dynamics of social groups, and they may be
modulated by circumstances beyond self-experiences and interests23–25, such as the transition to parenthood. Empathy comprises the “capacity to share and understand the subjective experience of others in reference to one-
self”23. This complex construct involves at least three primary components: (a) an affective response to another
person (i.e., sharing the other person’s emotional state); (b) a cognitive capacity to take the perspective of other
individual; and (c) regulatory mechanisms that monitor the origins of self- and other-feelings26,27. Specifically,
empathy may be heightened in PNs as it favors caregiving and the perception of social cues from infants15,22. Furthermore, higher empathy levels in parents increase secure attachment in children22. g
y
Also, substantial changes might be expected in the realm of social emotions. Unlike non-social emotions,
social emotions depend on other people’s feelings, actions or thoughts28–30. Besides, non-social emotions are often
provoked by stimuli with a direct physiological relevance, while social emotions appear in social interactions31. Envy and Schadenfreude–pleasure at others’ misfortunes– are two social emotions of which experience may be
modulated by the biobehavioral changes characterizing transition to parenthood. Envy refers to the discomfort associated with another person’s good fortune, while Schadenfreude denotes
the perceiver’s pleasure at another’s unfortunate situation32,33. www.nature.com/scientificreports/ www.nature.com/scientificreports/ and postpartum involve cognitive reorganizations10 and an increase in maternal sensitivity7,8. These changes affect
how women respond to emotional information in their environment and how they shape their behaviors in
response to exclusive stimuli of motherhood or mother-baby interactions8. In fact, increased emotional reactivity
reflects the sensitivity of the mother to external stimuli and has a direct effect on the way the mother responds to
her surroundings, in particular, to the signals from her baby10. g
p
g
y
In line with these findings, several studies have found changes in cortical volume associated to sexual steroid
hormones that regulate neuronal morphology7,11. Indeed, after birth and two years afterwards, women exhibit
reduced gray matter volumes in regions subserving social cognition (i.e., amygdala, insula, precuneus, superior
temporal and medial prefrontal areas)11. In addition, greater neural activity in these regions has been observed
in response to the visual signals of the babies. These brain changes predict measures of postpartum maternal
attachment, suggesting an adaptive process serving the transition into motherhood11. Moreover, functional neu-
roimaging studies have shown that hypo-oxytocinergic non-breastfeeding mothers as well as non-parents exhibit
decreased responses to visual signals of the babies in areas that have OXY receptors or direct connections to
oxytocin-sensitive areas7. Even though maternal caregiving involves neurobiological processes related to pregnancy and labor, the
father’s brain also adapts to the parental role as well12,13. In particular, testosterone and prolactin levels of fathers
are key mediators of paternal behavior: lower testosterone and higher prolactin levels have been associated with
higher sympathy and paternal alertness as well as positive responses to infant cries14. Immediately after birth, and
during the first few months, parents focus on the infants’ physical and psychological needs, increasing their pri-
mary maternal preoccupation, which will lead to developing emotional bonds while fostering empathy and emo-
tional recognition15. Research has shown an association of maternal and paternal OXY levels with parent-infant
synchrony16, showing increased levels during the first six months after birth13,17. OXY levels are related with
the parent-specific repertoire12; they have a crucial role in the regulation of pro-social and affiliative behavior
(e.g., mating, pair-bond formation, maternal/parental behavior, attachment), facilitating social cognition in
humans18,19. Moreover, PNs experience widespread changes in varied behavioral domains, such as contextual threat detec-
tion, emotional expression, and social attachment1,20,21. The unique social sense of
puerperium: Increased empathy
and Schadenfreude in parents of
newborns Hospital
Universitario San Ignacio, Bogotá, Colombia. 4Pontificia Universidad Javeriana, Departments of Physiology,
Psychiatry and Aging Institute, Bogotá, Colombia. 5Universidad de San Andrés, Buenos Aires, Argentina. 6National Scientific and Technical Research Council (CONICET), Buenos Aires, Argentina. 7Faculty of Education,
National University of Cuyo (UNCuyo), Mendoza, Argentina. 8Departamento de Lingüística y Literatura, Facultad
de Humanidades, Universidad de Santiago de Chile, Santiago, Chile. 9Department of Biomedical Engineering,
Universidad de los Andes, Bogotá, Colombia. 10Department of gynecology and obstetrics, Hospital Universitario
San Ignacio, Bogotá, Colombia. 11Universidad Autónoma del Caribe, Barranquilla, Colombia. 12Center for Social
and Cognitive Neuroscience (CSCN), School of Psychology, Universidad Adolfo Ibáñez, Santiago de Chile, Chile. 13Australian Research Council Center of Excellence in Cognition and its Disorders, Sydney, Australia. 14These authors
contributed equally: Ana-María Gómez-Carvajal, Hernando Santamaría-García and Sandra Baez. *email: sj.baez@
uniandes.edu.co entific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 Methods Participants. The study comprised 115 healthy adults from the same geographical area, namely: 55 PNs
(31 women, 24 men) and 60 controls (35 women, 25 men). We excluded four PNs and four control participants
because they not complete the totality of the protocol. Mothers or biological fathers of newborns were recruited
from Hospital Universitario San Ignacio (Bogota, Colombia). For each newborn, only one parent (the mother
or the father) participated in this study. They were evaluated after labor, within the first 6 and 24 hours after
childbirth and once breastfeeding had been initiated. No participants had a history of neurological or psychiatric
disorders, or of alcohol or drug abuse. Controls neither had children, a romantic relationship nor have had sexual
intercourse in the previous 48 hours. Also, all control women were in the luteal phase of the menstrual cycle or
using hormonal contraceptives. Participants in both groups were matched for sex, age, and depression symptoms
(Table 1). However, PNs and controls differed in educational level, executive functioning, perceived stress levels,
and hours of sleep during the previous week (Table 1). These differences were included as covariates in all social
cognition analyses. All subjects participated voluntarily and signed informed consent prior to the evaluation in
agreement with the Helsinki declaration. The study was approved by the Ethics Committees of Universidad de
Los Andes and Hospital Universitario San Ignacio. Instruments. The control group was evaluated in a laboratory room. The PNs were evaluated in a separate
quiet room at the Gynecology Service of the Hospital Universitario San Ignacio. Empathy. Empathy was assessed with the Interpersonal Reactivity Index (IRI)45. This self-report question-
naire measures four dimensions of affective and cognitive empathy, namely: (a) fantasy, i.e., the capacity or ten-
dency to identify with characters of novels and films; (b) perspective taking, i.e., the ability to understand the
position and arguments of others; (c) empathic concern, i.e., feelings of sympathy, compassion, and concern
(e.g., the capacity to feel what others feel); and (d) personal distress, i.e., feelings of anxiety or discomfort when
observing negative experiences to third parties. The IRI comprises 28 situations, seven for each subscale, which
are individually rated on a scale from 0 (does not describe me well) to 4 (describes me very well). Four scores were
derived from this scale, one for each dimension. Social emotions. www.nature.com/scientificreports/ Still,
no previous studies have compared EMG correlates of these two social emotions (envy and Schadenfreude) nor
assessed their modulations in PNs. elicit greater responses on the zygomaticus major when paired with negative (relative to positive) events42. Still,
no previous studies have compared EMG correlates of these two social emotions (envy and Schadenfreude) nor
assessed their modulations in PNs. Against these antecedents, we evaluated empathy and social emotions in mothers and fathers of newborns
(immediately postpartum, only a few hours after giving birth), compared to a control group. In addition, we
assessed whether empathy and social emotion outcomes in PNs were influenced by executive functions (EFs),
given that they have been associated both domains in other populations38,44. Also, considering that social cogni-
tion may be modulated by other experiential factors, we controlled for perceived stress levels, depression symp-
toms, and hours of sleep. In brief, this study aims to add new information for the emergent agenda regarding
socio-emotional adaptations during pregnancy and puerperium. www.nature.com/scientificreports/ These two social emotions are fundamental
in regulating social behavior and maintaining stability of social interactions34,35. Furthermore, both envy and
Schadenfreude are boosted when individuals make upward comparisons35. Also, these emotions are involved in
stabilizing tensions associated with experiencing inferior roles in hierarchical social contexts32,34,36. Moreover,
both emotions are related. Specifically, Schadenfreude is more likely to appear when a misfortune happens to an
envied person33,37. Besides, these emotions are manifested in the affective appraisal of situations that foreground
other people’s fortune relative to social norms, notions of justice, and social welfare38. In puerperal stages, PNs
increase their capacity to signal and understand social and emotional cues that could affect the baby’s well-being22
–an effect that might, in turn, modulate the experience of social emotions. f
g
p
To trigger envy or Schadenfreude, previous studies e.g.33,34,38–40, have used experimental tasks in which fortu-
nate or unfortunate events happen to fictional individuals. In these tasks, after reading the scenarios, participants
report (by means of scales) how much envy or pleasure (Schadenfreude) they felt for the characters. Besides,
facial electromyography (EMG) has been used to detect micro-movements related to the expression of envy and
Schadenfreude), given its relevance to overcome social desirability biases proper to subjective social emotion
instruments41–43. Schadenfreude expressions have been related with increased activity in the zygomaticus major41,42
and the orbicularis oculi41, alongside decreased activity in the corrugator supercilii41. In particular, envied targets Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 www.nature.com/scientificreports/ PNs (n = 55)
Controls
(n = 60)
PNs vs. controls
95% CI
Mean/SD
Mean/SD
p-value
Lower
Upper
Demographics
Age (years)
28.40/5.16
26.97/6.38
0.202
−0.779
3.646
Sex (F:M)
31:24
35:25
0.831
Education (years)
14.16/3.99
16.22/2.60
0.001
−3.287
−0.819
Executive functions assessment
Total IFS score
23.42/3.06
26.0/1.87
<0.001
−3.509
−1.655
Relevant variables
Hours of Sleep
5.31/1.86
6.03/1.29
0.014
−1.286
−0.149
ZDS
35.13/8.48
35.82/7.48
0.644
−3.64
2.260
PSS
26.28/7.80
22.45/8.34
0.013
0.822
6.834
Table 1. Demographic data, EFs and other relevant variables. PNs: parents of newborns; IFS: INECO Frontal
Screening battery; ZDS: Zung Depression Scale; PSS: Perceived Stress Scale. Table 1. Demographic data, EFs and other relevant variables. PNs: parents of newborns; IFS: INECO Fronta
Screening battery; ZDS: Zung Depression Scale; PSS: Perceived Stress Scale. elicit greater responses on the zygomaticus major when paired with negative (relative to positive) events42. Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 Methods The corrugator
supercilii and depressor supercilii enable crinkling of the eyebrow, the orbicularis oculi closes the eyelids and allows
the person to blink, and the zygomaticus major helps in smiling by pulling the perioral muscles upward46.h p
yg
j
p
g y p
g
p
p
Electrodes were placed in accordance with the Guidelines for Human Electromyographic Research47. The
ground electrode was placed at the midline approximately 3–4 cm superior to the upper borders of the inner
brows. For the corrugator supercilii, one electrode was affixed above the brow and the other electrode was posi-
tioned 1 cm lateral to, and slightly superior to, the first one on the border of the eyebrow. The depressor supercilii
shares a muscle with the corrugator supercili and thus and electrode, the first one mentioned above. We used a
jumper in order to use a single electrode for this same muscle. The second electrode for the depressor supercilii was
placed at a 1-cm distance on the side of the nasal bone. The first electrode in the orbicularis oculi was affixed 1 cm
inferior to the commissure of the eye fissure, and the other one was located 1 cm medial to, and slightly inferior to
the first, so that the electrode pair is parallel to the lower edge of the eyelid. Finally, to measure the electric activity
of the zygomaticus major, we placed one electrode midway along an imaginary line joining the cheilion and the
preauricular depression, and the second electrode was placed 1 cm inferior and medial to the first one along the
same imaginary line47. To hold the electrodes, we used Ten20 Conductive Paste and hypoallergenic tape. The
signals were converted to digital data at a sampling rate of 256 Hz48.f g
g
p
g
Several pilot tests were done to verify there was no electrical interference that could affect the EMG signal. Participants were asked to move as little as possible so as not to affect the EMG signal through body movements. Data analysis. Behavioral data. Demographic, cognitive state, and behavioral data were compared among
groups via independent sample t-tests. Chi-square tests were applied to analyze categorical variables (i.e., sex). All reported results were adjusted with Benjamini-Hochberg correction for multiple comparisons49. Methods We employed a previously reported38–40 computerized task involving sentences designed
to trigger envy (e.g., “She/he managed to get accepted at the university because she/he is the son/daughter of
the Dean”) and Schadenfreude (e.g., “She/he was discovered as being corrupt and he/she was denounced”) by
depicting everyday justice-related situations. The task was divided into two blocks: the first one comprised sen-
tences that evoked envy, and the second one contained sentences that evoked Schadenfreude –this sequencing
was adopted given that envy and Schadenfreude are interdependent and the former could promote the latter33. Furthermore, five neutral events were included in each block for control purposes. In each block, participants
read 20 different sentences presented in pseudorandomized order on a computer screen. After reading each sen-
tence, participants indicated having read the full sentence by pressing the space bar. Then, they reported, with Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 www.nature.com/scientificreports/ the keyboard, the intensity of their displeasure (envy) or pleasure (Schadenfreude) using a 9-point Likert scale
ranging from 1 (low emotional intensity) to 9 (high emotional intensity). Thus, the inter-stimulus interval was
~8 seconds (M = 7.90, SD = 2.40). (
,
)
Envy and Schadenfreude stimuli had similar linguistic properties in terms of length of the sentences, complex-
ity, and grammatical structure (see details in S2 and Supplementary Table 1). Electromyography recordings. Following reported procedures41, we used EMG to detect
micro-movements of facial expressions related to envy and Schadenfreude in a subsample of participants (45 PNs
and 23 controls). Registers of remaining participants were excluded because they had completed less than 80% of
the trials due to technical problems and/or the presence of excessive signal noise. We used a Micromed SD LTM
EXPRESS 64 device. Before electrode placement, the skin was cleansed with alcohol and rubbed with electrode
paste. Four bipolar electrodes were used to measure electrical activity while participants performed the exper-
imental task. As in previous EMG studies on social emotions41,42, the electrodes were located on the left side of
the participants’ face, over four muscles: (a) corrugator supercili, (b) depressor supercilii, (c) orbicularis oculi, and
(d) zygomaticus major. Additionally, one electrode was placed on the forehead as a ground signal. Methods Given that
biobehavioral changes associated to parenthood differ between women and men8,9, and considering that mode of
delivery may affect hormonal levels (e.g., OXY) associated with maternal behavior50, dependent variables yielding
significant between-group differences were subjected to additional subgroup analyses comparing: (a) mothers vs. fathers, (b) women with vaginal delivery vs. C-section, and (c) women with and without induced labor. Alpha
levels were set at 0.05 for all analyses. Effect sizes were calculated through Cohen’s d, with cut-offs of 0.20, 0.50,
and 0.80 for small, middle, and large effects, respectively51. gf
p
y
A post hoc sensitivity analysis was performed using G*power52. Results of this analysis showed that, assuming
a power of 0.80 and α level of 0.05, our sample size was sufficient to detect a medium effect size (d = 0.52, critical
t = 1.98). Given that educational level, executive functioning, perceived stress levels and hours of sleep may be con-
founding variables, we included these measures in analyses of covariance (ANCOVA) to control for their influ-
ence on empathy and social emotion outcomes. Effect sizes for these analyses were calculated through partial eta
squared (η2). Also, we conducted multiple regression analyses to explore whether empathy and social emotion out-
comes abilities were associated with educational level, EFs, sex, perceived stress levels or mean hours of sleep
during the previous week. Specifically, we estimated regression models in which measures yielding significant
between-group differences were considered as dependent variables. Group, sex, IFS scores, hours of sleep, and
perceived stress levels were included as predictive factors in all models. The latter two measures were included as
predictors, given that we found differences between groups in perceived stress levels and hours of sleep during
the previous week (see details in S3). EMG data. Following previous procedures53, we filtered raw data using a high-pass filter of 20 Hz to eliminate
low-frequency noise. Similarly, a notch-filter of 60 Hz was implemented to attenuate electromagnetic noise47. After the filtering process, the response to each stimulus was represented as the average signal of a two-second
post-trigger time interval (2 seconds after the stimuli presentation). As in previous EMG studies54, all signals were
standardized considering baseline activity levels elicited through neutral stimuli. The neutral response activity
from each time bin was subtracted from the corresponding activity levels triggered in envy and Schadenfreude
situations. Methods We did not consider traditional pre-trigger muscle activity because, in our case, it corresponded to the
rating of the previous sentence and it could add undesired facial expressions.t EMG data. Following previous procedures53, we filtered raw data using a high-pass filter of 20 Hz to eliminate
low-frequency noise. Similarly, a notch-filter of 60 Hz was implemented to attenuate electromagnetic noise47. After the filtering process, the response to each stimulus was represented as the average signal of a two-second
post-trigger time interval (2 seconds after the stimuli presentation). As in previous EMG studies54, all signals were
standardized considering baseline activity levels elicited through neutral stimuli. The neutral response activity
from each time bin was subtracted from the corresponding activity levels triggered in envy and Schadenfreude
situations. We did not consider traditional pre-trigger muscle activity because, in our case, it corresponded to the
rating of the previous sentence and it could add undesired facial expressions.t g
p
p
After baseline correction, and in line with previous procedures55–57, EMG data were transformed into z-scores
to remove variability and allow for direct comparisons between conditions. For each participant, z-scores were
calculated based on the participants’ averages and the mean and standard deviation of the whole sample. If the Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 www.nature.com/scientificreports/ Figure 1. (A) Empathy levels in both groups, as tapped through the IRI subscales. (B) Social emotion ratings in
both groups, for envy and Schadenfreude. Figure 1. (A) Empathy levels in both groups, as tapped through the IRI subscales. (B) Social emotion ratings in
both groups, for envy and Schadenfreude. igure 1. (A) Empathy levels in both groups, as tapped through the IRI subscales. (B) Social emotion ratings in
oth groups, for envy and Schadenfreude. z-score of a participant’s muscle activity was deviant (i.e., more than 2 SD away from the sample’s mean), the asso-
ciated EMG data was excluded. Whole data from six PNs and three control participants were excluded using this
data cleaning. After this procedure, the final sample size for EMG data was 39 PNs and 20 controls.h z-score of a participant’s muscle activity was deviant (i.e., more than 2 SD away from the sample’s mean), the asso-
ciated EMG data was excluded. Whole data from six PNs and three control participants were excluded using this
data cleaning. Methods After this procedure, the final sample size for EMG data was 39 PNs and 20 controls.h gti
Then, we conducted 2 (condition: envy and Schadenfreude) × 2 (group: PNs and controls) ANOVAs to iden-
tify muscular regions that reacted differently to envy compared with Schadenfreude and their differences between
groups. We conducted an independent analysis for each muscular region. Tukey’s HSD post-hoc tests were used
(when appropriate) to examine differences within each condition. Effect sizes were calculated through partial eta
squared (η2). Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 Results
h Empathy. PNs showed higher scores than controls in empathic concern (t = 3.26, p = 0.004, d = 0.61). This
difference remained significant after adjusting for educational level (F (1,112) = 9.99, p = 0.002, η2 = 0.082), exec-
utive functioning (F (1,112) = 7.35, p = 0.008, η2 = 0.061), stress levels (F (1,111) = 11.818, p < 0.001, η2 = 0.094)
and hours of sleep (F (1,109) = 12.846, p < 0.001, η2 = 0.104). Also, a marginal between-group difference emerged
in the personal distress subscale (t = 2.08, p = 0.08, d = 0.38). This effect was significant after controlling for hours
of sleep (F (1,109) = 4.888, p = 0.029, η2 = 0.043). However, the difference disappeared after controlling for execu-
tive functioning (F (1,112) = 2.21, p = 0.140, η2 = 0.019), educational level (F (1,12) = 2.61, p = 0.109, η2 = 0.022)
and stress levels (F (1,111) = 2.539, p = 0.114, η2 = 0.022) Fantasy (t = −1.62, p = 0.13, d = −0.10) and perspective
taking (t = −0.01, p = 0.98, d = 0.24) subscales yielded non-significant differences between groups. See Fig. 1A
and Supplementary Table 2.ffi pp
y
Affective empathy levels did not differ significantly between mothers and fathers or as a function of gyneco-
logical and obstetric variables measured (see details in S4 and S5 and Supplementary Tables 3, 4 and 5). Social emotions. Behavioral results. Compared to controls, PNs exhibited higher Schadenfreude ratings
(t = 3.125, p = 0.003, d = 0.60), a pattern that remained significant after adjusting for educational level (F (1,112)
= 11.92, p < 0.001, η2 = 0.095), executive functioning (F (1,112) = 5.85, p = 0.017, η2 = 0.049), stress levels (F
(1,111) = 8.229, p = 0.005, η2 = 0.068), and hours of sleep F (1,109) = 8.982, p = 0.003, η2 = 0.076). Nevertheless,
no significant between-group differences were observed in ratings of envy (t = −0.28, p = 0.77, d = −0.05) or
neutral situations (t = 0.98, p = 0.45, d = 0.18) –see Fig. 1B and Supplementary Table 2. Results
h Analysis of envy scores
revealed one outlier participant (2 SDs below the group’s mean), but results remained highly similar upon removal
of this subject (see details in S6).fi j
(
)
Finally, Schadenfreude ratings did not differ significantly between mothers and fathers or as a function of
gynecological and obstetric variables measured (see details in S4 and S5 and Supplementary Tables 3, 4 and 5). To establish how specific our results were to PNs, and how independent they are to the testing context, we j
Finally, Schadenfreude ratings did not differ significantly between mothers and fathers or as a function of
gynecological and obstetric variables measured (see details in S4 and S5 and Supplementary Tables 3, 4 and 5).i gy
g
pp
y
To establish how specific our results were to PNs, and how independent they are to the testing context, we
performed a complementary analysis comparing PNs vs. a second control group (n = 34) evaluated in the same
experimental context as the experimental group. Results showed that, relative to both control groups, PNs Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 www.nature.com/scientificreports/ Figure 2. Differences between groups in z-scores of EMG activity for each muscle and emotion. Asterisk
indicates significant differences between groups. Figure 2. Differences between groups in z-scores of EMG activity for each muscle and emotion. Asterisk
indicates significant differences between groups. exhibited higher EC, PD, and Schadenfreude levels. The results of these complementary analyses have been
reported in “Supplementary Data” (S7). xhibited higher EC, PD, and Schadenfreude levels. The results of these complementary analyses have been
eported in “Supplementary Data” (S7). EMG results. A significant effect of emotion showed higher activity in the depressor region for envy relative to
Schadenfreude (F (1,57) = 10.77, p = 0.001, η2 = 0.16). No significant effect of group was observed (F (1,57) =
0.21, p = 0.64, η2 = 0.003). Besides, there was no significant interaction between emotion and group (F (1,57) =
1.40, p = 0.24, η2 = 0.02).f p
η
A tendency for an effect of emotion was observed for the corrugator supercilii region (F (1,57) = 3.51, p = 0.06,
η2 = 0.06), showing a higher activity of this region for Schadenfreude compared to envy. We found a main effect
of group (F (1,57) = 20.91, p = 0.00002, η2 = 0.27) showing that PNs had higher activity on this muscle than con-
trols. Association between social cognition outcomes and relevant experiential variables.
We
d f
l
l
d l
d
h d
f
d
d h
ff
h
b Association between social cognition outcomes and relevant experiential variables. We esti-
mated four multiple regression models considering Schadenfreude, envy and the two affective empathic subscales
as dependent variables. We included group, sex, educational level, IFS scores, hours of sleep, and perceived stress
levels as predictive factors in the four models. A first model including Schadenfreude as dependent variable (F (6,
104) = 2.512, p = 0.026, R2 = 0.076) revealed that group (p = 0.029) was the only significant predictor. The second
model, including envy scores as dependent variable was not significant (F (6, 104) = 1.521, p = 0.179, R2 = 0.028)
(see Supplementary Table 6).h pp
y
The two subsequent models included empathic concern (F(6, 104) = 2.680, p = 0.019, R2 = 0.084) and per-
sonal distress (F(6, 104) = 1.683, p = 0.132, R2 = 0.036) as dependent variables. The only significant predictor was
group for emphatic concern with p = 0.002 (see Supplementary Table 7). Correlations between empathy and social emotions. No association was found between empathy
and Schadenfreude, including all participants and each group separately. Neither a correlation was found between
empathy and envy, considering all participants, PNs, and control groups (see Supplementary Tables 8 and 9). Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 Results
h We also found an interaction between group and emotion (F (1,57) = 5.14, p = 0.02, η2 = 0.08). A post-hoc
analysis (Tukey HSD, MS = 0.35, df = 110.22) revealed that, for Schadenfreude, PNs showed higher activity in
the corrugator supercilii region than controls (p = 0.0001) (see Fig. 2B). No group difference was found for envy
(p=0.99). (p
)
For the zygomaticus major we found a significant effect of emotion (F (1,57) = 10.64, p = 0.001, η2 = 0.15)
showing that activity in this region was higher for Schadenfreude than envy. There were no significant effect of
group (F (1,57) = 0.12, p = 0.72, η2 = 0.002) or interaction between emotion and group (F (1,57) = 2.24, p = 0.13,
η2 = 0.03). The orbicularis oculi region did not show any significant modulations for emotion (F (1,57) = 0.14, p = 0.70,
η2 = 0.002), group (F(1,57) = 0.60, p = 0.43, η2 = 0.01) or their interaction (F (1,57) = 0.03, p = 0.85, η2 = 0.0006). p
For the zygomaticus major we found a significant effect of emotion (F (1,57) = 10.64, p = 0.001, η2 = 0.15)
showing that activity in this region was higher for Schadenfreude than envy. There were no significant effect of
group (F (1,57) = 0.12, p = 0.72, η2 = 0.002) or interaction between emotion and group (F (1,57) = 2.24, p = 0.13,
η2 = 0.03).hi η
The orbicularis oculi region did not show any significant modulations for emotion (F (1,57) = 0.14, p = 0.70,
η2 = 0.002), group (F(1,57) = 0.60, p = 0.43, η2 = 0.01) or their interaction (F (1,57) = 0.03, p = 0.85, η2 = 0.0006). Discussionhi Consistent
with previous research41, this finding suggests that participants seem to exhibit a subtle contortions similar to
those involved in the act of smiling when a misfortune happens to another person. In addition, we found that
in control participants the depressor muscle activity was higher for envy than Schadenfreude. Depressor supercilii
activity show increased activity in response to negative facial stimuli (i.e., angry faces)66. Increased activity of this
muscle may be explained by the fact the envy stimuli employed here involve situations related to negative feelings
of deservingness (e.g., a young man got a better test score for being the son of a professor) or morality/legality
(e.g., a politician takes a vacation using taxpayers’ money). Furthermore, EMG results revealed that implicit mus-
cular correlates of Schadenfreude involve higher activity in the corrugator supercilii for PNs than controls. Note
that modulation of the corrugator supercilii indexes the disapproval of an action54, a process noticeably involved
in Schadenfreude responses. Considering that linguistic properties of stimuli may affect the zygomaticus major
and corrugator supercilii activities67, sentences for envy and Schadenfreude conditions were controlled in terms of
length, complexity, and grammatical structure. Thus, our behavioral and EMG results can hardly be attributed to
differences in the linguistic properties of both conditions stimuli.f f
g
p
p
Taken together, our results suggest that affective empathy and emotional reactivity to unfair or threating social
situations (Schadenfreude) are increased in PNs. Accordingly, social cognition changes seem present in mothers
and fathers of newborns, irrespective of type of delivery. In general, PNs seem more sensitive to the influence of
others and to salient social cues, which are crucial for parental bonding. These patterns align with previous studies
showing that the neural circuits underlying emotions in response to socially valued scenarios are partly targeted
by the oxytocinergic system65. In fact, exogenous OXY levels correlate positively with levels of empathy68,69 and
Schadenfreude61. Note, in this sense, that elevated OXY levels in PNs13,17 may selectively facilitate social cognition
in certain conditions68,69 and increase the salience of social cues61. Consistent with previous suggestions70,71, it has
been proposed that OXY has a dual effect on parental behavior, insofar as it inhibits aggression towards the off-
spring while promoting territoriality as well as aggressive and defensive behaviors against outsiders. Discussionhi A possible explanation for the selective differences in Schadenfreude observed in PNs might be the
pleasurable nature of this emotion33,59 and its strong relationship to reward mechanisms40, indexed by increased
engagement of the ventral striatum33. In fact, this brain region, along with others (e.g., thalamus, hippocampus
and amygdala), is crucially involved in oxytocinergic dynamics33,60. Previous studies suggest that the neurohor
mone OXY may partly account for variations in parent-infant interactions7. Higher OXY levels may be associ-
ated a wide range of emotions and social behaviors, such as raising children, trusting others, attacking potential
outsiders and competing with rivals, which can lead to trust and generosity, but at the same time to increased
Schadenfreude61. Null differences in envy might be explained by the fact that, unlike Schadenfreude, this is a
non-gratifying emotion that implies feelings of dissatisfaction with another person’s good fortune40. In fact, envy
implies greater neuronal activity in pain circuits rather than in the reward and pleasure systems62. Promisingly,
this new hypothesis, derived from our behavioral results, paves the way for new cross-methodological studies. Future studies should include neuroimaging measures as well as OXY and other hormones levels in order to test
this interpretation. p
Additionally, our social emotion task comprised a group of justice-related scenarios. Accordingly, the higher
Schadenfreude scores in PNs could reflect an enhanced sensitivity to track unfair situations and respond to sce-
narios in which those situations are punished. In fact, Schadenfreude might play a positive role when unfair social
situations are sanctioned63, which aligns with a widespread human trend to punish unfair or social inappropriate
situations –namely, altruistic punishment64, a behavior that is likely underpinned by negative emotions towards
defectors. Note, in this sense, that higher OXY levels seem to increase altruistic punishment behavior, by render-
ing cooperation and promoting cohesion in social groups65. Arguably, PNs exhibited higher Schadenfreude for
unfair or threatening scenarios as an expression of an increased sensitivity to track social threats. Conversely,
although the envy situations described unfair and inappropriate social situations, the lack of differences between
PNs and controls might reflect the role of control mechanisms in the former, favoring proactive punishment over
mere unpleasantness in the face of unfair social scenarios.h p
These interpretations are further supported by our EMG results. In line with previous EMG studies41,42, we
found that activity of the zygomaticus major activity was higher for Schadenfreude than envy responses. Discussionhi This is the first study investigating social cognition abilities in PNs. We found that, compared to controls, PNs
exhibited higher levels of affective empathy and Schadenfreude, the latter pattern being accompanied by increased
EMG modulations of the corrugator supercilii. These results further our understanding of social cognition changes
during the puerperal period.f g
p
p
p
As expected, PNs showed higher scores than controls in both affective empathy subscales (i.e., empathic con-
cern and personal distress), even after adjusting for executive functioning, educational levels, perceived stress
levels and hours of sleep. Conversely, non-significant differences were observed between groups in cognitive
empathy. Our results are consistent with previous suggestions7 that empathy is a key aspect of parenting, espe-
cially because babies’ needs are expressed non-verbally. Specifically, empathic concern and personal distress levels
are highly related with the social cognition abilities required to recognize and care for others people’s feelings,
and even turn to their aid45. In line with our findings, in the first stage of bonding, affective empathy is more
important and essential than cognitive empathy58. Higher affective empathy levels are involved in better emo-
tional communication, social attachment, and motivation to cooperate58. Increased parental empathy7 facili-
tates emotional communication, social attachment, parental caring58, and motivation to protect and care for the Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 www.nature.com/scientificreports/ newborn1. Notably, given the nature of our empathy measure, our results suggest that higher affective empathy
levels observed in PNs are not limited to parent-baby interactions, but are also present in scenarios involving
other individuals. newborn1. Notably, given the nature of our empathy measure, our results suggest that higher affective empathy
levels observed in PNs are not limited to parent-baby interactions, but are also present in scenarios involving
other individuals Regarding social emotions, our results showed increased Schadenfreude levels in PNs, which were not
explained by executive functioning, educational levels, stress levels or hours of sleep. By contrast, envy levels
were similar between groups. This pattern may be associated with the multiple hormonal, emotional, and bio-
logical changes that take place during pregnancy and puerperium. However, as endocrine, physiological or other
biological measures were not included in this study, interpretations about the relevance of these factors should
be cautions. Scientific Reports | (2020) 10:5760 | https://doi.org/10.1038/s41598-020-62622-7 www.nature.com/scientificreports/ in which depression and stress modulate the experience of Schadenfreude. These covariation analyses did not
reach significant effects suggesting that increased Schadenfreude in PNs is not directly explained by the mediation
of other emotional or cognitive changes occurred at afterbirth stages. In addition, puerperium is considered as
a particular intense emotional milestone in PNs’ life, usually associated with emotional changes and stress73,74. However, this milestone could be also accompanied by happy mood and the experience of positive emotions
such as joy, contempt or happiness. A potential limitation in our study was that we did not measure the role of
positive emotions and happy mood in the experience of Schadenfreude. To date, the state-of-art of studies assess-
ing Schadenfreude has shown dissociable neurocognitive and behavioral mechanisms underlying Schadenfreude
and positive emotions33,34,63,75. Furthermore, note that this is arguably one of the reasons why previous studies
on Schadenfreude have not controlled for the effects of joy or happy mood33,36,42,43,59,76. However, previous studies in which depression and stress modulate the experience of Schadenfreude. These covariation analyses did not
reach significant effects suggesting that increased Schadenfreude in PNs is not directly explained by the mediation
of other emotional or cognitive changes occurred at afterbirth stages. In addition, puerperium is considered as
a particular intense emotional milestone in PNs’ life, usually associated with emotional changes and stress73,74. However, this milestone could be also accompanied by happy mood and the experience of positive emotions
such as joy, contempt or happiness. A potential limitation in our study was that we did not measure the role of
positive emotions and happy mood in the experience of Schadenfreude. To date, the state-of-art of studies assess-
ing Schadenfreude has shown dissociable neurocognitive and behavioral mechanisms underlying Schadenfreude
and positive emotions33,34,63,75. Furthermore, note that this is arguably one of the reasons why previous studies
on Schadenfreude have not controlled for the effects of joy or happy mood33,36,42,43,59,76. However, previous studies
have revealed the complexity of positive emotions and its influences on secondary emotions77. Those influences
could also impact on the experience of social emotions, including Schadenfreude. New studies should assess the
extent to which dispositional emotions or instant and evoked emotional states could affect the intensity and expe-
rience of social emotions and social cognition in particular biological states as puerperium or pregnancy. www.nature.com/scientificreports/ Besides,
the group of effects on social emotions and empathy observed in PNs could also be affected by general changes on
emotional reactions including fear, anger, and happiness among other emotional manifestations. Future studies
also should control the effects of primary emotions on the social emotions and empathy. Finally, the difference
in the experimental testing contexts between PNs and controls represents a limitation of our study. However, the
results of the complementary analyses (with a control group evaluated in the same setting as the experimental
groups) suggest that our pattern of results is not explained by differences in testing sites. Future studies should use
specific designs to evaluate the potential impact of different contextual variables on performance.f pi
g
p
pf
p
In sum, this report offers unprecedented evidence that PNs exhibit increased emotional reactivity, charac-
terized by an exacerbation of affective empathy and Schadenfreude. These results open a new agenda to examine
changes in social cognition and their relationship with neuroendocrine phenomena. Data availability
Th d
h The data that support the findings of this study are available from the corresponding author upon reasonable
request. Received: 30 May 2019; Accepted: 5 March 2020;
Published: xx xx xxxx Received: 30 May 2019; Accepted: 5 March 2020;
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This work was partially supported by Universidad de los Andes, grants from CONICET, CONICYT/
FONDECYT Regular (1170010), FONCyT-PICT 2017-1818, FONCyT-PICT 2017-1820, FONDAP 15150012,
and the Programa Interdisciplinario de Investigación Experimental en Comunicación y Cognición (PIIECC),
Facultad de Humanidades, USACH. The authors are grateful to patients and workers of the Department of
gynecology and obstetrics, Hospital Universitario San Ignacio (Bogotá, Colombia), for supporting the data
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The authors declare no competing interests. Author contributions S.B., A.M.G.C., H.S.G., J.S., A.M.G., and A.I. conceived and designed the project, designed the analysis,
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2. Cikara, M., Bruneau, E. G. & Saxe, R. R. Us and Them: Intergroup Failures of Empathy. Curr. Dir. Psychol. Sci. 20, 149–153
10.1177_0956797610397667 (2011).itf 73. Kendell, R. E., McGuire, R. J., Connor, Y. & Cox, J. L. Mood changes in the first three weeks after childbirth. J Affect Disord 3,
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74. Turton, P. et al. Psychological impact of stillbirth on fathers in the subsequent pregnancy and puerperium. Br J Psychiatry 188,
165–172, https://doi.org/10.1192/bjp.188.2.165 (2006). 165–172, https://doi.org/10.1192/bjp.188.2.165 (2006). 5. van Dijk, W. W. © The Author(s) 2020 Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-62622-7. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-62622-7. Correspondence and requests for materials should be addressed to S.B. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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Household Saving, Class Identity, and Conspicuous Consumption
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http://www.american.edu/cas/econ/workpap.htm Copyright © 2008 by Jon D. Wisman. All rights reserved. Readers may make verbatim
copies of this document for non-commercial purposes by any means, provided that this
copyright notice appears on all such copies. Copyright © 2008 by Jon D. Wisman. All rights reserved. Readers may make verbatim
copies of this document for non-commercial purposes by any means, provided that this
copyright notice appears on all such copies. HOUSEHOLD SAVING, CLASS IDENTITY, AND CONSPICUOUS CONSUMPTION* By Jon D. Wisman** “In modern civilized communities the lines of demarcation
between social classes have grown vague and transient, and
wherever this happens the norm of reputability imposed by the
upper class extends its coercive influence with but slight hindrance
down through the social structure to the lowest strata. The result is
that the members of each stratum accept as their ideal of decency
the scheme of life in vogue in the next higher stratum, and bend
their energies to live up to that ideal. On pain of forfeiting their
good name and their self-respect in case of failure, they must
conform to the accepted code, at least in appearance....No class of
society, not even the most abjectly poor, foregoes all customary
conspicuous consumption” (Veblen 1899: 84, 85). “There is much to be said for regarding the explanation of the
content of preferences…as outside the economist’s competence. Nevertheless, there is also a danger with such delimitation of
responsibility, namely, that the problems excluded may not be
studied at all” (Houthakker 1961: 734). ABSTRACT The saving rate for U.S. households has long been low relative to those in other wealthy
countries and in recent decades this rate has plummeted. Most studies of household
saving behavior are based on the life-cycle theory of saving. However, there is doubt as
to whether these studies adequately explain the low and declining rate in the U.S. This
study explores two hypotheses that depart from the life-cycle explanatory framework. The first hypothesis examines the possibility that the low rate of household saving in the
U.S. is related to Americans’ strong belief that vertical mobility in the U.S. is readily
possible and hence their relatively weak sense of class identity. A second corollary
hypothesis is that in an economy in which a high degree of vertical mobility is thought
possible, a high degree of inequality in the distribution of income and wealth may
reinforce the tendency to save little. 2 2 1 g ,
g y,
p
** Professor of Economics at American University, Washington, D.C. Research assistance
provided by Talip Kilic, former Ph.D. Candidate in Economics at American University is
gratefully acknowledged. Special thanks to Martha Starr for her many insightful comments.
Helpful comments were also provided by Chris Brown, Maria Floro, Thomas Hungerford,
Walter Park, Sikander Rahim, Howard Wachtel, and two anonymous referees. * Forthcoming in the Journal of Economic Issues, March 2009. An earlier version of this paper
was presented at the annual meetings of the Association for Institutionalist Thought, Western
Social Science Association Meetings, Calgary, 13 April 2007. * Forthcoming in the Journal of Economic Issues, March 2009. An earlier version of this paper
was presented at the annual meetings of the Association for Institutionalist Thought, Western
Social Science Association Meetings, Calgary, 13 April 2007.
** Professor of Economics at American University, Washington, D.C. Research assistance
provided by Talip Kilic, former Ph.D. Candidate in Economics at American University is
gratefully acknowledged. Special thanks to Martha Starr for her many insightful comments.
Helpful comments were also provided by Chris Brown, Maria Floro, Thomas Hungerford,
Walter Park, Sikander Rahim, Howard Wachtel, and two anonymous referees. ABSTRACT The saving rate for U.S. households has long been low relative to those in other wealthy
countries and in recent decades this rate has plummeted. Most studies of household
saving behavior are based on the life-cycle theory of saving. However, there is doubt as
to whether these studies adequately explain the low and declining rate in the U.S. This
study explores two hypotheses that depart from the life-cycle explanatory framework. The first hypothesis examines the possibility that the low rate of household saving in the
U.S. is related to Americans’ strong belief that vertical mobility in the U.S. is readily
possible and hence their relatively weak sense of class identity. A second corollary
hypothesis is that in an economy in which a high degree of vertical mobility is thought
possible, a high degree of inequality in the distribution of income and wealth may
reinforce the tendency to save little. 2 The saving rate for U.S. households1 has long been low relative to those in other wealthy
countries and in recent decades this rate has plummeted, from 10.4 percent in 1980-84 to 7.7
percent in 1985-89,2 to 6.5 percent in 1990-94, 3.8 percent in 1995-99, and 2.1 percent in 2000-
04 (Garner 2006: 8). In 2005 and 2006, the rates were negative, -0.4 and -1.0 percent
respectively, the lowest since the Great Depression years of 1932-33.3 Although the relatively low saving rate of Americans and the recent decline in saving
rates have been extensively investigated by the economics profession, the explanations that have
been generated have not been found adequate. After reviewing recent explanations of declining
saving rates and finding none of them compelling, Massimo Guidolin and Elizabeth A. La
Jeunesse conclude that “the U.S. personal saving rate remains a puzzle” (2007: 512).4 This article draws upon the work of Thorstein Veblen to suggest that a fuller
understanding of the propensity to save requires that socio-cultural factors be taken into account. Accordingly, this article explores two hypotheses, both motivated by Veblen=s conception of
conspicuous consumption, which holds that above a certain level of subsistence, humans
consume in part to demonstrate social status. The first hypothesis is that the low rate of household saving in the U.S. is related to
Americans’ relatively strong belief that vertical mobility is readily possible and thus their
relatively weak sense of class identity. ABSTRACT To a lesser extent than in other relatively wealthy
societies, Americans do not view their social status as given by their birth. They more strongly
believe that if they are willing to put forth sufficient effort, they may improve their status. The first hypothesis is that the low rate of household saving in the U.S. is related to
Americans’ relatively strong belief that vertical mobility is readily possible and thus their
relatively weak sense of class identity. To a lesser extent than in other relatively wealthy
societies, Americans do not view their social status as given by their birth. They more strongly
believe that if they are willing to put forth sufficient effort, they may improve their status. Through adequate dedication and effort, anyone can move up, even to the very highest levels of
social status. It is the individual’s responsibility. It depends upon the individual’s willingness to
study and work hard. One’s social status is not given, but earned. Through adequate dedication and effort, anyone can move up, even to the very highest levels of
social status. It is the individual’s responsibility. It depends upon the individual’s willingness to
study and work hard. One’s social status is not given, but earned. 3 However, how hard one works is generally not directly observable. What more readily
catches attention is how much one can consume, which can stand, more or less, as a proxy for
how hard one has worked. Thus, because Americans believe more strongly than do peoples of
other wealthy nations that they are individually responsible for their own social standing, they
feel more strongly compelled to demonstrate status and hence class identity through
consumption. The second and corollary hypothesis is that in an economy in which a high degree of
vertical mobility is thought possible, a higher degree of inequality in the distribution of income
and wealth may reinforce the tendency to save little. Great inequality means that consumers
must stretch further to emulate the consumption standards above them. Veblenian conspicuous consumption manifests itself in two dimensions. Consumption
that permits “invidious comparison” is meant to demonstrate one’s status to be above those
below. “Pecuniary emulation,” on the other hand, refers to the practice of imitating the
consumption standards of those of higher status with the intent of appearing to also possess that
status. ABSTRACT Veblen claimed that AWith the exception of the instinct of self-preservation, the
propensity for emulation is probably the strongest and most alert and persistent of the economic
motives proper…[and] the propensity for emulation – for invidious comparison – is of ancient
growth and is a pervading trait of human nature” (Veblen 1899: 110; 109). Veblen believed that humans need the respect of others: “The usual basis of self-respect
is the respect accorded by one’s neighbors. Only individuals with an aberrant temperament can
in the long run retain their self-esteem in the face of the disesteem of their fellows” (1899: 39). And, AIn order to gain and to hold the esteem of men it is not sufficient merely to possess wealth
or power. The wealth or power must be put in evidence, for esteem is awarded only on 4 4
evidence@ (Veblen 1899: 36-37). Veblen is hardly alone in identifying self-esteem as centrally
important to humans. Indeed, John Rawls suggested that it is “perhaps the most important
primary good” such that without it nothing else has much value (1971: 440). This article examines the relevance of Veblen’s theory of consumer behavior for
understanding the relatively low and recently declining saving rate in the U.S.5 The next section
outlines the saving puzzle. Attention is then given to the historical evolution of the potential for
vertical mobility and its social implications. This is followed by an exploration of the first
hypothesis that the low rate of household saving in the U.S. is related to Americans’ relatively
strong belief that vertical mobility in the U.S. is readily possible. The following section
examines the corollary hypothesis that in an economy in which a high degree of vertical mobility
is thought possible, a high degree of inequality in the distribution of income and wealth may
reinforce the tendency to save little, and thus help explain the recent collapse in saving rates. The Saving Puzzle The low and falling rate of personal saving has drawn a considerable amount of attention
for four principal reasons: At the macroeconomic level, it is feared that too little saving will
mean too little investment and thus sluggish growth in the future. A corollary of this concern is
that the U.S. has become overly dependent on foreign financing of its investment needs. A
shorter-term macroeconomic concern is that a sharp reversal in the saving rate could precipitate
or deepen a recession.6 At the microeconomic level, it is lamented that a low saving rate, and
especially its decline, threatens future financial distress for many households (Bryant 2001). A
fourth concern is the issue of intergenerational fairness. Due to the huge bulge of baby boomers
entering retirement over the next 25 years, the rising costs of Social Security and Medicare
programs will burden future workers with the earlier spend-thrift behavior of the retired.7 5 Most mainstream economic studies of households’ decisions to save are based explicitly
or implicitly on the life-cycle theory of saving, according to which households save or dis-save
in order to even out their consumption over their life spans (consumption smoothing). However,
these studies on saving behavior have generated little consensus and the very ability of this
model to explain saving behavior has been challenged. Richard Thaler (1994), for instance,
argues that the life-cycle models of saving fail to capture actual household saving behavior in
three ways: It is unreasonable to assume that households possess adequate knowledge to solve a
“multiperiod dynamic maximization problem.” They have bounded rationality. Second, humans
often lack self-control. And third, households react differently depending upon “the source or
location of wealth” (whether coming in the form of current income or as changes in asset
values).8 Because of these inadequacies, and because the life-cycle theory of saving does not
seem to accord well with recent experience, it has led to “an increasing frustration in the
economics community about personal saving” (1994: 186). Axel Börsch-Supan concludes that
“Household saving is still little understood” (2001: 1). The life-cycle hypothesis views individuals (or households) as rationally allocating their
lifetime income so as to maintain a consistent standard of living. In doing so, it abstracts from
other social or cultural forces that might help explain the motivation to save/consume. The Saving Puzzle This is in
keeping with the general practice of mainstream economists of viewing the formation of
preference functions as exogenous to the science. The approach followed here, by contrast, is to view preference functions as at least
partially endogenous -- to some extent, socially created. Preferences are a consequence of both
our genetic heritage and our cultural conditioning. Thus any ability to rationally allocate our
lifetime income so as to maintain a consistent standard of living would be genetically given. 6 6
That we would actually do so is likely a consequence of our particular culture. By viewing
preferences as in part socially created, the arguments developed in this article are aligned with
James Duesenberry’s view that a “real understanding of the problem of consumer behavior must
begin with a full recognition of the social character of consumption patterns” (1967: 19). It is
also in keeping with Barry Bosworth’s suggestion that because international differences in
saving rates are not well explained by traditional economic variables, they may to some extent
reflect differences in culture (1993). The hypotheses explored here are cultural in the sense that ill-defined class distinctions
and a socially generated view of a high degree of vertical mobility suggest that individuals are
responsible for their social status. To demonstrate success, households emulate the behavior of
those socially above them, thereby generating a high level of conspicuous consumption.9 This
seems to be precisely what Veblen had in mind when he wrote: “in any community in which class distinctions are somewhat vague, all canons of
reputability and decency, and all standards of consumption, are traced back by insensible
gradations to the usages and habits of thoughts of the highest social and pecuniary class --
the wealthy leisure class” (1899: 104).10 “in any community in which class distinctions are somewhat vague, all canons of Moreover, Veblen viewed participation in conspicuous consumption as strongly socially
compelled “though popular insistence on conformity to the accepted scale of expenditure as a
matter of propriety, under pain of disesteem and ostracism.” Thus the consumer’s “motive is a
wish to conform to established usage, to avoid unfavourable notice and comment, to live up to
the accepted canons of decency...” (1899: 111; 115). So powerful is this motive that “No class
of society, not even the most abjectly poor, foregoes all customary conspicuous consumption. The Saving Puzzle [and] Very much of squalor and discomfort will be endured before the last trinket or the last 7 pretence of pecuniary decency is put away (1899: 85). For Veblen, conspicuous consumption is
not so much consciously pursued as undertaken to maintain respectability: “For the great body of the people in any modern community, the proximate ground of
expenditure in excess of what is required for physical comfort is not a conscious effort to
excel in the expensiveness of their visible consumption, so much as it is a desire to live
up to the conventional stand of decency in the amount and grade of goods consumed”
(1899: 102). Curiously, the pursuit of status might be theoretically crafted to fit the life-cycle model. If, to acquire higher status, too much is spent on present consumption, then it could be difficult to
maintain status in later years. However, current spending on status could also be seen as
partially investment, insofar as greater present status might enhance future income and thus
future status. Not only would such a model suffer from the same hyper-rationality of other life-
cycle models, any promise for empirically testing it seems slim. The Grounding of Social Status: From Ascriptive to Performative Status In pre-agricultural (hunter-gatherer) societies, status was acquired by success in
procuring food and skills in war. But with the adoption of agriculture and the subsequent
evolution of highly organized societies, status became ascriptive, ascribed by birth, by one=s
parents= status. Little vertical mobility existed in such societies. Everyone had their Aborn-to@
status. As Aristotle put it, AFrom the hour of their birth, some are marked out for subjection,
some for command@ (Politics 1962, Book I). This was their identity. Not only were there
practically no avenues for raising one=s status, but any attempt to do so was discouraged, if not
punished. Conspicuous consumption did occur in such societies, but it would largely be within
classes. And because only the very elite typically had significant discretionary income (more 8 than required for mere subsistence), demonstrating status through luxury consumption was a
privilege generally reserved to the aristocracy. The rise and spread of capitalism created greater potential for vertical social mobility. As
a rising commercial class, a bourgeoisie, began to accumulate wealth, it also sought social
recognition commensurate with its new command on wealth. It began to petition for equal status
with the aristocracy.11 Its success in doing so entailed that the grounding for status was shifting
from one=s birth to one=s achievement. Ascriptive status began to yield to performative status. Through diligent hard work and cleverness, one might rise in status. Vertical mobility became
increasingly possible. Vertical mobility would become most fully developed first in those capitalist countries
that were essentially composed of immigrants who had left behind worlds of more rigid status
barriers. Thus not surprisingly, considerable vertical mobility was found in countries populated
by immigrants such as the U.S., Canada, Australia, and Israel (Tyree et. al. 1979: 415). But in
terms of people’s behavior, more important than the actual degree of vertical mobility is what is
believed to be the case. That is, people’s behavior can be expected to correlate more with their
belief in the extent of vertical mobility than with what might actually exist. Accordingly, their
perception of each individual=s potential for changing his or her social status is more a
consequence of their understanding, regardless of whether true or false, of the degree of
Aequality of opportunity@ in their society. The Grounding of Social Status: From Ascriptive to Performative Status As Veblen pointed out (note epigram at the head of this article), in societies in which the
potential for vertical mobility is believed to be relatively high, class distinctions become blurred. Although individuals may have a general sense of where they fall in terms of their society=s
distribution of income or wealth, they have little sense of belonging to a class. Indeed, the extent 9 to which this is true in the U.S. is suggested by the fact that most Americans describe themselves
as middle class, “a concept that conveys not only one’s economic status but also the sense of
being a full participant in a fluid society” (Kuznet, et. al. 2006). The perceived potential for vertical mobility inculcates a sense that one is responsible for
one=s social status.12 If one works hard and diligently, one might move up. Therefore,
individuals are more prone to internalize responsibility for their successes or failures. This
places considerable pressure on people to demonstrate high status. To the extent they succeed in
doing so, they appear to possess the virtues of hard work. And the easiest and most practical
way to signal this success is through one=s consumption, as Veblen noted: “To sustain one’s
dignity – and to sustain one’s self-respect – under the eyes of people who are not socially one’s
immediate neighbors, it is necessary to display the token of economic worth, which practically
coincides pretty closely with economic success” (Veblen, 1919: 393). Further, “One’s neighbours, mechanically speaking, often are socially not one’s neighbours, or
even acquaintances; and still their transient good opinion has a high degree of utility. “One’s neighbours, mechanically speaking, often are socially not one’s neighbours, or
even acquaintances; and still their transient good opinion has a high degree of utility. The only practicable means of impressing one’s pecuniary ability on these unsympathetic
observers of one’s everyday life is an unremitting demonstration of ability to pay” (1999:
86-87).13 If a household consumes at the level of those with higher status, then it might acquire that status
and the good reputation that accompanies it.14 If a household consumes at the level of those with higher status, then it might acquire that status
and the good reputation that accompanies it.14 Where income and wealth inequality are greater, the amount that must be consumed to
create the impression of higher status is greater. The Grounding of Social Status: From Ascriptive to Performative Status But inequality would only have this effect
where a belief in the potential for vertical mobility is strong. In societies where relatively little
potential for vertical mobility is believed to exist, individuals understand their class status as 10 more fixed and known. They possess the status of their parents, which was that of their
grandparents and so on. Should their status be low, it is not the result of a personal failing. Moreover, if an individual were to attempt to show greater status through consumption, it would
more readily be viewed as negative, as “show-off” behavior.15 Rather than signaling virtue, it
would signal a character flaw. Also stimulating consumption is the fact that if identity is less given by inherited status
(class membership, community, ethnicity, religion, and even gender), then individuals feel more
responsible for their lives and self-identity. There is a greater sense of individuality, and self-
identity becomes a project. Consumption acts as a signaling device for identity, a means to
define one’s self and to project this definition to others. Maintaining if not improving this
identity is a never-ending project. Saving Rates and Perceptions of the Potential for Vertical Mobility in Vario Alexis de Tocqueville and Karl Marx both noted an exceptionally high degree of vertical
mobility in the U.S. and termed it “American exceptionalism.” Whatever might have been the
case in nineteenth century America, today such exceptionalism no longer seems valid. A recent
study (Jäntti, et. al. 2006) of vertical mobility in six wealthy countries (Denmark, Finland,
Norway, Sweden, the United Kingdom, and the United States) has found that the U.S. has less
mobility than the other five. This choice of countries is noteworthy in that it compares mobility
in two of the most laissez-faire economies with that in four of the most social-welfare-oriented. Alexis de Tocqueville and Karl Marx both noted an exceptionally high degree of vertical
mobility in the U.S. and termed it “American exceptionalism.” Whatever might have been the
case in nineteenth century America, today such exceptionalism no longer seems valid. A recent
study (Jäntti, et. al. 2006) of vertical mobility in six wealthy countries (Denmark, Finland, The authors conclude that “The only crystal-clear result is that there is less
intergenerational mobility in the U.S. than in the other countries” (17). Middle-class mobility
appears to be somewhat similar across all these countries, but there is higher probability in the
U.S. that children of the fathers in the poorest quintile will remain in that quintile. Also, children 11 11
of fathers in the highest quintile are more likely to remain there in the U.S. than in the other five
countries.16 The findings of Jäntti, et. al. are supported by a recent OECD (d’Addio 2007) study
that finds upward mobility between generations to be lower in the U.S. than in Canada, Sweden,
Germany, Spain, Denmark, Austria, Norway, Finland, and France. Yet other studies also lend
support to the claim that the U.S. is no longer the exceptional land of great equality of
opportunity (see Hertz 2007; Mishel, Bernstein, and Allegretto 2007; Mazumder 2005; Solon
1992). Nevertheless, the general view in the U.S. continues to be that it is the exceptional land of
opportunity.17 This view has deep roots in the socio-cultural values of America’s founding and
subsequent history. Such deep-rooted socio-cultural values are resistant to change and may
endure over very long periods of time (Dolfsma 2002; Campbell 1987), even when the
conditions on which they were founded no longer exist. Saving Rates and Perceptions of the Potential for Vertical Mobility in Vario Average household saving rates (saving as a percent of disposable household income) for
the United States over the period 1988-2007 were lower, and in most instances far lower, than in
sixteen other wealthy industrialized countries. Only three countries, two of them Scandinavian,
had lower saving rates, and curiously, as noted earlier, actual vertical mobility is greater in these
two Scandinavian countries (Denmark and Finland) than in the U.S. (Jäntti et. al.2006). 12 Norway
5.20
Sweden
6.36
Canada
6.71
United Kingdom (Gross Savings)
7.16
Japan
9.18
Austria
9.87
Switzerland
10.23
Portugal (Gross Savings)
10.47
Germany
11.03
France
11.42
Netherlands
11.47
Spain (Gross Savings)
13.01
Belgium (Gross Savings)
13.91
Korea
14.81
Italy
15.12
OECD Online Database Norway
5.20
Sweden
6.36
Canada
6.71
United Kingdom (Gross Savings)
7.16
Japan
9.18
Austria
9.87
Switzerland
10.23
Portugal (Gross Savings)
10.47
Germany
11.03
France
11.42
Netherlands
11.47
Spain (Gross Savings)
13.01
Belgium (Gross Savings)
13.91
Korea
14.81
Italy
15.12
OECD Online Database However, average household final consumption expenditures as a percent of GDP over the
period 1988-2004 was higher in the U.S. than in all nineteen other wealthy countries in this
sample. This likely reflects, at least in part, the fact that U.S. households must bear a greater
share of education and health care costs. TABLE II Share of Respondents Saying It is "Essential", "Very Important",
or "Fairly Important" to Come From a Wealthy Family to Get Ahead Share of Respondents Saying It is "Essential", "Very Important",
or "Fairly Important" to Come From a Wealthy Family to Get Ahead
Country
Essential
Very important
Fairly important
Total
Cyprus
22.3
38.5
26.9
87.7
Poland
18.3
38.9
25.5
82.7
Spain
7.9
45.4
26.5
79.8
Bulgaria
28.3
25.5
24.8
78.6
Slovakia
22.1
24.8
27.9
74.8
Portugal
19
35.8
18.8
73.6
Israel
18.6
32.2
22.7
73.5
Latvia
9.5
27.1
33
69.6
Russia
19.3
22.3
27.3
68.9
Germany East
4.1
26.8
37.2
68.1
Hungary
14.7
20.4
31.2
66.3
Philippines
13.6
30.8
19.8
64.2
Slovenia
5.8
20.4
35.9
62.1
Germany West
4.6
18.3
37.9
60.8
Austria
8.2
19.8
31.6
59.6
Australia
2.2
18
39.2
59.4
Chile
13
27.4
18.5
58.9
Sweden
3.1
14.9
39.4
57.4
Japan
1.8
9.7
42.5
54 14 14
North Ireland
5.2
18.6
28.2
52
New Zealand
3.6
12.8
35
51.4
Norway
0.9
9.7
39.9
50.5
Great Britain
3.3
15.4
30.6
49.3
France
1.9
8.1
37.8
47.8
United States
2.8
16.2
26.6
45.6
Canada
2.7
10.3
32.1
45.1
Czech Republic
8.6
10.4
23.6
42.6
Source: International Social Survey Programme 1999: Social Inequality III (ISSP 1999) The second proxy examines the extent to which people believe that hard work pays off. Only in the Philippines do a larger percentage of people than in the U.S. strongly agree or agree
that people are rewarded for effort (column two in Table IV below). A view that effort can make
a difference in what one receives in society is suggestive of a belief that vertical mobility is
possible. The third proxy refers to the extent that people are rewarded in terms of their skill levels. It is widely believed in the U.S. that individuals are personally responsible for their own
educational attainment. They are free to accumulate the level of human capital (skills) that they
wish. Thus a view that individuals are rewarded for skills suggests that vertical mobility is
possible. More people in the U.S. strongly agree or agree that people are rewarded for skill than
do peoples in all other 26 countries in this 1999 sample (column 3 in Table IV below). The fourth proxy addresses the extent to which people believe that material rewards in
society are justly distributed. TABLE II Household Final Consumption Expenditure (% of GDP)
Country
Average (1988- 2004)
United States
68.00
United Kingdom
64.53
Portugal
62.91
Spain
59.91
Switzerland
59.66
Australia
59.26
Italy
59.14
Germany
58.25
Austria
56.93
France
56.52
Canada
56.50
Japan
55.16
Belgium
54.68
Finland
51.79
Czech Republic
50.85
Netherlands
49.66
Sweden
49.38
Denmark
49.29 13 13 Although no comprehensive international survey has been undertaken of peoples’ beliefs
as to the degree of vertical mobility in their respective societies, there are survey results on a
number of other beliefs that are suggestive of views of vertical mobility and that might serve as
proxies. A 1999 sample of 27 countries provides five such possible proxies (International Social
Survey Programme 1999). The first proxy shows that only in the Czech Republic and Canada was the view weaker
than in the U.S. that to get ahead it is essential, very important, or fairly important to come from
a wealthy family (See Table III below). A belief that family wealth is important for rising in
status suggests that mobility is not thought to be highly fluid. The fact that relative to peoples in
most other countries, Americans do not believe that wealth is so important for getting ahead
suggests that they are more likely to believe that vertical mobility is possible. TABLE II If it is believed that people receive in income what they deserve in
terms of their efforts, then it is more likely that the distribution of income in that society will be
viewed as appropriate or fair. People get their just desserts. In the above mentioned sample,
fewer in the U.S. than in any of the other 26 countries strongly agree or agree that income
differences are too high (column 4 in Table IV below). 15 15
The fifth and final proxy relates to whether government should play a role in making
income distribution more just. If the distribution of income is viewed as just in terms of what
people deserve for their productive contributions to society, then it may be less likely that they
would wish their government to intervene to reduce income differences. And indeed, far fewer
in the U.S. than in any of the other 26 countries strongly agree or agree that government should
reduce income differences (column 5 in Table IV below). These five proxies provide strong support for the contention that Americans believe their
country provides greater potential for changing social status through personal effort than do
peoples of other wealthy or moderately wealthy countries. To a greater extent than in these other
countries, Americans believe their society is one of equal opportunity. These five proxies provide strong support for the contention that Americans believe their
country provides greater potential for changing social status through personal effort than do
peoples of other wealthy or moderately wealthy countries. To a greater extent than in these other
countries, Americans believe their society is one of equal opportunity. Share of Respondents Who Say They "Strongly Agree" or "Agree"
with the Indicated Statements, Referring to Their Own Country
Country
People get
rewarded for
their effort
People get
rewarded for
their intelligence
and skills
Differences
in income
are too large
It is the responsibility
of the govt. to reduce
income differences
Australia
58.2
65.2
68.3
47.6
Austria
41.8
50.3
83.5
69.1
Bulgaria
5.4
4.9
94.7
81.4
Canada
48.2
54.2
68.5
45.6
Chile
36.9
39.3
90.4
74.7
Cyprus
29.5
33.9
64
56.3
Czech Republic
15.7
23
87.2
69.4
France
20.3
32.9
86.6
65.6
Germany East
40.1
56.3
91.6
73.2
Germany West
53.5
64.5
72.3
47.2
Great Britain
32.5
48
79.7
65
Hungary
9
24.4
92.3
78.4
Israel
35.8
37.6
89.4
80.5
Japan
41.1
54.5
63.8
47.4
Latvia
15
20.4
96
75.5
New Zealand
40.6
50.7
70.5
46.7
North Ireland
43.2
53.9
64.5
62.7
Norway
30.9
37.9
71.5
59.9
Philippines
63.2
69.3
64.8
58.4
Poland
21.8
31.9
84.9
80.3
Portugal
36.1
44.6
95.1
88.7 Share of Respondents Who Say They "Strongly Agree" or "Agree"
with the Indicated Statements, Referring to Their Own Country 16
Russia
8
8.8
93
82
Slovakia
5.9
8.5
93.1
71.8
Slovenia
12.5
19.7
89.4
83.3
Spain
37.2
41.4
88.3
77
Sweden
34.1
38
70
57.4
United States
60.7
69.4
61.7
32.6
Source: International Social Survey Programme 1999: Social Inequality III (ISSP 1999) 16 Given the deep cultural embeddedness of Americans’ belief in fluid status mobility, it is
not surprising that it continues to find support in more recent surveys. A March 2005 New York
Times poll found that whereas 82 percent of Americans identified themselves as working class,
middle class, or lower class, 45 percent believed that it is very or somewhat likely that they will
become wealthy in the future. A striking 80 percent believe it is possible to start out poor and
become rich through hard work. Forty percent believe that it is easier to move up today than 30
years ago, whereas 23 percent believe it more difficult, and 35 percent believe there is no
change. These five proxies provide strong support for the contention that Americans believe their
country provides greater potential for changing social status through personal effort than do
peoples of other wealthy or moderately wealthy countries. To a greater extent than in these other
countries, Americans believe their society is one of equal opportunity. In 2008, in spite of negative views concerning the future of the economy, the majority
of Americans remained optimistic concerning their own futures. For instance, according to a
March 2008 Pew survey, whereas 56 percent of Americans rated the economy as poor, 55
percent believed that their own financial situation would improve over the coming year (Pew
Research Center 2008). A June-July 2008 nationwide poll by the Washington Post found that
low-wage workers (accounting for about one-quarter of all U.S. adults) “remain inspired by the
American dream” (Fletcher and Cohen 2008: A1). Although nearly half believed that their
financial situation deteriorated during the Bush administrations, 69 percent are hopeful of their
financial situation. When asked, “What do you think is more likely over the next few years in
terms of your social class?”, 58 percent believe they will move up, versus 14 percent who believe
they will slip backward and 24 percent who believe they will remain the same. Moreover, six 17 17
out of 10 believe they are responsible for their own financial situation (Fletcher and Cohen 2008:
A13). Yet other evidence corroborates the contention that, more than their counterparts abroad,
Americans see their country as offering the potential for upward mobility. It might be expected
that in a nation in which there is a relatively strong belief in the possibility of vertical mobility
and in which income distribution is highly unequal, individuals would not only readily take
credit for their economic successes, but also readily view the less fortunate as responsible for
their economic failures. This seems to be the case. Alesina, Glaeser, and Sacerdote report that
the World Values Survey found that 71 percent of Americans versus 40 percent of Europeans
believe that the poor could work their way out of poverty. “…54 percent of Europeans believe
that the poor are unlucky, whereas only 30 percent of Americans share that belief.” And “Sixty
percent of American respondents, but only 26 percent of Europeans say that the poor are lazy”
(2001: 237; 242; 243). These differences may also help explain Americans’ stronger embrace of
laissez-faire political ideology (see also, Lipset 1998). Alesina and La Ferrara find that in the
U.S., those individuals who believe they will experience future income growth more readily
oppose measures that would redistribute income in favor of the less-well off (2001). These five proxies provide strong support for the contention that Americans believe their
country provides greater potential for changing social status through personal effort than do
peoples of other wealthy or moderately wealthy countries. To a greater extent than in these other
countries, Americans believe their society is one of equal opportunity. Other
evidence also suggests that to a greater extent Americans hold individuals responsible for their
own fates. For instance, Alesina, Glaeser, and Sacerdote report that they “find an extremely
strong relationship in the United States between supporting capital punishment and opposing
welfare” (2001: 242). The data presented above (as well as that provided by Alesina and La Ferrara 2000)
suggest that there is “American exceptionalism,” but that it relates not to the actual degree of
vertical mobility in American society, but Americans’ exceptionally strong belief that they live 18 18
in a land of highly fluid vertical mobility, a land of relatively equal opportunity. If they study,
work hard and diligently, they can improve their social status. Each is responsible for his or her
own status. Possessing high status is thus a consequence of virtue. This places a premium on
showing higher status. One option for doing so is to struggle to consume at the level of those
with higher status and thereby improve one’s reputability. This special pressure to demonstrate
higher status through consumption may help account for the exceptionally low personal saving
rate in the U.S. Thus, there seems to be considerable support for the hypothesis that the low rate of
household saving in the U.S. is related to Americans’ strong belief that vertical mobility in the
U.S. is readily possible and hence their relatively weak sense of class identity. It might be noted that the hypothesis explored above is not in conflict with a long-
entertained cultural hypothesis constructed upon the fact that Americans have been targeted more
than peoples of other cultures by advertising, and that this bombardment has become ever more
intense.18 For instance, Frank Levy and Richard C. Michel argue that our low savings is due to
Aa culture that bombards us with messages to buy things – not save – every day. In this context,
the key to increased savings is a change in national outlook@ (1990: C2). They go on to suggest
that a national campaign such as that to reduce smoking or drunk driving or casual sex would
work. This general thesis was suggested a half-century ago by John Kenneth Galbraith=s
Adependency effect,@ advanced in his The Affluent Society in 1958. It was also the thesis of pop
sociologist Vance Packard=s 1957 The Hidden Persuaders. Clearly this Aadvertising-assault@ thesis is not in conflict with the hypotheses that are
explored here. These five proxies provide strong support for the contention that Americans believe their
country provides greater potential for changing social status through personal effort than do
peoples of other wealthy or moderately wealthy countries. To a greater extent than in these other
countries, Americans believe their society is one of equal opportunity. Any persuasive advertising would potentially guide and possibly augment a
conspicuous consumption effect.19 19 9
This section has found substantial support for this paper’s first hypothesis that the low
rate of household saving in the U.S. is related to American’s relatively weak sense of class
identity, and their relatively strong belief that vertical mobility is readily possible. The next
section will explore the second and corollary hypothesis that in an economy in which a high
degree of vertical mobility is thought possible, a high degree of inequality in the distribution of
income and wealth may reinforce the tendency to save little. Increasing Inequality and the Collapse of Household Saving The collapse in personal saving in the U.S. over the past quarter century has drawn a
great deal of attention.20 Most economists who have addressed the issue have done so by
drawing upon the life-cycle model of saving. However, as noted earlier, this model has been
found theoretically inadequate. It has also not fared well empirically (Courant, Gramlich, and
Laitner 1984; Mokhtari 1990; Thaler 1990). After examining the major “theories/explanations”
for the fall in saving rates (wealth effects, “new economy” effect, financial innovation, social
security programs and macroeconomic stability, demographics, Ricardian equivalence, and
trends in the way companies compensate shareholders), Guidolin and La Jeunesse conclude that
“none of them provides a compelling explanation” (2007: 491; See also Attanasio and Paiella
2001: 110). Similarly, Munnell, Golub-Sass and Varani note that “Economists have spent a lot
of energy attempting to explain the precipitous drop, but with little success” (2005: 2). Examining an earlier fall in the saving rate, Barry Bosworth concluded that “there is no simple
explanation for the collapse in saving.” He went on to suggest that to some extent some of the
explanation may be found in culture. Consonant with Bosworth’s suggestion, the culturally grounded hypothesis explored here
is that if there is a strong, widespread belief that a high degree of vertical mobility is possible, 20 then a high degree of inequality in the distribution of income and wealth may reinforce the
tendency to save little as the distance between consumption levels is greater. And a further
increase in inequality would put more pressure on individuals to consume yet more to attain their
status targets, thereby saving less. Keynes noted in the General Theory that personal saving is related to income and wealth
distribution. However, the second hypothesis set forth here – that greater inequality increases
consumption (lowers the saving rate) – is contrary to Keynes’ view that increasing equality
would raise consumption, by raising the average marginal propensity to consume (Keynes 1936:
372-75). The suggestion offered here is that the fact that the very wealthy have become so much
wealthier has meant not only that they themselves have dramatically increased their own
conspicuous consumption, but that because the gap between their incomes and those below them
is so much greater, those below must spend ever-more to achieve their status goals. Since the early 1970s, income inequality has become much greater in the U.S. Increasing Inequality and the Collapse of Household Saving The
poorest 20 percent of Americans saw their share of total income decline from 5.5 percent in 1973
to 4.0 percent in 2005. Over the same period, the second poorest 20 percent saw their share drop
from 11.9 to 9.6 percent, the middle 20 percent from 17.5 to 15.3. Meanwhile, the share of the
richest 20 percent rose from 41.1 to 48.1 percent. And the richest five percent saw their share
climb from 15.5 to 21.1 percent (Table 1.9, Mishel et. al. 2007). The rise in the Gini Coefficient
depicted in the chart below provides a graphic glimpse of this growing inequality. Inequality in 21 wealth ownership is yet far greater, and has also greatly increased (Wolff 2002). Figure 1: Gini Ratio for U.S. Households
Source: Bureau of the Census, Historical Income Table H-4
0.40
0.42
0.44
0.46
0.48
80
85
90
95
00
05
Year
http://www.census.gov/hhes/www/income/histinc/h04.html Figure 1: Gini Ratio for U.S. Households
Source: Bureau of the Census, Historical Income Table H-4
0.40
0.42
0.44
0.46
0.48
80
85
90
95
00
05
Year
http://www.census.gov/hhes/www/income/histinc/h04.html http://www.census.gov/hhes/www/income/histinc/h04.html It has been estimated that on the eve of the American Revolution, the top one percent of
households held about 15 percent of all wealth. By the end of the Civil War, they may have held
25 percent (DeLong 1997). As the figure below indicates, there have been three major
expansions in the share of wealth held by the super-rich: The first occurred between the end of
the Civil War and lasted until about 1900. It was this explosion in inequality and the behavior
that accompanied it that led Veblen to write his most noted work, Theory of The Leisure Class,
in which he unfolds his theory of conspicuous consumption. The second major expansion in the
share of wealth held by the super-rich occurred following World War I and lasted until the late
1920s. The last expansion began in the early 1970s and continues to the present. The first two
periods of rapidly rising inequality were called, respectively, the “Gilded Age,” and the “Roaring
Twenties,” suggesting the consumption orgies that accompanied these periods. The last period, 22 the present one, has yet to acquire a sobriquet that has stuck. Like the two earlier periods, it has
also witnessed extravagant consumption by the very rich, which has been emulated by those
further down the ladder. Source: DeLong, J. Bradford. 1997. Source: DeLong, J. Bradford. Increasing Inequality and the Collapse of Household Saving 1997. In a society in which individuals generally feel responsible for their own status and
struggle to demonstrate status through consumption, this substantial increase in inequality might
be expected to prompt households to potentially respond in three manners: People might
consume more, forcing them to save less; they might become more indebted to enable greater
consumption; and they might increase the hours they work to enable them to increase their
consumption levels. Evidence indicates that U.S. households have done all three. As inequality has increased over the past quarter century, the personal saving rate has
fallen, as noted earlier, from 10.4 percent in 1980-84, to 7.7 percent in 1985-89, to 6.5 percent in
1990-94, to 3.8 percent in 1995-99, to 2.1 percent in 2000-04; and in 2005 and 2006, the rates 23 were negative, -0.4 and -1.0 percent, respectively (See also figure 2 below). Where there is a
strong perception that vertical mobility is possible, growing inequality might put additional
pressure on households to struggle ever harder to demonstrate higher status through
consumption. Increasing inequality means the status standard is ever higher.21 A “free-to-choose” interpretation would not adequately capture the dynamics of this
intensified struggle. In his last major work, The Revolt of the Elites (1996), Christopher Lasch
noted that as economic elites take an ever-greater share of income and wealth, they tend to
isolate themselves in social enclaves such as gated communities, exclusive clubs, and private
schools. They tend to work in jobs, live in neighborhoods, and move in circles where they
literally do not see those struggling to stay on their feet in the economy. Because of elites’
disproportionate political power, this withdrawal from wider society and from direct contact with
the concerns of other citizens erodes support for public services on which those further down the
economic ladder depend—services such as schools, parks, transportation, even public safety. The decay of public services encourages those beneath the elites to do what is necessary – reduce
saving (see figure 2 below), become more indebted, or increase work hours – to enable them to
send their children to decent schools or to safe country clubs.22 And, of course, as those who can
afford to consume the private provision of these services opt out of consuming the public ones,
political support for, and the quality of, the latter continue to deteriorate. Increasing Inequality and the Collapse of Household Saving A vicious cycle
appears set in motion promising increasingly inferior public goods and ever-greater pressure to
increase consumption. 24 Figure 3: Personal Savings Rate Source: Bureau of Economic Analysis -4
0
4
8
12
16
80
85
90
95
00
05
Year -4
0
4
8
12
16
80
85
90
95
00
05
Year Year As inequality has increased, households have taken on more debt. Average consumer
debt in 2003 dollars for Americans over 15 years of age has increased from $712 in 1980 to
$3,261 in 2003 (Adkisson and McFerrin 2005: 447). This increased indebtedness holds for
households in all income quintiles. However, the indebtedness of lower and middle income
households has grown significantly more relative to income than has that of wealthier As inequality has increased, households have taken on more debt. Average consumer
debt in 2003 dollars for Americans over 15 years of age has increased from $712 in 1980 to
$3,261 in 2003 (Adkisson and McFerrin 2005: 447). This increased indebtedness holds for
households in all income quintiles. However, the indebtedness of lower and middle income
households has grown significantly more relative to income than has that of wealthier
households (See Table V below). Although there are other hypotheses for why indebtedness of
those in the lower part of the income distribution has increased (e.g., Weller 2007), the rise in
indebtedness for the rich and poor alike fits the hypothesis set forth here, that in a society in
which vertical mobility is believed to be highly fluid, increasing gaps in income all along the
spectrum would stimulate everyone but those at the very top to struggle harder to meet their
consumption status targets.23 households (See Table V below). Increasing Inequality and the Collapse of Household Saving Although there are other hypotheses for why indebtedness of
those in the lower part of the income distribution has increased (e.g., Weller 2007), the rise in
indebtedness for the rich and poor alike fits the hypothesis set forth here, that in a society in
which vertical mobility is believed to be highly fluid, increasing gaps in income all along the
spectrum would stimulate everyone but those at the very top to struggle harder to meet their
consumption status targets.23 It is noteworthy that increasing income and wealth inequality have occurred even in the 25 25
highest income decile, with the consequence that even those in this decile have, on average,
become increasingly indebted.24 The greatest gains in wealth and income have been for the
super-rich, the top 0.1 percent or one tenth of the top one percent (Wolff 2002), dramatically
increasing the consumption of extremely expensive goods and services (private jets, mansions,
Hirschian positional goods, etc.). These super-rich households are ever in competition with each
other for the very pinnacle of status, with the consequence that “In the consumer race the
finishing line always moves faster than the fastest of runners” (Bauman 2000: 72). This has put
pressure on the lesser rich who also wish to be seen as at the very top. This pressure is likely
reinforced by the advertising and programming that continually keep the consumption standards
of the rich and famous on public display (See Schor 1998; Frank 1999). TABLE V
Ratio of the Mean Value of Outstanding Debts to
Mean Family Income by Percentile
Year
Income
Percentile
1989
1992
1995
1998
2001
2004
All Families
0.88
1.08
1.09
1.20
1.05
1.47
Less than 20
0.89
0.33
1.85
1.84
1.68
2.31
20-39.9
0.86
0.35
1.12
1.23
1.12
1.62
40.59.9
0.85
0.42
1.03
1.19
1.18
1.61
60-79.9
0.96
0.71
1.14
1.32
1.16
1.56
80-89.9
0.84
0.85
1.08
1.14
1.12
1.47
90-100
0.60
0.62
0.76
0.79
0.68
0.99 Source: Survey of Consumer Finances If, as a consequence of rising inequality, individuals must spend ever more to attain their
status targets, then it might also be expected that they would increase their work hours to be
better able to do so. Indeed, during basically this same period – between 1970 and 2002 – work
hours per capita rose 20 percent in the U.S. By contrast, in the European Union where income
inequality has not much changed, work hours fell 12 percent (OECD 2004, Chapter 1). Indeed, 26 6
a study by Samuel Bowles and Yongjin Park has found “that increased inequality induces people
to work longer hours [and] …the underlying cause is the Veblen effect of the consumption of the
rich on the behaviour of those less well off” (2005: F410). It is also notable that the U.S. is the
only wealthy country that does not legally require employers to provide paid leave (Ray and
Schmitt 2007: 1). Even when U.S. workers held far more political power than is the case today,
they did not strongly pressure government to make leave mandatory. It is conceivable that the
relatively high conspicuous consumption pressures in the U.S. may help explain why. This is
conformable to Veblen’s contention that increased productivity would lead to greater
conspicuous consumption rather than fewer work hours: “As increased efficiency makes it possible to procure the means of livelihood with less
labour, the energies of the industrious members of the community are bent to the
compassing of a higher result in conspicuous expenditure, rather than slackened to a more
comfortable pace” (1899: 111). 25 Summary The low and falling U.S. personal saving rate has been an enigma within the economics
profession. Attempts to solve this enigma, most drawing upon the life-cycle theory of saving,
have not been successful. This study has found considerable support for two hypotheses that may help explain both
why U.S. household saving is low relative to rates in other relatively wealthy countries and why
they have declined so radically over the past quarter century. The first is that the low rate of
household saving in the U.S. is related to Americans’ relatively strong belief that vertical
mobility is readily possible. Evidence strongly suggests that Americans believe status to be
more fluid in the U.S. than do their foreign counterparts for their respective countries. 27 27
Consequently, Americans are more compelled to internalize responsibility for their own status. Their status is a consequence of their own actions. If they work hard and diligently they might
move up. However, the most ready way to flash one’s status is through consumption. A higher
level of consumption generally correlates with a higher level of income which in turn can be
understood to be the consequence of hard work. Thus, Americans feel more compelled to strive
for status through conspicuous consumption than do people in other relatively wealthy countries
in which belief in the potential for vertical mobility is weaker. The result is a lower rate of
saving. The second hypothesis is that in an economy in which a high degree of vertical mobility
is thought possible, a high degree of inequality in the distribution of income and wealth can be
expected to reinforce the tendency to save little. Great inequality means that consumers must
stretch further in their emulation of the consumption standards above them. The evidence
examined above demonstrates that this is indeed what they have done, resulting in a falling rate
of saving. These two hypotheses are founded upon the fact that social status is critically important
to people and thus strongly affects their behavior, where social status is meant in the broad sense
whereby an individual is motivated, as Karl Polanyi put it “to safeguard his social standing, his
social claims, his social assets” (1944: 46). The human preoccupation with status or relative
social position is understandable from an evolutionary perspective. Summary Those with higher status,
whatever its source, would possess disproportionate access to resources and members of the
opposite sex, thus permitting more and better cared-for progeny. A proclivity for seeking status
would thus be naturally selected. Or, as Robert Frank has put it, “falling behind one’s local
rivals can be lethal” (2005: 183). 28 Given the complexity of the motivation to save, it is not surprising that mainstream
theories crafted to explain saving behavior have not been compelling, leaving the issue a
“puzzle.” Bosworth was right to suggest that we might well turn to culture for a fuller
understanding. That is what this study does. Although the hypotheses explored here are not
meant to fully explain saving behavior, they are strongly supported by the evidence examined
above. The substantial support for these hypotheses also points to the continuing validity and fertility of Veblen’s rich socio-economic theory of consumer behavior. fertility of Veblen’s rich socio-economic theory of consumer behavior. fertility of Veblen’s rich socio-economic theory of consumer behavior. Bernt Bratsberg & Knut Røed & Oddbjørn Raaum & Robin Naylor & Markus Jäntti and Tor
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in J. Davis, W. Dolfsma, E. Veblen, Thorstein. 1919. “Some Neglected Points in The Theory of Socialism,” in The Place of
Science in Modern Civilization. New York: B.W. Huebsch, 387-408. NOTES 1 The national income and product accounts (NIPA) personal saving rate is computed by the Bureau of Economic
Analysis. It includes households and nonprofit institutions. It is the remainder of personal income after current
outlays for personal consumption and taxes, non-mortgage interest payments, and net transfers to government or
abroad. 2 Curiously, for the five years following the 1981 tax cuts that were to spur personal and private saving, the average
household saving rate continued to fall (Summers and Carroll 1987: 607). Summers and Carroll point out that “In
1986, American net national saving was below 2 percent of GNP, less than half the rate in Britain, less than 30
percent of the rate in France and Germany, and only 10 percent the rate in Japan” (1987: 607). 3 This could also be seen in terms of the ratio of consumption to GDP. While roughly constant between 1950 and
1980, over the following two decades it rose by about six percent (Parker 1999: 1). 3 This could also be seen in terms of the ratio of consumption to GDP. While roughly constant between 1950 and
1980, over the following two decades it rose by about six percent (Parker 1999: 1). It should be noted that this negative savings rate is not fully representative of household conditions. Asset
values have increased substantially in recent years, especially for real estate. It should be noted that this negative savings rate is not fully representative of household conditions. Asset
values have increased substantially in recent years, especially for real estate. 4 Their use of the term puzzle is highly Kuhnian: “In economics, a phenomenon is said to represent a puzzle when
standard and generally accepted economic principles and theories fail to provide a quantitatively satisfactory
explanation for a set of empirical regularities” (2007: 508). 4 Their use of the term puzzle is highly Kuhnian: “In economics, a phenomenon is said to represent a puzzle when
standard and generally accepted economic principles and theories fail to provide a quantitatively satisfactory
explanation for a set of empirical regularities” (2007: 508). 5 Although Veblen’s theory of consumer behavior has been the target of numerous attacks, Andrew Trigg argues
convincingly that they “misrepresent Veblen’s original conception of conspicuous consumption and take it out of
context in relation to his overall framework” (2001: 100). REFERENCES Oughton and J. Wheelock, eds., Elgar Handbook of Socio-
Economics. Starr, Martha. 2007. “Spending, Saving and Self-Control,” Review of Radical Political
Economy, Vol. 39 (2), Forthcoming. Stigler, George and Gary Becker. 1977. “De Gustibus Non Est Disputandum,” American
Economic Review, Vol. 67, No. 2 (March), pp. 76-90. Summers, Lawrence, and Chris Carroll. 1987. “Why is U.S. National Saving so Low?”
Brookings Papers on Economic Activity, 1987 (2), 607-42. Thaler, Richard H. 1990. “Anomalies: Savings, Fungibility, and Mental Accounts,” Journal of
Economic Perspectives, 4 (1), Winter: 193-205. Thaler, Richard H. 1994. “Psychology and Saving Policies,” The American Economic Review,
84 (2), May, 186-92. Trigg, Andrew B. 2001. “Veblen, Bourdieu, and Conspicuous Consumption,” Journal
of Economic Issues, XXXV (1), March: 99-115. Twitchell, James B. 2002. Living it Up: America's Love Affair with Luxury New York: Simon
and Schuster. Tyree, Andrea and Moshe Semyonov and Robert W. Hodge. 1979. AGaps and Glissandos:
Inequality, Economic Development, and Social Mobility in 24 Countries,@ American
Sociological Review, 44, June: 410-24. Veblen, Thorstein. 1899. The Theory of the Leisure Class. New York: The Modern Library,
1934. Veblen, Thorstein. 1919. “Some Neglected Points in The Theory of Socialism,” in The Place of
Science in Modern Civilization. New York: B.W. Huebsch, 387-408. 34 Weller, Christian E. 2007. “Need or Want: What Explains the Run-up in Consumer Debt?”
Challenge, XLI (2), June: 583-91. Weller, Christian E. and Edward Wolff. 2005. Retirement Income: The Crucial Role of Social
Security. Washington, D.C.: Economic Policy Institute. Weller, Christian E. and Edward Wolff. 2005. Retirement Income: The Crucial Role of Social
Security. Washington, D.C.: Economic Policy Institute. Wolff, Edward. 2002. Top Heavy: A Study of Increasing Inequality of Wealth in America. New
York: Twentieth Century Fund Press. 8 For a listing of other criticisms of the life-cycle model, see Starr 2006b: 1. NOTES Note the extreme case of India=s Auntouchables,@ who, until fairly recently in their history,
generally believed that their grim existence at the bottom of the social structure was just, due to poor karma in an
earlier incarnation. 13 There are, of course, other, aesthetically more sophisticated means by which those successful in earning a good
deal of money can demonstrate their virtue and status. But, as Veblen noted, the “cultivation of the aesthetic faculty
requires time and application, and the demands made upon the gentleman in this direction therefore tend to change
his life of leisure into a more or less arduous application to the business of learning how to live a life of ostensible
leisure in a becoming way” (1899: 75-75). The consequence, as Alan Shipman notes, is that “Those who have got
rich quick have an understandably low tolerance for the time and tuition needed to gain cultural accomplishment. So they aim to let depth of pocket prevail over depth of discernment, and shift the battlegound from unearned
income to unashamed expenditure” (2004: 279). 14 It should be noted, however, that despite the decline in the Protestant ethic, status today is understood to be far
more the consequence of hard work than was the case at the time Veblen was writing. Writing at the end of the 19th
century, Veblen viewed people as struggling to exhibit leisure status, that is, to be above work. He noted, for
instance, that “wealth acquired passively by transmission from ancestors or other antecedents presently becomes
even more honorific than wealth acquired by the possessor’s own effort.” And, “The leisure class stands at the head
of the social structure in point of reputability; and its manner of life and its standards of worth therefore afford the
norm of reputability for the community” (1899: 29; 84). The reason for this, he claimed, is that “During the
predatory culture labour comes to be associated in men’s habits of thought with weakness and subjection to a
master. It is therefore a mark of inferiority, and therefore comes to be accounted unworthy of man in his best estate. By virtue of this tradition labour is felt to be debasing, and this tradition has never died out” (1899: 36). In the U.S. today, these attitudes toward work and leisure are no longer prevalent. Instead, everyone feels
compelled to work. Even the very rich work. NOTES 6 This might be triggered by the collapse of real estate values that may have already begun. Sierminska and
Takhtamanova speculate that because “the housing wealth effect dominates the financial wealth effect [on saving],
at least in some countries, then the effects of a softening in the housing market in a number of industrialized
countries could have effects more dramatic than the historic stock market declines that began in 2000” (2007: 20). 7 A study by Douglas Bernheim (1993) finds that baby-boomers are saving only a third of what they will need to
maintain their standard of living in retirement. Weller and Wolff (2005) find that over 20 percent of workers
between the ages of 55 and 64 have no retirement savings other than Social Security. For a listing of other criticisms of the life-cycle model, see Starr 2006b: 1. 8 For a listing of other criticisms of the life-cycle model, see Starr 2006b: 1. 9 The possible effect of conspicuous consumption on household saving rates does not appear to have been explored. It has, however, been noted. For instance, Lusardi, Skinner, and Venti, after recognizing that the wealth effect
resulting from the surge in stock market values is only directly relevant to the well-off, speculate that “Conspicuous
consumption of the rich may create imitation effects for the rest of the population” (2001: 9). They do not, however,
follow up on this speculation. 35 10 Duesenberry also identified a relationship between belief in social mobility and consumption: “…recognition of
upward mobility as a social goal converts the drive for self-esteem into a desire for high social status [which]
requires the maintenance of a high consumption standard” (1949: 31). He did not, however, develop this
relationship in the direction of the hypotheses set forth here. 11 Understandably, the aristocracy would struggle to block this quest, even in the domain of conspicuous
consumption. Sumptuary laws B especially those laws proscribing lower classes from wearing the garments of those
classes above them B proliferated in the late Middle Ages in Europe. There were even special courts enforcing these
dress codes. 12 By contrast, in traditional societies in which status was ascribed, high status individuals felt their status was their
right. Indeed, they generally understood themselves to be in fact superior, whereas those below felt themselves to be
in fact inferior. NOTES It is through work that social certification occurs. Some of this
change has taken place over the past 40 years. In the 1960s, people revered the leisured so-called Ajet-setters.@
Today, they would be more readily looked upon as flawed, or even debauched. 15 Note the negative judgment frequently made of the consumption practices of the self-made wealthy, the so-called
nouveau riche. The French have an especially cutting and naughty put-down for such behavior: AIl pete plus haut
que son cul.@ 16 Thus Gary Becker’s view that “low earnings as well as high earnings are not strongly transmitted from fathers to
sons” appears contradicted (1988:10). Other recent studies also challenge Becker’s view. See Bowles, Gintis, and
Groves, eds. 2005. 17 Jäntti et. al. speculate that “One reason that may account for the widespread belief is that it is the middle classes
who primarily hold this belief” and their data suggest that “the U.S. middle classes are quite as likely to be mobile as
those in the U.K. or the Nordic countries” and “Because this substantial fraction of the U.S. population includes the
median voter, such attitudes might help explain why there is not more political pressure for mobility-promoting
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High-throughput quantum-mechanics/molecular-mechanics (ONIOM) macromolecular crystallographic refinement with<i>PHENIX</i>/<i>DivCon</i>: the impact of mixed Hamiltonian methods on ligand and protein structure
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ISSN 2059-7983 Oleg Borbulevych, Roger I. Martin and Lance M. Westerhoff* Received 19 March 2018
Accepted 12 September 2018 Received 19 March 2018
Accepted 12 September 2018 Received 19 March 2018
Accepted 12 September 2018 QuantumBio Inc., 2790 West College Avenue, State College, PA 16801, USA. *Correspondence e-mail:
lance@quantumbioinc.com Edited by R. J. Read, University of Cambridge,
England Conventional macromolecular crystallographic refinement relies on often
dubious stereochemical restraints, the preparation of which often requires
human validation for unusual species, and on rudimentary energy functionals
that are devoid of nonbonding effects owing to electrostatics, polarization,
charge transfer or even hydrogen bonding. While this approach has served
the crystallographic community for decades, as structure-based drug design/
discovery (SBDD) has grown in prominence it has become clear that these
conventional methods are less rigorous than they need to be in order to produce
properly predictive protein–ligand models, and that the human intervention that
is required to successfully treat ligands and other unusual chemistries found
in SBDD often precludes high-throughput, automated refinement. Recently,
plugins to the Python-based Hierarchical ENvironment for Integrated Xtallo-
graphy (PHENIX) crystallographic platform have been developed to augment
conventional methods with the in situ use of quantum mechanics (QM) applied
to ligand(s) along with the surrounding active site(s) at each step of refinement
[Borbulevych et al. (2014), Acta Cryst D70, 1233–1247]. This method (Region-
QM) significantly increases the accuracy of the X-ray refinement process, and
this approach is now used, coupled with experimental density, to accurately
determine protonation states, binding modes, ring-flip states, water positions and
so on. In the present work, this approach is expanded to include a more rigorous
treatment of the entire structure, including the ligand(s), the associated active
site(s) and the entire protein, using a fully automated, mixed quantum-
mechanics/molecular-mechanics (QM/MM) Hamiltonian recently implemented
in the DivCon package. This approach was validated through the automatic
treatment of a population of 80 protein–ligand structures chosen from the Astex
Diverse Set. Across the entire population, this method results in an average
3.5-fold reduction in ligand strain and a 4.5-fold improvement in MolProbity
clashscore, as well as improvements in Ramachandran and rotamer outlier
analyses. Overall, these results demonstrate that the use of a structure-wide
QM/MM Hamiltonian exhibits improvements in the local structural chemistry of
the ligand similar to Region-QM refinement but with significant improvements
in the overall structure beyond the active site. Keywords: X-ray crystallography; quantum-
mechanics refinement; PM6 semiempirical
method; QM/MM; ONIOM macromolecular
refinement; molecular mechanics; stereo-
chemical restraints; ligand strain; MolProbity
clashscore; high-throughput crystallography. Supporting information: this article has
supporting information at journals.iucr.org/d research papers High-throughput quantum-mechanics/
molecular-mechanics (ONIOM) macromolecular
crystallographic refinement with PHENIX/DivCon:
the impact of mixed Hamiltonian methods on ligand
and protein structure research papers bond lengths and angles, and a lack of intermolecular inter-
actions in conventional functionals (Read et al., 2011). Efforts
have been made in recent years to improve the automatic
generation of ligand-restraint libraries for ligands in order to
address these problems. The eLBOW tool (Moriarty et al.,
2009) found within the Python-based Hierarchical Environ-
ment for Integrated Xtallography (PHENIX) package (Adams
et al., 2010) is capable of creating restraints based on quantum-
mechanics optimization, and the AceDRG tool from CCP4
provides similar capabilities (Nicholls, 2017; Long et al., 2017). Alternatively, the publicly available Grade webserver (http://
grade.globalphasing.org) along with the commercial Mogul
package (Bruno et al., 2004), which are both based on the
Cambridge Structural Database (CSD; Groom et al., 2016),
use small-molecule X-ray structural information to determine
target values. Finally, the AFITT program (Janowski et al.,
2016) produced by OpenEye Inc. works to improve the ligand
geometry based on the Merck Molecular Mechanics Force
Field (MMFF94). Regardless of the accuracy of the CIF,
however, conventional methods are unable to accurately
account for crucial binding influences on both the ligand and
the surrounding active site arising from coordination, bond
making/breaking, hydrogen bonding, electrostatics and other
nonbonding interactions (Borbulevych et al., 2014; Janowski et
al., 2016; Read et al., 2011). This problem is further exacer-
bated when such species are covalently bound to the macro-
molecule. drug candidates. In recent years, X-ray crystallography has
become routine thanks to advances in data collection and
processing, structure solution and refinement automation. However, protein crystal models are still subject to significant
uncertainties in atomic coordinates and other structural errors
(Davis et al., 2003, 2007), and these errors negatively impact
the very ligand-binding affinity estimations (Davis et al., 2003)
that are critical to SBDD/FBDD applications. This has led to
the development of structure-validation metrics, including
Ramachandran, clashscore and MolProbity score, the latter of
which is a composite of the clashscore and Ramachandran plot
and rotamer outliers (Ramachandran et al., 2011; Read et al.,
2011; Chen et al., 2010; MacCallum et al., 2009). In particular,
the median clashscore, which is the number of clashes per 1000
atoms, for all X-ray structures deposited in the Protein Data
Bank (PDB) and the worldwide PDB (wwPDB) (Berman et
al., 2003, 2007) since 1990, and determined at a resolution of
1.5 A˚ or better, is 8.8 units. Furthermore, this median score
deteriorates as the resolution decreases (Read et al., 2011). research papers The prevalence of problematic geometries observed in
deposited PDB structures suggests that conventional refine-
ment methods are not sufficiently rigorous to represent the
chemistry within the protein–ligand complex (Kleywegt, 2007;
Pozharski et al., 2013; Smart et al., 2018). Overall, this problem
stems from an intrinsic limitation of macromolecular X-ray
crystallographic refinement, which is its reliance on an insuf-
ficient ratio of observed reflections to refined parameters, as
typically observed at moderate and low resolutions (Rupp,
2009). In order to overcome this limitation, conventional
refinement methods use a priori information about the
structure in the form of stereochemical restraints (for example
bond lengths, bond angles and bond torsion angles, as well as
chirality and group planarity information) for all components
included within the protein–ligand complex. For standard
amino acids, these fixed stereochemical restraints are based on
the ideal Engh and Huber parameters (Engh & Huber, 1991),
and these restraints often lead to significant structural
deficiencies (Moriarty et al., 2014). In these situations, the
backbone geometry can deviate significantly from these ideal
values for high-resolution models (Vlassi et al., 1998), and this
problem becomes even more pronounced when small mole-
cules and ions (for example, ligands, inhibitors and/or metallic
or nonmetallic cofactors) are bound to the protein in question
(Kleywegt, 2007). Surveys of the PDB indicate that the
percentage of ligands with questionable geometric parameters
in deposited macromolecular structures could be as high as
60% (Gore et al., 2011; Liebeschuetz et al., 2012). Taking a different route, in 2014 our laboratory introduced
(Borbulevych et al., 2014) a plugin to the PHENIX package to
treat the active site or the entire protein using our DivCon
linear-scaling, semiempirical quantum-mechanics (SE-QM)
implementation (Dixon & Merz, 1996, 1997) and the PM6
Hamiltonian (Stewart, 2009; Rˇ eza´cˇ et al., 2009). The advantage
of this approach is that interactions such as hydrogen bonding,
dispersion, electrostatics, polarization and charge transfer
between the ligand and the protein are taken into account
(Diller et al., 2010; Zhang et al., 2010). While the DivCon
implementation can be applied to structures with thousands or
even tens of thousands of atoms, the plugin was designed to
optionally focus the QM method on one or more user-
definable regions (for example active sites, ligands, key resi-
dues etc.) during the refinement (Region-QM), leaving the rest
of the macromolecule dependent on conventional stereo-
chemical restraints. 1. Introduction X-ray crystallography is a popular technique that is used to
determine the three-dimensional atomic structures of bio-
molecular systems, which serve as three-dimensional templates
for structure-based drug discovery (SBDD) and fragment-
based drug discovery (FBDD). The quality of the model is
crucial for the overall success of high-throughput screening,
docking and scoring (for example rank ordering) of potential https://doi.org/10.1107/S2059798318012913
1063 Acta Cryst. (2018). D74, 1063–1077 research papers 2. Methods 2.1. PHENIX refinement and the QM/MM methodology Typical biomacromolecular systems, such as those including
protein, DNA and/or RNA, are usually quite large and ab
initio or density functional theory (DFT) QM methods are too
expensive to treat these structures quickly and efficiently on
the timescales demanded by industrial practitioners. The
DivCon Discovery Suite (QuantumBio, 2017) employs divide-
and-conquer (D&C), linear scaling, semiempirical quantum-
mechanics (SE-QM) methods described previously (Dixon &
Merz, 1996, 1997; Van der Vaart, Gogonea et al., 2000; Van der
Vaart, Suarez et al., 2000; Wang et al., 2007) to characterize all-
atom structures of tens or even hundreds of thousands of
atoms using the traditional AM1 (Dewar et al., 1985) or PM3
(Stewart, 1989) SE-QM Hamiltonians, as well as the more
modern PM6 Hamiltonian (Stewart, 2009; Rˇ eza´cˇ et al., 2009). Over the last two decades, this approach has been applied to a
number of key SBDD applications including QMScore (Diller
et al., 2010; Merz & Raha, 2011; Raha & Merz, 2005; Zhang et
al., 2010) and NMRScore (Wang et al., 2004, 2007; Williams et
al., 2009), QM-based quantitative structure–activity relation-
ship (QSAR) models (Dixon et al., 2005; Peters & Merz, 2006;
Zhang et al., 2010) and X-ray refinement (Borbulevych et al.,
2014, 2016; Li et al., 2010; Yu et al., 2005). There are generally two QM/MM coupling schemes in
common use in the computational chemistry field today:
additive (Liu et al., 2014) and subtractive (Vreven et al., 2003). Additive QM/MM represents the energy of the system as the
sum of three terms, EQM=MM
system
¼ EQM
region þ EMM
region þ EQM=MM
interactions:
ð1Þ ð1Þ The first two terms describe the energies of the QM and MM
regions, respectively, and the third term explicitly expresses
interactions (coupling) between the QM and MM subsystems
in the form of an additional, one-electron QM Hamiltonian
describing the electrostatic coupling interactions between the
two layers (Brooks et al., 1983; Field et al., 1990). This coupling
term leads to greater complexity in the Hamiltonian, and
calculating this term accurately can be particularly difficult
given the inclusion of link atoms and electrostatic perturba-
tions in the QM Hamiltonian (Plotnikov et al., 2011). research papers In the present work, we explore a
‘complete functional’ representation for macromolecular
refinement which uses a mixed quantum-mechanics/molecular-
mechanics (QM/MM) Hamiltonian based on the ONIOM
(Our own N-layered Integrated molecular Orbital and mole-
cular Mechanics) method (Vreven et al., 2003) as recently
implemented in DivCon Discovery Suite build-7.1.1-b4015.17
(QuantumBio, 2017). We use SE-QM for the high-level theory,
‘region layer’ [including ligands(s) and active site(s)], while
the remainder of the biomolecule, called the ‘system layer’, is
treated using our implementation of the Assisted Model
Building with Energy Refinement (AMBER) molecular-
mechanics force field (Case et al., 2014). In addition to vali-
dating the ONIOM refinement method against our previous These conventional methods rely on a detailed description
of the molecular geometry for each species to be refined, and
an accurate library or Crystallographic Information File (CIF)
is important to the ultimate success of the effort. Unfortu-
nately, the creation and validation of accurate CIFs is a non-
trivial task which requires significant human intervention and
often leads to bound ligand structures of less than desirable
quality. These deficiencies are owing to the great variety of
ligand chemistries and structures (Kleywegt, 2007), incom-
plete or inaccurate a priori understanding of in situ bound 1064
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement Acta Cryst. (2018). D74, 1063–1077 research papers Region-QM method, the results of conventional refinement as
provided by the PHENIX platform are also discussed. these structures as it allows one to treat the region of interest,
such as an active site, at an SE-QM level of theory, while the
remaining residues outside this region are treated at a faster,
more approximate molecular-mechanics (MM) level of theory. This approach combines these different levels of theory in a
way which significantly improves the speed of the calculation
versus treating the entire structure at the higher level, but with
a greater accuracy than if the entire structure were treated at
the lower level (Chung et al., 2015). 2. Methods 2. Methods Subtractive QM/MM, on the other hand, represents the
energy of a system through the following equation (Vreven et
al., 2003), While the DivCon D&C implementation is faster than
conventional semiempirical implementations, density func-
tional theory (DFT) and ab initio QM methods (Dixon &
Merz, 1996, 1997), linear-scaling SE-QM methods can still
be time-consuming for large biomacromolecular structures
(especially within an industrial environment, where a quicker
turnaround time is often required). Therefore, the mixed QM/
MM Hamiltonian concept provides a reasonable tradeoff for EQM=MM
ONIOM ¼ EQM
region þ EMM
all EMM
region;
ð2Þ ð2Þ where the EMM
all
term is the MM energy calculated for the
entire system, the EMM
region term is the MM energy for a region
and EQM
region is the energy of the region computed using the
QM method. As per Vreven et al. (2003), QM/MM ONIOM Acta Cryst. (2018). D74, 1063–1077
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement
1065
Figure 1
A flowchart of protein–ligand file treatment in ONIOM calculations. Figure 1
A flowchart of protein–ligand file treatment in ONIOM calculations. Figure 1
A flowchart of protein–ligand file treatment in ONIOM calculations. Figure 1
A flowchart of protein–ligand file treatment in ONIOM calculations. Borbulevych et al. High-throughput QM/MM (ONIOM) refinement
1065 Acta Cryst. (2018). D74, 1063–1077 research papers gradients in the subtractive scheme are computed using (3),
which is similar to (2), approximate the interactions between the QM region and the
MM region in a way that does not adversely impact on the
convergence of the QM calculation. rxQM=MM
ONIOM ¼ rxQM
region þ rxMM
all rxMM
region;
ð3Þ ð3Þ Traditionally, with the explosion of different approaches
and implementations, both general QM/MM varieties are
often difficult to use depending upon the application and the
desired outcomes of the investigator (Sousa et al., 2016; Cao &
Ryde, 2018). They can exhibit problems with convergence and
performance which make the routine use of the methods
expensive (Hu et al., 2011), they are often limited to a single,
compact QM region (Case et al., 2018), they require significant
atom-type and charge preparation of any unknown species
(for example ligands, cofactors, nonstandard amino acids etc.)
and protonation (Chung et al., 2015), and/or they rely on the
ability of a user to correctly define the QM atoms/residues
along with any link atoms needed to complete broken bonds
(Sousa et al., 2016). As depicted in Fig. 1, the QM/MM
implementation in DivCon addresses these problems through
the inclusion of the following key features. and in which the gradients of the QM region(s) include
contributions from both the QM and the MM functionals. While standard ONIOM does not include electrostatic
perturbations of the QM density matrix by the atoms within
the MM region, the lack of a coupling term representing the
interactions between these two regions in subtractive QM/MM
leads to generally faster and more convergent calculations,
along with the ability to treat multiple QM regions (such as
those with multiple active sites or sites of interest or those with
multiple copies). This makes the method particularly well
suited to fast, routine, high-throughput QM/MM-based crys-
tallographic refinement. With the use of the gradients repre-
sented in (3), which utilize both QM and MM terms, we can Figure 2
(a) Schematic view of the ONIOM two-layer (MM/QM) concept). (b) A
PDB structure with two ligand regions to illustrate the ONIOM
refinement concept (i) The pervasive use of modern, QM energy-convergence
algorithms. (ii) Automatic perception and characterization of ‘unknown
species’ (for example ligands, cofactors and ions) along with
any closed-shell metal ions supported by our implementation
of the PM6 SE-QM Hamiltonian (Stewart, 2009; Rˇ eza´cˇ et al.,
2009). g
(a) Schematic view of the ONIOM two-layer (MM/QM) concept). (b) A
PDB structure with two ligand regions to illustrate the ONIOM
refinement concept. research papers Table 1
MolProbity statistics after ONIOM, Region-QM and conventional PHENIX refinements of 80 Astex PDB structures. rota_out is the percentage of side chains with rotamer outliers, rama_fav is the percentage of amino acids in the ‘favored’ region of the Ramachandran plot and
rama_iffy is the percentage of amino acids not in the ‘favored’ region of the Ramachandran plot. ONIOM
Region-QM
PHENIX: no QM
PDB
code
Res. research papers (A˚ )
MPscore
Clash-
score
rama_
fav
rama_
iffy
rota_
out
MPscore
Clash-
score
rama_
fav
rama_
iffy
rota_
out
MPscore
Clash-
score
rama_
fav
rama_
iffy
rota_
out
1g9v
1.85
1.23
0.66
98.23
0
4.76
1.57
2.64
98.94
0
4.76
1.54
2.42
98.94
0
4.76
1gkc
2.30
1.47
1.82
97.43
0
3.56
1.77
6.28
96.78
0
1.98
1.54
3.24
96.78
0
1.98
1gpk
2.10
1.03
0.61
95.24
0.19
0.45
1.56
4.36
95.05
0.19
0.68
1.48
3.63
95.24
0.38
0.68
1hnn
2.30
1.75
1.47
97.33
0.38
9.22
2.52
9.55
95.99
0.38
9.46
2.48
8.08
95.99
0.38
10.17
1hp0
2.10
1.29
1.23
97.16
0
2.44
1.97
6.86
96.84
0
3.38
1.85
4.92
96.68
0
3.19
1hq2
1.25
0.74
0.77
98.72
0
0.00
0.83
1.15
99.36
0
0.00
0.74
0.77
99.36
0
0.00
1hvy
1.90
1.02
0.58
96.64
0.18
1.40
1.76
4.87
96.29
0.27
2.20
1.75
4.13
95.94
0.18
2.30
1hwi
2.16
1.15
0.30
96.60
0.20
2.62
1.74
3.61
96.60
0.20
3.03
1.75
3.05
96.21
0.13
3.36
1hww
1.87
0.89
0.86
97.33
0.20
0.44
1.49
5.36
96.74
0.40
0.44
1.36
4.81
97.43
0.40
0.55
1ia1
1.72
1.62
1.70
97.63
0.26
6.61
2.01
5.25
97.37
0.26
6.32
1.98
4.63
97.37
0.26
6.61
1ig3
1.90
0.98
0.25
96.55
0
1.60
1.66
5.32
96.55
0.20
1.60
1.65
5.44
96.75
0.41
1.60
1j3j
2.30
1.53
1.70
94.43
0.46
1.97
2.29
8.81
92.39
0.84
2.86
2.23
8.43
93.13
0.84
2.76
1jd0
1.50
0.82
1.10
98.25
0
0.43
1.09
2.70
97.86
0
0.43
1.04
2.33
97.86
0
0.43
1jje
1.29
1.39
0.29
95.65
0.23
4.24
2.24
4.86
95.42
0.92
7.69
2.12
3.86
95.42
0.46
6.63
1jla
2.50
1.66
0.89
95.53
0.53
5.83
2.78
11.98
91.28
1.28
7.81
2.74
10.52
91.38
0.96
8.04
1k3u
1.70
0.70
0.40
97.72
0.15
0.78
1.15
2.99
97.72
0.15
0.78
1.16
3.09
97.72
0.15
0.58
1ke5
2.00
0.96
1.97
98.55
0
0.00
1.49
6.99
97.45
0
0.41
1.43
6.99
97.82
0
0.82
1kzk
1.09
0.85
0.93
98.45
0
1.22
1.17
3.10
98.97
0
1.22
1.10
3.10
98.45
0
0.61
1l2s
1.94
0.60
0.27
98.02
0
0.89
1.04
2.10
97.74
0
0.71
1.05
2.01
97.74
0
1.07
1l7f
1.80
1.06
0.80
95.60
0
0.00
1.34
3.04
96.37
0
0.88
1.36
3.04
96.11
0
0.88
1lpz
2.41
1.53
0.45
95.41
0.35
5.33
2.33
5.36
95.05
0.71
8.20
2.19
4.25
95.05
0.71
6.97
1lrh
1.90
0.80
1.04
98.10
0
0.52
1.39
4.65
98.10
0
1.56
1.30
3.23
98.42
0
1.74
1meh
1.95
1.04
0.73
96.81
0.29
1.39
1.73
5.48
96.23
0.29
1.74
1.72
5.29
96.23
0.29
1.74
1mmv
2.00
0.87
0.60
97.52
0
1.24
1.82
6.20
96.16
0.12
1.92
1.73
4.86
96.16
0.12
1.92
1mzc
2.00
0.70
0.43
97.77
0
0.94
1.48
3.15
97.07
0.14
1.89
1.45
2.89
97.07
0.14
1.89
1n1m
2.50
2.23
3.74
92.34
1.17
5.98
2.83
14.82
91.52
1.66
7.13
2.76
13.06
92.07
1.45
7.13
1n2j
1.80
0.70
0.23
98.42
0
1.41
1.39
4.02
98.77
0
1.87
1.39
4.02
98.95
0
1.87
1n2v
2.10
1.52
1.21
96.49
0
3.95
2.31
10.74
95.14
0.27
3.62
2.18
8.49
95.68
0.27
3.62
1n46
2.20
1.99
3.83
97.27
0.42
7.93
2.67
11.60
95.81
1.05
11.42
2.55
9.69
96.44
1.05
11.42
1nav
2.48
1.64
1.26
93.93
0.40
3.24
2.12
5.03
93.52
0.40
3.70
2.10
4.53
93.12
0.40
3.70
1of1
1.95
0.89
0.94
98.34
0
1.40
1.59
6.05
97.19
0.50
1.40
1.55
5.11
97.35
0.50
1.60
1of6
2.10
1.82
1.89
95.96
0.51
6.06
2.09
3.71
95.34
0.51
6.28
2.05
3.59
95.60
0.44
6.15
1opk
1.80
0.88
0.28
97.32
0.22
1.53
1.61
2.35
96.20
0.45
2.81
1.51
1.94
96.42
0.45
2.56
1oq5
1.50
1.13
0
95.67
0
2.73
1.62
2.71
96.46
0
2.73
1.59
2.47
96.46
0
2.73
1owe
1.60
0.77
0.52
97.53
0
0.94
1.48
3.88
97.53
0
1.88
1.48
3.10
97.53
0
2.35
1oyt
1.67
1.03
1.30
97.45
0
1.21
1.21
3.04
97.45
0
0.40
1.13
2.39
97.45
0
0.40
1p2y
2.28
1.47
1.08
97.28
0
4.83
1.90
4.31
97.28
0
5.40
1.84
3.39
97.04
0
5.11
1p62
1.90
1.05
0.26
96.44
0
1.94
1.26
1.32
97.78
0
2.91
1.08
1.06
97.78
0
1.94
1q1g
2.02
1.18
1.27
97.37
0
1.86
1.81
6.46
96.54
0.07
2.02
1.64
3.97
96.68
0
2.10
1q41
2.10
1.05
1.00
96.73
0.74
1.16
1.64
3.92
95.83
1.19
1.66
1.51
3.01
95.83
1.19
1.50
1q4g
1.98
1.50
1.54
97.82
0.09
5.53
1.94
5.25
97.73
0
6.25
1.84
4.61
98.00
0
6.25
1r1h
1.95
1.98
3.76
97.12
0.14
7.44
2.32
9.21
97.12
0.43
7.60
2.30
8.41
96.97
0.43
7.60
1r55
1.59
0.71
0.63
98.01
0.5
0
1.39
4.43
97.01
0.50
0.00
1.37
4.11
97.01
0.5
0.00
1r58
1.90
1.34
1.04
94.28
1.91
1.58
1.92
8.13
94.01
1.91
1.26
1.99
8.13
94.01
1.91
1.58
1r9o
2.00
1.69
1.21
94.65
1.11
4.38
2.41
8.06
93.99
2.00
5.60
2.35
6.85
93.99
2
5.60
1s19
2.00
0.77
0
96.02
0
0.43
1.20
1.71
96.02
0
0.43
1.06
0.98
96.02
0
0.43
1s3v
1.80
1.57
1.95
97.28
0
4.17
2.18
4.87
95.65
0.54
6.55
2.13
4.87
96.20
1.09
6.55
1sg0
1.50
1.06
1.08
96.27
0
0.77
1.53
6.32
96.93
0
0.52
1.51
5.52
96.71
0
0.52
1sj0
1.90
1.70
2.27
97.07
0.84
5.07
2.61
13.09
95.82
0.84
8.29
2.36
9.06
96.65
0.84
7.37
1sq5
2.00
0.77
0.87
98.30
0.17
0.67
1.52
4.62
97.11
0.09
1.44
1.36
3.70
97.62
0.17
1.44
1t40
1.80
1.20
0.58
99.04
0
4.63
1.68
3.11
98.41
0.32
5.69
1.68
2.91
98.41
0
6.05
1t46
1.60
1.25
1.67
98.29
0
2.73
1.52
2.71
97.61
0
3.12
1.54
2.92
97.61
0
3.12
1t9b
2.20
0.94
1.32
98.38
0
1.26
1.24
4.03
98.29
0
1.16
1.18
3.14
98.63
0
1.26
1tow
2.00
1.37
0.96
96.12
0.78
2.61
1.62
4.78
96.90
0
1.74
1.77
3.82
96.90
0.78
3.48
1tt1
1.93
0.81
0.62
97.99
0
1.36
1.41
3.45
97.99
0
2.27
1.30
2.46
97.99
0
2.27
1tz8
1.85
0.97
1.61
98.45
0
1.21
1.36
4.53
98.23
0
1.51
1.24
3.95
98.23
0
1.21
1u1c
2.20
1.49
1.06
97.30
0.54
5.37
1.86
3.71
97.71
0.47
6.88
1.93
4.29
97.51
0.61
6.46
1u4d
2.10
1.32
1.20
96.84
0.20
2.45
2.08
5.75
96.25
0.40
4.68
1.96
4.31
96.25
0.59
4.45
1uml
2.50
2.44
3.40
92.51
2.31
12.83
3.00
12.52
91.35
2.31
14.47
3.03
12.16
90.78
2.31
15.46
1unl
2.20
1.71
0.77
95.77
0.69
7.66
2.56
6.74
93.59
1.26
10.05
2.56
6.88
93.48
1.14
9.67
1uou
2.11
1.54
1.85
97.69
0
4.92
2.13
8.31
97.69
0.23
6.46
2.12
8.78
97.92
0
6.77
1v0p
2.00
1.28
0.91
98.09
0.19
4.65
2.17
4.77
96.18
0.38
7.40
2.22
6.48
96.56
0.38
6.98
1v48
2.20
1.15
0.49
97.25
0.39
2.82
1.39
3.46
97.25
0
1.41
1.30
3.21
97.65
0.39
1.41
1v4s
2.30
1.42
1.00
95.52
0.45
2.60
2.37
7.99
92.60
0.67
4.17
2.31
6.71
93.50
0.67
4.69
1vcj
2.39
1.72
0.83
95.61
0
7.38
2.43
7.98
96.12
0
9.23
2.37
6.15
95.87
0.26
9.54 ter ONIOM, Region-QM and conventional PHENIX refinements of 80 Astex PDB structures. Acta Cryst. (2018). D74, 1063–1077 research papers (iii) Integrated protonation methods which include effects
owing to pH, hydrogen bonding, clashes and ring-flip states. (iv) Support for multiple QM region(s) through automatic
residue-based
selection,
expansion
and
broken-bond
completion. (v) Automatic typing of crystallographically truncated
residues and covalently bound residues and ligands. The DivCon Discovery Suite build-7.1.1-b4015.17 was used
for all QM/MM (ONIOM) calculations in this project. This
package includes implementations of the SE-QM Hamilton-
ians AM1 (Dewar et al., 1985), PM3 (Stewart, 1989) and PM6
(Stewart, 2009; Rˇ eza´cˇ et al., 2009) along with an imple-
mentation of the AMBER MM force field (Case et al., 2014). In the present project, we employed a two-layer ONIOM
configuration as depicted in Figs. 2(a) and 2(b) where, for
each characterized structure, the ligand(s) along with the
surrounding active site was (were) treated using the PM6 SE-
QM Hamiltonian and the remainder of the protein was treated
using the 2014 parameter set of the AMBER MM force field. Both PM6 and AMBERFF14 were chosen as they are the
most advanced methods available in the DivCon Discovery
Suite at this time and they include a large coverage of atoms
and atom types (for example, PM6 includes support for
upwards of 70 elements). Furthermore, while newer SE-QM
methods are available in the literature in other packages, such
as PM7 (Stewart, 2013), recent benchmarks indicate similar
performance characteristics between PM6 and PM7, with PM6
often demonstrating superior results (Hostasˇ et al., 2013). Given these observations, the impact of the choice of SE-QM
Hamiltonian on the results observed in the present study
would be negligible. g
(a) Schematic view of the ONIOM two-layer (MM/QM) concept). (b) A
PDB structure with two ligand regions to illustrate the ONIOM
refinement concept. 1066
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement Acta Cryst. (2018). D74, 1063–1077 research papers Borbulevych et al.
High-throughput QM/MM (ONIOM) refinement
1067 research papers research papers Table 1 (continued)
ONIOM
Region-QM
PHENIX: no QM
PDB
code
Res. (A˚ )
MPscore
Clash-
score
rama_
fav
rama_
iffy
rota_
out
MPscore
Clash-
score
rama_
fav
rama_
iffy
rota_
out
MPscore
Clash-
score
rama_
fav
rama_
iffy
rota_
out
1w1p
2.10
0.98
1.34
97.78
0
1.24
1.58
5.06
97.17
0.10
1.61
1.54
4.54
97.48
0.1
1.86
1w2g
2.10
0.83
0.86
97.96
0.26
1.20
1.69
6.01
96.68
1.02
1.59
1.30
3.43
97.45
0.51
1.20
1x8x
2.00
0.70
0.59
99.38
0
0.75
1.12
2.95
99.06
0
1.12
1.12
2.95
99.06
0
1.12
1xm6
1.90
1.01
0.65
97.68
0.15
1.99
1.64
3.88
97.68
0.31
3.31
1.48
3.05
97.99
0.31
3.15
1xoq
1.83
0.61
0.28
98.29
0
1.01
1.07
1.77
98.44
0
1.52
0.97
1.40
98.60
0
1.35
1xoz
1.30
1.10
0.38
99.69
0
4.12
1.52
2.44
99.69
0
4.47
1.54
2.81
99.69
0
4.12
1y6b
2.10
0.88
0.46
97.27
0
1.29
1.24
3.68
98.05
0
1.29
1.47
4.38
97.66
0
1.72
1ygc
2.00
1.09
0.63
97.03
0
1.91
1.66
4.44
97.03
0
2.29
1.56
2.75
97.03
0
2.67
1yv3
1.99
0.57
0.19
98.27
0
0.91
1.08
1.68
98.12
0
1.64
1.15
1.86
97.98
0
1.82
1yvf
2.50
1.21
0.56
94.84
0.71
1.67
2.49
13.11
90.57
1.96
2.71
2.51
12.33
90.75
1.96
3.12
1ywr
1.90
2.3
3.08
95.18
0.9
13.76
2.88
11.43
95.18
0.9
18.79
2.87
10.88
94.88
0.6
18.46
1z95
1.80
1.53
3.03
98.29
0
3.70
1.81
6.82
98.72
0
3.70
1.85
6.82
98.72
0
4.17
2bm2
2.20
2.09
1.39
93.54
0
11.11
2.87
11.12
92.81
0.42
13.04
2.73
8.01
92.6
0.42
12.44
2br1
2.00
1.72
1.13
95.52
0.75
5.86
2.41
5.87
94.03
1.12
7.95
2.34
5.19
94.03
1.12
7.53
2bsm
2.05
1.35
0.3
94.17
0.97
2.82
1.83
2.73
91.75
0.97
2.26
1.79
2.73
92.72
0.97
2.26 H atoms were added to each structure, including all water
molecules, using Protonate3D (Labute, 2009) as implemented
in MOE2016 from Chemical Computing Group Inc. Likewise,
CIFs for any unsupported species were automatically gener-
ated using Scientific Vector Language (SVL) extensions to
MOE2016 provided in the DivCon Discovery Suite. For each
structure in the set, every copy of each ligand specified in
Table 1 was chosen as one or more QM region centers. The
QM region(s) of each structure was (were) extended 3.0 A˚
from each center to include all amino-acid residues, ions and
crystal waters within each pocket. research papers (2014),
(4) is extended in order to calculate the ONIOM QM/MM
gradients on each atom with coordinates x according to where xray and geom are weights assigned to X-ray data and
geometry (QM/MM ONIOM) restraints, respectively, and
wcxscale is an additional scale factor implemented in PHENIX
(Afonine et al., 2012). geom is typically set to 1, while xray is a
variable weight determined using an automatic procedure in
PHENIX (Adams et al., 1997). Mimicking the Region-QM
refinement framework detailed in Borbulevych et al. (2014),
(4) is extended in order to calculate the ONIOM QM/MM
gradients on each atom with coordinates x according to ðrxiÞtotal ¼ wcxscale xray ðrxiÞxray þ geom ðrxQM=MM
ONIOM Þ; ðrxiÞtotal ¼ wcxscale xray ðrxiÞxray þ geom ðrxQM=MM
ONIOM Þ;
ð5Þ ðrxiÞtotal ¼ wcxscale xray ðrxiÞxray þ geom ðrxQM=MM
ONIOM Þ;
ð5Þ ð5Þ where rxQM=MM
ONIOM corresponds to the ONIOM gradients deter-
mined using (3), where any ligands and surrounding binding
pockets is are defined as part of the QM region and the
remainder of the structure is designated as the MM region. Under this regime, unlike in our prior work, all stereochemical
restraint gradients are replaced by QM/MM gradients. research papers Table 1
MolProbity statistics after ONIOM, Region-QM and conventional PHENIX refinements of 80 Astex PDB structures. rota_out is the percentage of side chains with rotamer outliers, rama_fav is the percentage of amino acids in the ‘favored’ region of the Ramachandran plot and
rama_iffy is the percentage of amino acids not in the ‘favored’ region of the Ramachandran plot. rotamer outliers, rama_fav is the percentage of amino acids in the ‘favored’ region of the Ramachandran plot and
t in the ‘favored’ region of the Ramachandran plot. Acta Cryst. (2018). D74, 1063–1077 research papers The balance of residues and
crystal waters were defined as part of the MM region and
capping link atoms were automatically added to the QM
region edges to satisfy covalent bonds that were cut in the
process. In order to compare the new QM/MM refinement
with older methods, we also refined the structures using both
conventional (i.e. non-QM PHENIX) refinement and the
Region-QM approach as described in our previous work
(Borbulevych et al., 2014). The same input PDB files were
used in all three types of refinement, and in order to char-
acterize automated refinement, only default parameters and
automatically determined X-ray weights (Adams et al., 1997,
2010) were used for phenix.refine. The aforementioned CIF
files were used in the conventional refinement and they were
provided as input to PHENIX in the Region-QM and
ONIOM refinements in order to satisfy the internal ‘error-
trapping’
mechanism
of
the
phenix.refine
executable. Certainly, we could spend a significant amount of time
manually manipulating the input parameters, weights and
restraints in order to ‘tune’ the conventional refinement for
each of the 80 structures of the Astex Diverse Set; however,
this approach could arguably no longer be considered high-
throughput. Furthermore, from a scientific perspective, with
too much ‘hand manipulation’ one would need to ask how
much investigator bias could be introduced into the final
model. Therefore, the approach utilized in the present study
works to minimize investigator bias so that the final models
are based solely on the combination of the experimental data Building on the QM-based plugin that we described in
detail in Borbulevych et al. (2014), the ONIOM QM/MM
method was integrated with the PHENIX package v.1.11.1-
2575 (Adams et al., 2010). The typical refinement protocol in
PHENIX involves fitting bulk-solvent parameters and aniso-
tropic scaling, reciprocal-space atomic coordinate refinement,
atomic displacement parameter (ADP) refinement and occu-
pancy refinement. The overall refinement target Etotal in
PHENIX is presented as Etotal ¼ wcxscale xray Exray þ geom EQM=MM
ONIOM ;
ð4Þ ð4Þ where xray and geom are weights assigned to X-ray data and
geometry (QM/MM ONIOM) restraints, respectively, and
wcxscale is an additional scale factor implemented in PHENIX
(Afonine et al., 2012). geom is typically set to 1, while xray is a
variable weight determined using an automatic procedure in
PHENIX (Adams et al., 1997). Mimicking the Region-QM
refinement framework detailed in Borbulevych et al. MAD ¼ median½jXi medianðXÞj;
ð6Þ where Xi represents data point i and X is the array of data. where Xi represents data point i and X is the array of data. 2.3.1. Local ligand-strain energy calculations. Local ligand-
strain energy is the difference in the conformational energy of
the isolated ligand conformation and the protein-bound ligand
conformation. This metric serves as a quality indicator of
protein–ligand structures as it shows how much strain the
ligand must take on or ‘accept’ in order to bind to the protein,
and lower strain energy is preferred to higher strain energy
(Fu et al., 2011; Janowski et al., 2016; Mobley & Dill, 2009;
Perola & Charifson, 2004). Previously, we used ligand strain to
validate Region-QM refinement and we validated the method
against a repertoire of 50 quasi-randomly chosen PDB struc-
tures (Borbulevych et al., 2014); we went on to use this metric
as a critical component of our XModeScore method (Borbu-
levych et al., 2016). As detailed in Fu et al. (2011), the ligand-
strain energy Estrain is computed as Z½ðrÞ ¼
ðrÞ
½ðrÞ ;
ð9Þ ð9Þ where [(r)] is the standard deviation of the difference
density (mFo DFc) maps and corresponds to the random
error of the model and is pure precision, while the Z score of
the difference density is a measure of the residual, nonrandom
error and is pure accuracy. In order to limit the impact of
outliers or noise on the final value, while at the same time
preserving information, we assume that the difference density
Z values should approach a normal distribution of random
errors with zero mean and unit standard deviation as the
quality of the model, as measured by 2, improves. The subset
of values of x2
(i) that maximize the probability pmax over k are
summed, Estrain ¼ Exray
ligand Eoptimized
ligand
;
ð7Þ ð7Þ where Exray
ligand is the single point energy computed for the ligand
X-ray geometry and Eoptimized
ligand
is the energy of the optimized
ligand that corresponds to the local minimum. 2.2. Structure preparation and refinement 2.2. Structure preparation and refinement Coordinates and structure factors for all 80 structures from
the Astex Diverse Set (Hartshorn et al., 2007; Table 1,
Supplementary Table S1) were downloaded from the PDB. Ligands(s), solvent molecules, metals and/or anions (e.g. Cl)
were included in each of the refinements. Since QM/MM is an
‘all-atom’ method (requiring protons as well as heavy atoms), 1068
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement Acta Cryst. (2018). D74, 1063–1077 research papers and the initial placement of each structure as published, along
with the Hamiltonian used for the refinement. restraints and approximate molecular-mechanics parameters
with more accurate QM gradients which significantly reduce
the method-induced ligand strain, as shown in our previous
work (Borbulevych et al., 2014). In order to address the
Eplacement
strain
term in (8), additional side-chain sampling and/or
ligand re-docking would need to be performed. These steps
are beyond the scope of the present work, and the observed
results are attributable to localized changes (for example
improvements in bond lengths, torsions, rotations and trans-
lations) within the radius of convergence of the input
conformation. 2.3. Validation metrics In order to validate the performance of ONIOM refinement
in comparison to other refinement types (conventional and
Region-QM), we employed two groups of metrics: ligand
quality, consisting of both the strain energy and Z score of the
difference density (ZDD; Tickle, 2012) assessed using DivCon
(Borbulevych et al., 2014; QuantumBio, 2017), and overall
structure quality including MolProbity metrics assessed using
the MolProbity program (Chen et al., 2010) as distributed
within the PHENIX package. 2.3.2. Difference density as a measure of the accuracy of
density around a ligand. The conventional quality metric used
to communicate agreement between the model and the X-ray
(or neutron) density is the real-space correlation coefficient
(RSCC; Bra¨nde´n & Jones, 1990). However, in 2012 Tickle
demonstrated that the RSCC correlates with both the accu-
racy and the precision of the structure model, and described a
more sophisticated quality indicator, the real-space Z score of
difference density (ZDD), which measures the accuracy of
the model alone (Tickle, 2012; Borbulevych et al., 2016). A
detailed mathematical description of ZDD can be found in
Borbulevych et al. (2016) and Tickle (2012), but briefly the
Z score for a point difference density value is expressed by Since the histograms depicted in the present study (Figs. 3,
4, 6 and 9) show that the results are skewed and deviate from
the normal distribution, instead of standard deviations (SDs)
to show the spread of the data in the sections below, we
employed the median absolute deviation (MAD; Sachs, 1984)
calculated as ð6Þ MAD ¼ median½jXi medianðXÞj;
ð6Þ MAD ¼ median½jXi medianðXÞj;
ð6Þ pmax ¼ maxk p 2
k P
N
i¼k
x2
ðiÞ
’ maxk P 1
2
P
N
i¼k
x2
ðiÞ; ðN þ 1 kÞ=2
If2½xðkÞ 1; k 1; N þ 1 kg;
ð10Þ When discussing ligand strain, it should be noted that it can
be thought of as a combination of a number of different
factors, as represented qualitatively by ð10Þ Estrain ’ Etarget
strain þ Eplacement
strain
þ Emethod
strain :
ð8Þ ð8Þ where the function P is the lower normalized gamma function
representing the cumulative distribution function (CDF) of k
2. where the function P is the lower normalized gamma function
representing the cumulative distribution function (CDF) of k
2. The second function, I, is also computed as the complement in
practice and is the normalized incomplete beta function (CDF
of a normal-order statistic; Gibbons & Chakraborti, 2010)
which accounts for the ‘multiple comparisons’ correction
(Yuriev & Ramsland, 2013). In this equation, Etarget
strain is the ‘natural’ or ‘target-induced’
strain associated with changes in ligand geometry/conforma-
tion owing to binding, Eplacement
strain
is the strain associated with
initial ligand placement (e.g. docking) and Emethod
strain
is the strain
related to the underlying method or force field (restraints/CIF,
functional or Hamiltonian) being used in the refinement. Ideally, Estrain would equal Etarget
strain. The PHENIX/DivCon
plugin is primarily designed to address the Emethod
strain
term
through
the
replacement
of
inaccurate
stereochemical The second function, I, is also computed as the complement in
practice and is the normalized incomplete beta function (CDF
of a normal-order statistic; Gibbons & Chakraborti, 2010)
which accounts for the ‘multiple comparisons’ correction
(Yuriev & Ramsland, 2013). ZDD is evaluated as the two-tailed normal Z score corre-
sponding to the maximal value pmax over k of the cumulative
probability of k
2 derived from (10), Borbulevych et al. High-throughput QM/MM (ONIOM) refinement 1069 Acta Cryst. (2018). D74, 1063–1077 research papers ZDD ¼ 1½ð1 maxÞ=1; ð11Þ effects, one would expect that clashscore should be a parti-
cularly indicative metric. where the function is the CDF of the normal distribution,
2(|Z|) 1 is the CDF of the half-normal distribution of the
absolute value of a normal variate Z, and 1 is the inverse
function or the value of Z corresponding to a given prob-
ability. ð12Þ ð12Þ Thus, ZDD as used below is always positive and lower values
correspond to a lower amount of residual difference density. Tickle (2012) provided further guidance to interpreting ZDD,
such as a magnitude of over 3 indicates significant difference
density peaks. 3.2. Overall structure-quality metrics 3.2.1. MolProbity: Ramachandran and rotamer scores. Fig. 3 depicts a histogram of MPScores for all 80 Astex
structures involved in the current study, in which the average
MPScore of ONIOM-refined structures is 1.23 0.32 units. This average is lower (better) than the corresponding values
for Region-QM (1.81 0.32 units) and conventional (1.75
0.34 units) refinements. Furthermore, unlike the Region-QM
and conventional refinements, ONIOM refinement shows a
bimodal distribution in which the first peak is at 0.75 units and
covers about 50% of the population, and the second peak is at
1.6 units and coincides with peaks that are also observed for
conventional and Region-QM data. This second peak has a
long tail for these less sophisticated methods, with 25% of
conventional and Region-QM structures distributed in the
2.0+ unit bin. 2.3.3. Overall structure-quality metrics: MolProbity score
and clashscore. MolProbity, which is included as a module in
PHENIX, is a software tool that includes several macro-
molecular model-validation metrics using multiple quality
criteria (Chen et al., 2010). The MolProbity score (MPScore)
represents overall structure quality and is a logarithm-based
score combining three key component metrics: clashscore,
Ramachadran plot outliers (MacCallum et al., 2009) and
rotamer outliers (Hintze et al., 2016; Lovell et al., 2000). The
lower the value of the MPScore, the better the quality of the
model. In particular, an important component of the MPScore
is the clashscore, which is the number of clashes per 1000
atoms; it is determined through nonbonded atom contacts
derived using a rolling-probe algorithm employed by the
program Probe (Word et al., 1999). A clash occurs when the
dot surface around one atom overlaps the dot surface around
another by greater than 0.4 A˚ (Davis et al., 2007). Generally, a
chemically incorrect model will yield a high number of clashes
(Chen et al., 2010). Since the stereochemical restraint function
does not explicitly include electrostatics and other nonbonded
interactions for attraction and repulsion, while AMBER and
PM6 do include these attractive, and in particular repulsive, An analysis of the individual Ramachadran and rotamer
components that comprise MPScore indicates that ONIOM
refinement leads to models which exhibit improved statistics
versus the models yielded by both conventional and Region-
QM refinements. MAD ¼ median½jXi medianðXÞj;
ð6Þ The set of negative density values, owing to incorrectly
positioned atoms, yields ZDD. Likewise, the set of positive
density values, owing to missing atoms, yields ZDD+. The final
ZDD is the maximum of the absolute values of ZDD and
ZDD+ as defined using where the function is the CDF of the normal distribution,
2(|Z|) 1 is the CDF of the half-normal distribution of the
absolute value of a normal variate Z, and 1 is the inverse
function or the value of Z corresponding to a given prob-
ability. The set of negative density values, owing to incorrectly
positioned atoms, yields ZDD. Likewise, the set of positive
density values, owing to missing atoms, yields ZDD+. The final
ZDD is the maximum of the absolute values of ZDD and
ZDD+ as defined using 3.1. R-factor analysis As shown in Supplementary Table S2, the ONIOM method
yields an average Rwork of 0.177 0.02 and an average Rfree of
0.218 0.02. Similarly, conventional PHENIX refinement
produces averages of 0.171 0.02 and 0.217 0.02, respec-
tively, and Region-QM refinement yields averages of 0.174
0.02 and 0.218 0.02, respectively. Together, these results
show that the ONIOM methodology does not negatively
impact the overall agreement between the experimental data
and the atomic structure models. ZDD ¼ maxðjZDD j; ZDDþÞ:
ð12Þ research papers This residue in the conventionally refined
structure adopts an m-80
rotamer conformation, with the 1
and 2 torsion angles both being 84
. On the other hand,
ONIOM refinement yields a 1 angle in Asn413 which is
increased by 15
, making this torsion angle (69
) very close
to the ideal value of 71
for the m-80
rotamer (Lovell et al.,
1999). Interestingly, this structural shift leads to the removal of
both of the above-noted bad clashes and to an improvement in
the Asn41 OD1–Wat1098 O bond distance (which approaches
a typical hydrogen-bond distance). Specifically, when accom-
panied by the rotation of the Wat1098 water molecule
depicted in Fig. 5, a hydrogen bond is indeed formed between
Asn413 OD1 and Wat1098 O, as shown by the interatomic
distance
of
2.73 A˚
and
the
Wat1098 O–Wat1098 H1
Asn413 OD1 bond angle of 161
observed after ONIOM
refinement. 3 2 3 MolProbity: Cb deviations and r m s bond and angle
es refined with three Figure 4 Figure 4
Histogram of MolProbity clashscore distributions for 80 Astex structures refined with three
methods: ONIOM, Region-QM and conventional. component. As shown in Table 1, for the 80 Astex models
studied the average clashscore is 1.10 0.41 units for the
ONIOM models, which is 4.5–5.0-fold lower (better) than the
average clashscores for the conventional (4.83 1.2 units) and
Region-QM (5.54 1.6 units) models. The clashscore histo-
gram (Fig. 4) shows a clear peak around 0.5 units which
comprises 90% of the ONIOM models, while a peak repre-
senting both conventional and Region-QM model data is
located around 3.5 units. Furthermore, around 50% of the data
in the conventional and Region-QM histograms are found in
the tails of the respective peaks and are distributed in histo-
gram bins of 4.5+ units and above, while no ONIOM data are
found in this range. This observation suggests that the
ONIOM QM/MM method utilized in this study exhibits
greater consistency over the range of structures studied versus
the use of stereochemical restraints alone in an automated
(high-throughput) regime with default phenix.refine settings. Furthermore, since the Region-QM and conventional refine-
ments yield similar results for the bulk of the protein structure,
this would suggest that much of the improvement in clashscore
is attributable to the use of the QM/MM Hamiltonian on the
entire structure. research papers Interestingly, this structural shift leads to the removal of
both of the above-noted bad clashes and to an improvement in
the Asn41 OD1–Wat1098 O bond distance (which approaches
a typical hydrogen-bond distance). Specifically, when accom-
panied by the rotation of the Wat1098 water molecule
depicted in Fig. 5, a hydrogen bond is indeed formed between
Asn413 OD1 and Wat1098 O, as shown by the interatomic
distance
of
2.73 A˚
and
the
Wat1098 O–Wat1098 H1
Asn413 OD1 bond angle of 161
observed after ONIOM
refinement. 3.2.3. MolProbity: Cb deviations and r.m.s. bond and angle
es refined with three The
crystal
structure
of
human
estrogen
receptor
ligand-binding
domain in complex with the antagonist
ligand 4-D determined at 1.9 A˚ resolu-
tion (PDB entry 1sj0; Kim et al., 2004)
has been chosen as a representative
example in order to demonstrate the
sort of improvements that we have
observed in treatment of the Astex
Diverse Set with the QM/MM method. An initial clashscore for the deposited
structure was calculated as 18.64 units. While all three refinements led to a
noticeable reduction (improvement) in
clashscore, ONIOM refinement exhib-
ited the largest improvement, with a
clashscore of 2.27 units compared with
9.06 units for conventional refinement and 13.09 units for
Region-QM refinement. As shown in Supplementary Table S3,
the poorer score of the conventional refinement is owing to
the 36 bad clashes that remained after refinement (compared
with 74 bad clashes in the originally downloaded file). 28 of
those 36 clashes were not observed in the ONIOM model, and
no additional clashes were introduced with ONIOM. Inter-
estingly, owing to the addition of six clashes at the boundary of
the buffer region, Region-QM refinement yielded a higher
(worse) clashscore than both ONIOM and conventional
refinement. Among the bad clashes observed after conven-
tional refinement, ONIOM refinement leads to an average
improvement of 0.25 0.12 A˚ , while some significant short
contacts were improved by as much as 0.65 A˚ . A notable
example is depicted in Fig. 5, where the intermolecular
distance between Asn413 ND and Wat1098 O in conventional
refinement yields a clash distance of 2.41 A˚ , while ONIOM
refinement yields a more reasonable 3.23 A˚ . Further, struc-
tural rearrangement in this region after ONIOM refinement is
mostly attributable to the movement of the side chain of
Asn413 (Fig. 5). 3.2. Overall structure-quality metrics For example, when comparing conventional
refinement and ONIOM refinement, the average percentage
of Ramachadran plot outliers decreases from 0.40% to 0.26%,
while the residue population in the favorable regions of the
Ramachadran plot slightly increases
from 96.46% to 96.90%. In over 91% of
the cases studied ONIOM leads to
models with a Ramachandran plot and
rotamer angles that are as good or
better when compared with those from
conventional refinement, demonstrating
that use of the ONIOM plugin does not
break or otherwise damage the final
model. An analysis of the individual Ramachadran and rotamer
components that comprise MPScore indicates that ONIOM
refinement leads to models which exhibit improved statistics
versus the models yielded by both conventional and Region-
QM refinements. For example, when comparing conventional
refinement and ONIOM refinement, the average percentage
of Ramachadran plot outliers decreases from 0.40% to 0.26%,
while the residue population in the favorable regions of the pu at o
t e avo ab e eg o s o t e
Ramachadran plot slightly increases
from 96.46% to 96.90%. In over 91% of
the cases studied ONIOM leads to
models with a Ramachandran plot and
rotamer angles that are as good or
better when compared with those from
conventional refinement, demonstrating
that use of the ONIOM plugin does not
break or otherwise damage the final
model. Figure 3
Histogram of MPScore distributions for 80 Astex structures refined with three methods: ONIOM,
Region-QM and conventional. 3.2.2. MolProbity: clashscore. While
a portion of the observed improvement
in
the
MPScore
is
attributable
to
improvements in the Ramachandran
and rotamer components, the largest
improvement is seen for the clashscore Figure 3
Histogram of MPScore distributions for 80 Astex structures refined with three methods: ONIOM,
Region-QM and conventional. Figure 3
Histogram of MPScore distributions for 80 Astex structures refined with three methods: ONIOM,
Region-QM and conventional. 1070
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement 1070
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement 1070
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement Acta Cryst. (2018). D74, 1063–1077 research papers Figure 4
Histogram of MolProbity clashscore distributions for 80 Astex structures refined with three
methods: ONIOM, Region-QM and conventional. The
crystal
structure
of
human
estrogen
receptor
ligand-binding
domain in complex with the antagonist
ligand 4-D determined at 1.9 A˚ resolu-
tion (PDB entry 1sj0; Kim et al., 2004)
has been chosen as a representative
example in order to demonstrate the
sort of improvements that we have
observed in treatment of the Astex
Diverse Set with the QM/MM method. An initial clashscore for the deposited
structure was calculated as 18.64 units. While all three refinements led to a
noticeable reduction (improvement) in
clashscore, ONIOM refinement exhib-
ited the largest improvement, with a
clashscore of 2.27 units compared with
9.06 units for conventional refinement and 13.09 units for
Region-QM refinement. As shown in Supplementary Table S3,
the poorer score of the conventional refinement is owing to
the 36 bad clashes that remained after refinement (compared
with 74 bad clashes in the originally downloaded file). 28 of
those 36 clashes were not observed in the ONIOM model, and
no additional clashes were introduced with ONIOM. Inter-
estingly, owing to the addition of six clashes at the boundary of
the buffer region, Region-QM refinement yielded a higher
(worse) clashscore than both ONIOM and conventional
refinement. Among the bad clashes observed after conven-
tional refinement, ONIOM refinement leads to an average
improvement of 0.25 0.12 A˚ , while some significant short
contacts were improved by as much as 0.65 A˚ . A notable
example is depicted in Fig. 5, where the intermolecular
distance between Asn413 ND and Wat1098 O in conventional
refinement yields a clash distance of 2.41 A˚ , while ONIOM
refinement yields a more reasonable 3.23 A˚ . Further, struc-
tural rearrangement in this region after ONIOM refinement is
mostly attributable to the movement of the side chain of
Asn413 (Fig. 5). This residue in the conventionally refined
structure adopts an m-80
rotamer conformation, with the 1
and 2 torsion angles both being 84
. On the other hand,
ONIOM refinement yields a 1 angle in Asn413 which is
increased by 15
, making this torsion angle (69
) very close
to the ideal value of 71
for the m-80
rotamer (Lovell et al.,
1999). research papers Figure 5
An example of resolving a bad clash between Asn413 and a water
molecule in PDB entry 1sj0 after ONIOM refinement (green). The
conventional refined structure is shown in magenta. The A-weighted
2mFo DFc electron-density map is contoured at 1. Figure 5 3.2.3. MolProbity: Cb deviations and r.m.s. bond and angle
deviations. In addition to the aforementioned Ramachandran,
clashscore
and
rotamer
components,
for
the
sake
of
completeness
the
C
deviations
are
also
reported
in Figure 5
An example of resolving a bad clash between Asn413 and a water
molecule in PDB entry 1sj0 after ONIOM refinement (green). The
conventional refined structure is shown in magenta. The A-weighted
2mFo DFc electron-density map is contoured at 1. Borbulevych et al. High-throughput QM/MM (ONIOM) refinement
1 1071 Acta Cryst. (2018). D74, 1063–1077 research papers
Figure 6
Histogram of ligand-strain energy distributions for 141 ligand instances from 80 Astex structures
refined with three methods: ONIOM, Region-QM and conventional. research papers types in MM and the captured atom–
atom interactions in both methods. types in MM and the captured atom–
atom interactions in both methods. 3.3. Ligand-quality metrics
3.3.1. Local
ligand-strain
energy. Ligand strain is a method to explore
refined ligand structural models (Fu et
al., 2011; Janowski et al., 2016; Mobley &
Dill, 2009; Perola & Charifson, 2004),
and ligand strain is a key metric which
we have used previously to evaluate the
quality
of
the
region
refinement
(Borbulevych et al., 2012, 2014). For the
present study, we find that the average
strain
energies
calculated
over
141
ligands from 80 Astex structures are
similar
in
ONIOM
(9.95
3.77 kcal mol1) and Region-QM (10.49
4.52 kcal mol1)
refinements. As
shown in the ligand-strain histogram
(Fig. 6), we also see similar distributions
between both ONIOM refinement and Region-QM refine-
ment
in
that
both
methods
exhibit
peaks
around
3.0 kcal mol1 which account for approximately three quarters
of the models in the set. This is compared with conventional
refinement using automatically generated CIFs, which yields a
population of structures which are more evenly distributed in
a broad range from 10 to 40 kcal mol1 and 30% of the data
are in the last bin of >50 kcal mol1. research papers This finding is consistent
with our previous work, in which we demonstrated that QM
refinement across a diverse population of structures yields a
tighter strain energy range versus conventional methods
(Borbulevych et al., 2014). In addition to exhibiting a wider
strain range, the average ligand-strain energy after conven-
tional refinement of the Astex set is 35.64 9.35 kcal mol1 or
about 3.5-fold higher than in the QM-driven refinements. This
average improvement in strain energy is consistent with the
3.4-fold
average
improvement
observed
in
Region-QM
refinements in our previous study (Borbulevych et al., 2014). While beyond the scope of the present work, which is focused
on automated, high-throughput methods, arguably one could
potentially manipulate these CIFs ‘by hand’ in order to yield
ligand structures with lower strain energy or even which mimic
the capture of atom–atom interactions (for example slightly
elongated/shortened
bond
lengths,
rotations
etc.)
auto-
matically observed in QM/MM refinement. However, with
over 80 species considered, these manipulations would come
at a significant cost in investigator time with more opportu-
nities for inclusion of investigator bias. Further, the success or
failure of each structure would be much more greatly
dependent on investigator proficiency. Refinement of the crystal structure of the vitamin D
receptor (VDR) ligand-binding domain bound to calcipotriol
˚
m 80 Astex structures 3.3. Ligand-quality metrics 3.3.1. Local
ligand-strain
energy. Ligand strain is a method to explore
refined ligand structural models (Fu et
al., 2011; Janowski et al., 2016; Mobley &
Dill, 2009; Perola & Charifson, 2004),
and ligand strain is a key metric which
we have used previously to evaluate the
quality
of
the
region
refinement
(Borbulevych et al., 2012, 2014). For the
present study, we find that the average
strain
energies
calculated
over
141
ligands from 80 Astex structures are
similar
in
ONIOM
(9.95
3.77 kcal mol1) and Region-QM (10.49
4.52 kcal mol1)
refinements. As
shown in the ligand-strain histogram
(Fig 6) we also see similar distributions Figure 6
Histogram of ligand-strain energy distributions for 141 ligand instances from 80 Astex structures
refined with three methods: ONIOM, Region-QM and conventional. Supplementary Table S2. Generally, C deviations are defined
as abnormalities in bond-angle distributions around the C
atom. Deviations
larger
than
0.25 A˚
typically
indicate
incompatibility between main-chain and side-chain confor-
mations (Davis et al., 2007). research papers As indicated in Supplementary
Table S2, the number of C deviations is similar in all three
refinement types and over 90% of structures are free of this
aberration. Furthermore, the average r.m.s.d. in bond length is
the same for ONIOM (0.014 0.002 A˚ ), Region-QM (0.014
0.002 A˚ ) and conventional (0.013 0.002 A˚ ) refinements
(Supplementary Table S2). However, the average r.m.s.d. in
angles is slightly lower for conventional refinement (1.30
0.20
) compared with QM-driven refinements (1.86 0.20
for
ONIOM and 1.53 0.20
for Region-QM), suggesting greater
variability in the QM and MM methods. This deviation is
likely to be caused by different target bond angles in the
AMBER functional together with the greater number of atom Supplementary Table S2. Generally, C deviations are defined
as abnormalities in bond-angle distributions around the C
atom. Deviations
larger
than
0.25 A˚
typically
indicate
incompatibility between main-chain and side-chain confor-
mations (Davis et al., 2007). As indicated in Supplementary
Table S2, the number of C deviations is similar in all three
refinement types and over 90% of structures are free of this
aberration. Furthermore, the average r.m.s.d. in bond length is
the same for ONIOM (0.014 0.002 A˚ ), Region-QM (0.014
0.002 A˚ ) and conventional (0.013 0.002 A˚ ) refinements
(Supplementary Table S2). However, the average r.m.s.d. in
angles is slightly lower for conventional refinement (1.30
0.20
) compared with QM-driven refinements (1.86 0.20
for
ONIOM and 1.53 0.20
for Region-QM), suggesting greater
variability in the QM and MM methods. This deviation is
likely to be caused by different target bond angles in the
AMBER functional together with the greater number of atom Figure 7
Superimposition of the ligand calcipotriol (ligand ID MC9) in PDB entry
1s19 refined with the ONIOM (green), Region-QM (yellow) and
conventional (magenta) methods. The A-weighted 2mFo DFc
electron-density map is contoured at 1. Refinement of the crystal structure of the vitamin D
receptor (VDR) ligand-binding domain bound to calcipotriol
(ligand ID MC9) determined at 2.1 A˚ resolution (PDB entry
1s19; Tocchini-Valentini et al., 2004) is chosen as an illustrative
example. Conventional refinement of PDB entry 1s19 leads to g
Superimposition of the ligand calcipotriol (ligand ID MC9) in PDB entry
1s19 refined with the ONIOM (green), Region-QM (yellow) and
conventional (magenta) methods. research papers Table 2
Strain-energy (kcal mol1) and ZDD values for 141 ligands after
ONIOM, Region-QM and conventional PHENIX refinements of 80
Astex PDB structures. Table 2
Strain-energy (kcal mol1) and ZDD values for 141 ligands after
ONIOM, Region-QM and conventional PHENIX refinements of 80
Astex PDB structures. Astex PDB structures. ONIOM
Region-QM
Conventional
PDB
code
Res. research papers (A˚ )
Ligand
Strain
energy
ZDD
Strain
energy
ZDD
Strain
energy
ZDD
1g9v
1.85
RQ3_A_801
5.11
2.5
4.74
2.1
27.01
2.1
1g9v
RQ3_C_802
3.65
3.0
4.29
2.9
31.72
3.5
1gkc
2.30
NFH_A_1448
10.36
1.0
11.78
0.9
22.10
1.1
1gkc
NFH_B_1449
10.80
4.6
10.35
4.7
27.53
3.3
1gpk
2.10
HUP_A_1540
2.61
1.1
3.87
0.3
10.13
0.4
1hnn
2.30
SKF_A_3001
5.48
1.5
9.07
1.2
12.84
0.8
1hnn
SKF_B_3002
8.43
3.1
9.03
3.9
15.67
4.0
1hp0
2.10
AD3_A_1315
17.65
5.9
15.83
5.6
25.79
5.9
1hp0
AD3_B_1316
15.42
2.4
14.32
2.9
18.03
2.6
1hq2
1.25
PH2_A_181
9.98
3.2
11.17
4.3
27.93
4.3
1hvy
1.90
D16_A_414
9.29
4.9
10.20
3.7
41.92
4.4
1hvy
D16_B_415
8.10
3.8
9.81
4.8
49.22
4.4
1hvy
D16_C_416
8.73
5.4
9.42
3.8
35.70
5.5
1hvy
D16_D_417
9.62
2.5
8.36
2.8
49.21
3.0
1hwi
2.16
115_A_2
6.14
1.5
5.58
0.5
22.29
0.8
1hwi
115_B_1
29.81
1.5
14.69
1.2
29.92
1.5
1hwi
115_C_4
10.86
1.5
15.65
2.5
31.13
2.0
1hwi
115_D_3
16.54
1.7
10.32
1.5
24.88
1.1
1hww
1.87
SWA_A_1103
29.64
2.4
16.36
0.9
13.07
0.1
1ia1
1.72
TQ3_A_194
2.01
1.2
1.65
1.4
9.99
3.0
1ia1
TQ3_B_196
2.57
1.7
2.92
1.7
10.87
2.4
1ig3
1.90
VIB_A_502
4.37
1.8
6.16
2.8
14.81
3.5
1ig3
VIB_B_501
2.72
4.4
4.78
5.3
13.38
6.0
1j3j
2.30
CP6_A_609
30.44
4.7
22.57
6.0
64.86
5.0
1j3j
CP6_B_709
1.07
1.6
1.71
1.4
89.56
8.8
1jd0
1.50
AZM_A_1400
6.23
6.4
5.58
7.7
37.34
6.5
1jd0
AZM_B_2401
12.73
4.4
14.09
7.8
34.54
4.5
1jje
1.29
BYS_A_250
28.44
2.8
29.37
4.1
31.76
3.5
1jje
BYS_B_250
31.08
4.0
32.20
5.1
46.87
4.4
1jla
2.50
TNK_A_999
68.96
1.3
71.30
2.7
187.39
2.6
1k3u
1.70
IAD_A_801
20.12
3.7
20.90
4.3
28.03
5.7
1ke5
2.00
LS1_A_299
11.98
2.5
8.84
2.5
28.62
4.5
1kzk
1.09
JE2_A_701
16.31
0.9
10.94
1.6
19.18
1.7
1l2s
1.94
STC_A_1115
4.27
1.6
6.71
2.3
11.07
2.8
1l2s
STC_B_2115
2.98
3.7
4.60
3.0
13.00
3.1
1l2s
STC_B_3115
8.30
13.3
6.07
13.9
29.20
15.4
1l7f
1.80
BCZ_A_801
8.52
1.5
9.04
1.8
21.23
1.9
1lpz
2.41
CMB_B_301
11.11
3.6
12.73
2.8
69.81
3.0
1lrh
1.90
NLA_A_5190
4.51
3.6
5.23
3.8
6.80
3.9
1lrh
NLA_B_6190
4.13
1.9
5.26
2.5
7.33
1.9
1lrh
NLA_C_7190
3.91
1.9
4.23
2.4
6.12
1.9
1lrh
NLA_D_8190
4.23
0.7
5.23
1.1
6.87
1.1
1meh
1.95
MOA_A_600
3.75
1.8
2.85
1.4
19.07
1.5
1mmv
2.00
3AR_A_1785
29.88
1.5
27.67
1.4
37.18
1.6
1mmv
3AR_B_2785
35.22
1.3
33.81
1.2
34.36
1.9
1mzc
2.00
BNE_B_1003
3.85
1.1
4.13
0.9
23.42
2.5
1n1m
2.50
A3M_A_954
4.90
1.6
9.72
1.8
14.36
0.8
1n1m
A3M_B_955
6.60
1.5
16.58
2.2
27.12
0.9
1n2j
1.80
PAF_A_1001
6.10
1.6
4.78
1.6
6.93
0.9
1n2j
PAF_B_1002
6.57
2.1
4.92
1.5
8.99
0.5
1n2v
2.10
BDI_A_900
10.50
1.2
15.59
1.4
13.29
1.4
1n46
2.20
PFA_A_462
31.06
0.1
23.01
0.6
68.42
0.9
1n46
PFA_B_463
30.63
0.7
27.54
1.2
72.57
0.8
1nav
2.48
IH5_A_600
7.37
0.5
9.33
1.1
39.41
2.0
1of1
1.95
SCT_A_400
2.86
1.1
2.64
0.9
16.10
1.5
1of1
SCT_B_500
5.31
0.7
4.49
1.0
15.90
1.3
1of6
2.10
DTY_A_1370
8.28
1.7
7.51
3.4
151.20
2.0
1of6
DTY_B_1370
10.02
0.7
8.94
2.1
149.66
2.1
1of6
DTY_C_1371
7.28
1.1
7.62
2.3
155.81
2.1 Figure 8
The A-weighted mFo DFc difference electron-density map peaks
around the ligand calcipotriol (ligand ID MC9) in PDB entry 1s19 refined
with the ONIOM (a), Region-QM (b) and conventional (c) methods. Acta Cryst. (2018). D74, 1063–1077 Borbulevych et al.
High-throughput QM/MM (ONIOM) refinement
1073 research papers (A˚ )
Ligand
Strain
energy
ZDD
Strain
energy
ZDD
Strain
energy
ZDD
1g9v
1.85
RQ3_A_801
5.11
2.5
4.74
2.1
27.01
2.1
1g9v
RQ3_C_802
3.65
3.0
4.29
2.9
31.72
3.5
1gkc
2.30
NFH_A_1448
10.36
1.0
11.78
0.9
22.10
1.1
1gkc
NFH_B_1449
10.80
4.6
10.35
4.7
27.53
3.3
1gpk
2.10
HUP_A_1540
2.61
1.1
3.87
0.3
10.13
0.4
1hnn
2.30
SKF_A_3001
5.48
1.5
9.07
1.2
12.84
0.8
1hnn
SKF_B_3002
8.43
3.1
9.03
3.9
15.67
4.0
1hp0
2.10
AD3_A_1315
17.65
5.9
15.83
5.6
25.79
5.9
1hp0
AD3_B_1316
15.42
2.4
14.32
2.9
18.03
2.6
1hq2
1.25
PH2_A_181
9.98
3.2
11.17
4.3
27.93
4.3
1hvy
1.90
D16_A_414
9.29
4.9
10.20
3.7
41.92
4.4
1hvy
D16_B_415
8.10
3.8
9.81
4.8
49.22
4.4
1hvy
D16_C_416
8.73
5.4
9.42
3.8
35.70
5.5
1hvy
D16_D_417
9.62
2.5
8.36
2.8
49.21
3.0
1hwi
2.16
115_A_2
6.14
1.5
5.58
0.5
22.29
0.8
1hwi
115_B_1
29.81
1.5
14.69
1.2
29.92
1.5
1hwi
115_C_4
10.86
1.5
15.65
2.5
31.13
2.0
1hwi
115_D_3
16.54
1.7
10.32
1.5
24.88
1.1
1hww
1.87
SWA_A_1103
29.64
2.4
16.36
0.9
13.07
0.1
1ia1
1.72
TQ3_A_194
2.01
1.2
1.65
1.4
9.99
3.0
1ia1
TQ3_B_196
2.57
1.7
2.92
1.7
10.87
2.4
1ig3
1.90
VIB_A_502
4.37
1.8
6.16
2.8
14.81
3.5
1ig3
VIB_B_501
2.72
4.4
4.78
5.3
13.38
6.0
1j3j
2.30
CP6_A_609
30.44
4.7
22.57
6.0
64.86
5.0
1j3j
CP6_B_709
1.07
1.6
1.71
1.4
89.56
8.8
1jd0
1.50
AZM_A_1400
6.23
6.4
5.58
7.7
37.34
6.5
1jd0
AZM_B_2401
12.73
4.4
14.09
7.8
34.54
4.5
1jje
1.29
BYS_A_250
28.44
2.8
29.37
4.1
31.76
3.5
1jje
BYS_B_250
31.08
4.0
32.20
5.1
46.87
4.4
1jla
2.50
TNK_A_999
68.96
1.3
71.30
2.7
187.39
2.6
1k3u
1.70
IAD_A_801
20.12
3.7
20.90
4.3
28.03
5.7
1ke5
2.00
LS1_A_299
11.98
2.5
8.84
2.5
28.62
4.5
1kzk
1.09
JE2_A_701
16.31
0.9
10.94
1.6
19.18
1.7
1l2s
1.94
STC_A_1115
4.27
1.6
6.71
2.3
11.07
2.8
1l2s
STC_B_2115
2.98
3.7
4.60
3.0
13.00
3.1
1l2s
STC_B_3115
8.30
13.3
6.07
13.9
29.20
15.4
1l7f
1.80
BCZ_A_801
8.52
1.5
9.04
1.8
21.23
1.9
1lpz
2.41
CMB_B_301
11.11
3.6
12.73
2.8
69.81
3.0
1lrh
1.90
NLA_A_5190
4.51
3.6
5.23
3.8
6.80
3.9
1lrh
NLA_B_6190
4.13
1.9
5.26
2.5
7.33
1.9
1lrh
NLA_C_7190
3.91
1.9
4.23
2.4
6.12
1.9
1lrh
NLA_D_8190
4.23
0.7
5.23
1.1
6.87
1.1
1meh
1.95
MOA_A_600
3.75
1.8
2.85
1.4
19.07
1.5
1mmv
2.00
3AR_A_1785
29.88
1.5
27.67
1.4
37.18
1.6
1mmv
3AR_B_2785
35.22
1.3
33.81
1.2
34.36
1.9
1mzc
2.00
BNE_B_1003
3.85
1.1
4.13
0.9
23.42
2.5
1n1m
2.50
A3M_A_954
4.90
1.6
9.72
1.8
14.36
0.8
1n1m
A3M_B_955
6.60
1.5
16.58
2.2
27.12
0.9
1n2j
1.80
PAF_A_1001
6.10
1.6
4.78
1.6
6.93
0.9
1n2j
PAF_B_1002
6.57
2.1
4.92
1.5
8.99
0.5
1n2v
2.10
BDI_A_900
10.50
1.2
15.59
1.4
13.29
1.4
1n46
2.20
PFA_A_462
31.06
0.1
23.01
0.6
68.42
0.9
1n46
PFA_B_463
30.63
0.7
27.54
1.2
72.57
0.8
1nav
2.48
IH5_A_600
7.37
0.5
9.33
1.1
39.41
2.0
1of1
1.95
SCT_A_400
2.86
1.1
2.64
0.9
16.10
1.5
1of1
SCT_B_500
5.31
0.7
4.49
1.0
15.90
1.3
1of6
2.10
DTY_A_1370
8.28
1.7
7.51
3.4
151.20
2.0
1of6
DTY_B_1370
10.02
0.7
8.94
2.1
149.66
2.1
1of6
DTY_C_1371
7.28
1.1
7.62
2.3
155.81
2.1 Table 2
Strain-energy (kcal mol1) and ZDD values for 141 ligands after
ONIOM, Region-QM and conventional PHENIX refinements of 80
Astex PDB structures. research papers The A-weighted 2mFo DFc
electron-density map is contoured at 1. g
Superimposition of the ligand calcipotriol (ligand ID MC9) in PDB entry
1s19 refined with the ONIOM (green), Region-QM (yellow) and
conventional (magenta) methods. The A-weighted 2mFo DFc
electron-density map is contoured at 1. 1072
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement Acta Cryst. (2018). D74, 1063–1077 research papers research papers based refinements (Figs. 8a and 8b). These peaks generally
indicate that the ligand conformation adopted is likely to be
incorrectly placed within the density after conventional
refinement. based refinements (Figs. 8a and 8b). These peaks generally
indicate that the ligand conformation adopted is likely to be
incorrectly placed within the density after conventional
refinement. a strain energy of 28.62 kcal mol1 for the ligand MC9 (Table
1). However, QM-driven refinement yields a ligand structural
model in which ligand strains are 3.8–3.5-fold lower or
7.52 kcal mol1 for ONIOM and 8.28 kcal mol1 for Region-
QM. Closer examination of the geometry of this ligand after
the conventional and QM-driven refinements reveals that the
key difference is related to the orientation of the hydroxyl-
propene fragment at the junction with the cyclopropyl ring
described by the torsion angle C22—C23—C24—C25, which is
35
for conventional refinement, 119
for ONIOM
refinement and 128
for Region-QM refinement (Fig. 7). Further, the conventional model exhibits positive and negative
density peaks (Fig. 8c) which are not observed in the two QM- QM. Closer examination of the geometry of this ligand after
the conventional and QM-driven refinements reveals that the
key difference is related to the orientation of the hydroxyl-
propene fragment at the junction with the cyclopropyl ring
described by the torsion angle C22—C23—C24—C25, which is
35
for conventional refinement, 119
for ONIOM
refinement and 128
for Region-QM refinement (Fig. 7). Further, the conventional model exhibits positive and negative
density peaks (Fig. 8c) which are not observed in the two QM-
Figure 8
The A-weighted mFo DFc difference electron-density map peaks
around the ligand calcipotriol (ligand ID MC9) in PDB entry 1s19 refined
with the ONIOM (a), Region-QM (b) and conventional (c) methods. The
difference density is drawn at the 3 level. Table 2
Strain-energy (kcal mol1) and ZDD values for 141 ligands after
ONIOM, Region-QM and conventional PHENIX refinements of 80
Astex PDB structures. ONIOM
Region-QM
Conventional
PDB
code
Res. research papers research papers Table 2 (continued)
ONIOM
Region-QM
Conventional
PDB
code
Res. (A˚ )
Ligand
Strain
energy
ZDD
Strain
energy
ZDD
Strain
energy
ZDD
1of6
DTY_D_1370
11.18
0
8.19
0.8
146.43
0.2
1of6
DTY_E_1370
6.58
0.2
8.08
0.2
154.67
0.4
1of6
DTY_F_1370
8.00
0.0
8.40
1.0
163.72
0.9
1of6
DTY_G_1369
8.35
0.4
6.51
2.4
160.73
1.4
1of6
DTY_H_1369
9.36
0.8
9.67
1.6
150.23
1.1
1opk
1.80
P16_A_2
2.10
5.6
1.84
6.0
35.02
6.2
1oq5
1.50
CEL_A_701
11.81
3.2
15.60
3.7
19.44
4.2
1owe
1.60
675_A_1001
8.71
2.6
11.22
1.9
14.06
2.0
1oyt
1.67
FSN_H_501
8.40
3.5
8.85
3.4
27.29
4.2
1p2y
2.28
NCT_A_440
2.08
1.6
1.05
2.1
13.39
1.6
1p62
1.90
GEO_B_302
11.64
0.2
9.97
0.4
19.44
2.0
1q1g
2.02
MTI_A_301
15.92
3.2
15.04
3.4
32.53
3.9
1q1g
MTI_B_302
13.60
2.1
13.15
1.7
30.40
2.8
1q1g
MTI_C_303
15.61
3.5
14.22
2.9
29.91
5.5
1q1g
MTI_D_304
18.04
2.6
17.90
2.8
26.51
3.5
1q1g
MTI_E_305
14.46
1.1
18.81
0.7
31.12
1.9
1q1g
MTI_F_306
15.61
2.3
13.54
2.4
26.37
5.4
1q41
2.10
IXM_A_451
1.37
2.7
1.59
3.4
36.35
3.4
1q41
IXM_B_452
1.59
6.3
1.92
5.6
35.60
6.5
1q4g
1.98
BFL_A_701
2.55
3.2
3.97
5.2
6.53
3.9
1q4g
BFL_B_1701
2.47
6.5
3.76
6.2
7.92
5.6
1r1h
1.95
BIR_A_2001
13.81
1.0
17.32
1.9
33.90
1.7
1r55
1.59
097_A_518
21.70
1.5
16.20
1.6
29.71
2.0
1r58
1.90
AO5_A_501
41.38
3.9
62.41
3.9
68.73
2.9
1r9o
2.00
FLP_A_501
4.58
1.8
2.32
1.6
9.40
1.2
1s19
2.00
MC9_A_500
7.52
1.1
8.28
1.6
28.62
4.3
1s3v
1.80
TQD_A_187
8.60
0.6
9.10
0.8
29.76
1.4
1sg0
1.50
STL_A_501
2.55
10.2
2.88
10.1
12.51
12.0
1sg0
STL_B_502
4.41
4.8
5.96
5.6
14.94
4.5
1sj0
1.90
E4D_A_600
13.33
2.7
18.99
3.8
33.95
2.7
1sq5
2.00
PAU_A_6001
6.06
1.6
6.12
1.2
17.33
3.2
1sq5
PAU_B_6003
9.28
4.6
10.13
5.5
21.19
4.8
1sq5
PAU_C_6002
9.53
4.6
10.26
5.7
23.71
4.5
1sq5
PAU_D_6004
7.67
2.3
7.54
3.6
16.22
3.3
1t40
1.80
ID5_A_320
13.61
1.6
6.41
1.3
16.32
0.8
1t46
1.60
STI_A_3
16.69
3.5
17.06
4.3
59.53
3.2
1t9b
2.20
1CS_A_695
3.32
4.3
4.09
4.7
21.14
4.6
1t9b
1CS_B_1695
3.05
4.1
4.75
4.5
33.33
4.9
1tow
2.00
CRZ_A_501
7.25
1.1
3.74
0.7
10.99
0.5
1tt1
1.93
KAI_A_998
7.04
1.2
23.20
1.1
22.77
1.6
1tt1
KAI_B_999
6.31
1.1
22.03
0.8
22.88
1.2
Table 2 (continued)
ONIOM
Region-QM
Conventional
PDB
code
Res. research papers (A˚ )
Ligand
Strain
energy
ZDD
Strain
energy
ZDD
Strain
energy
ZDD
1tz8
1.85
DES_B_128
5.09
3.2
3.98
2.7
62.82
13.9
1tz8
DES_C_129
0.24
3.8
0.82
4.3
56.87
9.1
1tz8
DES_D_128
1.11
0.8
0.28
0.7
46.17
10.0
1u1c
2.20
BAU_A_5400
7.88
4.1
13.09
4.2
22.15
3.5
1u1c
BAU_B_5011
8.83
1.3
15.44
2.2
21.01
0.8
1u1c
BAU_C_5021
4.66
2.4
18.31
2.7
22.46
3.0
1u1c
BAU_D_5031
7.33
2.4
20.52
2.5
22.11
2.2
1u1c
BAU_E_5041
7.62
1.1
11.21
0.5
18.81
1.2
1u1c
BAU_F_5051
5.87
0.7
17.19
1.0
23.16
1.2
1u4d
2.10
DBQ_A_398
5.44
0.1
5.66
0.4
21.99
0.1
1u4d
DBQ_B_401
5.46
0.8
3.54
1.2
18.50
1.8
1uml
2.50
FR4_A_1001
10.24
2.1
11.81
2.5
26.14
3.8
1unl
2.20
RRC_A_1293
25.22
3.2
27.55
3.2
83.26
4.0
1uou
2.11
CMU_A_1481
4.73
0.8
5.04
1.0
20.52
1.5
1v0p
2.00
PVB_A_1287
6.68
1.1
8.45
1.7
36.39
1.8
1v0p
PVB_B_1287
6.26
2.9
5.70
2.0
37.24
2.4
1v48
2.20
HA1_A_290
29.71
0.3
22.82
0.8
38.12
0.4
1v4s
2.30
MRK_A_501
2.99
2.1
2.79
2.0
27.23
2.4
1vcj
2.39
IBA_A_1
14.97
1.1
15.13
1.3
27.21
2.8
1w1p
2.10
GIO_A_1518
4.21
1.0
1.74
1.6
14.11
1.2
1w1p
GIO_B_1501
3.49
2.2
1.53
2.0
17.21
1.5
1w2g
2.10
THM_A_1210
3.03
1.0
7.16
1.1
10.95
1.5
1w2g
THM_B_1210
4.21
1.3
5.25
1.9
14.94
1.7
1x8x
2.00
TYR_A_952
14.47
0.1
4.77
0.1
101.28
0.1
1xm6
1.90
5RM_A_1003
0.71
0.4
1.32
0.6
12.15
0.9
1xm6
5RM_B_1003
2.67
1.0
2.22
0.4
15.49
1.4
1xoq
1.83
ROF_A_502
2.92
0.8
3.00
0.9
20.01
1.1
1xoq
ROF_B_501
2.55
1.3
2.97
1.1
21.02
1.4
1xoz
1.30
CIA_A_501
3.60
0.6
3.19
1.6
18.75
1.1
1y6b
2.10
AAX_A_201
6.88
4.9
6.89
5.4
19.13
5.5
1ygc
2.00
905_H_1
17.62
0.9
22.37
1.8
44.66
1.9
1yv3
1.99
BIT_A_800
3.51
2.8
2.75
3.2
12.91
2.7
1yvf
2.50
PH7_A_800
1.94
1.3
2.95
2.4
30.45
2.8
1ywr
1.90
LI9_A_361
17.66
4.1
21.03
4.8
52.45
3.9
1z95
1.80
198_A_501
5.73
1.3
6.55
1.1
28.01
3.3
2bm2
2.20
PM2_A_3211
1.43
1.4
2.27
1.4
14.88
1.8
2bm2
PM2_B_3211
1.87
0.9
1.96
1.1
21.68
2.3
2bm2
PM2_C_3211
1.56
1.0
1.38
1.4
12.97
1.9
2bm2
PM2_D_3211
1.40
1.8
1.41
1.1
13.31
1.7
2br1
2.00
PFP_A_1277
5.63
0.9
7.29
0.6
21.90
2.2
2bsm
2.05
BSM_A_1224
19.15
1.3
10.32
1.6
21.54
1.8
Figure 9
Histogram of ligand ZDD distributions for 141 ligand instances from 80 Astex structures refined
with three methods: ONIOM, Region-QM and conventional. i 3.3.2. Ligand ZDD. The histogram for
ZDD (Fig. research papers (A˚ )
Ligand
Strain
energy
ZDD
Strain
energy
ZDD
Strain
energy
ZDD
1of6
DTY_D_1370
11.18
0
8.19
0.8
146.43
0.2
1of6
DTY_E_1370
6.58
0.2
8.08
0.2
154.67
0.4
1of6
DTY_F_1370
8.00
0.0
8.40
1.0
163.72
0.9
1of6
DTY_G_1369
8.35
0.4
6.51
2.4
160.73
1.4
1of6
DTY_H_1369
9.36
0.8
9.67
1.6
150.23
1.1
1opk
1.80
P16_A_2
2.10
5.6
1.84
6.0
35.02
6.2
1oq5
1.50
CEL_A_701
11.81
3.2
15.60
3.7
19.44
4.2
1owe
1.60
675_A_1001
8.71
2.6
11.22
1.9
14.06
2.0
1oyt
1.67
FSN_H_501
8.40
3.5
8.85
3.4
27.29
4.2
1p2y
2.28
NCT_A_440
2.08
1.6
1.05
2.1
13.39
1.6
1p62
1.90
GEO_B_302
11.64
0.2
9.97
0.4
19.44
2.0
1q1g
2.02
MTI_A_301
15.92
3.2
15.04
3.4
32.53
3.9
1q1g
MTI_B_302
13.60
2.1
13.15
1.7
30.40
2.8
1q1g
MTI_C_303
15.61
3.5
14.22
2.9
29.91
5.5
1q1g
MTI_D_304
18.04
2.6
17.90
2.8
26.51
3.5
1q1g
MTI_E_305
14.46
1.1
18.81
0.7
31.12
1.9
1q1g
MTI_F_306
15.61
2.3
13.54
2.4
26.37
5.4
1q41
2.10
IXM_A_451
1.37
2.7
1.59
3.4
36.35
3.4
1q41
IXM_B_452
1.59
6.3
1.92
5.6
35.60
6.5
1q4g
1.98
BFL_A_701
2.55
3.2
3.97
5.2
6.53
3.9
1q4g
BFL_B_1701
2.47
6.5
3.76
6.2
7.92
5.6
1r1h
1.95
BIR_A_2001
13.81
1.0
17.32
1.9
33.90
1.7
1r55
1.59
097_A_518
21.70
1.5
16.20
1.6
29.71
2.0
1r58
1.90
AO5_A_501
41.38
3.9
62.41
3.9
68.73
2.9
1r9o
2.00
FLP_A_501
4.58
1.8
2.32
1.6
9.40
1.2
1s19
2.00
MC9_A_500
7.52
1.1
8.28
1.6
28.62
4.3
1s3v
1.80
TQD_A_187
8.60
0.6
9.10
0.8
29.76
1.4
1sg0
1.50
STL_A_501
2.55
10.2
2.88
10.1
12.51
12.0
1sg0
STL_B_502
4.41
4.8
5.96
5.6
14.94
4.5
1sj0
1.90
E4D_A_600
13.33
2.7
18.99
3.8
33.95
2.7
1sq5
2.00
PAU_A_6001
6.06
1.6
6.12
1.2
17.33
3.2
1sq5
PAU_B_6003
9.28
4.6
10.13
5.5
21.19
4.8
1sq5
PAU_C_6002
9.53
4.6
10.26
5.7
23.71
4.5
1sq5
PAU_D_6004
7.67
2.3
7.54
3.6
16.22
3.3
1t40
1.80
ID5_A_320
13.61
1.6
6.41
1.3
16.32
0.8
1t46
1.60
STI_A_3
16.69
3.5
17.06
4.3
59.53
3.2
1t9b
2.20
1CS_A_695
3.32
4.3
4.09
4.7
21.14
4.6
1t9b
1CS_B_1695
3.05
4.1
4.75
4.5
33.33
4.9
1tow
2.00
CRZ_A_501
7.25
1.1
3.74
0.7
10.99
0.5
1tt1
1.93
KAI_A_998
7.04
1.2
23.20
1.1
22.77
1.6
1tt1
KAI_B_999
6.31
1.1
22.03
0.8
22.88
1.2 3.3.2. Ligand ZDD. The histogram for
ZDD (Fig. 9) exhibits similar distribu-
tions for all three refinement types, with
a
rather
broad
peak
at
1.4 units. research papers However, the proportion of ONIOM-
and Region-QM-refined models in the
first three bins, which cover the range of
values from 0 to 1.2 ZDD units, is higher
than
the
number
of
conventional
models in the same range. Thus, the
average
ZDD
for
the
ligands
in
ONIOM-refined
structures
(2.3
0.8 units) is slightly lower (better) than
that after conventional refinement (2.9
1.1 units). Region-QM refinement
yields a set of models which are in the
middle (2.6 0.9 units) (Table 2). Overall, the ZDD distribution differs
significantly from that observed in the
1074
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement
Acta Cryst. (2018). D74, 1063–1077
Table 2 (continued)
ONIOM
Region-QM
Conventional
PDB
code
Res. research papers (A˚ )
Ligand
Strain
energy
ZDD
Strain
energy
ZDD
Strain
energy
ZDD
1of6
DTY_D_1370
11.18
0
8.19
0.8
146.43
0.2
1of6
DTY_E_1370
6.58
0.2
8.08
0.2
154.67
0.4
1of6
DTY_F_1370
8.00
0.0
8.40
1.0
163.72
0.9
1of6
DTY_G_1369
8.35
0.4
6.51
2.4
160.73
1.4
1of6
DTY_H_1369
9.36
0.8
9.67
1.6
150.23
1.1
1opk
1.80
P16_A_2
2.10
5.6
1.84
6.0
35.02
6.2
1oq5
1.50
CEL_A_701
11.81
3.2
15.60
3.7
19.44
4.2
1owe
1.60
675_A_1001
8.71
2.6
11.22
1.9
14.06
2.0
1oyt
1.67
FSN_H_501
8.40
3.5
8.85
3.4
27.29
4.2
1p2y
2.28
NCT_A_440
2.08
1.6
1.05
2.1
13.39
1.6
1p62
1.90
GEO_B_302
11.64
0.2
9.97
0.4
19.44
2.0
1q1g
2.02
MTI_A_301
15.92
3.2
15.04
3.4
32.53
3.9
1q1g
MTI_B_302
13.60
2.1
13.15
1.7
30.40
2.8
1q1g
MTI_C_303
15.61
3.5
14.22
2.9
29.91
5.5
1q1g
MTI_D_304
18.04
2.6
17.90
2.8
26.51
3.5
1q1g
MTI_E_305
14.46
1.1
18.81
0.7
31.12
1.9
1q1g
MTI_F_306
15.61
2.3
13.54
2.4
26.37
5.4
1q41
2.10
IXM_A_451
1.37
2.7
1.59
3.4
36.35
3.4
1q41
IXM_B_452
1.59
6.3
1.92
5.6
35.60
6.5
1q4g
1.98
BFL_A_701
2.55
3.2
3.97
5.2
6.53
3.9
1q4g
BFL_B_1701
2.47
6.5
3.76
6.2
7.92
5.6
1r1h
1.95
BIR_A_2001
13.81
1.0
17.32
1.9
33.90
1.7
1r55
1.59
097_A_518
21.70
1.5
16.20
1.6
29.71
2.0
1r58
1.90
AO5_A_501
41.38
3.9
62.41
3.9
68.73
2.9
1r9o
2.00
FLP_A_501
4.58
1.8
2.32
1.6
9.40
1.2
1s19
2.00
MC9_A_500
7.52
1.1
8.28
1.6
28.62
4.3
1s3v
1.80
TQD_A_187
8.60
0.6
9.10
0.8
29.76
1.4
1sg0
1.50
STL_A_501
2.55
10.2
2.88
10.1
12.51
12.0
1sg0
STL_B_502
4.41
4.8
5.96
5.6
14.94
4.5
1sj0
1.90
E4D_A_600
13.33
2.7
18.99
3.8
33.95
2.7
1sq5
2.00
PAU_A_6001
6.06
1.6
6.12
1.2
17.33
3.2
1sq5
PAU_B_6003
9.28
4.6
10.13
5.5
21.19
4.8
1sq5
PAU_C_6002
9.53
4.6
10.26
5.7
23.71
4.5
1sq5
PAU_D_6004
7.67
2.3
7.54
3.6
16.22
3.3
1t40
1.80
ID5_A_320
13.61
1.6
6.41
1.3
16.32
0.8
1t46
1.60
STI_A_3
16.69
3.5
17.06
4.3
59.53
3.2
1t9b
2.20
1CS_A_695
3.32
4.3
4.09
4.7
21.14
4.6
1t9b
1CS_B_1695
3.05
4.1
4.75
4.5
33.33
4.9
1tow
2.00
CRZ_A_501
7.25
1.1
3.74
0.7
10.99
0.5
1tt1
1.93
KAI_A_998
7.04
1.2
23.20
1.1
22.77
1.6
1tt1
KAI_B_999
6.31
1.1
22.03
0.8
22.88
1.2
Table 2 (continued)
ONIOM
Region-QM
Conventional
PDB
code
Res. research papers The
difference density is drawn at the 3 level. Figure 8
The A-weighted mFo DFc difference electron-density map peaks
around the ligand calcipotriol (ligand ID MC9) in PDB entry 1s19 refined
with the ONIOM (a), Region-QM (b) and conventional (c) methods. The
difference density is drawn at the 3 level. Acta Cryst. (2018). D74, 1063–1077 research papers (A˚ )
Ligand
Strain
energy
ZDD
Strain
energy
ZDD
Strain
energy
ZDD
1tz8
1.85
DES_B_128
5.09
3.2
3.98
2.7
62.82
13.9
1tz8
DES_C_129
0.24
3.8
0.82
4.3
56.87
9.1
1tz8
DES_D_128
1.11
0.8
0.28
0.7
46.17
10.0
1u1c
2.20
BAU_A_5400
7.88
4.1
13.09
4.2
22.15
3.5
1u1c
BAU_B_5011
8.83
1.3
15.44
2.2
21.01
0.8
1u1c
BAU_C_5021
4.66
2.4
18.31
2.7
22.46
3.0
1u1c
BAU_D_5031
7.33
2.4
20.52
2.5
22.11
2.2
1u1c
BAU_E_5041
7.62
1.1
11.21
0.5
18.81
1.2
1u1c
BAU_F_5051
5.87
0.7
17.19
1.0
23.16
1.2
1u4d
2.10
DBQ_A_398
5.44
0.1
5.66
0.4
21.99
0.1
1u4d
DBQ_B_401
5.46
0.8
3.54
1.2
18.50
1.8
1uml
2.50
FR4_A_1001
10.24
2.1
11.81
2.5
26.14
3.8
1unl
2.20
RRC_A_1293
25.22
3.2
27.55
3.2
83.26
4.0
1uou
2.11
CMU_A_1481
4.73
0.8
5.04
1.0
20.52
1.5
1v0p
2.00
PVB_A_1287
6.68
1.1
8.45
1.7
36.39
1.8
1v0p
PVB_B_1287
6.26
2.9
5.70
2.0
37.24
2.4
1v48
2.20
HA1_A_290
29.71
0.3
22.82
0.8
38.12
0.4
1v4s
2.30
MRK_A_501
2.99
2.1
2.79
2.0
27.23
2.4
1vcj
2.39
IBA_A_1
14.97
1.1
15.13
1.3
27.21
2.8
1w1p
2.10
GIO_A_1518
4.21
1.0
1.74
1.6
14.11
1.2
1w1p
GIO_B_1501
3.49
2.2
1.53
2.0
17.21
1.5
1w2g
2.10
THM_A_1210
3.03
1.0
7.16
1.1
10.95
1.5
1w2g
THM_B_1210
4.21
1.3
5.25
1.9
14.94
1.7
1x8x
2.00
TYR_A_952
14.47
0.1
4.77
0.1
101.28
0.1
1xm6
1.90
5RM_A_1003
0.71
0.4
1.32
0.6
12.15
0.9
1xm6
5RM_B_1003
2.67
1.0
2.22
0.4
15.49
1.4
1xoq
1.83
ROF_A_502
2.92
0.8
3.00
0.9
20.01
1.1
1xoq
ROF_B_501
2.55
1.3
2.97
1.1
21.02
1.4
1xoz
1.30
CIA_A_501
3.60
0.6
3.19
1.6
18.75
1.1
1y6b
2.10
AAX_A_201
6.88
4.9
6.89
5.4
19.13
5.5
1ygc
2.00
905_H_1
17.62
0.9
22.37
1.8
44.66
1.9
1yv3
1.99
BIT_A_800
3.51
2.8
2.75
3.2
12.91
2.7
1yvf
2.50
PH7_A_800
1.94
1.3
2.95
2.4
30.45
2.8
1ywr
1.90
LI9_A_361
17.66
4.1
21.03
4.8
52.45
3.9
1z95
1.80
198_A_501
5.73
1.3
6.55
1.1
28.01
3.3
2bm2
2.20
PM2_A_3211
1.43
1.4
2.27
1.4
14.88
1.8
2bm2
PM2_B_3211
1.87
0.9
1.96
1.1
21.68
2.3
2bm2
PM2_C_3211
1.56
1.0
1.38
1.4
12.97
1.9
2bm2
PM2_D_3211
1.40
1.8
1.41
1.1
13.31
1.7
2br1
2.00
PFP_A_1277
5.63
0.9
7.29
0.6
21.90
2.2
2bsm
2.05
BSM_A_1224
19.15
1.3
10.32
1.6
21.54
1.8 Table 2 (continued)
ONIOM
Region-QM
Conventional
PDB
code
Res. 4. Discussion Protein crystallography continues to play a central role in drug
discovery as SBDD remains a critical technique for ligand
design and optimization, high-throughput screening and often
FDA
approval
(Blundell,
2017). However,
the
overall
lackluster quality of ligands within deposited protein–ligand
complexes raises serious concerns. Unfortunately, these errors
in the ligand geometry, placement and protonation states
often lead to the misperception of protein–ligand interactions
and to problems in binding-mode determination, thus dimin-
ishing the relevance of such models for SBDD (Borbulevych et
al., 2012, 2014, 2016; Cooper et al., 2011; Malde & Mark, 2011;
Reynolds, 2014). These issues have been acknowledged
(Debreczeni & Emsley, 2017), and the community has made
significant methodological improvements in the generation of
higher quality restraint (CIF) ligand dictionaries (Nicholls,
2017; Long et al., 2017; Janowski et al., 2016). However, these
improvements still lead to a static dictionary file which is
created for an isolated ligand without explicit consideration of
the in situ impact of the protein and the ligand on one another. In our previous work (Borbulevych et al., 2014), we introduced
an approach for macromolecular refinement within the
PHENIX
package
for
Region-QM
refinement. In
this
approach, the quality of the CIF is immaterial, and the entire
user-defined region including both the ligand(s) and the active
site(s) are treated as one QM system, thus capturing inter-
molecular interactions (for example electrostatics, charge
transfer, polarization, dispersion and hydrogen bonding) at
each refinement step. This work has led to significant
improvements in ligand strain and ligand ZDD upon QM
refinement. In cases where significant strain is still observed,
manual building of the model may still be necessary to fix large
model errors or rotamer outliers since any gradient-driven
refinement cannot make changes beyond its radius of
convergence. When considering the distribution of ligand ZDD values
(Table 1, Fig. 9), it is worth noting that ONIOM refinement
leads to a smaller (better) average ZDD (2.29) when
compared with the corresponding average for conventional
refinement (2.94). However, this improvement is smaller in
magnitude than that observed for ligand-strain energy. ZDD
values generally correspond to the amount of difference
density around the ligands (Borbulevych et al., 2016; Tickle,
2012), and previously we have shown that ZDD is very
sensitive to protomeric/tautomeric states (Borbulevych et al.,
2016) or ligand poses (Borbulevych & Westerhoff, 2018). 4. Discussion However, in the present study the input PDB files including
ligand states/positions were the same for all three types of
refinement and therefore we would expect that the ZDD
distributions would likewise be similar for those refinements
(Fig. 9). research papers 9) exhibits similar distribu-
tions for all three refinement types, with
a
rather
broad
peak
at
1.4 units. However, the proportion of ONIOM-
and Region-QM-refined models in the
first three bins, which cover the range of
values from 0 to 1.2 ZDD units, is higher
than
the
number
of
conventional
models in the same range. Thus, the
average
ZDD
for
the
ligands
in
ONIOM-refined
structures
(2.3
0.8 units) is slightly lower (better) than
that after conventional refinement (2.9
1.1 units). Region-QM refinement
yields a set of models which are in the
middle (2.6 0.9 units) (Table 2). Overall, the ZDD distribution differs
significantly from that observed in the Figure 9
Histogram of ligand ZDD distributions for 141 ligand instances from 80 Astex structures refined
with three methods: ONIOM, Region-QM and conventional. 1074
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement 1074
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement 1074
Borbulevych et al. High-throughput QM/MM (ONIOM) refinement Acta Cryst. (2018). D74, 1063–1077 research papers ligand strain, and the square of the Pearson correlation
coefficient (R2) between ZDD and ligand strain is zero for all
three refinements, demonstrating that these two metrics are
uncorrelated. average factor of 4.5–5.0 upon ONIOM refinement compared
with both Region-QM and conventional PHENIX refine-
ments (Table 1), demonstrating that ONIOM is able to correct
bad clashes. The cause of these improvements can be
explained when one considers how MM works. Specifically,
since any residues outside the QM region are described at the
MM level (in this case using the AMBER forcefield as
implemented in DivCon), any reduction of unfavorable short
clashes arises from the 6-12 Lennard–Jones potential for van
der Waals interactions. Furthermore, the electrostatic inter-
actions captured by the qiqj/rij term of the AMBER functional
also play an essential role, as shown by the example shown in
Fig. 5. In this case, the bad clash between Asn413 ND2 and
Wat1098 O that was found in the original structure, and that
was not corrected by conventional and Region-QM refine-
ment, was not only corrected by ONIOM but the interaction
was also converted to an electrostatically favorable hydrogen
bond. Acknowledgements The authors wish to acknowledge the continued support of the
PHENIX Consortium, in particular Drs Nigel Moriarty, Pavel
Afonine and Paul Adams, for maintaining the application
programming interface (API) hooks to our software within the
PHENIX distribution and for helpful discussion and feedback. The authors would also like to thank Gregory Warren for
helpful discussion along with the editor and reviewers for their
suggestions and feedback which have lead to a much improved
paper. The DivCon plugin to PHENIX is provided by Quan-
tumBio Inc. and it is available at no cost to academic users at
http://www.quantumbioinc.com/products/software_licensing. Lee, T. S., LeGrand, S., Li, P., Lin, C., Liu, J., Luchko, T., Luo, R., Mermelstein, D. J., Merz, K. M., Miao, Y., Monard, G., Nguyen, C., Roitberg, A., Sagui, C., Schott-Verdugo, S., Shen, J., Simmerling, C. L., Smith, J., Salomon-Ferrer, R., Swails, J., Walker, R. C., Wang, J., Wei, H., Wolf, R. M., Wu, X., Xiao, L., York, D. M. & Kollman,
P. A. (2018). AMBER 2018. University of California, San
Francisco, USA. Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson,
D. C. (2010). Acta Cryst. D66, 12–21. Chung, L. W., Sameera, W. M. C., Ramozzi, R., Page, A. J., Hatanaka,
M., Petrova, G. P., Harris, T. V., Li, X., Ke, Z., Liu, F., Li, H.-B.,
Ding L & Morokuma K (2015) Chem Rev 115 5678–5796 Ding, L. & Morokuma, K. (2015). Chem. Rev. 115, 5678–5796. Funding information Cooper, D. R., Porebski, P. J., Chruszcz, M. & Minor, W. (2011). Exp. Opin. Drug. Discov. 6, 771–782. The research reported in this publication was supported by the
National Institute of General Medical Sciences of the National
Institutes of Health under Award Nos. R44GM112406 and
R44GM121162. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Institutes of Health. Davis, A. M., Teague, S. J. & Kleywegt, G. J. (2003). Angew. Chem. Int. Ed. 42, 2718–2736. Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J.,
Wang, X., Murray, L. W., Arendall, W. B., Snoeyink, J., Richardson,
J. S. & Richardson, D. C. (2007). Nucleic Acids Res. 35, W375–
W383. Debreczeni, J. E´ . & Emsley, P. (2017). Acta Cryst. D73, 77–78. Dewar, M. J. S., Zoebisch, E. G., Healy, E. F. & Stewart, J. J. P. (1985). J. Am. Chem. Soc. 107, 3902–3909. 5. Conclusions Recently, numerous new programs and approaches to create
high-quality ligand restraints have been published (Steiner &
Tucker, 2017). These methods generally suffer from a critical,
fundamental flaw in that they do not explicitly capture the in
situ interactions between the protein and the ligand during
refinement. In the present work, we demonstrate an entirely
new methodology to perform X-ray refinement using the two-
layer ONIOM method as implemented in the QuantumBio
DivCon package. Using this concept, ligands and corre-
sponding active-site residues are treated at the QM level,
while the rest of molecule is represented using the MM
functional. Both functionals are then combined to derive the
ONIOM energy, and associated gradients, of the system. In the
present work, the ONIOM approach for the X-ray refinement
has been validated against 80 protein–ligand structures from
the Astex Diverse Set using both MolProbity metrics and
ligand-quality metrics. We established that ONIOM refine-
ment excels in both sets of metrics, resulting in a superior
overall quality of the protein–ligand model compared with
conventional refinement. Combined with a fully automatic The present study takes this improvement to macro-
molecular refinement further through the development and
integration of a high-throughput and fully automated two-
layer mixed QM/MM ONIOM module applied to the entire
structure. With this fully automated approach, any user-
chosen ligands, metal ions and cofactors, together with the
surrounding residues, comprise a QM layer, while the rest of
the atoms in the structure comprise the MM layer and inter-
actions between the two layers are addressed. ONIOM
refinement exhibits all benefits of the previously developed
DivCon Region-QM refinement versus conventional refine-
ment, as measured by ligand-strain energy and ligand ZDD
(Table 1, Figs. 6 and 9), while at the same time showing marked
improvements in overall structure quality as measured by
MPScore (Table 1, Fig. 3). In particular, we observed an
improvement in the clashscore component of MPScore by an Borbulevych et al. High-throughput QM/MM (ONIOM) refinement 1075 Acta Cryst. (2018). D74, 1063–1077 research papers structure-preparation protocol and fast, convergent QM/MM
calculations, we believe that the ONIOM refinement devised
in this paper sets a new paradigm for fast, accurate and user-
friendly macromolecular X-ray refinement. Luo, R., Madej, B., Merz, K. M., Paesani, F., Roe, D. R., Roitberg,
A., Sagui, C., Salomon-Ferrer, R., Seabra, G., Simmerling, C. L.,
Smith, W., Swails, J., Walker, R. C., Wang, J., Wolf, R. M., Wu, X. &
Kollman, P. A. (2014). AMBER 14. University of California, San
Francisco, USA. Case, D. A., Ben-Shalom, I. Y., Brozell, S. R., Cerutti, D. S.,
Cheatham, I. T. E., Cruzeiro, V. W. D., Darden, T. A., Duke, R. E.,
Ghoreishi, D., Gilson, M. K., Gohlke, H., Goetz, A. W., Greene, D.,
Harris, R., Homeyer, N., Izadi, S., Kovalenko, A., Kurtzman, T.,
Lee, T. S., LeGrand, S., Li, P., Lin, C., Liu, J., Luchko, T., Luo, R.,
Mermelstein, D. J., Merz, K. M., Miao, Y., Monard, G., Nguyen, C.,
Nguyen, H., Omelyan, I., Onufriev, A., Pan, F., Qi, R., Roe, D. R.,
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1077 Borbulevych et al. High-throughput QM/MM (ONIOM) refinement
1077 Acta Cryst. (2018). D74, 1063–1077
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Technology for cleaning glass substrates in glow discharge plasma for microfluidics applications
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1 Introduction In the current development of micro-, nano- and microelectronics, contamination of
substrate surfaces is becoming commensurate with the size of the micro- and nanostructures
being formed, which leads to the need for thorough cleaning and tight control over the
degree of surface cleanliness. Microfluidic chips have quickly gained widespread adoption at the point of care,
offering advantages of smaller sample volume, fast detection, high throughput, low
contamination and easy integration. Microfluidic chips often consist of a polymer cap assembly and a glass substrate with
microchannels (Fig. 1), produced by photolithography and wet etching. Therefore, it is
important to prepare the glass substrate before applying the functional layers. Technology for cleaning glass substrates in
glow
discharge
plasma
for
microfluidics
applications
D.M. Rabotyazheva1*
1Bauman Moscow Technical University, ul. Baumanskaya 2-ya, 5/1, 105005 Moscow, Russia Abstract. The production of microfluidic chips is increasing every year
because the technology using the chip allows you to make an express test
to obtain the analysis, as well as economically viable. Often microfluidic
chips are based on glass in which the microchannels are formed. Accurate
production of a microfluidic device requires careful preparation of the
glass substrate to improve adhesion for further application of functional
layers. The choice of cleaning mode strongly affects the output product, so
a clear understanding of what factors affect the quality of cleaning in HF
plasma is needed. Mathematical modeling of dependence between plasma
power of the glow discharge, time and gas medium first of all gives
understanding which cleaning mode is optimal and which parameter
affects stronger very relevant for development of this potentially promising
technology of quality products. * Corresponding author: pryanichnikovadm@student.bmstu.ru E3S Web of Conferences 413, 02001 (2023)
INTERAGROMASH 2023 E3S Web of Conferences 413, 02001 (2023)
INTERAGROMASH 2023 https://doi.org/10.1051/e3sconf/202341302001 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative
Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). Fig. 1. Schematic diagram of a microfluidic chip The implementation of plasma treatment in microfluidic devices represents a promising
approach to the development of compact, relatively inexpensive, sensitive and effective
diagnostic tools for clinical practice. * Corresponding author: pryanichnikovadm@student.bmstu.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative
Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 413, 02001 (2023)
INTERAGROMASH 2023 https://doi.org/10.1051/e3sconf/202341302001 Fig. 1. Schematic diagram of a microfluidic chip 2 Materials and methods Plasma treatment of glass is currently a common method in various fields of industry [1]. Plasma treatment can achieve the best cleaning of surfaces compared to other methods. An important element of research for a microfluidic chip is the preparation of a glass
substrate prior to subsequent operations [2], developed at the Department of ME11 of the
Bauman Moscow State Technical University. An experiment, using the MSS plasma unit
(Fig. 2) and a goniometer to measure the wetting angle (Fig. 3), deriving a mathematical
model to determine the best processing mode within the ME11 laboratory, showed good
results for their subsequent use. Plasma treatment of glass is currently a common method in various fields of industry [1]. Plasma treatment can achieve the best cleaning of surfaces compared to other methods. Fig. 2. Purification scheme in glow discharge plasma: 1 - substrate, 2 - electrode, 3 - plasma, 4 - gas
injection, 5 – pumping Fig. 2. Purification scheme in glow discharge plasma: 1 - substrate, 2 - electrode, 3 - plasma, 4 - gas
injection, 5 – pumping 2 2 E3S Web of Conferences 413, 02001 (2023)
INTERAGROMASH 2023 https://doi.org/10.1051/e3sconf/202341302001 INTERAGROMASH 2023 Fig. 3. Goniometer LK-1 Fig. 3. Goniometer LK-1 Thus, the plasma treatment of glass substrate surface before coating in vacuum leads not
only to removal of surface impurities, such as organic impurities, oxides and physical
inclusions, but also to modification of surface properties towards increasing its adsorption
and adhesion activity, i.e. to creating active adsorption and adhesion centers of atomized
particles on substrate surface, and to surface hardening [3]. The most effective way to
prepare the glass substrate surface for subsequent operations in a vacuum chamber is the
method combining degreasing and glow discharge treatment. Alcohol was used as a solvent
for degreasing. As a result of desorption, the contaminant molecules pass from the surface
to be cleaned into the near-surface liquid layer and then diffuse into its volume. 2 Materials and methods Contaminants on the surface of substrates are classified, as a rule, according to their
physical and chemical properties physical contaminants (dust particles, lint, abrasives, silicates, silica dust and other
foreign particles); physical contaminants (dust particles, lint, abrasives, silicates, silica dust and other
foreign particles); chemically unrelated to the surface of wafers and substrates; contaminants chemically bound to the surface of wafers and substrates (oxides, nitrides
and other compounds); organic contaminants (fats, oils, silicones and other impurities); water-soluble contaminants (salts, acids, etchant residues, fluxes, etc.); gases adsorbed by the surface of wafers and substrates. Various types of contaminants can be present on the surface of the wafers at the same
time. The most difficult to remove are organic and chemically bound contaminants, as well
as contaminants from abrasive materials, polar gases and ions embedded in the surface
layer of the plates. Using a pure gas environment is not the optimal cleaning method, so a mixture of gases
should be created in a vacuum chamber. Argon is an inert and chemically neutral gas and is
effectively used for physical cleaning. The oxygen environment is good for removing
organic contaminants, resulting in gaseous compounds that are evacuated by the vacuum
chamber. The mixture of oxygen and argon gases will not only physically dislodge
contaminants, but also chemically clean the surface from contaminants. Analysis of scientific articles has shown that the parameters of the glow discharge
plasma on which the efficiency of substrate cleaning depends are: - processing time; - discharge power; - residual gas pressure in the chamber; - location of the substrate (this determines the treatment zone); - composition of the gas medium. These parameters are related quantities that interact with each other. 3 E3S Web of Conferences 413, 02001 (2023)
INTERAGROMASH 2023 https://doi.org/10.1051/e3sconf/202341302001 3 Results The main purpose of the experiment is to develop a mathematical model adequately
describing the influence of process parameters on the value of the wetting angle. Simulation
of the selected process is necessary to determine which of the input factors most
significantly affect the output parameter, and which influence the output parameter to a
lesser extent. Table 1. Results of glass cleaning on the MSS unit
№
Time,
sec
Power,
Watt
Ar
concentration
in О2, %
Angle
1
60
100
15
10,4
2
180
100
15
4,9
3
60
200
15
8,3
4
180
200
15
13,9
5
60
200
30
12,4
6
180
100
30
7,2
7
60
100
30
12,9
8
180
200
30
4,3 Table 1. Results of glass cleaning on the MSS unit The pressure in the working chamber was set depending on the stable operation of the
generator and monitored by a wide-range sensor. Power was adjusted and set on the
generator unit. Concentration of working gas was set by the gas flow regulator and
controlled by the control scale on it. It should be noted that the studied parameter in this experiment is the wetting angle of
the substrate surface. The treated surface is inherently hydrophilic (Θ<90°o). A quantitative
measure of surface wettability is the wetting angle (Fig. 4) - the angle between the tangent
to the surface of a liquid drop at the point of contact of the three phases (solid, liquid and
gaseous) and the surface of a solid, measured inside the liquid phase [5]. The wetting angle was measured using a goniometer. The procedure for measuring the
wetting angle is as follows: the sample under study is placed on the goniometer slide, a
drop of distilled water is applied to the sample surface using a special syringe [4]. X3 - normalized power of HF discharge from 100 watts to 200 watts;
bili
l Fig. 4. Drop profile projection The calculation is as follows: 𝑉𝐼𝐶=
𝑗∙1 ∙40
1,6 ∙10−19 ∙6,02 ∙1026 ∙2500 = 0,166 ∙10−9 𝑗 𝑚/𝑠𝑒𝑐 = 0,166𝑗 𝑛𝑚/𝑠𝑒𝑐 The density of the ionic current is determined by the formula: 𝑗=
𝐼
𝑆 , А/𝑚2,
where the current strength varied from 0.75 to 1 A; The density of the ionic current is determined by the formula: 𝑗=
𝐼
𝑆 , А/𝑚2, where the current strength varied from 0.75 to 1 A;
S=0,062 m2 - cross-sectional area. Based on the formula, ion current density will be
determined by the interval from 12,1 to 16,1 А/m2. S=0,062 m2 - cross-sectional area. Based on the formula, ion current density will be
determined by the interval from 12,1 to 16,1 А/m2. On the basis of calculation, a graph of ion cleaning in glow discharge plasma was
plotted: Fig. 4. Graph of ionic purification
4 Conclusion Fig. 4. Graph of ionic purification Fig. 4. Drop profile projection Fig. 4. Drop profile projection For better understanding of the relationship between the factors under study and the
output parameter of the cleaning quality dependence on the composition of the gas medium,
time and power of low-pressure HF plasma, a mathematical model was made:
y=9,2903 - 1,7022X1 + 2,2452X2 - 0,0909X3 + 0,9789X1X2 - 1,7535X1X3 - 1,7535X2X3 -
1,7949X1X2X3 where Х1 - normalized substrate processing time of 60 to 180 seconds; X2 - normalized percentage of Ar in the O2 gas medium from 15% to 30%; X3 - normalized power of HF discharge from 100 watts to 200 watts;
y - wettability angle y - wettability angle. 4 E3S Web of Conferences 413, 02001 (2023)
INTERAGROMASH 2023 https://doi.org/10.1051/e3sconf/202341302001 Also, an analysis of the ion purification rate from the ion current density was carried
out, which gives a direct understanding of the interaction of these parameters. Ion purification is realized using argon Ar+ noble gas ions, which atomize atoms of the
target material due to the high (over 1 keV) energy of the ions. In ion purification, the purification rate (physical atomization of the material) is:
A
e
i
IC
N
q
SM
j
V
h
j
12 1 16 1 А/ 2 i
t d
it In ion purification, the purification rate (physical atomization of the material) is:
i
IC
SM
j
V
A
e
i
IC
N
q
SM
j
V
where ji =12,1..16,1 А/m2– ion current density; where ji =12,1..16,1 А/m2– ion current density; S = 1 atom/ion - atomization factor; S = 1 atom/ion - atomization factor; M = 40 kg/kmol - molar mass; g
qe =1, 6 ⸱10-19 Кл – Cl - electron charge; g
qe =1, 6 ⸱10-19 Кл – Cl - electron charge;
NA =6,02⸱1026 molecules/kmol - Avogadro num NA =6,02⸱1026 molecules/kmol - Avogadro = 2500 kg/m3 - density of the material. The calculation is as follows: = 2500 kg/m3 - density of the material. = 2500 kg/m3 - density of the material. 4 Conclusion As a result of investigation of the influence of low-pressure HF plasma on the surface of
glass substrates, eight variants of MSS unit operation modes were selected, based on their 5 5 E3S Web of Conferences 413, 02001 (2023)
INTERAGROMASH 2023 E3S Web of Conferences 413, 02001 (2023)
INTERAGROMASH 2023 https://doi.org/10.1051/e3sconf/202341302001 results, a mathematical model of the surface wetting angle was derived. From the
experimental data obtained, we can conclude that the smallest angle of wettability of the
surface has the mode at 30% argon in oxygen medium, the maximum power of 200 watts
delivered to the electrodes and a time of 180 seconds. At these process parameters, the
wetting angle is 4,3о. Also, it can be noted that mode 4 at maximum power and time, but minimal argon
content in the oxygen environment indicates that the physical knockout of particles has a
greater impact on the surface to be treated. 5 Acknowledgements The research was carried out as part of the ME11 research project at the MSS facility. The research was carried out as part of the ME11 research project at the MSS facility. References 1. Br. J. Appl, The cleaning of glass in a glow discharge L. Holland, F.Inst.P., Research
Laboratory, Edwards High Vacuum Ltd., Crawley, Sussex doi:10.1088/0508-
3443/9/10/306 2. X. Su, Y. Xu, H. Zhao, S. Li, L. Chen, Design and preparation of centrifugal
microfluidic chip integrated with SERS detection for rapid diagnostics, Talanta, 194
(2019) doi.org/10.1016/ 3. А. G. Luchkin, G. S. Luchkin, Cleaning the surface of substrates for coating by
vacuum-plasma methods, UDC 533.599, 621.793, 539.23. 4. S. A. Borodin, A. V. Volkov, N. L. Kazanskiĭ, Device for analyzing nanoroughness
and contamination on a substrate from the dynamic state of a liquid drop deposited on
its surface. Journal of Optical Technology, 76(7), 42-47 (2009) 5. V. A. Sojfer, N. L. Kazansky, V. A. Kolpakov, A. I. Kolpakov, V. V. Podlipnov,
Method of measuring substrate surface purity, No 2380684 (2008) 6. L. I. Maissel, R. Glang, P. P. Budenstein, Handbook of thin film technology. Journal of
The Electrochemical Society, 118(4), 768 (1977) 7. K. Yu. Vukolov, E. N. Andreenko, I. I. Orlovskiy, Radiation tests of the prototypes of
the fiber-optic collector for ITER plasma diagnostics, Fusion Engineering and Design,
170, 112465 (2021) https://doi.org/10.1016/j.fusengdes.2021.112465 6
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Implementasi Model Pembelajaran Problem Based Learning (PBL) Tentang Materi Biologi Untuk Meningkatkan Keterampilan Pemecahan Masalah: Literature Review
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Jurnal dedikasi pendidikan/Dedikasi: Jurnal Pendidikan dan Keguruan Universitas Abulyatama
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EDITORIAL TEAM
JURNAL DEDIKASI PENDIDIKAN
ISSN 2548-8848 (Online)
Editor in Chief
Putri Dini Meutia, M.Pd. (Universitas Abulyatama)
Editors
Dr. Syarifah Rahmi Muzanna, M.Pd. (Universitas Abulyatama)
Dr. Silvi Puspa Widya Lubis, M.Pd(Universitas Abulyatama)
Riki Musriandi, M.Pd. (Universitas Abulyatama)
Hasanah, M.A. (Universitas Abulyatama)
Suryani M.Pd (Universitas Abulyatama)
Safriana, M.Pd. (Universitas Malikulsaleh)
Rita Sari, M.Pd. (Institut Agama Islam Negeri Langsa)
Cut Mawar Helmanda, M.Pd. (Universitas Muhammadiyah Aceh)
Reviewers
Dr. Abdul Haliq, S.Pd. M.Pd. (Universitas Negeri Makassar)
Dr. Anwar, M.Pd. (Universitas Samudra)
Dr. Hendrik A.E. Lao (Institut Agama Kristen Negeri Kupang)
Dr. Asanul Inam, M.Pd., Ph.D (Universitas Muhammadiyah Malang)
Dr. Baiduri (Universitas Muhammadiyah Malang)
Septhia Irnanda, S.Pd., MTESOL., Ph.D. (Universitas Serambi Mekkah)
Dr. Tuti Marjan Fuadi, M.Pd. (Universitas Abulyatama)
Ugahara M, M.TESOL., Ph.D (Universitas Abulyatama)
Murni, S.Pd., M.Pd., Ph.D (Universitas Abulyatama)
Marina, M.Ed. (Universitas Malikulsaleh)
Mauloeddin Afna, M.Pd, (Institut Agama Islam Negeri Langsa) JURNAL DEDIKASI PENDIDIKAN
ISSN 2548-8848 (Online)
Editor in Chief
Putri Dini Meutia, M.Pd. (Universitas Abulyatama)
Editors
Dr. Syarifah Rahmi Muzanna, M.Pd. (Universitas Abulyatama)
Dr. Silvi Puspa Widya Lubis, M.Pd(Universitas Abulyatama)
Riki Musriandi, M.Pd. (Universitas Abulyatama)
Hasanah, M.A. (Universitas Abulyatama)
Suryani M.Pd (Universitas Abulyatama)
Safriana, M.Pd. (Universitas Malikulsaleh)
Rita Sari, M.Pd. (Institut Agama Islam Negeri Langsa)
Cut Mawar Helmanda, M.Pd. (Universitas Muhammadiyah Aceh)
Reviewers
Dr. Abdul Haliq, S.Pd. M.Pd. (Universitas Negeri Makassar)
Dr. Anwar, M.Pd. (Universitas Samudra)
Dr. Hendrik A.E. Lao (Institut Agama Kristen Negeri Kupang)
Dr. Asanul Inam, M.Pd., Ph.D (Universitas Muhammadiyah Malang)
Dr. Baiduri (Universitas Muhammadiyah Malang)
Septhia Irnanda, S.Pd., MTESOL., Ph.D. (Universitas Serambi Mekkah)
Dr. Tuti Marjan Fuadi, M.Pd. (Universitas Abulyatama)
Ugahara M, M.TESOL., Ph.D (Universitas Abulyatama)
Murni, S.Pd., M.Pd., Ph.D (Universitas Abulyatama)
Marina, M.Ed. (Universitas Malikulsaleh)
Mauloeddin Afna, M.Pd, (Institut Agama Islam Negeri Langsa)
Alamat Sekretariat/Redaksi :
LPPM Universitas Abulyatama
Jl. Blang Bintang Lama Km. 8,5 Lampoh Keude Aceh Besar
Website : http://jurnal.abulyatama.ac.id/
Email : jurnal_dedikasi@abulyatama.ac.id
Telp/fax : 0651-23699 JURNAL DEDIKASI PENDIDIKAN Editor in Chief
Putri Dini Meutia, M.Pd. (Universitas Abulyatama) Volume 7, No. 2, Juli 2023 Volume 7, No. 2, Juli 2023 ISSN. 2548-8848 (Online) ISSN. 2548-8848 (Online) DAFTAR ISI 1. Implementasi Model Pembelajaran Problem Based Learning (PBL) Tentang
Materi Biologi Untuk Meningkatkan Keterampilan Pemecahan Masalah:
Literature Review
(Putri Silmi Nurul Fadila, Fitri Arsih, Ganda Hijrah Selaras, Heffi Alberida)
347-354
2. Pola Pendidikan Agama Kristen Dalam Keluarga Petani Di Desa O’Baki
Kecamatan Kokbaun Kabupaten Timor Tengah Selatan
(Nofriana Baun, Sumeriani Tsu, Amelia Wila)
355-366
3. Persepsi Guru PAUD Tentang Pentingnya Pelatihan Kurikulum Merdeka
(Chairun Nisa Fadillah, Munawarah, Reza Aulia)
367-374
4. Manajemen Sarana Dan Prasarana Di SMK Plus Al-Aitaam Kabupaten Bandung
(Deti Rostini, Wiwik Dyah Aryani, Muhammad Danil, Raden Riki Barkah
Zulfikar, Rohma)
375-382
5. Analisis Strategi Guru Dalam Pelaksanaan Pengelolaan Kelas Oleh Guru Kelas V
SD Swasta Assisi Medan
(Antonius Remigius Abi, Lona Medita Lingga, Saut Mahulae, Syafri Fadhilah
Marpaung, Hambali)
383-392
6. Analisis Bentuk Manajemen Peserta Didik Di SMTK Rote Timur Kabupaten
Rote Ndao
(Yonatan Foeh)
393-402
7. Penerapan Strategi Predict, Organize, Rehearse, Practice And Evaluate (PORPE)
Untuk Meningkatkan Keterampilan Membaca Pemahaman Siswa Sekolah Dasar
(Mhd. Iqbal Maulana, Nurhaswinda, Rizki Amalia, Putri Hana Pebriana,
Fadhilaturrahmi)
403-414
8. Pengembangan Media Audio Visual Dalam Pembelajaran PPKn Dengan
Pendekatan Problem Based Learning Di Kelas VI Sekolah Dasar
(Devita Eka Rahmadani, Linda Zakiah, Adi Putra)
415-428
9. Penerapan Model Pembelajaran Questioning Untuk Meningkatkan Keterampilan
Membaca Pemahaman Siswa Sekolah Dasar
(Bagas Rianto, Putri Hana Pebriana, Nurhaswinda, Sumianto, Fadhilaturrahmi)
429-442
10. Urgensi Membangun Literasi Pada Anak Usia Dini
(Munawarah, Chairun Nisa Fadhilah, Reza Aulia, Nur Cahyati Ngaisah, Firman
Friyo Suhasto)
443-450
11. Manajemen Stres Kerja Dan Konflik Kerja: Pengaruhnya Terhadap Kinerja Guru
(Nikmatullaili, Nurhizrah Gistituati, Sufyarma Marsidin)
451-458 1. Implementasi Model Pembelajaran Problem Based Learning (PBL) Tentang
Materi Biologi Untuk Meningkatkan Keterampilan Pemecahan Masalah:
Literature Review
(Putri Silmi Nurul Fadila, Fitri Arsih, Ganda Hijrah Selaras, Heffi Alberida)
347-354 2. Pola Pendidikan Agama Kristen Dalam Keluarga Petani Di Desa O’Baki
Kecamatan Kokbaun Kabupaten Timor Tengah Selatan
(Nofriana Baun, Sumeriani Tsu, Amelia Wila)
355-366 3. Persepsi Guru PAUD Tentang Pentingnya Pelatihan Kurikulum Merdeka
(Chairun Nisa Fadillah, Munawarah, Reza Aulia)
367-374 4. Manajemen Sarana Dan Prasarana Di SMK Plus Al-Aitaam Kabupaten Bandung
(Deti Rostini, Wiwik Dyah Aryani, Muhammad Danil, Raden Riki Barkah
Zulfikar, Rohma)
375-382 5. Analisis Strategi Guru Dalam Pelaksanaan Pengelolaan Kelas Oleh Guru Kelas V
SD Swasta Assisi Medan
(Antonius Remigius Abi, Lona Medita Lingga, Saut Mahulae, Syafri Fadhilah
Marpaung, Hambali)
383-392
6. Analisis Bentuk Manajemen Peserta Didik Di SMTK Rote Timur Kabupaten
Rote Ndao
(Yonatan Foeh)
393-402 5. DAFTAR ISI Analisis Strategi Guru Dalam Pelaksanaan Pengelolaan Kelas Oleh Guru Kelas V
SD Swasta Assisi Medan
(Antonius Remigius Abi, Lona Medita Lingga, Saut Mahulae, Syafri Fadhilah
Marpaung, Hambali)
383-392 6. Analisis Bentuk Manajemen Peserta Didik Di SMTK Rote Timur Kabupaten
Rote Ndao
(Yonatan Foeh)
393-402 6. Analisis Bentuk Manajemen Peserta Didik Di SMTK Rote Timur Kabupaten
Rote Ndao
(Yonatan Foeh)
393-402 7. Penerapan Strategi Predict, Organize, Rehearse, Practice And Evaluate (PORPE)
Untuk Meningkatkan Keterampilan Membaca Pemahaman Siswa Sekolah Dasar
(Mhd. Iqbal Maulana, Nurhaswinda, Rizki Amalia, Putri Hana Pebriana,
Fadhilaturrahmi)
403-414 8. Pengembangan Media Audio Visual Dalam Pembelajaran PPKn Dengan
Pendekatan Problem Based Learning Di Kelas VI Sekolah Dasar
(Devita Eka Rahmadani, Linda Zakiah, Adi Putra)
415-428 9. Penerapan Model Pembelajaran Questioning Untuk Meningkatkan Keterampilan
Membaca Pemahaman Siswa Sekolah Dasar
(Bagas Rianto, Putri Hana Pebriana, Nurhaswinda, Sumianto, Fadhilaturrahmi)
429-442 10. Urgensi Membangun Literasi Pada Anak Usia Dini
(Munawarah, Chairun Nisa Fadhilah, Reza Aulia, Nur Cahyati Ngaisah, Firman
Friyo Suhasto)
443-450 11. Manajemen Stres Kerja Dan Konflik Kerja: Pengaruhnya Terhadap Kinerja Guru
(Nikmatullaili, Nurhizrah Gistituati, Sufyarma Marsidin)
451-458 12. Konsep Manajemen Perguruan Tinggi Keagamaan Islam (PTKI)
(Ali Mustopa Yakub Simbolon, Ira Yanti, Weni Sumarni, M. Arif)
459-476 (Ali Mustopa Yakub Simbolon, Ira Yanti, Weni Sumarni, M. Arif)
459-476
13. Pengaruh Kepemimpinan Kepala Sekolah Dan Kinerja Guru Terhadap Mutu
Pendidikan Pada SMP Swasta Binaan Di Kupang
(Isak Ano Marthen Kolihar, Hendrik A.E.Lao, Yakobus Adi Saingo)
477-492
14. Pengaruh
Pemberian
Reinforcement
Dan
Self-Efficacy
Siswa
Dalam
Meningkatkan Prestasi Belajar Siswa
(Roberto Y. Liufeto, Hendrik A E.Lao, Umar Ali)
493-502
15. Analisis Kesalahan Leksikal Dan Sintaksis Dalam Menulis Teks Eksposisi Pada
Siswa Kelas X
(Hayatun Rahmi, S. Nofiana, Muhammad Iqbal)
503-516
16. Implementasi Kurikulum Merdeka Berbasis Literasi Pada Sekolah Penggerak Di
SD Gmit Airnona 1 Kota Kupang
(Yesli Ivana Seran, Hendrik A.E Lao, Umar Ali)
517-528
17. Pengaruh Pendekatan Realistic Mathematics Education (RME) Dengan Media
Dakon Pada Materi Perkalian Terhadap Hasil Belajar Peserta Didik
(Rizkina Maulisa, Linda Vitoria, Aida Fitri)
529-540
18. Analisis Keterampilan Berbicara Siswa Kelas V Pada Pembelajaran Bahasa
Indonesia SDN Karang Tengah 06
(Dini Utami, Boy Dorahman, Dilla Fadhillah)
541-552
19. Kajian Retorika Yang Berkembang Pada Masa Pandemi Covid-19 Di Indonesia
(Erfinawati, Ismawirna, Harunun Rasyid, Nisa Ayu Lestri, Eli Nurliza)
553-564
20. Penerapan Model Problem-Based Learning Dengan Pembelajaran Berdiferensiasi
Untuk Meningkatkan Kemampuan Berpikir Kritis Pada Pelajaran Ekonomi
(Mahmudah, Retno Dewi Mustika, Mochamad Sohibul Anhar)
565-580
21. DAFTAR ISI Analisis Kesalahan Penulisan Huruf Kapital Dan Penggunaan Tanda Baca Pada
Karangan Deskripsi
(Rezki Amelia Agustin, Dilla Fadhillah, Moh. Iqbal Firdaus)
631-636 26. Analisis Kesalahan Penulisan Huruf Kapital Dan Penggunaan Tanda Baca Pada
Karangan Deskripsi
(Rezki Amelia Agustin, Dilla Fadhillah, Moh. Iqbal Firdaus)
631-636
27. Strategi Kepemimpinan Kepala Sekolah Dalam Meningkatkan Motivasi Kerja
Guru
(Helsi Febrianti, Umy Nadrah Simatupang, Nurhizrah Gistituati)
637-644
28. Manajemen Pembiyaan Pendidikan Di Sekolah Dasar
(Arjunaini, Dahliawati, Yuni Revita, Hadiyanto, Yahya)
645-658
29. Analisis Nilai Sosiokultural Dalam Novel Laksamana Malahayati Sang
Perempuan Keumala Karya Endang Moerdopo
(Eli Nurliza, Erfinawati, Cut Nurul Fahmi, Faudi, Nursafiah, Ismawirna)
659-668
30. Hubungan Kegiatan Literasi Dasar Dengan Minat Baca Siswa Kelas V SD Negeri
53 Banda Aceh
(Noni Zahara, Maulidar, Indah Suryawati, Rifaatul Mahmuzah, Tri Putri Utami)
669-680
31. The Impact Of Religious Beliefs Among Acehnese EFL Pre-Service Teachers
(Rahmi)
681-692
32. Kebijakan Merdeka Belajar Sebagai Strategi Peningkatan Mutu Pendidikan
(Rizki Ananda, Wulandari Citra Wibisono, Anugrah Kisvanolla, Pris Ajeng
Purwita)
693-708
33. Analisis Kompetensi Guru Wali Kelas Terhadap Penggunaan Media Audio Visual
Pembelajaran SD
(Aisyah, Fitri Zuliana, Siti Aminah, Rizki Ananda)
709-718
34. Dynamic Equivalence: Translation Theory
(Lina Farsia, Sarair)
719-726
35. Analisis Tingkat Kemampuan Komunikasi Matematis Siswa
(Irvandi, Riki Musriandi, Rahmi, Irma Aryani, Anzora, Rini Susiani)
727–732
36. The Impact Of Native Speakerism On The Identity Construction Of ‘English
Teacher As An English Speaker’: Voices From Indonesia
(Ugahara, Suryani)
733-743
37. Strategi Guru PJOK Meningkatkan Minat Siswa Dalam Olahraga Di SMPN 18
Banda Aceh
(Syahrianursaifi, Zulheri Is, Safrizal, Musran, Erizal Kurniawan)
745-752
38. Peran Guru Dalam Meningkatkan Communication Skill Peserta Didik Abad 21
(Ammar Zaki1, Akhyar, Samsuar, Syarifah Farissi Hamama, Dwi Wahyu
Kartikasari, Ade Irfan)
753-760
39. Pemahaman Mahasiswa Terhadap MBKM: Pelaksanaan Dan Program MBKM
(Yulinar, Weniang Nugraheni, Agus Taufiq, Yusi Riksa Yustina, Silvi Puspa Widya
Lubis)
761-774
40. Identifying Factors Contributing To Students’ Obstacles In Understanding
Reading Descriptive Text
(Rahmayanti Rini Susiani Putri Dini Meutia Ferlya Elyza Ema Dauyah)
775-784 27. Strategi Kepemimpinan Kepala Sekolah Dalam Meningkatkan Motivasi Kerja
Guru
(Helsi Febrianti, Umy Nadrah Simatupang, Nurhizrah Gistituati)
637-644 36. The Impact Of Native Speakerism On The Identity Construction Of ‘English
Teacher As An English Speaker’: Voices From Indonesia
(Ugahara, Suryani)
733-743 37. Strategi Guru PJOK Meningkatkan Minat Siswa Dalam Olahraga Di SMPN 18
Banda Aceh
(Syahrianursaifi, Zulheri Is, Safrizal, Musran, Erizal Kurniawan)
745-752 38. DAFTAR ISI Penerimaan Berita Hoaks Melalui Media Sosial Sebagai Literasi Informasi
Dikalangan Remaja Di Kota Banda Aceh
(Furqan, Muhammad Syarif, Syukur Kholil)
581-592
22. Implementasi
Blended
Learning
Melalui
Aplikasi
Whatsapp
Dalam
Meningkatkan Listening Siswa Di SMA Negeri 2 Lhokseumawe
(Rahmati)
593-602
23. Kepraktisan
Model
E-STEM
PjBL
Dalam
Pembelajaran
IPA
Untuk
Meningkatkan Kemampuan Pemecahan Masalah Siswa SMP (Syarifah Rahmiza
Muzana, Silvi Puspa Widya Lubis, Hasanah, Rahmati, Wirda, Nurlaila)
603-610
24. Penerapan Model Pembelajaran Project-Based Learning (PjBL) Untuk
Meningkatkan Keaktifan Peserta Didik Pada Mata Pelajaran Ekonomi
(Nurul Farahdilla, Albrian Fiky Prakoso, Nurul Fahimah)
611–620
25. Etnomatematika Pada Kue Khas Aceh Sebagai Bahan Pembelajaran
Matematika 13. Pengaruh Kepemimpinan Kepala Sekolah Dan Kinerja Guru Terhadap Mutu
Pendidikan Pada SMP Swasta Binaan Di Kupang
(Isak Ano Marthen Kolihar, Hendrik A.E.Lao, Yakobus Adi Saingo)
477-492 14. Pengaruh
Pemberian
Reinforcement
Dan
Self-Efficacy
Siswa
Dalam
Meningkatkan Prestasi Belajar Siswa
(Roberto Y. Liufeto, Hendrik A E.Lao, Umar Ali)
493-502 15. Analisis Kesalahan Leksikal Dan Sintaksis Dalam Menulis Teks Eksposisi Pada
Siswa Kelas X
(Hayatun Rahmi, S. Nofiana, Muhammad Iqbal)
503-516 16. Implementasi Kurikulum Merdeka Berbasis Literasi Pada Sekolah Penggerak Di
SD Gmit Airnona 1 Kota Kupang
(Yesli Ivana Seran, Hendrik A.E Lao, Umar Ali)
517-528 17. Pengaruh Pendekatan Realistic Mathematics Education (RME) Dengan Media
Dakon Pada Materi Perkalian Terhadap Hasil Belajar Peserta Didik
(Rizkina Maulisa, Linda Vitoria, Aida Fitri)
529-540 17. Pengaruh Pendekatan Realistic Mathematics Education (RME) Dengan Media
Dakon Pada Materi Perkalian Terhadap Hasil Belajar Peserta Didik
(Rizkina Maulisa, Linda Vitoria, Aida Fitri)
529-540
18. Analisis Keterampilan Berbicara Siswa Kelas V Pada Pembelajaran Bahasa 21. Penerimaan Berita Hoaks Melalui Media Sosial Sebagai Literasi Informasi
Dikalangan Remaja Di Kota Banda Aceh
(Furqan, Muhammad Syarif, Syukur Kholil)
581-592 22. Implementasi
Blended
Learning
Melalui
Aplikasi
Whatsapp
Dalam
Meningkatkan Listening Siswa Di SMA Negeri 2 Lhokseumawe
(Rahmati)
593-602 23. Kepraktisan
Model
E-STEM
PjBL
Dalam
Pembelajaran
IPA
Untuk
Meningkatkan Kemampuan Pemecahan Masalah Siswa SMP (Syarifah Rahmiza
Muzana, Silvi Puspa Widya Lubis, Hasanah, Rahmati, Wirda, Nurlaila)
603-610 24. Penerapan Model Pembelajaran Project-Based Learning (PjBL) Untuk
Meningkatkan Keaktifan Peserta Didik Pada Mata Pelajaran Ekonomi
(Nurul Farahdilla, Albrian Fiky Prakoso, Nurul Fahimah)
611–620 25. Etnomatematika Pada Kue Khas Aceh Sebagai Bahan Pembelajaran
Matematika
(Asmaul Husna, Samsul Bahri, Rahmat )
621-630 26. Analisis Kesalahan Penulisan Huruf Kapital Dan Penggunaan Tanda Baca Pada
Karangan Deskripsi
(Rezki Amelia Agustin, Dilla Fadhillah, Moh. Iqbal Firdaus)
631-636 26. Kata kunci : PBL, Biologi, Keterampilan Pemecahan Masalah Kata kunci : PBL, Biologi, Keterampilan Pemecahan Masalah Kata kunci : PBL, Biologi, Keterampilan Pemecahan Masalah dalam menghadapi berbagai kemajuan zaman. Hal
ini selaras dengan pendapat Bilik (2021) pendidikan
dapat
membentuk
manusia
yang
mampu
membangun dirinya sendiri dan bangsanya, karena
itulah
perlu
dilakukan
peningkatan
mutu Putri Silmi Nurul Fadila1*, Fitri Arsih2, Ganda Hijrah Selaras3, Heffi Alberida4 Putri Silmi Nurul Fadila1*, Fitri Arsih2, Ganda Hijrah Selaras3, Heffi Alberida4
1,2,3,4 Program Studi Pendidikan Biologi, Departemen Biologi, FMIPA, Universitas Negeri Padang,
Padang, 25131, Indonesia. *E
il k
d
i
t i il i133@
il
1 *Email korespondensi : putrisilmi133@gmail.com1 *Email korespondensi : putrisilmi133@gmail.com1 Keywords : Problem Based Learning, Biology, Problem Solving Skills. Abstrak: Pembelajaran Biologi dipersiapkan untuk mendorong peserta didik agar mampu memecahkan
masalah dalam kehidupan sehari-hari. Untuk dapat mewujudkan hal tersebut, dibutuhkan model pembelajaran
yang dapat menkonstruksikan pengetahuan peserta didik sehingga mampu memecahkan masalah. Penelitian
ini bertujuan untuk melihat bagaimana pengimplementasian model pembelajaran Problem Based Learning
(PBL) sebagai upaya meningkatkan keterampilan pemecahan masalah peserta didik di SMA terutama pada
pembelajaran Biologi. Metode yang digunakan dalam penelitian ini adalah literature review. Sumber bacaan
yang digunakan dari artikel nasional dan artikel internasional yang relevan. Teknik analisis data dilakukan
secara deskriptif dengan menganalisis dan mengidentifikasi sumber bacaan sehingga mengahasilkan suatu
ringkasan yang padat, jelas, dan informatif. Hasil penelitian menunjukkan bahwa implementasi model
pembelajaran PBL dapat meningkatkan keterampilan pemecahan masalah peserta didik khususnya tentang
materi biologi Diterima Desember 2022; Disetujui Juli 2023; Dipublikasi 31 Juli 2023 Abstract: Biology learning is prepared to encourage students to be able to solve problems in everyday life. To
be able to realize this, a learning model is needed that can construct students' knowledge so that they are able
to solve problems. This study aims to see how to implement the Problem Based Learning (PBL) learning model
as an effort to improve students' problem solving skills in high school, especially in Biology learning. The
method used in this research is literature review. The reading sources used are from relevant national and
international articles. Data analysis techniques were carried out descriptively by analyzing and identifying
reading sources so as to produce a concise, clear and informative summary. The results showed that the
implementation of the PBL learning model could improve students' problem-solving skills, especially regarding
biology material. Keywords : Problem Based Learning, Biology, Problem Solving Skills. DAFTAR ISI Peran Guru Dalam Meningkatkan Communication Skill Peserta Didik Abad 21
(Ammar Zaki1, Akhyar, Samsuar, Syarifah Farissi Hamama, Dwi Wahyu
Kartikasari, Ade Irfan)
753-760 39. Pemahaman Mahasiswa Terhadap MBKM: Pelaksanaan Dan Program MBKM
(Yulinar, Weniang Nugraheni, Agus Taufiq, Yusi Riksa Yustina, Silvi Puspa Widya
Lubis)
761-774 40. Identifying Factors Contributing To Students’ Obstacles In Understanding
Reading Descriptive Text
(Rahmayanti, Rini Susiani, Putri Dini Meutia, Ferlya Elyza, Ema Dauyah)
775-784 41. Design Pembelajaran Online Berbasis Authentik Bagi Siswa Sekolah Dasar
(Abna Hidayati, Vevi Sunarti, Reza Gusmanti)
785-789
Jurnal Dedikasi Pendidikan, Vo. 7, No. 2, Juli 2023: 347-354 Learning (PBL) Sofyan, dkk (2017) menyatakan bahwa
sebenarnya sejarah PBL dimulai tahun 1920,
dimana waktu itu Celestine Freinet seorang guru SD
kembali ke kampung halamannya dibagian
Tenggara Perancis tepatnya setelah perang dunia-I. Beliau tidak sanggup bersuara lantang dan banyak
berbicara, sehingga beliau menggunakan metode
baru dan meninggalkan metode tradisional yang
dipakai saat itu. Beliau meminta peserta didiknya
belajar secara mandiri dan menemukenali sendiri, ia
hanya mengawasi dan memberikan fasilitas saja. Itulah awal pertama model Problem Based
Learning
(PBL)
diperkenalkan. Namun,
sebenarnya pada tahun 1916 John Dewey seorang
pengajar juga telah merancang metode yang tidak
jauh berbeda dengan problem based learning ini. PENDAHULUAN Pendidikan merupakan sesuatu yang harus
dikembangkan, karena pendidikan menjadi syarat
mutlak untuk meningkatkan kualitas manusia Pendidikan merupakan sesuatu yang harus
dikembangkan, karena pendidikan menjadi syarat
mutlak untuk meningkatkan kualitas manusia 347 Jurnal Dedikasi Pendidikan, Vol. 7, No. 2, Juli 2023 : 347-354
http://jurnal.abulyatama.ac.id/index.php/dedikasi secara umum pendidikan bertujuan menjadikan
peserta didik memperoleh pengalaman yang dapat
digunakan dalam memecahkan masalah dalam
kehidupan
baik
secara
individu
maupun
bermasyarakat. Oleh karena itu, mata pelajaran
Biologi dikembangkan agar peserta didik mampu
menyelesaikan masalah-masalah yang berkaitan
dengan kehidupan sehari-hari di lingkungannya. Dalam proses pembelajaran tentunya dibutuhkan
model pembelajaran untuk dapat mencapai tujuan
pembelajaran. Hal yang sama diungkapkan oleh
Pitaloka dan Slamet (2019) bahwa pembelajaran
biologi memiliki kaitan yang erat dengan makhluk
hidup dan segala peristiwa kehidupan manusia
sehingga dapat ditemui berbagai macam persoalan
yang dapat dikaji dalam pembelajaran. pendidikan. Salah satu upaya pemerintah untuk
merealisasikan hal tersebut adalah membuat
perubahan pada kurikulum yang digunakan. Di
Indonesia sendiri pendidikan diatur dalam undang-
undang No. 20 Tahun 2003 terkait sistem
pendidikan
Nasional
yang
menyebutkan
“kurikulum adalah seperangkat rencana pengaturan
mengenai tujuan, isi, dan bahan pelajaran serta cara
yang digunakan sebagai pedoman penyelenggaraan
kegiatan pembelajaran untuk mencapai tujuan
pembelajaran tertentu”. Sejalan dengan itu, menurut
Yulisman, dkk (2019) kurikulum memiliki
keterkaitan yang erat dengan landasan pendidikan,
yang mana landasan pendidikan ini bertujuan
membantu peserta didik dalam mengikuti setiap
proses pembelajaran yang memuat psikologi,
kultural,
filosofis,
sosiologi,
dan
etnografi. Kurikulum yang baik adalah kurikulum yang dapat
mengaplikasikan semua landasan pendidikan, serta
mampu mengarahkan peserta didik melakukan
pendekatan ilmiah yang berpusat pada diri peserta
didik itu sendiri (student center) untuk menemukan
konsep dan teori yang dipelajari, termasuk pada
mata pelajaran Biologi. Dalam proses pembelajaran tentunya dibutuhkan
model pembelajaran untuk dapat mencapai tujuan
pembelajaran. Hal yang sama diungkapkan oleh
Pitaloka dan Slamet (2019) bahwa pembelajaran
biologi memiliki kaitan yang erat dengan makhluk
hidup dan segala peristiwa kehidupan manusia
sehingga dapat ditemui berbagai macam persoalan
yang dapat dikaji dalam pembelajaran. Pembelajaran Biologi Pembelajaran Biologi dirancang dengan
tujuan memberikan peluang kepada peserta didik
untuk dapat menemukan fakta, konsep, dan nilai
baru melalui setiap proses. Menurut Bilik (2020)
materi dan konsep-konsep yang terdapat dalam
pembelajaran biologi memiliki hubungan yang erat
dengan fenomena dan gejala alam yang dapat
ditemukan
dalam
kehidupan
sehari-hari. Ditambahkan oleh Anwar dalam Haka (2021) Dalam pembelajaran Biologi sendiri, model ISSN 2548-8848 (Online) ISSN 2548-8848 (Online) 348 Jurnal Dedikasi Pendidikan, Vo. 7, No. 2, Juli 2023: 347-354 Problem Based Learing (PBL) dapat digunakan. Menurut Jamil (2021) model PBL dapat
mengembangkan keterampilan berpikir tingkat
tinggi peserta didik, seperti berpikir kritis, analitis,
dan mampu memecahkan masalah terhadap
persoalan-persoalan
yang
dihadapkan
dalam
kehidupan sehari-hari peserta didik. Sejalan dengan
pendapat Dewi dalam Adinia (2022) penggunaan
model pembelajaran berbasis masalah dapat
meningkatkan keterampilan berpikir tingkat tinggi
peserta didik sehingga membuat hasil belajar
peserta didik meningkat lebih baik. Selain itu,
model PBL dapat mendorong keterampilan lainnya
dalam diri peserta didik, seperti yang dinyatakan
Adinia (2022) model PBL yang digunakan dalam
proses pembelajaran dapat menumbuhkan jiwa
kreatif dan kolaboratif, meningkatkan pemahaman,
mengembangkan
kemandirian
belajar,
meningkatkan keterampilan memecahkan masalah
sehingga tercapainya tujuan pembelajaran. Maka
model
PBL
ini
dapat
digunakan
dalam
pembelajaran Biologi sebagai upaya dalam
meningkatkan berbagai keterampilan belajar yang
harus dimiliki peserta didik, termasuk keterampilan
pemecahan masalah tersebut. Keterampilan Pemecahan Masalah Keterampilan pemecahan masalah (problem
solving)
merupakan
keterampilan
dalam
mengimplementasikan proses berpikir individu
sehingga mampu memecahkan suatu masalah. Menurut Rahma, dkk (2020) Pemecahan masalah
adalah
pembelajaran
yang
dimulai dengan
menyajikan sebuah masalah yang mampu membuat
peserta didik mempelajari konsep sekaligus prinsip
untuk memecahkan masalah tersebut. Dimana
bentuk pembelajaran ini menciptakan jawaban
terhadap masalah (produk) serta bagaimana cara
memecahkan
permasalahan
(proses). Dalam
memecahkan suatu permasalahan dapat dilakukan
dengan
pengumpulan
fakta,
menganalisis
informasi, menyusun alternatif pemecahan, serta
memilih pemecahan masalah yang cocok dan
efektif. Sebagaimana yang dinyatakan Cheng
(2017) ada lima komponen utama dalam menyusun
perencanaan
pemecahan
masalah,
yaitu
mengidentifikasi konsep atau informasi yang
diperoleh, memunculkan strategi berupa langkah-
langkah terperinci untuk memperoleh solusi,
melakukan evaluasi terhadap solusi sendiri,
melakukan strategi tersebut, menunjukkan bukti
berupa penjelasan sesuai dengan hasil yang didapat
dari tindakan yang dilakukan. Model pembelajaran PBL merupakan salah
satu model pembelajaran yang didukung oleh teori
kontruktivisme, hal tersebut berkaitan erat dengan
proses pembelajaran yang membuat peserta didik
mampu memecahkan berbagai persoalan di
lingkungan belajar peserta didik itu sendiri, pada
abad-21
ini
disebut
dengan
keterampilan
pemecahan masalah (problem solving). Karmana,
dkk (2019) menyatakan bahwa keterampilan
pemecahan masalah menjadi bagian esensial yang
harus ditanamkan dalam diri setiap peserta didik. Menurut Lavoie dan Hall (dalam Rahmah,
2020) biologi sebagai bagian dari ruang lingkup
sains memiliki tujuan meningkatkan keterampilan
berpikir kritis, mampu merespons sesuatu secara
logis, dan meningkatkan keterampilan pemecahan
masalah. Karena hal tersebut, model pembelajaran
yang digunakan dalam proses pembelajaran harus
diperhatikan agar dapat menunjang terbentuknya
berbagai keterampilan abad-21 peserta didik Implementasi Model Pembelajaran Problem.... (Fadila, Arsih, Selaras, & Alberida, 2023) 349 Jurnal Dedikasi Pendidikan, Vol. 7, No. 2, Juli 2023 : 347-354
http://jurnal.abulyatama.ac.id/index.php/dedikasi Jurnal Dedikasi Pendidikan, Vol. 7, No. 2, Juli 2023 : 347-354
http://jurnal.abulyatama.ac.id/index.php/dedikasi terutama
keterampilan
pemecahan
masalah. Namun, yang sering terjadi di lapangan ialah
adanya ketidak sesuaian antara model pembelajaran
yang digunakan dengan perkembangan peserta
didik yang mengikuti alur kemajuan zaman. Dikuatkan oleh Saepudin (2018) hal tersebut
menyebabkan peserta didik beranggapan bahwa
pelajaran biologi hanya hafalan yang monoton,
sehingga dapat menurunkan semangat belajar
peserta didik. Oleh karena itu, perlu dilakukannya
penerapan model pembelajaran yang sesuai
khususnya pada pembelajaran Biologi. diperoleh beberapa artikel pendukung. diperoleh beberapa artikel pendukung. HASIL DAN PEMBAHASAN
PEMBAHASAN Berdasarkan penelitian Azizi (2019) yang
diperoleh dari hasil belajar dan tingkat keterampilan
pemecahan masalah peserta didik tentang materi
biologi dapat dilihat pada tabel berikut ini. Tabel 1. Data Hasil Kemampuan Pemecahan
Masalah Peserta Didik Kelas PBL dan Kelas
kontrol Tabel 1. Data Hasil Kemampuan Pemecahan
Masalah Peserta Didik Kelas PBL dan Kelas
kontrol
Kelas
Jumlah
peserta
didik
Nilai
terendah
Nilai
tertinggi
Nilai
rata-rata
PBL
Bermain
Peran (BP)
30
70
90
86,18
PBL
28
70
87
82,8 Dari permasalahan di atas maka peneliti
bermaksud ingin meneliti mengenai implementasi
model pembelajaran PBL pada materi biologi untuk
meningkatkan keterampilan pemecahan masalah
peserta didik. Dari tabel di atas dapat dilihat bahwa antara
model pembelajaran PBL bermain peran dengan
model pembelajaran PBL saja memiliki nilai rata-
rata dalam kemampuan memecahkan masalah tidak
jauh berbeda (hampir sama), yaitu selisih 2,83 nilai
saja. Meskipun nilai tersebut tidak signifikan
perbedaannya, kita dapat memahami bahwa rata-
rata nilai yang menggunakan model pembelajaran
PBL
dengan
bermain
peran
lebih
tinggi
dibandingkan kelas yang hanya menggunakan
model pembelajaran PBL. METODE PENELITIAN Metode yang digunakan dalam penelitian ini
yaitu literatur review. Menggunakan beberapa
sumber bacaan, seperti artikel nasional dan artikel
internasional. Sumber bacaan akan diolah dengan
tiga tahap yaitu 1) analisis deskriptif, yaitu
mengumpulkan dan mengaanalisis data; 2) analisis
isi, yakni memanfaatkan prosedur tertentu untuk
memperoleh kesimpulan; 3) analisis kritis, yaitu
mengkritik fakta yang ditemukan melalui studi
kepustakaan, serta menyikapi makna dari fenomena
secara ilmiah. Dari penelitian Pitaloka dan Slamet (2019)
serta Bilik (2020) diketahui bahwa model
pembelajaran PBL tentang materi biologi di sekolah
dapat meningkatkan keterampilan pemecahan
masalah peserta didik. Data yang diperoleh
menunjukkan bahwa nilai hasil posttest kelas
eksperimen yaitu kelas yang menerapkan model
PBL dengan bantuan audio visual mencapai rata-
rata 82 dan ini termasuk dalam ketegori tinggi. Sedangkan pada kelas kontrol atau kelas yang tidak Dalam mencari sumber bacaan, peneliti
menggunakan berbagai platform seperti Google
Cendekia dan Google Scholar, dengan kata kunci
“Implementasi model PBL pada pembelajaran
Biologi”. Peneliti juga mencari secara umum
mengenai “keterampilan pemecahan masalah” dan ISSN 2548-8848 (Online) ISSN 2548-8848 (Online) 350 Jurnal Dedikasi Pendidikan, Vo. 7, No. 2, Juli 2023: 347-354 pendapat Bilik (2020) masalah-masalah yang
disajikan oleh guru ataupun permasalahan yang
terjadi di lingkungan peserta didik dapat
memunculkan perilaku internalisasi dan retensi
konsep terhadap sumber belajar. Hal tersebut
memberikan umpan balik bagi peserta didik
sehingga ia merasa tertantang untuk belajar dan
menambah
usaha
dalam
menyelesaikan
permasalahan-permasalahan
yang
ia
temui,
sehingga peserta didik mendapatkan pengetahuan
yang lebih bermakna. Rizki dan Miza (2022)
menyatakan bahwa pada dasarnya model PBL
mendukung kegiatan mandiri, aktif, dan kritis,
sehingga dapat dijadikan dasar untuk kegiatan
pembelajaran selanjutnya. menerapkan model pembelajaran PBL diperoleh
nilai rata-rata 66 dan termasuk dalam kategori
rendah. Hal
tersebut
menunjukkan
bahwa
pengimplementasian model pembelajaran PBL
dapat meningkatkan kemampuan pemecahan
masalah bagi peserta didik khususnya tentang
materi-materi biologi. Karena pada hakikatnya
penerapan
model
PBL
ini
dalam
proses
pembelajaran
mendorong
peserta
didik
menganalisa
fakta-fakta
dan
memunculkan
keterampilan
pemecahan
masalah
terhadap
berbagai persoalan yang dihadapinya, sehingga
peserta
didik
nantinya
berani
dalam
mengemukakan
pendapatnya
berdasarkan
pengalaman. Dilanjutkan oleh penelitian Murdiyah, dkk
(2020) dan Mukharomah, dkk (2021) pembelajaran
berbasis masalah dengan teknik pemetaan konsep
terbukti memberikan pengaruh yang signifikan
terhadap pencapaian hasil belajar peserta didik. Dikuatkan oleh penelitian Haka dan Diana (2021)
diperoleh data adanya peningkatan kemampuan
pemecahan masalah peserta didik di Bandar
Lampung
dengan
menggunakan
model
pembelajaran PBL. METODE PENELITIAN Hal tersebut dapat dibuktikan
berdasarkan hasil uji hipotesis, yaitu menggunakan
uji One sample T test dengan perolehan nilai sig
0,000 dan pada T-hitung diperoleh nilai 41,990. Dari data tersebut secara langsung dapat menjawab
permasalahan yang telah ditentukan sebelumnya. Menurut Amalia, dkk (2017) dengan adanya
keterlibatan keterampilan pemecahan masalah
dalam proses pembelajaran, peserta didik akan
mengerahkan semua usaha untuk menemukan
jawaban atau solusi dari permasalahan. Dikuatkan
oleh Baysal (2017) dalam mengembangkan
keterampilan pemecahan masalah memberikan
sejumlah manfaat bagi peserta didik, yaitu: 1. Memunculkan keterampilan peserta didik
dalam
memecahkan
masalah
yang
ditemuinya. 2. Mengasah kemampuan peserta didik dalam
memecahkan masalah yang dihadapkan. 3. Meningkatkan keterampilan berpikir peserta
didik. Maka
pengimplementasian
model
pembelajaran PBL dapat melatih peserta didik
untuk berkembang dan mengeksplor masalah
dengan meningkatkan kesadaran cara berpikir dan
memberikan solusi yang dapat menyelesaikan atau
menjawab permasalahan tersebut. Implementasi Model Pembelajaran Problem.... (Fadila, Arsih, Selaras, & Alberida, 2023)
Salah satu hal yang perlu diperhatikan dalam
proses pembelajaran khususnya tentang materi
biologi
adalah
kemampuan
guru
dalam
memberikan kesempatan kepada semua peserta
didik untuk mereka mampu mengembangkan
keterampilan pemecahan masalah. Selaras dengan 351 Jurnal Dedikasi Pendidikan, Vol. 7, No. 2, Juli 2023 : 347-354
http://jurnal.abulyatama.ac.id/index.php/dedikasi Jurnal Dedikasi Pendidikan, Vol. 7, No. 2, Juli 2023 : 347-354
http://jurnal.abulyatama.ac.id/index.php/dedikasi Problem Based learning (PBL) dapat mengaitkan
antara teori dan praktek serta mampu meningkatkan
kompetensi peserta didik, seperti keterampilan
pemecahan masalah. Pada penelitian beliau juga
dapat membuktikan jika model pembelajaran PBL
memiliki pengaruh yang nyata terhadap pencapaian
nilai kognitif peserta didik, hal ini dapat dilihat dari
sebelum dan sesudah diberikan perlakuan oleh
peneliti. Dikuatkan oleh Sahyar dan Rika (2017)
model pembelajaran berbasis masalah dapat
memberikan efek baik kepada peserta didik, seperti
mendorong mereka lebih terlibat aktif dalam proses
pembelajaran atas informasi yang diterima. Sehingga dapat dipahami bahwa keterampilan
peserta didik dalam memecahkan masalah bukan
hanya saat di lapangan, tapi juga dapat menguasai
konsep untuk dikembangkan dengan tujuan
memecahkan masalah. Menurut pendapat Phungsuk, dkk (2017) PBL
merupakan cara yang efisien bagi peserta didik
untuk belajar keterampilan pemecahan masalah
dasar dan cara yang aktif untuk memperoleh
pengetahuan melalui interaksi dengan orang lain, ini
merupakan keterampilan utama yang dituntut oleh
hampir semua pekerjaan atau kegiatan di
lingkungan. Pandangan yang sama dari Khoiriyah
dan Husamah (2018) data dari hasil uji yang
dilakukan menunjukkan metode PBL dapat
meningkatkan pengetahuan siswa, termasuk dari
segi keterampilan pemecahan masalah peserta didik
tentang pembelajaran biologi. METODE PENELITIAN Ngongo dan Ismail
(2021) menyatakan bahwa peserta didik yang
terbiasa dihadapkan pada sebuah masalah dalam
proses pembelajaran, maka akan dapat membentuk
kondisi mental yang lebih baik. Oleh karena itu,
penerapan
model
pembelajaran
PBL
ini
mengintegrasikan
pendalaman
materi
atas
permasalahan yang muncul. Kesimpulan Peran guru sendiri tentunya tidak kalah
penting, guru sebagai fasilitator dituntut untuk
mampu meningkatkan kemampuan berpikir peserta
didik
sehingga
terbentuk
keahlian
dalam
memecahkan masalah tersebut. Sebagaimana yang
dikemukakan oleh Yusuf (2018) kemampuan guru
dalam memfasilitator peserta didik menjadi penentu
dari kualitas dan keberhasilan peserta didik. Selain
itu, guru harus dapat meyakinkan peserta didik
bahwa setiap permasalahan yang muncul, selalu ada
jalan penyelesaiannya serta dapat dipecahkan
bersama kelompoknya. Berdasarkan
hasil
penelitian
dapat
disimpulkan
bahwa
implementasi
model
pembelajaran
PBL
dapat
meningkatkan
kemampuan pemecahan masalah peserta didik,
khususnya tentang materi biologi. Dengan adanya
kebutuhan akan keterampilan pemecahan masalah,
membuat peserta didik mengerahkan segala
usahanya untuk menganalisis permasalahan yang
didapat serta mampu memberikan solusi terhadap
permasalahan tersebut. Hal itu dapat menjadikan
peserta didik berperan aktif bukan hanya saat
beraktivitas di lapangan, tapi juga mampu
menguasai konsep dengan tujuan memecahkan
masalah sehingga proses pembelajaran akan lebih
bermakna. Mukharomah, dkk (2021) meyakini bahwa
para peneliti mengakui model pembelajaran ISSN 2548-8848 (Online) ISSN 2548-8848 (Online) 352
Jurnal Dedikasi Pendidikan, Vo. 7, No. 2, Juli 2023: 347-354 Masalah pada Materi Sistem Reproduksi. Edulab: Majalah Ilmiah Laboratorium
Pendidikan, 6 (1), 15-28. Saran Adapun saran yang peneliti berikan adalah
model pembelajaran Problem Based Learning
(PBL) dapat diimplementasikan oleh guru dalam
kegiatan pembelajaran biologi sebagai upaya untuk
meningkatkan keterampilan peserta didik diabad-21
ini, khususnya keterampilan pemecahan masalah. Diharapkan peserta didik lebih berperan aktif dalam
menemukan solusi atas permasalahan yang
diberikan, sehingga didapatkan aktivitas belajar
lebih bermakna dan membuat hasil belajar peserta
didik lebih baik. Cheng, S-C., She, H-C., & Huang, L-Y. (2017). The
Impact
of
Problem-Solving
Instruction on Middle School Students’
Physical Science Learning: Interplays of
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Journal
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Mathematics, Science and Technology
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Problem
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Learning
Dengan
Metode
Scaffolding
Terhadap
Kemampuan Pemecahan Masalah Dan
Self Directed Learning Peserta Didik
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Pembelajaran
Aktif
Berbantuan LKPD Problem Solving
Terhadap
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Pemecahan
Masalah dan Penguasaan Konsep Biologi
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Kopi. Jurnal Inovasi Pembelajaran
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Efektivitas Kemampuan Berpikir Tingkat
Tinggi Pada Mata Pelajaran Biologi
Menggunakan Model Problem Based
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learning (PBL) in mathematics problem
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students. Internation Journal of Advance
Research and Innovative Ideas in
Education, 3(2), 3402–3406. Karmana, W, I., Ibrahim, M., & Susanti, E. (2019). Development
of
Karmana-
Problem Based Learning Modelto Train
Problem Solving Skills and Concept
Mastery of Biology Teacher Candidates. Journal of Physics. Conf. Series 1227. 012002. Azizi, As. (2019). Implementasi Problem Based
Learning (PBL) Dengan Bermain Peran
(BP)
Terhadap
Kemampuan
Memecahkan
Masalah. Jurnal
Pendidikan Mandala, 4(5), 188-194. Khoiriyah, A. J., & Husamah. (2018). Problem-
Based Learning: Creative Thinking
Skills,
ProblemSolving
Skills, And
Learning Outcome Of Seventh Grade
Students. Indonesia Journal of Biology
Education, 4(2), 151-160. Baysal, Z. N. (2017). The problem-based
learning
process:
Reflections
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Kartika,
A. (2021). Pengaruh
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Terhadap
Kemampuan Kognitif Mahasiswa Pada
Mata Kuliah Pengetahuan Lingkungan. Jurnal Biologi dan Pendidikan Biologi,
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Pemecahan Implementasi Model Pembelajaran Problem.... (Fadila, Arsih, Selaras, & Alberida, 2023) 353 Jurnal Dedikasi Pendidikan, Vol. 7, No. 2, Juli 2023 : 347-354
http://jurnal.abulyatama.ac.id/index.php/dedikasi Jurnal Dedikasi Pendidikan, Vol. 7, No. 2, Juli 2023 : 347-354
http://jurnal.abulyatama.ac.id/index.php/dedikasi Dalam Kurikulum 2013. Yogyakarta:
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H. (2023). Implementasi
Model Pembelajaran Problem Based
Learning (PBL) Tentang Materi Biologi
Untuk
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529 529 Saepudin, A. (2018). Analisis Keterampilan
Menjelaskan
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Jigsaw. Jurnal Edukasi Matematika dan
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Problem-Based Learning Model (PBL)
and Adversity Quotient (Q) on Problem-
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Educational Research, 5(2), 179-183. Sofyan, H.(2017). Problem Based Learning 354 ISSN 2548-8848 (Online)
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Damping of Low-Frequency Oscillations in Power Systems by Large-Scale PV Farms: A Comprehensive Review of Control Methods
|
IEEE access
| 2,021
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cc-by
| 15,352
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Damping of Low-Frequency Oscillations in
Power Systems by Large-Scale PV Farms:
A Comprehensive Review of Control Methods MAHDI SAADATMAND
1, GEVORK B. GHAREHPETIAN
2, (Senior Member, IEEE),
ALI MOGHASSEMI
3, JOSEP M. GUERRERO
4, (Fellow, IEEE),
PIERLUIGI SIANO
5, (Senior Member, IEEE), AND
HASSAN HAES ALHELOU
6, (Senior Member, IEEE)
1Department of Electrical Engineering, Science and Research Branch, Islamic Azad University, Tehran 14778-93855, Iran
2Department of Electrical Engineering, Amirkabir University of Technology, Tehran 15916-34311, Iran
3Department of Energy Technology, Aalborg University, 6700 Esbjerg, Denmark
4Center for Research on Microgrids (CROM), Department of Energy Technology, Aalborg University, 9220 Aalborg, Denmark
5Department of Management and Innovation Systems, University of Salerno, 84084 Fisciano, Italy
6School of Electrical and Electronic Engineering, University College Dublin, Dublin 4, D04 V1W8, Ireland Corresponding author: Hassan Haes Alhelou (alhelou@ieee.org) The work of Hassan Haes Alhelou was supported in part by the Science Foundation Ireland (SFI) through the SFI Strategic Partnership
Programme under Grant SFI/15/SPP/E3125, and in part by the University College Dublin (UCD) Energy Institute. ABSTRACT Global warming and the desire to increase the use of clean energy have led to increasing the
installation and operation of renewable energy power plants (REPPs), especially large-scale photovoltaic
(PV) farms (LPFs). Given that the LPFs are added to power system or replace conventional power plants,
they must be able to perform the basic tasks of synchronous generators (SGs). One of these tasks is the
ability to mitigate the low-frequency oscillation (LFO) risk. Also, one of the LPFs problems is reducing
the power system inertia and increasing the risk of LFOs. Therefore, these types of power plants must damp
the LFOs through a power oscillation damping controller (PODC), similar to the performance of power
system stabilizers (PSSs) in the SGs. This paper represents an overview of the different PODCs and control
methods for LFOs damping by LPF. It seems that it can be a driver for future studies. Different studies show
that the application of PODCs for LPFs can play an effective role to damp the LFOs and increase the power
system stability. INDEX TERMS Low-frequency oscillation (LFO), first generation generic model (FGGM), large-scale
PV farm (LPF), power oscillation damping controller (PODC), second generation generic model (SGGM),
small-signal stability (SSS). INDEX TERMS Low-frequency oscillation (LFO), first generation generic model (FGGM), large-scale
PV farm (LPF), power oscillation damping controller (PODC), second generation generic model (SGGM),
small-signal stability (SSS). Received April 24, 2021, accepted May 4, 2021, date of publication May 10, 2021, date of current version May 20, 2021. Received April 24, 2021, accepted May 4, 2021, date of publication May 10, 2021, date of current version May 20, 2021. Digital Object Identifier 10.1109/ACCESS.2021.3078570 Digital Object Identifier 10.1109/ACCESS.2021.3078570 I. INTRODUCTION Due to the challenge of global warming and increasing air
pollution in the world, in recent years, much attention has
been paid to the use of renewable energy resources. One
of the most important resources is solar energy [1]. Studies
have revealed that the earth’s surface receives approximately
1.8×1011 MW of power from solar radiation at each instant. This is much more than the total power consumption in
the world [2]. Also, studies have shown that the electrical
power demand of Europe, the North African region, and the
Mediterranean can be supplied by building solar facilities
in the Sahara Desert [3]. Figure 1 indicates the world solar
energy potential map. As shown in the figure, most of the
countries have a high potential for solar power generation [4]. Accordingly, there is a strong desire to install large-scale
photovoltaic (PV) farms (LPFs) (>100 MW), and their pen-
etration level is increasing every day [5]. It is estimated that
by 2030, the power generation capacity of LPFs worldwide
will be more than 3000 TWh [6]. Based on these two issues, different studies have been done
to introduce the control mechanisms to mitigate and damp
the LFOs by LPFs based on PODCs design [19]–[28]. The
primary aim of this paper is to investigate the LFOs damping
by LPFs in power systems and introduce the control mech-
anisms. To motivate the research on this important research
area, a complete overview on the power system stability is
introduced focusing on the rotor angle stability in which
the stability of LFOs is one of the important topics. The
mathematical representation of small-signal stability (SSS)
is then presented. This paper also introduces the LPF models
that can be used for LPF modeling in power systems dynamic
studies. Furthermore, the structures of used PODCs in LPF
to damp LFO are surveyed and discussed. Moreover, this
paper highlights the main challenges and research gaps to
motivate research on this area to improve the stability of
the power systems with the high penetration level of LPFs. Also, problems and challenges related to the design and
industrialization of the controllers have been investigated. The opportunities to improve the stability of the power system
based on control methods implemented based on wide-area
monitoring systems are also highlighted. LPFs have a different structure from conventional power
plants. Damping of Low-Frequency Oscillations in
Power Systems by Large-Scale PV Farms:
A Comprehensive Review of Control Methods NOMENCLATURE
AVR
Automatic voltage regulator
BESS
Battery energy storage system
DAE
Differential-algebraic equations
DFIG
Doubly-fed induction generator
EPRI
Electric Power Research Institute
FACTS
Flexible AC transmission systems
FGGM
First generation generic model
FFR
Fast frequency response
FOPID
Fractional-order PID
The associate editor coordinating the review of this manuscript and
approving it for publication was Derek Abbott
. GE
General Electric
GrHDP
Goal representation heuristic dynamic
programming
HVDC
High voltage direct current
HVRCM
High voltage reactive current management
ITAE
Integral of time-weighted absolute error
IBPP
Inverter-based power plant
LLC
Lead-lag compensator
LPF
Large-scale PV farm
LQG
Linear–quadratic–Gaussian
LQR
Linear-quadratic regulator
LSSM
Linearized small-signal model
LTI
Linear time-invariant
LTV
Linear time-varying
VOLUME 9, 2021
This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/
72183 72183 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs LFO
Low-frequency oscillation
LVACM
Low voltage active current management
MMAC
Multiple model adaptive control
NERC
North American Electric Reliability
Corporation
PCC
Point of common coupling
PSO
Particle swarm optimization
PSS
Power system stabilizer
PID
Proportional-integral-derivative
PV
Photovoltaic
PV1E
Electrical control model
PV1G
PV generator/ converter model
PODC
Power oscillation damping controller
REPP
Renewable energy power plant
REEC_B
Renewable energy electrical
control_version B
REGC_A
Renewable energy generator/
convertor_version A
REPC_A
Renewable energy plant
controller_version A
SG
Synchronous generator
SGGM
Second generation generic model
SSS
Small-signal stability
STATCOM
Static synchronous compensator
VSG
Virtual synchronous generator
WAMS
Wide-area measurement system
WECC
Western Electricity Coordinating Council TABLE 1. Comparison between LPFs and conventional power plants. TABLE 1. Comparison between LPFs and conventional power plants. the effects of LPFs on power system stability [9]–[18]. The
results of studies show that LPFs can strongly increase the
risk of low-frequency oscillations (LFOs) and power system
instability, by reducing the power system inertia. Some mechanisms indicating the indirect effect of the LPF
on LFOs are as follows [8]: • Replacing LPF instead of synchronous generator (SG). • Impact on synchronization forces due to the effect of
LPF on the main transmission lines. • Interaction between the LPF controls and damping
torques of large SGs. So, with increasing the penetration level of LPFs, two basic
issues are raised: • Considering that LPFs reduce the system inertia, so it
is necessary to create inertia through an additional
mechanism. Damping of Low-Frequency Oscillations in
Power Systems by Large-Scale PV Farms:
A Comprehensive Review of Control Methods • Due to the increasing penetration level of LPFs, they
must be able to do the basic tasks of SGs such as LFOs
mitigation by a power oscillation damping controller
(PODC). I. INTRODUCTION These types of power plants are based on the
inverter and they are classified as inverter-based power plants
(IBPPs) [7], [8]. LPF does not have rotating mechanical
components. Therefore, they do not have inherent inertia and
can reduce the power system inertia [8]. Table 1 shows a com-
parison between LPF and conventional power plants. Given
the different behavior of these types of power plants than con-
ventional power plants, many studies have been conducted on The rest of this paper is organized as follows. In Section 2,
a brief overview of the power system stability concept and the
mathematical basis of SSS are presented. The LPF dynamic
models for stability analysis are presented in Section 3. Then, 72184 72184 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 1. World solar energy potential map. FIGURE 1. World solar energy potential map. in Section 4 the basis of the LPF damping controller perfor-
mance is presented. Designed LPF damping controllers are
introduced and discussed in Section 5. Also, comparisons and
discussions of the types of PODCs are provided in Section 6. The research gaps and opportunities are stated in Section 7. Moreover, the challenges and research gaps are presented in
Section 8. Finally, the conclusions are given in Section 9. doubly-fed induction generator (DFIG) controls on elec-
trical resonance stability are expressed in the resonance
stability [34], [35]. As shown in Figure 3, rotor angle stability is catego-
rized into two various categories: transient stability and
SSS [36]–[40]. Transient stability is the power system’s
ability to keep the synchronism when it’s exposed to a
severe disturbance [29], [30]. The impact of small distur-
bances on the power system variables such as low varia-
tions in load and power generation [32], [36] is defined
in SSS studies [36], [37]. The SSS is classified into two
different classes. Oscillatory state due to lack of damping
torque and non-oscillatory state due to lack of synchronizing
torque [36], [37]. The damping torque is the component
of torque that is in phase with the speed deviation. Also,
the synchronizing torque is the component of the torque that
is in phase with the rotor angle deviation. II. POWER SYSTEM STABILITY The capability of the power system to keep the balance during
normal conditions and to restore equilibrium after distur-
bances is considered as power system stability [29]–[33]. Power system stability is categorized based on the system
response to a disturbance as shown in Figure 2. This classification can be expressed as follows: This classification can be expressed as follows: • Rotor angle stability is the ability of the SGs to
keep or restore the balance between electromagnetic
torque and mechanical torque. The problem of non-oscillatory states has been largely
solved using an Automatic Voltage Regulator (AVR) in the
excitation system of SG [36], [37]. Also, oscillatory states are
usually damped using PSS. In oscillatory states, oscillation
with a frequency between 0.1 Hz to 2 Hz is called LFO [36]. This oscillation can be divided into two general categories as
follows [36]: • Frequency stability is defined as the power system’s
capability to recover the equilibrium between system
generation and load demand. • Voltage stability refers to the power system’s capability
to keep the steady-state of all bus voltages under normal
conditions and after disturbances. • Inter-area oscillation with a frequency range of 0.1-
1.0 Hz. It should be noted that these expressions are the classic
classification of power system stability [30], [33]. Recently,
in [34], [35], the classic classification has been expanded,
and the following two new classes have been added to the
classification of power systems stability: • Local oscillation with a frequency range of 1.0-2.0 Hz. Inter-area oscillation is caused by the oscillation of a group
of generators or power plants in an area relative to genera-
tors or power plants in another area, while local oscillation
is caused by the oscillation of a generator or a power plant
relative to a generator or power plant in the same area [36]. • Converter- driven stability • Resonance stability Converter- driven stability involves dynamic interactions
between control systems of power electronic-based systems
and the power system devices [34], [35]. Also, the impact
of high voltage direct current (HVDC) and flexible AC
transmission systems (FACTS) on torsional and effect of VOLUME 9, 2021 A. CONCEPT OF SSS AND MATHEMATICAL BACKGROUND Power systems are non-linear dynamic systems that are
considered by a set of non-linear differential-algebraic 72185 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 2. Power system stability classification. FIGURE 3. Classification of the rotor angle stability. FIGURE 4. Schematic structure of the LPF. FIGURE 5. The simple aggregated model for PF. FIGURE 2. Power system stability classification. FIGURE 2. Power system stability classification. FIGURE 2. Power system stability classification. FIGURE 3. Classification of the rotor angle stability. FIGURE 4. Schematic structure of the LPF. FIGURE 3. Classification of the rotor angle stability. FIGURE 5. The simple aggregated model for PF. equations (DAE) [41], [42]. To power system analysis these
equations usually explained using state-space equations as
follows [28], [29]: Based on the first method of Lyapunov [43], [44], the linear
system in (3) and (4) is stable if and only if: |arg(eig(A))| > π
2
(6) ˙x = f (x, u) = Ax + Bu
(1)
y = g (x, u) = Cx + Du
(2) |arg(eig(A))| > π
2
(6) (1)
(2) (6) (2) where eig(A) indicates the eigenvalue of the system matrix. Therefore, by evaluating the eigenvalues, the power system
stability can be determined. It should be noted that in some
cases due to the use of some non-linear elements and devices
in controllers such as limiters and dead-bands, the power
system model is strongly non-linear and the values related
to the linear approximation do not give the correct results. In this case, the use of modal analysis is impossible and the
non-linear time-domain analysis is used. where eig(A) indicates the eigenvalue of the system matrix. Therefore, by evaluating the eigenvalues, the power system
stability can be determined. It should be noted that in some
cases due to the use of some non-linear elements and devices
in controllers such as limiters and dead-bands, the power
system model is strongly non-linear and the values related
to the linear approximation do not give the correct results. In this case, the use of modal analysis is impossible and the
non-linear time-domain analysis is used. where eig(A) indicates the eigenvalue of the system matrix. Th
f
b
l
i
h
i
l
h where x = [x1, . . . , xn]T is the vector of the state vari-
ables, y = [y1, . . . (3)
(4) where 1 remarks a small variation around the operating
point. Based on modal analysis, the power system stability
can be investigated by calculating the eigenvalues of state
matrix. It should be noted that, the eigenvalues are the roots
of the system characteristic equation det (sI −A) = 0, where
det is the determinant. Each eigenvalue can be expressed as
follows: where 1 remarks a small variation around the operating
point. Based on modal analysis, the power system stability
can be investigated by calculating the eigenvalues of state
matrix. It should be noted that, the eigenvalues are the roots
of the system characteristic equation det (sI −A) = 0, where
det is the determinant. Each eigenvalue can be expressed as
follows: A. CONCEPT OF SSS AND MATHEMATICAL BACKGROUND , yn]T is the vector of the system output,
u = [u1, . . . , un]T is the vector of the system input, A is
the state matrix, B is the input matrix, C is the output matrix
and, D is a matrix describing the direct connection between
input and output matrices. Also, f and g are the vectors of
non-linear functions. A popular method for SSS analysis is the modal analy-
sis or eigenvalues analysis. To use this method, it is necessary
to linearize the power system. Therefore, the power system
described by (1) and (2) is linearized around an operating
point [29], [30]. So, the power system can be stated as a
linear system: III. LPF MODELS FOR STABILITY ANALYSIS The LPF includes three basic parts, the PV array, inverter, and
the controller as shown in Figure 4. For power system stability
analysis, it is necessary to, the steady-state and dynamic mod-
els of LPF are available [5], [45]–[47]. So, it should describe
the LPF models for steady-state and dynamic analysis. 1˙x = A1x + B1u
(3)
1y = C1x + D1u
(4) (3)
(4) A. LPF MODEL FOR STEADY-STATE STUDIES To study the power system’s behavior in steady-state, the
steady-state models of all system devices must be defined. Given that renewable energy power plants (REPPs) are the
important components of modern power systems, so, having
a steady-state model of this type of power plant is essential. Accordingly, the LPF is modeled into a single generator
model for steady-state analysis. This model is called simple λi = σi ± jωi
(5) (5) VOLUME 9, 2021 72186 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 6. Overall structure of FGGM. FIGURE 6. Overall structure of FGGM. system [49], [56]. Since this was the first generation of the
LPF model, so the number 1 shows its generation. The PV1G
schematic is shown in Figure 7. When running this model,
the MVA rating of the LPF is equal to the total MVA rating
of the PVGs inside the PF. PV1G model is a simple display
of inverter protection and time-delay of the inverter control-
ling system. The 0.02-seconds time-delay provides a proper
approximation of control system delay [56]. aggregate model [5], [47]–[51]. The simple aggregated model
is shown in Figure 5. This model has a power rating equal to
the LPF power rating and connected to the point of common
coupling (PCC). Since these types of power plants have
reactive power generation/ absorption ability, the LPF, like
SG, is considered for steady-state analysis, i.e. its bus is a
PQ or PV bus [49], [50]. b: ELECTRICAL CONTROL MODEL (PV1E) The PV1E model sends the active and reactive currents com-
mand to the PV1G model. The schematic of this controller is
depicted in Figure 8 [49], [57]. 1) FGGM FOR LPF MODELING This model is actually an upgrade of the FGGM that
has a central control module. The SGGM is WECC
approved [48], [51], [61]. The schematic structure of SGGM
is depicted in Figure 9 [48]. This model includes three mod-
ules, inverter protection module named renewable energy
generator/convertor_version A (REGC_A), an electrical con-
troller module for local power control named renewable This subsection discusses the structure and functionalities
of FGGM. This model has two components as shown
in Figure 6 [49], [57]. B. LPF MODELS FOR DYNAMIC STUDIES The inverter of LPF is the current controlled device and
its performance is very dependent on current thermal lim-
its [56]. For this purpose, the high voltage reactive current
management (HVRCM) module is used to detect the injected
reactive current [48], [49]. If the terminal voltage of the
inverter is increased from the set Volim value, the HVRCM
module limits the increase in the reactive current injection by
decreasing the terminal voltage. Also, the low voltage active
current management (LVACM) module limits the increase in
the active current during the low voltage events, based on the
current limitations [59]–[63]. From the beginning of the 21st century, with the increasing
desire to install and operate LPFs, operators and owners of
LPFs needed to model the LPF to evaluate its performance in
different operating conditions [52]–[55]. Until then, a generic
and standard model was not available; therefore, they used
the user-written model files in many software tools such as
GE PSLF and Siemens PSSE [56]. For the first in 2010,
General Electric (GE) introduced a generic standard model
called the First Generation Generic Model (FGGM) [49]. Also, in a study in 2011 [57], the FGGM was examined. In 2012, Western Electricity Coordinating Council (WECC)
published a guide for the LPF dynamic model [58]. This
guide is considered to serve for the LPF’s model to be imple-
mented for power system analysis and simulations. Later this
model was named the Second Generation Generic Model
(SGGM). The SGGM is currently being developed in col-
laboration with WECC and Electric Power Research Institute
(EPRI) [48], [50]. It should be noted that this model can
only be used for the positive sequence in the steady-state
analysis [48], [59]. In the continuation of this section, these
two models are investigated. In other words, the HVRCM and LVACM modules are
considered for the thermal protection modeling of the
power switches (IGBT and diode). This type of protection
is based on the current-carrying capability of the power
switches [62]. FIGURE 8. PV1E model block diagram. A- Local active power control: This is a control loop that
provides the active current command as a command
signal to the REGC_A module. Note that the command
signal is subject to the thermal limitations of the power
electronic switches, as well as the priority between
active and reactive currents [48], [61]. energy electrical control_version B (REEC_B), and a central
control module for power control at plant-level named renew-
able energy plant controller_version A (REPC_A) [48], [61]. a: PV GENERATOR/ CONVERTER MODEL (PV1G) This model is equivalent to the LPF converters and
plays the interface role between the LPF and the power 72187 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 7. PV1G model block diagram. FIGURE 8. PV1E model block diagram. FIGURE 7. PV1G model block diagram. FIGURE 7. PV1G model block diagram. FIGURE 7. PV1G model block diagram. FIGURE 8. PV1E model block diagram. a: REGC_A MODULE The REGC_A is similar to the PV1G model [48], [61]. It combines a high bandwidth current regulator that injects the
command signals (Iq, Ip) into the inverter model in response
to command signals (Iqcmd, Ipcmd) from REEC_ B. This
module is depicted in Figure 10 [48], [61]. B- Local reactive power control: This is a control loop
that provides the reactive current command to the
REGC_A model. The command signal is subject to
current limiting. The following control states are con-
sidered [48], [61]: b: REEC_B MODULE
The REEC_B module includes two separate control loops. A local active power control loop and a local reactive power
control loop [48], [61]. c: REPC_A MODULE Due to the non-linearity of these models, they have exten-
sive and complex relationships. A more complete explanation
of these models is available in [62] and [63]. Typical values
of the parameters of these models and internal variables are
listed in the appendix [61]. The REPC_A model demonstrates the central controller
model behavior [48], [51], [61]. This model is optional
because not all LPFs are constructed with the central con-
troller. The REPC_A model provides the plant-level control
commands to the REEC_B. The schematic of this model is
depicted in Figure 12 [48], [51], [61]. This model transmits
the command signals to the inverters controllers. A general comparison of the capabilities of these models
is listed in Table 2. This model includes the features as below: This model includes the features as below: This model includes the features as below: b: REEC_B MODULE b: REEC_B MODULE
The REEC_B module includes two separate control loops. A local active power control loop and a local reactive power
control loop [48], [61]. • Constant power factor state, based on the inverter
power factor in steady-state. • Constant reactive power state, based either on
the
inverter
absolute
reactive
power
in
the VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 9. Overall structure of SGGM. FIGURE 10. Schematic structure of REGC_A model. FIGURE 9. Overall structure of SGGM. FIGURE 9. Overall structure of SGGM. FIGURE 10. Schematic structure of REGC_A model. • Provides governor response at plant-level based on the
frequency deviation of a remote bus. • Provides governor response at plant-level based on the
frequency deviation of a remote bus. steady-state or reactive power from the central
controller. It should be noted that, with different flags in the SGGM,
the PF can have many control strategies for various operating
conditions. The flag setting and input parameter settings for
the different strategies to control the active and reactive power
are described in [48], [61]. As shown in Figure 11, there are several flags in this
module that are used to determine different control strategies
in the local control mode [48], [61]. IV. THE BASIS OF THE LPF DAMPING CONTROLLER
PERFORMANCE • Regulation of remote bus voltage through the voltage
control loop. This is done by compensating for the line
drop. The LFO of the power system occurs mainly due to the
lack of equilibrium between electrical torque and mechanical
torque [29]–[33]. In this section, to show the effect of the • Regulation of the reactive power of the selected branch. 72189 72189 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 11. Schematic structure of REEC_B model. FIGURE 12. Schematic structure of REPC_A model. LPF based PODC for LFO damping, inter-area oscillation is
considered as LFO in a simple two-area system [64], [65]. For
his purpose, any area has been considered as an equivalent
SG. As shown in Figure 13 both areas are connected through a
transmission line. Also, each area contains a local load. In this
system, LPF is integrated with area 1. FIGURE 11. Schematic structure of REEC_B model. FIGURE 11. Schematic structure of REEC_B model. FIGURE 12. Schematic structure of REPC_A model. LPF based PODC for LFO damping, inter-area oscillation is
considered as LFO in a simple two-area system [64], [65]. For
this purpose, any area has been considered as an equivalent
SG. As shown in Figure 13 both areas are connected through a
transmission line. Also, each area contains a local load. In this
system, LPF is integrated with area 1. 72190
VOLUME 9 2021 FIGURE 12. Schematic structure of REPC_A model. SG. As shown in Figure 13 both areas are connected through a
transmission line. Also, each area contains a local load. In this
system, LPF is integrated with area 1. LPF based PODC for LFO damping, inter-area oscillation is
considered as LFO in a simple two-area system [64], [65]. For
this purpose, any area has been considered as an equivalent LPF based PODC for LFO damping, inter-area oscillation is
considered as LFO in a simple two-area system [64], [65]. For
this purpose, any area has been considered as an equivalent 72190 VOLUME 9, 2021 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs TABLE 2. General comparison between FGGM and SGGM. FIGURE 13. Simple structure of a two-area test system. TABLE 2. General comparison between FGGM and SGGM. TABLE 2. General comparison between FGGM and SGGM. synchronously in the two areas. IV. THE BASIS OF THE LPF DAMPING CONTROLLER
PERFORMANCE In this condition the gen-
erators rotor angle difference between the two areas δ12 is
constant, and the generators speed difference between the
two areas ω12 is equal to zero. However, when a distur-
bance occurs, the equilibrium between electrical power and
mechanical power of generators is lost, which may lead to
the inter-area oscillation between the two areas. Therefore,
to maintain the SSS, it is necessary to damp the LFOs quickly. As shown in (10), the LPFs can compensate for the reactive
power. Therefore, these types of power plants can control the
bus voltage. Therefore, LFOs can be damped by controlling
the bus voltage. It is done by injecting additional reactive
power to the grid in disturbances conditions. For this purpose,
an auxiliary controller can be used as a PODC, such as the
PSS operation in the SG excitation system. FIGURE 13. Simple structure of a two-area test system. V. LPF DAMPING CONTROLLERS As shown in the previous section, the use of an auxiliary
controller can be a good solution for LFO damping by LPFs. Many studies have been done on the effect of LPFs on
the power systems stability, but little study has been done
on the PODC design and LFO damping by LPFs. It seems
that in the future more advanced types of controllers will be
introduced as PODC. In the continuation of this section, the
introduced controllers are reviewed. FIGURE 13. Simple structure of a two-area test system. The dynamic performance without LPF can be explained
using the swing equation as follows [64], [65]: A. LEAD-LAG COMPENSATOR (LLC) The simplest and most common type of PODC is the LLC. These types of controllers are used as a common structure
of current PSSs. The conventional and popular type of these
controllers is the 2nd order single-input LLC, which its use is
common in the industry due to its simple structure and easy
tuning [67]–[70]. The control block diagram of this type of
controller is depicted in Figure 14 [67]–[69]. dδ12
dt
= ω12
(7)
dω12
dt
= 1
H1
(Pm1 −PL1) −1
H2
(Pm2 −PL2)
−
1
H1
+ 1
H2
V1V2
X
sin δ12
(8) (7) (8) where δ12 = (δ1 −δ2) and ω12 = (ω1 −ω2) represent the
generators rotor angle difference between the two areas and
generators speed difference between the two areas, respec-
tively. When the LPF is connected to area 1, (8) can be
considered as follows: FIGURE 14. The 2nd order single-input LLC. dω12
dt
= 1
H1
(Pm1 + PPV −PL1) −1
H2
(Pm2 −PL2)
−
1
H1
+ 1
H2
V1V2
X
sin δ12
(9) FIGURE 14. The 2nd order single-input LLC. where K is the gain of controller, Tw is the time constant of
washout filter, and T1, T2, T3, and T4 are the time constants. (9) In a study in 2017 [19], an LLC was proposed as PODC
for LPF. In the study, an adaptive PODC based on goal rep-
resentation heuristic dynamic programming (GrHDP) algo-
rithm was proposed. By GrHDP, the adaptive PODC does not
require a power system model and is compatible with various
operating conditions. Moreover, an adaptive delay compen-
sator is also employed for the proposed PODC to compensate
for the communication delay existing in the wide-area mea-
surement system (WAMS). The simulation results showed
that the proposed PODC can provide satisfactory damping
performance and compensate for the communication delay. In a study in 2017 [19], an LLC was proposed as PODC
for LPF. In the study, an adaptive PODC based on goal rep-
resentation heuristic dynamic programming (GrHDP) algo-
rithm was proposed. By GrHDP, the adaptive PODC does not
require a power system model and is compatible with various
operating conditions. Moreover, an adaptive delay compen-
sator is also employed for the proposed PODC to compensate
for the communication delay existing in the wide-area mea-
surement system (WAMS). A. LEAD-LAG COMPENSATOR (LLC) The simulation results showed
that the proposed PODC can provide satisfactory damping
performance and compensate for the communication delay. As shown in the third part of (9), the transmitted active
power from area 1 to area 2 is related to the angle difference
between the two areas. Also, the transmitted reactive power
is related to the voltage magnitude, as follows [65]–[67]: Qt = Qg1 −QL1 + QPV = V 2
2 −V1V2 cos δ12
X
(10) (10) where Qt is the transmitted reactive power from area 1 to
area 2, QPV is the reactive power injected from LPF to the
grid, and QL1 is the reactive power consumption by loads
of area 1. In the steady-state condition, the SGs operate 72191 VOLUME 9, 2021 VOLUME 9, 2021 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 15. FFR controller and LLC scheme in REPC_A module. FIGURE 15. FFR controller and LLC scheme in REPC_A module. Another study in 2019 [20] proposed a new fast fre-
quency response (FFR) and LFOs damping control by LPFs
controlled as static synchronous compensator (STATCOM),
termed PV-STATCOM, for simultaneously enhance fre-
quency regulation and SSS of power systems. As shown
in Figure 15 the study used the SGGM for LPF modeling. Moreover, an LLC based PODC proposed for LFOs damp-
ing in the voltage control loop of REPC_A. Also, a FFR
controller has been suggested for frequency control in the
active power control loop of REPC_A. Then, the LLC has
been tuned using a residue-based method [37]. The simu-
lation results showed the proper performance of the pro-
posed composite control to compensating for the frequency
deviation, damping the LFOs, and voltage regulation during
disturbances. Accordingly, in 2019 [22] a probabilistic method has been
proposed for PODC tuning under stochastic time delay and
under other power system uncertainties arising due to REPPs
and loads. In the study, the LLC has been proposed as a PODC
in LPF model. Also, the mitigation strategy has been used for
the objective function definition. Moreover, the optimization
method has been used for PODC design. The results showed
that tuning the PODC using the proposed method greatly
improves the SSS under various operating conditions. Also,
the tuned PODC is robust against time delay uncertainty and
other power system uncertainties. B. PROPORTIONAL-INTEGRAL-DERIVATIVE (PID)
CONTROLLER Schematic structure of MMAC strategy. Recently in 2020 [23], a general technique to damp the
LFOs by LPFs has been proposed. In the study, the opti-
mal PID controller was used as a PODC. For this purpose,
the PODC was optimally tuned by the particle swarm opti-
mization (PSO) algorithm [70] [78] [81] Finally the perfor
FIGURE 19. The
FIGURE 20. FOPI
(a) integer-order,
of operating c
compared wit
C. LINEAR-Q
The performa
minimization
state’s deviat
terms [83], [8
is to address
to minimize
effort at the m
nation of a lin
filter [84]. Th
time-invariant
(LTV) system
linear feedbac
As shown e
by (1) and (2)
process and s
can be written
where w and
respectively. it is necessary
the objective f M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 19. The FOPID controller structure. FIGURE 20. FOPID and PID controllers, from points to plane,
(a) integer-order, and (b) fractional-order. FIGURE 16. PID controller structure. FIGURE 19. The FOPID controller structure. FIGURE 20. FOPID and PID controllers, from points to plane,
(a) integer-order, and (b) fractional-order. FIGURE 16
PID
t
ll
t
t
FIGURE 19. The FOPID controller structure. FIGURE 19. The FOPID controller structure. FIGURE 19. The FOPID controller structure. FIGURE 19. The FOPID controller structure. FIGURE 16. PID controller structure. FIGURE 16. PID controller structure. FIGURE 17. LQG controller structure. FIGURE 20. FOPID and PID controllers, from points to plane,
(a) integer-order, and (b) fractional-order. of operating conditions. Also, in the study, the results were
compared with the performance of LLCs. FIGURE 17. LQG controller structure. FIGURE 17. LQG controller structure. FIGURE 18. Schematic structure of MMAC strategy. B. PROPORTIONAL-INTEGRAL-DERIVATIVE (PID)
CONTROLLER Recently, various types of controllers have been introduced in
power system applications. Among them the PID controller
is known as a simple and efficient controller [71]–[73]. This
type of controller has been widely used in industries because
of its simple structure and robust performance in different
operating conditions. The simple design and simple struc-
ture of the PID controller have led to its widespread use in
industries to improve dynamic response and reduce steady-
state error [74]–[78]. Its transfer function is in the form of: The proposed inverter control made effective utilization
of the PV inverter capacity and available solar power. Also,
it was shown to be superior to the conventional droop control
recommended by North American Electric Reliability Corpo-
ration (NERC) for generating plants. In another study in 2019 [21], the LFO damping was pro-
posed by an optimal LLC based PODC. The proposed PODC
structure was a single-input 2nd order LLC. In the study,
the SGGM was used for LPF modeling. Moreover, the PSO
algorithm has been used to determine the values of LLC
parameters. In fact, PODC was optimally designed. Then the
robustness of PODC was assessed in the different operating
and loading conditions. The simulation results demonstrated
the proper performance of the proposed PODC for the wide
range of operating conditions. Y (t) = KPR (t) + (KI
Z t
0
R (x) dx) + KD
dR (t)
dt
(11) (11) where KP, KI, and KD represent the proportional, integral,
and derivative gains, respectively [71]–[78]. The PID transfer
function in the Laplace domain is as follows: The communication delay that occurs inherently in the
WAMS negatively affects the SSS. This issue is stochastic
in nature and needs to be considered as one of the system
uncertainties in smart grids and future systems. Therefore,
this issue needs to be considered in PODC design. H (S) = Y (S)
R(S) = KP + KI
S + +KDS
(12) (12) where S is the complex frequency. The schematic block
diagram of this type of controller is shown in Figure 16. 72192 72192 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs
FIGURE 16. PID controller structure. FIGURE 17. LQG controller structure. FIGURE 18. Schematic structure of MMAC strategy. M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs
FIGURE 16. PID controller structure. FIGURE 17. LQG controller structure. FIGURE 18. C. LINEAR-QUADRATIC-GAUSSIAN (LQG) CONTROLLER FIGURE 18. Schematic structure of MMAC strategy. C. LINEAR-QUADRATIC-GAUSSIAN (LQG) CONTROLLER
The performance of the LQG controller is based on the
minimization of an objective function that penalizes the
state’s deviations and actuator’s actions during transient
terms [83], [84]. The basic idea of the LQG controller design
is to address the intrinsic compromise between an attempt
to minimize the error and an attempt to maintain control
effort at the minimum. This type of controller is the combi-
nation of a linear-quadratic regulator (LQR) with a Kalman
filter [84]. The LQG controllers can be applied to both linear
time-invariant (LTI) systems as well as linear time-varying
(LTV) systems [85]. Therefore, it is possible to design the
linear feedback controllers for non-linear uncertain systems. As shown earlier, the general state-space equations explain
by (1) and (2). By ignoring the D matrix and considering the
process and sensor noise inputs for a plant, these equations
can be written as follows [24], [25]: FIGURE 18. Schematic structure of MMAC strategy. ˙x = Ax + Bu + 0w
(13)
y = Cx + v
(14) (13)
(14) Recently in 2020 [23], a general technique to damp the
LFOs by LPFs has been proposed. In the study, the opti-
mal PID controller was used as a PODC. For this purpose,
the PODC was optimally tuned by the particle swarm opti-
mization (PSO) algorithm [70], [78]-[81]. Finally, the perfor-
mance of the proposed PODC was examined in a two-area
benchmark system [82]. The results of the study showed the
proper performance of the proposed PODC in the wide range where w and v are the process and sensor noise inputs,
respectively. To determine the LQG controller parameters
it is necessary to obtain an optimal control that minimizes
the objective function. The objective function is expressed as
bellow [24]: J = lim
T→∞
1
T E
Z T
0
xT Qx + uT Ru
dτ
(15) (15) 72193 72193 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 21. Structure of REPC_B module with FOPID controller. FIGURE 22. Schematic structure of two-area test system. where Q and R are weighting matrices such that QT = Q and
RT = R. C. LINEAR-QUADRATIC-GAUSSIAN (LQG) CONTROLLER By the separation principle, LQG can be divided
nto two following sub-problems:
• The LQR Problem or determine the optimal state-
f
db
k
t
l Thi i
i
i
b [20] [83] [85]
• LQE Problem or the required state’s estimation
Measuring all the states is impossible practically, thus,
a Kalman filter is employed to provide the required estimates
as an estimator. The Kalman filter structure is that of an
di
t t
ti
t
ith [24] FIGURE 21. Structure of REPC_B module with FOPID controller. FIGURE 21. Structure of REPC_B module with FOPID controller. FIGURE 21. Structure of REPC_B module with FOPID controller. FIGURE 21. Structure of REPC_B module with FOPID controller. FIGURE 22. Schematic structure of two-area test system. FIGURE 22. Schematic structure of two-area test system. • LQE Problem or the required state’s estimation • LQE Problem or the required state’s estimation The structure of the LQG controller is depicted in Figure 17
[83]–[85]. In 2013 [24], a minimax LQG-based controller was pro-
posed for use in LPFs as PODC. For this purpose, the FGGM
was used as an LPF dynamic model and, the two-area bench-
mark system was used for power system simulation. Then
the performance of the proposed PODC was evaluated con-
sidering feedback signal transmission delay. The simulation
results demonstrated that the proposed controller for LPF
provides sufficient damping to the LFOs for a wide range
of operating conditions and disturbances. This issue also has
been investigated in [25]. Using these residuals, the probability of each model rep-
resenting the actual system response is computed. Based on
the probabilities, proper weights are assigned to individual
control moves such that the less probable models carry less
weight [86]. This ensures that the controllers designed for less
probable models influence the final control move to a lesser
extent [86]. At each level of the recursive algorithm, primarily
two tasks are performed, i.e., calculation of probability using
a Bayesian approach and assigning suitable weights based on
the value of probability [86]. VOLUME 9, 2021 • LQE Problem or the required state’s estimation where Q and R are weighting matrices such that QT = Q and
RT = R. By the separation principle, LQG can be divided
into two following sub-problems: Measuring all the states is impossible practically, thus,
a Kalman filter is employed to provide the required estimates
as an estimator. The Kalman filter structure is that of an
ordinary state-estimator with [24]: • The LQR Problem or determine the optimal state-
feedback control. This issue is given by [20], [83]–[85]: ˙ˆx = Aˆx + Bu + Kf (y −C ˆx)
(19)
Kf = Pf CT V −1
(20) u = −KCx
(16)
KC = R−1BT PC
(17) (19)
(20) (16) (19) (17) (20) where PC is a symmetric positive semi-definite solution of
the Riccati equation, as follows: where Kf is the Kalman filter and Pf is a symmetric positive
semi-definite solution of the Riccati equation: where Kf is the Kalman filter and Pf is a symmetric positive
semi-definite solution of the Riccati equation: Pf AT + APf + 0w0T −Pf CT V −1CPf = 0
(21) AT PC + PCA + Q −PCR−1BT PC = 0 (18) (21) 72194 72194 VOLUME 9, 2021 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 23. Rotor angle of generator G1; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. FIGURE 23. Rotor angle of generator G1; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. Finally, the optimal control formula of LQG becomes: of the system after an event. Note that, each one of the LSSMs
must be located in the model bank. u = −K C ˆx
(22) (22) A general overview of the conventional MMAC strat-
egy is depicted in Figure 18 [86]. The recursive algorithm
uses a bank of linearized plant models such as LSSMs
in [86], to capture the possible system dynamics following
an event [86]. One separate controller is designed, a priori,
based on each model from the model bank. At each simulation
phase, the actual response is compared with the response of
the linearized models which are driven by the same control
input [87]. The differences in the response of each model
concerning the actual system response are used to generate
individual model residuals. The structure of the LQG controller is depicted in Figure 17
[83]–[85]. D. MULTIPLE MODEL ADAPTIVE CONTROL (MMAC)
STRATEGIES For a power system, different scenarios can be considered
for post-event conditions. Events include a severe fault in the
grid, the sudden outage of a big load, generator, or tie-line,
and etc. Then, based on each event, a linearized small-signal
model (LSSM) can be considered for the system status after
the event. In 2017 [26], the MMAC has been applied as a con-
trol strategy to mitigate the LFOs by LPFs. In the study,
the K-means clustering algorithm was used taking inter-area In a study in 2004 [86], a total of 12 LSSMs have been
considered to cover the whole space of anticipated response 72195 72195 VOLUME 9, 2021 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs p
g
y
y
FIGURE 24. Voltage magnitude at PCC; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. FIGURE 25. PCC Frequency; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. modes as features for operating condition clustering and,
a common damper was designed for each cluster to reduce the
scales of the model bank and the damper bank. Based on the
deviation between the output dynamic responses of the actua
system and models, the Bayesian approach was employed
to calculate the probability of each model representing the
72196
VOLUME 9 202 FIGURE 24. Voltage magnitude at PCC; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. FIGURE 24. Voltage magnitude at PCC; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. FIGURE 24. Voltage magnitude at PCC; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. FIGURE 25. PCC Frequency; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. FIGURE 25. PCC Frequency; (A) scenario I, (B) scenario II, (C) scenario III, and (D) scenario IV. deviation between the output dynamic responses of the actual
system and models, the Bayesian approach was employed
to calculate the probability of each model representing the deviation between the output dynamic responses of the actual
system and models, the Bayesian approach was employed
to calculate the probability of each model representing the modes as features for operating condition clustering and,
a common damper was designed for each cluster to reduce the
scales of the model bank and the damper bank. VI. SIMULATION RESULS AND COMPARISON Given that SSS analysis and simulation of LFOs are required,
a standard power system should be used for this purpose. Given that SSS analysis and simulation of LFOs are required,
a standard power system should be used for this purpose. There are several benchmark test systems for studying the
LFOs, the most common of which is the two-area test sys-
tem [82]. This system has also been used in most literature
studies. A smart two-area system has been used in this study
as a case study. The specifications of this system are shown
in Figure 22 and Table 3 [30]. Y (t) = KPR (t) + KID−λ
t
R (t) + KDDδ
t R (t)
(23) (23) TABLE 3. Test system specifications. TABLE 3. Test system specifications. Based on (23), the transfer function, H(s), in Laplace-
domain is as follows [91], [93]: Based on (23), the transfer function, H(s), in Laplace-
domain is as follows [91], [93]: H (s) = Y (s)
R(s) = KP + KIs−λ + KDsδ
(24) (24) where R(s) is the input signal, and Y(s) is the output signal. Moreover, KP, KI and KD present the proportional, integral,
and derivative gains. In addition, λ and δ show the orders
of integral and derivative. The schematic of the FOPID con-
troller in a control loop is shown in Figure 19 [93]. As shown in Figure 20, the orders of integral and deriva-
tive of this type of controller, unlike the PID controller,
have a wide range. This provides robustness and flexibil-
ity to the system and increases the range of power system
stability [93]–[95]. It should be noted that the difference in the genera-
tors speeds in the two areas (1ω) is considered as PODC
input signal [97], [98]. On the other hand, due to the fact
that the transmission of input signals is done through the
WAMS [66], [97], so it is necessary to define the constant
of time delay, Tm, for signal transmission [97]. In 2020 [27] the idea of the FOPID controller application in
the dynamic model of IBPPs was first proposed. In the study,
an LPF was used as a case study in a two-area test system [82]. Also, the SGGM is used for the LPF model. Then the PODC
tuning is performed based on the optimization method in
the time-domain. D. MULTIPLE MODEL ADAPTIVE CONTROL (MMAC)
STRATEGIES Based on the 72196 VOLUME 9, 2021 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs The results of the research showed the robustness of the
proposed PODC against a wide range of events and power
system uncertainties. actual system in real-time. The non-linear simulation results
indicated that the suggested control strategy can damp the
LFOs in unexpected operating conditions without any prior
knowledge about the post-disturbance state. It should be
noted that the FGGM has been used for LPF modeling in the
study. In both studies, the PODCs are considered in the Q/V
control loop of SGGM. As depicted in Figure 21, the study
proposed two various points for the PODC in the REPC_B
module. Each point can be considered based on the IBPP
control strategy. E. FRACTIONAL-ORDER PID (FOPID) CONTROLLER It should be noted that in the literature, the PODC has
been connected to the Q/V control loop, therefore the LPF
injects additional reactive power into the power system under
disturbance conditions. This is the LFO damping mechanism
by LPF, which is described in Section IV. This type of controller is the general form of a typical PID
controller. The mathematical structure of the FOPID con-
troller is based on the fractional-order calculus, which is
an effective tool for modeling many phenomena in engi-
neering [88]–[90]. These types of controllers provide robust
performance and wide range of stability area than the con-
ventional PID controllers due to the additional degree of
freedom caused by the fractional-orders of integral and
derivative [91]–[95]. Other advantages of this controller
include high flexibility in tuning, distortion rejection and high
reliability of the model in non-linear applications [88]–[90]. The FOPID controller is a new approach in electrical engi-
neering for robust controllers tuning with a wide stability
area. The standard form of the fractional differential equation
of this controller is as below [90]–[95]: VI. SIMULATION RESULS AND COMPARISON In the study, adjustment of FOPID con-
troller parameters was obtained using PSO optimization, and
the objective function was defined based on the integral of
time-weighted absolute error (ITAE) index [96]. The result of
the research indicated the better performance of the proposed
PODC than LLC and LQG controller. To evaluate the performance of the proposed PODCs, four
scenarios are considered. Although these scenarios are dif-
ferent in terms of disturbance severity, they all lead to LFOs
in the power system [30], [32]. These scenarios consider as
follows: • Scenario I: A three-phase fault at bus 8 at t = 1s for
170 ms. • Scenario II: Outage of line L78-1 at t = 1s for 67 ms. • Scenario III: Outage of generator G1 at t = 1s for 67 ms. Scenario IV: Outage of load L2 at t = 1s for 67 m Recently in 2021 [28], a method was proposed to the
coordinated tuning of FOPID-PODC controller with PSS
of SGs to damp the LFOs. The study also used SGGM
for LPF modeling. In addition, the coordinated tuning was
performed based on the PSO algorithm in the time-domain. Also, the robustness of the PODCs is evaluated in terms
of the time delay uncertainty of the PODC input signal. According to the defined scenarios, the simulation results are
as follows. 72197 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LP
FIGURE 26. Rotor angle of generator G1 for the scenario I for various time delays; (A) FOPID, (B) LQG, (C) MMAC strategy, (D) PID, and (E) LLC. As can be seen in Figures 23 to 25, the response of the
the PODC and cause them to malfunction and cause powe M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs of generator G1 for the scenario I for various time delays; (A) FOPID, (B) LQG, (C) MMAC strategy, (D) PID, and (E) LLC. FIGURE 26. Rotor angle of generator G1 for the scenario I for various time delays; (A) FOPID, (B) LQG, (C) MMAC strat As can be seen in Figures 23 to 25, the response of the
system to disturbances is different, using different PODCs. the PODC and cause them to malfunction and cause power
system instability. Therefore, controllers must have suffi-
cient robustness against this type of uncertainty in the power
systems. A. TIME DELAY UNCERTAINTY OF THE PODC INPUT
SIGNAL Results of scenario IV for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. controller and the MMAC are robust against time delay
uncertainty. controller and the MMAC are robust against time delay
uncertainty. In this section, a comparison is made between the ranges
of stability area of the PODCs proposed in the literature. For
this purpose, the previous scenarios in a longer period are
reviewed as follows: A. TIME DELAY UNCERTAINTY OF THE PODC INPUT
SIGNAL In this section, the robustness of the proposed PODCs in
the literature against different time delays is examined. Time delay uncertainty is one of the major challenges in using
the WAMS in smart grids. In these systems, control signals
may be received from long distances, so they naturally have
a time delay. This time delay can affect the performance of Accordingly, the performance of the proposed PODCs in
scenario I for different time delays is shown in Figure 26. As can be seen from the simulation results, the FOPID 72198 VOLUME 9, 2021 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs
FIGURE 27. Results of scenario I for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 28. Results of scenario III for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 29. Results of scenario IV for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs FIGURE 27. Results of scenario I for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 27. Results of scenario I for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 28. Results of scenario III for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 27. Results of scenario I for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 28. Results of scenario III for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 27. Results of scenario I for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 27. Results of scenario I for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 28. Results of scenario III for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. nerator G1, and (B) PCC frequency. FIGURE 28. Results of scenario III for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 29. Results of scenario IV for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. FIGURE 28. Results of scenario III for various PODCs; (A) Rotor angle of generator G1, and (B) PCC frequency. ; ( )
g FIGURE 29. B. STABILITY AREA OF THE PODCs • Scenario I within 380 ms. One of the indicators needed to compare the performance of
PODCs is the range of stability area. In other words, after a
large disturbance, a controller with a larger range of stability
area causes the system to return to stability more quickly. In this case, a PODC with a smaller range of stability area
may cause system instability. • Scenario III within 145 ms. • Scenario IV within 320 ms. The comparison between the proposed PODCs in the
literature in terms of the range of stability area is shown
in Figures 27 to 29. 72199 72199 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs TABLE 4. Summary of the performance of the proposed PODCs. TABLE 5. Summary of the proposed types of PODCs in studies. TABLE 6. Classification of LPF modeling in the studies. TABLE 7. Classification of LPF control strategy. TABLE 8. Summary of PODCs comparison. As can be seen in the figures, in all scenarios, LLC and
PID controllers become unstable quickly and can be said to
have small stability areas. It is clear that the FOPID controller
h
d t bilit
i
ll
i
d h
id TABLE 9. REGC_A and PV1G parameters, typical values and internal
variables. TABLE 9. REGC_A and PV1G parameters, typical values and internal
variables. TABLE 4. Summary of the performance of the proposed PODCs. TABLE 5. Summary of the proposed types of PODCs in studies. TABLE 8. Summary of PODCs comparison. TABLE 8. Summary of PODCs comparison. advantages. In some studies, such as [28], the controllers have
been compared and the benefits of each controller have been
described. On the other hand, each of the studies has used
one of the types of LPF models for simulations, as shown
in Table 6. Certainly, with the increasing development of
LPFs and the need for a central plant controller, model of this
controller is also needed in the LPF model. Therefore, it can
be said that with the development of modern power systems
and moving towards future power systems, modeling will also
lead to the use of the SGGM. The PODC design is generally
done using four methods: residue method [20], robust control
method [24], [25], optimization-based method [27], [28],
and adaptive method [19], [26]. B. STABILITY AREA OF THE PODCs Based on this, the design
method of the proposed PODCs in the studies can be sum-
marized in Table 7. Further studies are needed to com-
pare the performance of different PODCs. For example,
the industrialization and commercial aspects of some of these As can be seen in the figures, in all scenarios, LLC and
PID controllers become unstable quickly and can be said to
have small stability areas. It is clear that the FOPID controller
shows good stability in all scenarios and has a wide range
of stability area. Although the LQG controller is stable in
Scenario III, it becomes unstable quickly in the other two
scenarios. Regarding MMAC, it can be said that compared
to the LQG controller, it has a smaller stability area. This is
summarized in Table 4. VII. DISCUSSIONS AND REMARKS In the studies, several different control methods have been
proposed for LFOs damping using LPFs, which are summa-
rized in Table 5. Each of the proposed control methods has VOLUME 9, 2021 VOLUME 9, 2021 72200 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs TABLE 10. REEC_B and PV1E parameters, typical values and internal
variables. TABLE 10. REEC_B and PV1E parameters, typical values and internal
variables. TABLE 10. (Continued.) REEC_B and PV1E parameters, typical values and
internal variables. TABLE 10. (Continued.) REEC_B and PV1E parameters, typical values and
internal variables. VIII. CHALLENGES AND RESEARCH GAPS Modern power systems are moving toward renewable energy
resources to overcome problems related to climate change
and global warming. Therefore, REPPs such as LPFs are
highly deployed in modern power systems. The high pene-
tration level of LPFs highly reduces the total inertia which
affects the stability and security of power systems. So, these
types of power plants must be able to increase the power
system inertia as well as perform the basic tasks of SGs. For
this purpose, they must be able to damp the LFOs by PODCs. As shown in this paper, different control techniques have
been suggested to damp the LFOs by LPFs, but some prob-
lems affect the applicability of this issue. On the other hand,
it seems that there are still research gaps that need further
research. The main challenges and research gaps are as
follows: • Low capacity of the LPFs: One of the main challenges
is the low capacity of current LPFs. As long as the
LPFs do not have high capacity in power systems, they
are not effective for LFOs damping. It is important to
note that although LPFs based PODCs have acceptable
performance, it is necessary to develop the LPFs with
capacities above 500 MW in order to be effective for
LFOs damping. LFOs damping. • Uncertainty of power generation of LPFs: Due to the
lack of access to solar radiation at night and also the
stochastic behavior of sunlight during the day, power
generation stops at night and there is a sharp fluctuation
of power production during the day. Therefore, the high
intensity of power generation uncertainty has reduced
power system reliability. It seems that in this condition,
it is practically impossible to depend on this type of
power plant for LFOs damping. • Auto-tuning: Given the development of smart grids
and taking into account the requirements of modern
power systems, one of the most important issues is
the auto-tuning of controllers depending on the opera-
tion conditions. In fact, the proposed PODCs are now controllers have not yet been identified, but a brief com-
parison between the various controllers can be made and
summarized in Table 8. controllers have not yet been identified, but a brief com-
parison between the various controllers can be made and
summarized in Table 8. 72201 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs TABLE 11. (Continued.) REPC_A parameters, typical values and internal
variables. TABLE 11. (Continued.) REPC_A parameters, typical values and internal
variables. BLE 11. REPC_A parameters, typical values and internal variables. pre-configured and have fixed parameter values for a
operating conditions. It seems that in future system
th t
i
f PODC
h
ld b b
d
li
t
i TABLE 11. REPC_A parameters, typical values and internal variables. and auto-tuning. This can be a research suggestion for
future work. • Commercialization and industrialization of PODCs:
one of the important research gaps in this issue is the
examination of the capabilities of the proposed modern
PODCs such as the FOPID controller for commercial-
ization and industrialization. • Low
capacity
of
battery
energy
storage
sys-
tems (BESSs) and the impossibility of using virtual
SGs (VSGs) [99]–[102]: currently, one of the major
challenges in power systems is the low capacity of
BESSs. Due to the high power of LPFs, this makes
it impossible to use VSG and BESS to increase the
reliability of LPFs for operation and LFO damping. Despite the challenges and research gaps mentioned,
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operating conditions. It seems that in future systems,
the tuning of PODCs should be based on online tuning VOLUME 9, 2021 VOLUME 9, 2021 72202 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs performance of the proposed PODCs for LFOs damping by
LPFs, there are challenges in this area. It seems that with
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by the National Elites Foundation, as a Laureates of the Alameh Tabatabaei
Award, and was awarded the National Prize in 2008, 2010, 2018, 2018,
2019, and 2019, respectively. Based on the Web of Science database for the
period 2005–2019, he is among world’s top 1% Elite Scientists, according to
Essential Science Indicators (ESI) ranking systems. Since 2004, he has been
the Editor-in-Chief of the Journal of IAEEE. Dr. Gharehpetian is a Senior Member of CIGRE and IAEEE, and a
Distinguished Member of CIGRE, IEEE, and IAEEE. As a Ph.D. Student,
he has received scholarship from DAAD (German Academic Exchange
Service), from 1993 to 1996. He was selected by the Ministry of Science
Research and Technology (MSRT), as a Distinguished Professor of Iran,
by the Iranian Association of Electrical and Electronics Engineers (IAEEE),
as a Distinguished Researcher of Iran, by the Iran Energy Association (IEA),
as the Best Researcher of Iran in the field of energy, by the MSRT, as a
Distinguished Researcher of Iran, by the Academy of Science of the Islamic
Republic of Iran, as a Distinguished Professor of electrical engineering,
by the National Elites Foundation, as a Laureates of the Alameh Tabatabaei
Award, and was awarded the National Prize in 2008, 2010, 2018, 2018,
2019, and 2019, respectively. Based on the Web of Science database for the
period 2005–2019, he is among world’s top 1% Elite Scientists, according to
Essential Science Indicators (ESI) ranking systems. Since 2004, he has been
the Editor-in-Chief of the Journal of IAEEE. [91] I. Podlubny, ‘‘Fractional-order systems PIλDµ-controller,’’ IEEE Trans. Autom. Control, vol. 44, no. 1, pp. 208–214, Jan. 1999. [92] C. A. Monje, Y. Chen, B. M. Vinagre, D. REFERENCES Control Syst. Technol., vol. 15, no. 1, pp. 151–160, Jan. 2007. [61] WECC PV Power Plant Dynamic Modeling Guide, Western Electricity
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ber, IEEE) received the B.S. degree (Hons.) from
Tabriz University, Tabriz, Iran, in 1987, the M.S. degree (Hons.) from the Amirkabir University of
Technology (AUT), Tehran, Iran, in 1989, and
the Ph.D. degree (Hons.) from Tehran University,
Tehran, in 1996, all in electrical engineering. [86] B. Chaudhuri, R. Majumder, and B. C. Pal, ‘‘Application of multiple-
model adaptive control strategy for robust damping of interarea oscilla-
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vol. 21, no. 1, pp. 1–30, 2007. He was with the High Voltage Institute of
RWTH Aachen, Aachen, Germany. From 1997 to
2003, he was an Assistant Professor with AUT, He was with the High Voltage Institute of
RWTH Aachen, Aachen, Germany. From 1997 to
2003, he was an Assistant Professor with AUT,
where he was an Associate Professor, from 2004 to 2007, and has been
a Professor, since 2007. He has authored more than 1200 journal articles
and conference papers. His teaching and research interests include smart
grid, microgrids, FACTS, and HVDC systems, and monitoring of power
transformers and its transients. [88] K. S. Miller and B. Ross, An Introduction to the Fractional Calculus and
Fractional Differential Equations. New York, NY, USA: Wiley, 1993. ,
,
where he was an Associate Professor, from 2004 to 2007, and has been
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and conference papers. His teaching and research interests include smart
grid, microgrids, FACTS, and HVDC systems, and monitoring of power
transformers and its transients. [89] H. Singh, D. Kumar, and D. Baleanu, Methods of Mathematical Mod-
elling: Fractional Differential Equations. Boca Raton, FL, USA: CRC
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‘‘Small-signal modeling and parameters design for virtual synchronous
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pp. 2820–2824, Apr. 2018. JOSEP M. GUERRERO (Fellow, IEEE) received
the Ph.D. degree from the Technical Univer-
sity of Catalonia, Barcelona, Spain, in 2003. He is currently a Full Professor with the Depart-
ment of Energy Technology, Aalborg University,
Denmark, where he is the Director of the Center
for Research on Microgrids. His research interests
include distributed energy-storage systems, hier-
archical and cooperative control, energy manage-
ment systems, smart metering, and the Internet of
Things for ac/dc microgrid clusters. He is a member of the IEEE Industrial
Electronics Society. He was awarded the Institute for Scientific Information
Highly Cited Researcher by Thomson Reuters for six consecutive years, from
2014 to 2019, and a VILLUM Investigator, in 2019. MAHDI SAADATMAND received the M.Sc. degree
in
electrical
engineering
from
the
Amirkabir University of Technology, Tehran,
Iran, in 2013, and the Ph.D. degree in electri-
cal engineering from the Science and Research
Branch, Islamic Azad University, Tehran, in 2020. He has authored more than 15 scientific jour-
nal articles and conference papers. His research
interests include power system dynamics, smart
grids,
renewable
energy
technologies,
and
fractional-order control. Since 2019, he has been serving as a reviewer for
several high-quality journals. 72205 VOLUME 9, 2021 VOLUME 9, 2021 M. Saadatmand et al.: Damping of LFOs in Power Systems by LPFs HASSAN HAES ALHELOU (Senior Member,
IEEE) is currently with the School of Electrical
and Electronic Engineering, University College
Dublin, Dublin, Ireland. He is also a Faculty Mem-
ber of Tishreen University, Lattakia, Syria. He has
published more than 130 research articles in the
high quality peer-reviewed journals and more than
130 research papers in the high quality interna-
tional conferences. He has participated in more
than 15 industrial projects. His major research
interests include power systems, power system dynamics, power system
operation and control, dynamic state estimation, frequency control, smart
grids, micro-grids, demand response, load shedding, and power system
protection. REFERENCES He has also performed reviews for high prestigious journals,
including IEEE TRANSACTIONS ON INDUSTRIAL INFORMATICS, IEEE TRANSACTIONS
ON INDUSTRIAL ELECTRONICS, Energy Conversion and Management, Applied
Energy, International Journal of Electrical Power and Energy Systems. He was a recipient of the Best Young Researcher in the Arab Student Forum
Creative, among 61 researchers from 16 countries at Alexandria University,
Egypt, in 2011. He was a recipient of the Outstanding Reviewer Award from
Energy Conversion and Management Journal, in 2016, ISA Transactions
Journal, in 2018, Applied Energy Journal, in 2019, and many other awards. He is included in the 2018 and 2019 Publons list of the top 1% Best Reviewer
and researchers in the field of engineering. PIERLUIGI
SIANO PIERLUIGI
SIANO
(Senior Member, IEEE)
received the M.Sc. degree in electronic engineer-
ing and the Ph.D. degree in information and elec-
trical engineering from the University of Salerno,
Salerno, Italy, in 2001 and 2006, respectively. He is
currently a Professor and the Scientific Director
of the Smart Grids and Smart Cities Laboratory,
Department of Management and Innovation Sys-
tems, University of Salerno. In the research fields,
he has coauthored more than 500 articles, includ-
ing more than 300 international journal articles that received in Scopus,
more than 9450 citations with an H-index equal to 47. His research interests
include demand response, on energy management, on the integration of
distributed energy resources in smart grids, on electricity markets, and on
planning and management of power systems. He received the award as the
2019 Highly Cited Researcher by the ISI Web of Science Group. He has been
the Chair of the IES TC on Smart Grids. He is an Editor for the Power and
Energy Society Section of IEEE ACCESS, IEEE TRANSACTIONS ON INDUSTRIAL
INFORMATICS, IEEE TRANSACTIONS ON INDUSTRIAL ELECTRONICS, IEEE OPEN
JOURNAL OF THE INDUSTRIAL ELECTRONICS SOCIETY, and IET Renewable Power
Generation. 72206 VOLUME 9, 2021
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English
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A nondestructive method to estimate the chlorophyll content of Arabidopsis seedlings
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Plant methods
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cc-by
| 8,825
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© The Author(s) 2017. 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: Chlorophyll content decreases in plants under stress conditions, therefore it is used commonly as an
indicator of plant health. Arabidopsis thaliana offers a convenient and fast way to test physiological phenotypes of
mutations and treatments. However, chlorophyll measurements with conventional solvent extraction are not applica‑
ble to Arabidopsis leaves due to their small size, especially when grown on culture dishes. Results: We provide a nondestructive method for chlorophyll measurement whereby the red, green and blue (RGB)
values of a color leaf image is used to estimate the chlorophyll content from Arabidopsis leaves. The method accom‑
modates different profiles of digital cameras by incorporating the ColorChecker chart to make the digital negative
profiles, to adjust the white balance, and to calibrate the exposure rate differences caused by the environment so that
this method is applicable in any environment. We chose an exponential function model to estimate chlorophyll con‑
tent from the RGB values, and fitted the model parameters with physical measurements of chlorophyll contents. As
proof of utility, this method was used to estimate chlorophyll content of G protein mutants grown on different sugar
to nitrogen ratios. Conclusion: This method is a simple, fast, inexpensive, and nondestructive estimation of chlorophyll content of
Arabidopsis seedlings. This method lead to the discovery that G proteins are important in sensing the C/N balance to
control chlorophyll content in Arabidopsis. Keywords: Arabidopsis thaliana, C/N sensing, Chlorophyll content, ColorChecker chart, Heterotrimeric G protein
complex, Stress assay cotyledons. It is important to develop a non-destructive
method to estimate chlorophyll content for Arabidopsis
because it is a genetic model plant, however traditional
chlorophyll extraction is not useful due to the small size
of the Arabidopsis leaves grown on agar plates. Recently,
digital photographic imaging showed great promise for
quantitating plant phenotypes [2]. Indirect methods are
available but none are yet suitable for Arabidopsis. Sass
et al. [3] developed a protocol to convert the RGB values
of a color image into a hue saturation value (HSV), and
showed that the hue value was correlated to the chloro-
phyll content estimated by a destructive method. A simi-
lar color-image method was used to assess the nitrogen
status of rice under natural light condition [4]. Riccardi
et al. [5] found that an exponential function model dis-
plays the best correlation between the RGB values and
the chlorophyll content through single and multiple METHODOLOGY METHODOLOGY METHODOLOGY
Open Access Open Access © The Author(s) 2017. 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. *Correspondence: yajungao@nwsuaf.edu.cn; alan_jones@unc.edu
1 Department of Biology, The University of North Carolina at Chapel Hill,
Coker Hall, CB#3280, Chapel Hill, NC 27599‑3280, USA
2 College of Natural Resources and Environment, Northwest A&F
University, Yangling 712100, Shaanxi, China
Full list of author information is available at the end of the article Plant Methods Plant Methods Liang et al. Plant Methods (2017) 13:26
DOI 10.1186/s13007-017-0174-6 Note 1: This plugin was successfully tested in both ImageJ
1.48v and ImageJ 1.50i. Note 1: This plugin was successfully tested in both ImageJ
1.48v and ImageJ 1.50i. 6. Optional: Materials for chlorophyll extraction: 80%
acetone in water, spectrophotometer In this study, we describe a convenient and nondestruc-
tive method to estimate leaf chlorophyll of Arabidopsis
seedlings grown on agar plates using calibrated-RGB
images. We also provide instructions how to adapt it to
other small leave samples. We quantitated chlorophyll
content in small Arabidopsis seedlings grown on differ-
ent C: N ratios in the agar medium. The results indicated
that G proteins play important roles in sensing and/
or responding to the C/N balance in this chlorophyll
response. Plant growth 1. Grow Arabidopsis seedlings in the light on square
8 cm × 8 cm plastic Petri plates (VWR; Cat No. 60872-310) with a 40-mL layer of agar and media
suitable for plant growth. 2. Thirty-six seedlings are placed individually within the
36-gridded area of the plate; one seedling per grid. There should be no overlap of seedlings. This spacing
is important for the software to automatically detect
seedlings for chlorophyll calculations. There is also
an option described later to create a different grid
then the default 6 × 6 grid (step 8 of the protocol). Backgroundh The chlorophyll content of leaves is an indirect indica-
tor of the health and nutritional status of the plant [1]. Traditional methods to calculate the chlorophyll content
include a destructive chemical extraction and a non-
destructive measurement of chlorophyll fluorescence. The former method, while direct, is tedious and unsuit-
able for continuous monitoring individual plants because
of its destructive manner. The latter method needs expen-
sive instruments of which none are presently suitable
for small leaves such as the commonly used Arabidopsis *Correspondence: yajungao@nwsuaf.edu.cn; alan_jones@unc.edu
1 Department of Biology, The University of North Carolina at Chapel Hill,
Coker Hall, CB#3280, Chapel Hill, NC 27599‑3280, USA
2 College of Natural Resources and Environment, Northwest A&F
University, Yangling 712100, Shaanxi, China
Full list of author information is available at the end of the article Liang et al. Plant Methods (2017) 13:26 Page 2 of 10 DNG
converter
http://www.adobe.com
(public
source),
DNG profile editor http://www.adobe.com (public
source) and
PhotoShop or Lightroom http://www.adobe.com
(license required). Optional software: Matlab http://www.mathworks. com/(license required). regression in quinoa and amaranth leaves. No similar
color-image methods have been adapted for Arabidopsis
chlorophyll content, in particular Arabidopsis seedlings
grown on agar plates. The lack of a quantitative method
for measuring chlorophyll of plate-grown Arabidopsis
restricted previous studies on stress-induced phenotypes
to subjective assessment without quantitation [6, 7].f DNG profile editor http://www.adobe.com (public
source) and Chlorophyll content in leaves is affected by the car-
bon (C) and nitrogen (N) balance. Genetics studies
using Arabidopsis revealed that the C/N balance is reg-
ulated through multiple signaling cascades of abscisic
acid-insensitive 1 (ABI1), hexokinase 1 (HXK1), nitrate
transporters, glutamate receptor (AtGLR1.1) and het-
erotrimeric G proteins [6, 8–12]. Many mutant alleles in
these pathways confer altered chlorophyll content regu-
lated by the C/N balance in Arabidopsis [6, 9, 12], there-
fore these mutants are useful for validating the utility
of our digital image method. Heterotrimeric G proteins
consist of one canonical Gα subunit (GPA1), one Gβ sub-
unit (AGB1), three Gγ subunits (AGGs) and three atypical
extra-large Gα proteins (XLGs) [13–15] in Arabidopsis. G protein signaling pathway senses glucose levels in the
environment [16, 17], and is also involved in nitrogen use
efficiency in rice [10]. Regulator of G protein Signaling 1
protein (AtRGS1) is a component of the glucose sensor
[11, 12, 18]. This protein modulates the activation state of
G signaling. 5. Backgroundh Plug-in programs for ImageJ are provided here in
Additional file 1: S1, Additional file 2: S2, Additional
file 3: S3 and Additional file 4: S4. Additional file 1: S1
and Additional file 2: S2 are for chlorophyll calcula-
tion and Additional file 3: S3 and Additional file 4: S4
are for reading the RGB values of the images. Addi-
tional file 1: S1 and Additional file 3: S3 are the .java
source code used to generate the .class files (Addi-
tional file 2: S2 and Additional file 4: S4). In most
cases, only the .class files are needed, however the
corresponding .java files are provided for those want-
ing to examine or improve the programs. 5. 5. Plug-in programs for ImageJ are provided here in
Additional file 1: S1, Additional file 2: S2, Additional
file 3: S3 and Additional file 4: S4. Additional file 1: S1
and Additional file 2: S2 are for chlorophyll calcula-
tion and Additional file 3: S3 and Additional file 4: S4
are for reading the RGB values of the images. Addi-
tional file 1: S1 and Additional file 3: S3 are the .java
source code used to generate the .class files (Addi-
tional file 2: S2 and Additional file 4: S4). In most
cases, only the .class files are needed, however the
corresponding .java files are provided for those want-
ing to examine or improve the programs. Protocol 1. Photograph seedlings arranged on the square plates
and save as RAW files. Any digital camera that stores
images as RAW files can be used. 1. Photograph seedlings arranged on the square plates
and save as RAW files. Any digital camera that stores
images as RAW files can be used. Note 4: For this study, an Olympus digital camera
E-3 captured the RGB color images and the images
stored as ORF file, a type of RAW format. Settings for
the digital camera were the following: aperture = f/7,
shutter speed = 1/100 s, ISO = 400, quality = F, file
storage = RAW. Shutter speed should be adjusted
according to the actual ambient light condition. ii
4. Using the Checker.dcp files generated in step 3,
calibrate all seedling images using the DNG profile
through the software Lightroom or Photoshop. These
programs provide a step-by-step instructions for cali-
bration. After the adjustment, export the images in
jpg format labeled accordingly: IMAGE_ID.jpg. 5. Open the IMAGE_ID.jpg files in Adobe Photoshop. Clean or erase the background of the images with
the eraser or magic wand/delete tools (Fig. 1b). Label
files IMAGE_ID_cleaned.JPG The plugin will auto-
matically remove slightly gray or otherwise imper-
fect backgrounds, but use of this capability should be
carefully validated against test images with manually
cleaned backgrounds. 5. Open the IMAGE_ID.jpg files in Adobe Photoshop. Clean or erase the background of the images with
the eraser or magic wand/delete tools (Fig. 1b). Label
files IMAGE_ID_cleaned.JPG The plugin will auto-
matically remove slightly gray or otherwise imper-
fect backgrounds, but use of this capability should be
carefully validated against test images with manually
cleaned backgrounds. Note 5: Acquire images of the X-rite ColorChecker
classic chart both immediately before and after
acquiring images of the samples (Fig. 1a). The Color-
Checker chart is to make sure the final data compara-
ble despite different light conditions and cameras but
may not be necessary. Before and after images of the
chart are acquired to test whether the light condition
is consistent during the photographing. Check the
RGB values of the X-rite Colorchecker chart boards
as described below. Should you find that the starting
and ending values are different, it will be necessary to
stabilize the light environment and re-acquire sample
images. If the light condition is stable in the lab, it is
not necessary to acquire two images every time. Materials needed 1. Arabidopsis seedlings grown on agar plates. In
this study, we used the following T-DNA insertion
mutant alleles: agb1-2 [19], rgs1-2 [20], gpa1-3 [21],
xlg1xlg2xlg3 [13] which combines these alleles xlg1-
1 (SAIL_760H08) [13], xlg2-2 (SALK_062645), xlg3-
2 (SAIL_107656) [13], and xlg/gpa1 which combines
the xlg1-1, xlg2-1, xlg3-2 and gpa1-3 alleles above
[22] (in press). 1. Arabidopsis seedlings grown on agar plates. In
this study, we used the following T-DNA insertion
mutant alleles: agb1-2 [19], rgs1-2 [20], gpa1-3 [21],
xlg1xlg2xlg3 [13] which combines these alleles xlg1-
1 (SAIL_760H08) [13], xlg2-2 (SALK_062645), xlg3-
2 (SAIL_107656) [13], and xlg/gpa1 which combines
the xlg1-1, xlg2-1, xlg3-2 and gpa1-3 alleles above
[22] (in press). Note 2: It is not necessary to use a square petri dish on
which to arrange the seedlings; any rectangle background
with samples in a matrix format will work. It is also possi-
ble to treat seedlings in liquid culture or on some matrix
other than agar and then transfer them to the square agar
plates for photography. 2. A digital camera that captures images in RAW for-
mat. Note 3: For this study, seedlings were grown on the
indicated media arranged on square plates as described
above and photographed as will be described below. Spe-
cifically, Murashige and Skoog (MS) Modified Medium
w/o Nitrogen (Plantmedia; Cat No. 30630200-1) supplied 3. X-rite ColorChecker classic chart (http://xritephoto. com/colorchecker-classic). 4. Software:
ImageJ
https://imagej.nih.gov/ij/(public
source; imageJ 1.50i is recommended), Liang et al. Plant Methods (2017) 13:26 Page 3 of 10 lated by estimating the chlorophyll content of 36
individual seedlings on a plate placed in the center
and 4 corners of the field. with 0.8% phytoagar and 1 g/L MES was used. The pH
was adjusted to 5.7 with KOH. Filter-sterilized d-glucose
was added to the medium to adjust the glucose concen-
tration as indicated. A stock solution of 1 M KNO3 was
used as the nitrogen resource. The agar plates of sterilized
seed, sealed with a gas-permeable tape, were stratified
at 4 °C for 3–5 days in the dark. The plates were placed
horizontally under constant dim light (35–50 µEm−2 s−1)
at 23 °C for 12 days. Images were obtained on the 12th
day. Plants were also grown on soil in a long day cham-
ber (200 µEm−2 s−1, 16 h light/8 h dark) at 23 °C for
4–8 weeks as indicated in the experimental description. i
2. Materials needed Convert the RAW files to DNG format using the
DNG converter. There are many formats of RAW file;
one is a DNG. If your camera stores the files in DNG
format, nothing more is needed at this step.i 3. Generate a DNG profile of the X-rite ColorChecker
chart. This format can be used by Adobe PhotoShop
and Lightroom programs. Launch the DNG profile
editor, and then click on the ‘Chart’ tab. Then load the
DNG file checkerchart. Use the mouse to position the
four colored circles in the image at the centers of the
four corner color panels of the chart. The colors of the
circles should correspond to the colors of the patches. Leave the popup menu set at ‘Both Color Tables’ and
click ‘Create Color table’ button. Then export the profile
by selecting Export Profile in the File menu and name
the profile as ColorCheckerChart.dcp. Make sure the
file is saved in the CameraProfiles directory (default). Protocol It is
important to use the same settings and the same light
conditions for all the images to be compared. Note 8: Cleaning the background is an important step. Assure that shadows are completely eliminated and
only leaves remain in the image. An example of a per-
fectly cleaned image is provided as Additional file 6: S6. i
6. Open ImageJ and install the plugin Chloropyll_
Imager provided in the Additional file 2: S2. Addi-
tional file 1: S1 is the .java code which could also be
used if preferred. 7. Open ColorChecker.jpg in ImageJ and record the RGB
values designated r, g, b of the white background. The
r, g, and b values are obtained using the plugin labeled
‘RGB_measure provided in Additional file 4: S4. Addi-
tional file 3: S3 is the .java code which could also be used
if preferred. Select the white panel of the ColorChecker
chart and run the plug-in. The corresponding r, g, b val-
ues of the white panel will appear in a table. 7. Open ColorChecker.jpg in ImageJ and record the RGB
values designated r, g, b of the white background. The
r, g, and b values are obtained using the plugin labeled
‘RGB_measure provided in Additional file 4: S4. Addi-
tional file 3: S3 is the .java code which could also be used
if preferred. Select the white panel of the ColorChecker
chart and run the plug-in. The corresponding r, g, b val-
ues of the white panel will appear in a table. Note 6: The image size for the plate is the same
as for the ColorChecker chart, approximately
210 × 300 mm. The square plate is placed in the
center of this field for imaging. Note 7: The light should be uniformly distributed. Tests for position effect in this field were determined
to be a maximum of 4.8% (n = 36) (Additional file 5:
S5) of the chlorophyll values. This value was calcu- p
pp
8. Open IMAGE_ID_cleaned.jpg files in ImageJ and run
the plugin “Chorophyll_Image” which in step 6 you 8. Open IMAGE_ID_cleaned.jpg files in ImageJ and run
the plugin “Chorophyll_Image” which in step 6 you Liang et al. Plant Methods (2017) 13:26 Page 4 of 10 had saved in the ImageJ ‘plugins folder. A dialog box
will appear asking for the number of rows and col-
umns (Fig. 1c). Protocol Chlorophyll a (µg/mL) = 12.7 (A663) −2.69 (A645)
Chlorophyll b (µg/mL) = 22.9 (A645) −4.68 (A663)
Total chlorophyll (µg/mL) = 20.2 (A645) + 8.02 (A663) white panel recorded in step 7. Enter these values. By
clicking “OK”, you will generate a table of the chlo-
rophyll content as ng/mm2. The dialogue also asks
if you want to make a normalized grayscale image
(Fig. 1d). If so, click the box and a grey scale image
will also be presented as output (see Additional file 7:
S7 for an example). This greyscale image offer a spa-
tial map of chlorophyll on a leaf; i.e. 2-dimensional
information is provided. white panel recorded in step 7. Enter these values. By
clicking “OK”, you will generate a table of the chlo-
rophyll content as ng/mm2. The dialogue also asks
if you want to make a normalized grayscale image
(Fig. 1d). If so, click the box and a grey scale image
will also be presented as output (see Additional file 7:
S7 for an example). This greyscale image offer a spa-
tial map of chlorophyll on a leaf; i.e. 2-dimensional
information is provided. (2) (2) The area of the leaves were measured by software Image J
and chlorophyll content and the total chlorophyll content
per leaf area was expressed as ng/mm2. Protocol Enter these values or use the default
value of 6 rows × 6 columns. This step divides the
images into 36 parts with 6 rows × 6 columns. The
dialog box also will request the RGB values of the
ig. 1 Examples of images needed in converting RGB value to chlorophyll content. a The picture of the X-rite ColorChecker classic chart. b The
riginal picture of the Arabidopsis seedlings grown on the plates under 4% glucose and 2 mM nitrogen for 12 days. c The dialogue box of the plugin
ImageJ. d The grey scale pictures represent the chlorophyll content, which is calculated by the equation estimate the chlorophyll content Fig. 1 Examples of images needed in converting RGB value to chlorophyll content. a The picture of the X-rite ColorChecker classic chart. b The
original picture of the Arabidopsis seedlings grown on the plates under 4% glucose and 2 mM nitrogen for 12 days. c The dialogue box of the plugin
in ImageJ. d The grey scale pictures represent the chlorophyll content, which is calculated by the equation estimate the chlorophyll content Fig. 1 Examples of images needed in converting RGB value to chlorophyll content. a The picture of the X-rite ColorChecker classic chart. b The
original picture of the Arabidopsis seedlings grown on the plates under 4% glucose and 2 mM nitrogen for 12 days. c The dialogue box of the plugin
in ImageJ. d The grey scale pictures represent the chlorophyll content, which is calculated by the equation estimate the chlorophyll content Fig. 1 Examples of images needed in converting RGB value to chlorophyll content. a The picture of the X-rite ColorChecker classic chart. b The
original picture of the Arabidopsis seedlings grown on the plates under 4% glucose and 2 mM nitrogen for 12 days. c The dialogue box of the plugin
in ImageJ. d The grey scale pictures represent the chlorophyll content, which is calculated by the equation estimate the chlorophyll content had saved in the ImageJ ‘plugins folder. A dialog box
will appear asking for the number of rows and col-
umns (Fig. 1c). Enter these values or use the default value of 6 rows × 6 columns. This step divides the
images into 36 parts with 6 rows × 6 columns. The
dialog box also will request the RGB values of the Liang et al. Plant Methods (2017) 13:26 Page 5 of 10 B. Fitting parameters for the function and “Do it
yourself” validation for other types of chlorophyll
containing samples Note 9: The default coefficient values for chlorophyll estima-
tion are shown as a1, a2, a3, and a4 in the boxes of the plug-
in menu. These values can be changed to fit other types of
samples such as leaf pieces. Other coefficients can be deter-
mined as described in the “Do it yourself” section below. The default coefficient values used above are described
here in the event that the user needs to modify this tool
to obtain different coefficients for other types of chloro-
phyll samples such as leaf pieces. A least squares method
was used to search for the coefficients for the exponential
function equation to estimate chlorophyll contents from
RGB values. If the user desires to validate this method on
their own using their own images and chlorophyll sam-
ples, then follow these steps: 9. Validation using a test sample. A cleaned image with 6
rows × 6 columns seedlings is provided as Additional
file 6: S6. The RGB values of the white background for
this figure are 171.666, 171.297, and 171.256, respec-
tively. After running the plugin, you will obtain the
chlorophyll content of the 36 seedlings provided in
the test file. In order to confirm correct operation of
the program, compare your calculated results with the
values shown in Additional file 8: S8. 1. Read the RGB values of each sample. A plugin labeled
‘RGB_measure.java’ is provided in Additional file 4:
S4. 2. Measure the chlorophyll content using chemical
extraction (see “Chlorophyll extract” section). The
chlorophyll content should be expressed as total
chlorophyll per leaf area, ng/mm2. Chl = EXP
a1 ∗R ∗r/243 + a2 ∗G ∗g/243 + a3 ∗B ∗b/242 + a4 Chlorophyll extraction For validation purposes, we compared our method to
extracted chlorophyll. Chlorophyll content was estimated
by spectrophotometry of samples prepared by 80% ace-
tone extraction. The leaves were incubated at room tem-
perature in a 1.5-mL tube with 1 mL 80% acetone solution
for at least 24 h then clarified by centrifugation for 5 min
at 15,000g. In this study, absorbance of the supernatant
was measured at wavelengths 645, 646, and 663 nm (A645,
A646, and A663) with a Shimadzu UV-3000™ dual-wave-
length, double-beam spectrophotometer, although any
spectrophotometer is suitable. Complete spectra were
taken during development of this protocol in order to
assure that the predominant absorbance was from chlo-
rophyll; this is not routinely necessary. Samples having
absorbance greater than 1 were diluted by half with 80%
acetone and re-evaluated. Chlorophyll concentration was
estimated following the Lichtenthaler’s equations (A) [23]
and the Arnon’s equations (B) [24] as follows: 3. Combine the datasets of R, G and B values obtained
from step 1 with the chlorophyll content estimated
by chemical extraction from step 2 to search for the
coefficient with a least-squares method. An excel
file, Additional file 9: S9, labeled EXAMPLE.xlsx,
provides a sample dataset of RGB values with chlo-
rophyll content data. Replace your datasets with the
original ones. This spreadsheet provides the input
chlorophyll and color data used to fit the best coef-
ficients. i
4. Open Matlab and upload the appropriate ‘Leastsqua-
reequation’ script provided in Matlab code (Addi-
tional file 10: S10). The provided code finds the best
fit of the coefficients using the following equation (3) where R, G and B refer to the color of the plants read
from the plug-in described above, and r, g and b refer
to the corresponding RGB values of the white back-
ground. Before running the scripts, change the input
of r, g and b to these new values. A. Chlorophyll a (µg/mL) = −1.93 A646 + 11.93 A663
Chlorophyll b (µg/mL) = 20.36 A646 −5.50 A663
Total chlorophyll (µg/mL) = 6.43 A663 + 18.43 A646 A. Chlorophyll a (µg/mL) = −1.93 A646 + 11.93 A663
Chlorophyll b (µg/mL) = 20.36 A646 −5.50 A663
Total chlorophyll (µg/mL) = 6.43 A663 + 18.43 A646 Total chlorophyll (µg/mL) = 6.43 A663 + 18.43 A646 (1) Liang et al. Plant Methods (2017) 13:26 Page 6 of 10 Having the new coefficients enables you to estimate
chlorophyll in other samples nondestructively. From RGB value to chlorophyll content We adapted the method of Riccardi et al. [5] to Arabi-
dopsis seedlings grown on culture plates and compared
this method to biochemical extraction of chlorophyll. We
extracted chlorophylls with acetone, measured absorb-
ance spectra, then calculated chlorophyll content in the
solvent extract using both the Lichtenthaler’s [23] and
Arnon’s equations [24] (Eqs. 1, 2). Samples from 12-day-old Arabidopsis shoots from
seedlings grown under different C/N treatments on
square plates as described under plant growth were used
to search for the parameters for the exponential func-
tion model (see Additional file 11: S11). To assure that
the images taken by different cameras are comparable,
we incorporated a standard to enable comparison of pub-
lished data. The X-rite ColorChecker chart (Fig. 1a) was
used to make a DNG profile and to adjust the white bal-
ance. Another critical variable to account for is the light
intensity and color in the room, chamber, greenhouse or
field. The X-rite ColorChecker chart solves these prob-
lems and makes the assay applicable to artificial and
natural light. The software Image J was used to measure
average R, G and B values of individual leaves. We used
the exponential function model Results and discussion Fig. 2 Correlation of the chlorophyll content estimated by RGB value
and chemical extraction. a, b Comparison of extracted chlorophyll
calculated by the Lichthenther (a) and Arnon (b) methods to chloro‑
phyll content estimated by RGB value of Arabidopsis seedlings grown
in agar plates under different C/N treatment (n = 234, four independ‑
ent experiment). c, d Chlorophyll content estimated by RGB value
and chemical extraction in soil-grown-plant using the defaulted coef‑
ficients (c) versus the refitted coefficients (d), respectively (n = 15) Chlorophyll extraction Lichthenther
0
100
200
300
0
100
200
300
400
Arnon
0
100
200
300
R2=0.799
R2=0.811
Lichthenther
0
50
100
150
200
0
100
200
300
Lichthenther
0
50
100 150
200
R2=0.847
defaulted
R2=0.891
refitted
Chlorophyll content estimated by RGB value(ng/mm2)
a
b
d
c
Chlorophyll content estimated by chemical extraction (ng/mm2)
Fig. 2 Correlation of the chlorophyll content estimated by RGB value
and chemical extraction. a, b Comparison of extracted chlorophyll
calculated by the Lichthenther (a) and Arnon (b) methods to chloro‑
phyll content estimated by RGB value of Arabidopsis seedlings grown
in agar plates under different C/N treatment (n = 234, four independ‑
ent experiment). c, d Chlorophyll content estimated by RGB value
and chemical extraction in soil-grown-plant using the defaulted coef‑
ficients (c) versus the refitted coefficients (d), respectively (n = 15) Lichthenther
0
100
200
300
0
100
200
300
400
Arnon
0
100
200
300
R2=0.799
R2=0.811
Lichthenther
0
50
100
150
200
0
100
200
300
Lichthenther
0
50
100 150
200
R2=0.847
defaulted
R2=0.891
refitted
Chlorophyll content estimated by RGB value(ng/mm2)
a
b
d
c
Chlorophyll content estimated by chemical extraction (ng/mm2) Note 10: For Matlab, paste the provided MatLab script
‘Leastsquareequation.m’ (Additional file 10: S10) and
the EXAMPLE.xlsx (Additional file 9: S9) into the same
folder. Note 11: Other color charts may be used but the values
of white may be different than for the X-rite ColorChart. In this case, replace the values 243, 243, 242, respectively
in Eq. (3) with the corresponding white background
values. Chlorophyll content estimated by chemical extraction (ng/mm2) Robustness of the default parameters Our method is optimized for Arabidopsis seedlings
grown on agar plates, a common format for Arabidopsis
researchers. Since the default parameter values were gen-
erated using chlorophyll extracted seedlings grown under
one light condition, a concern is how well these values
apply to other growth conditions. To test this, we com-
pared chlorophyll estimation in two extreme growth con-
ditions. In our lab, the thickness of 4–7 week-old leaves
grown on soil under a long-day condition is almost twice
as seedling leaves grown on plates under constant low
light (176 vs. 100 µm, respectively). We compared chlo-
rophyll estimation of these thicker leaf pieces using the
default parameters to refitted parameters. The thicker
leaf pieces were imaged as described above and the
extracted chlorophyll used to refit the data to generate
new parameters: (4)
Chli = ea1ri+a2gi+a3bi+a4 Chli = ea1ri+a2gi+a3bi+a4 (4) to estimate the chlorophyll content where the (ri, gi, bi)
represents the R, G or B value for each sample where i
accounts for the sample index [5]. We used a biochemical
extraction with Lichtenthaler’s (Eq. 1) equations to meas-
ure the chlorophyll content and fitted coefficients for the
equation. We took samples from 4 independent experi-
ments (n = 234 samples; Additional file 11: S11) and
determined the coefficients: a1 through a4 in the equa-
tion (Eq. 4) using the least squares method in the MAT-
LAB environment (Fig. 2a, b). a1 = −0.2032,
a2 = 0.115409,
a3 = 0.044964,
a4 = 8.048844. The optimized parameters increased the R2 correlation
coefficient only from 0.85 to 0.89 (cf. Fig. 2c, d). This indi-
cates that the default values are robust with regard to dif-
ferent growth conditions that may affect leaf thickness. Nonetheless, when extreme accuracy is required, we rec-
ommend calculating the parameters by the “Do it your-
self” fitting method described above. Liang et al. Plant Methods (2017) 13:26 Page 7 of 10 Proof of utility: chlorophyll content of Arabidopsis
seedlings grown under different C/N ratios average leaf area changed. As quantitated in Additional
file 12: S12A, plant area at 0% glucose varied greatly but
was statistically unchanged. When the medium contains
glucose, plant area increased with increasing nitrogen. The optimal glucose concentration at the highest nitro-
gen concentration at 3 mM was 1–2% (Additional file 12:
S12 panel A).h In order to determine how plants respond to different
C/N ratios, we tested six different glucose concentrations
(0, 1, 2, 4, 5 and 6% d-glucose) and five nitrogen concen-
trations (0.1, 0.3, 0.5, 2 and 6 mM KNO3) in a matrix for-
mat. Figure 3 shows plant growth under these different
C/N ratios and Table 1 shows the plants area in response
to different C/N ratios. Plant area did not change in
response to nitrogen under 0% glucose, although the The chlorophyll content correlated with the nitrogen
concentration in the presence of carbon supply. We esti-
mated the chlorophyll content at different C/N ratio. As 0.1mM
0.3mM
0.5mM
2mM
6mM
0%
1%
2%
4%
5%
6%
NItrogen concentration
Glucose concentration
1cm
Fig. 3 Growth of the wild type Arabidopsis in response to C/N ratios. The image of the plate-grown plants under different C/N ratios, including six
different glucose concentration (0, 1, 2, 4, 5, and 6%) and five nitrogen (0.1, 0.3, 0.5, 2 and 6 mM KNO3) concentrations Fig. 3 Growth of the wild type Arabidopsis in response to C/N ratios. The image of the plate-grown plants under different C/N ratios, including six
different glucose concentration (0, 1, 2, 4, 5, and 6%) and five nitrogen (0.1, 0.3, 0.5, 2 and 6 mM KNO3) concentrations Liang et al. Plant Methods (2017) 13:26 Page 8 of 10 Table 1 Plant size (mm2) in response to glucose and nitro-
gen treatment
Growth condition and treatments are as described for Fig. 3. Data analysis is
performed by software SAS8.0. Single factor analysis (n = 12). Proof of utility: chlorophyll content of Arabidopsis
seedlings grown under different C/N ratios Capital letters
represent similarity groups (p > 0.05) among glucose treatment and lowercase
letters represent similarity groups among nitrogen treatments (p > 0.05)
Glucose
(w/v) (%)
Nitrogen concentration (mM)
0.1
0.3
0.5
2
6
0
8.94 a A
7.00 a A
13.59 a B
16.70 a DE
8.62 a D
1
5.93 d C
16.29 c AB
23.32 bc A
30.58 b BC
59.27 a A
2
6.74 d B
13.28 cd AB 21.20 c A
45.80 b A
55.73 a A
4
6.03 b B
9.02 b B
13.13 b B
31.85 a B
42.36 a B
5
6.75 b B
9.70 b AB
9.26 b B
22.32 a CD
23.60 a C
6
2.51 d D
6.20 cd C
8.15 bc B
11.59 b E
15.87 a CD Table 1 Plant size (mm2) in response to glucose and nitro-
gen treatment shown in Table 2, the chlorophyll content of the Col-0
seedlings grown on 0% glucose did not change with
increasing nitrogen in the medium. All the plates were
grown under continuous dim light (35–50 µEm−2 s−1)
at 23 °C, which decreases the photosynthesis. However,
even a slight amount of glucose dramatically changed
this relationship. For example, in the presence of 1% glu-
cose, the chlorophyll content increased in response to
the nitrogen concentration, and slightly decreased when
the nitrogen concentration was raised further. This indi-
cates that the chlorophyll content of the leaves is slightly
influenced by nitrogen concentration but highly influ-
enced by the carbon availability. Also, the chlorophyll
content increased as the glucose concentration increased;
the seedlings grown with 4% or 5% d-glucose had dark
green leaves. Based on plant area, optimal growth was at
1 and 2% glucose with 6 mM nitrogen. This condition did
not produce the highest chlorophyll content because the
seedlings were stressed. The highest concentration of 6%
glucose, with 0.1 mM nitrogen, stressed seedlings fur-
ther, as was apparent by the red color of leaves caused by
excessive anthocyanin pigments. Growth condition and treatments are as described for Fig. 3. Data analysis is
performed by software SAS8.0. Single factor analysis (n = 12). Capital letters
represent similarity groups (p > 0.05) among glucose treatment and lowercase
letters represent similarity groups among nitrogen treatments (p > 0.05) Table 2 Chlorophyll content (ng/mm2) in response to glu-
cose and nitrogen treatment
Data analysis is performed by software SAS8.0. Function of G protein signaling pathway in C/N sensing Function of G protein signaling pathway in C/N sensing
In order to analyze whether G protein signaling path-
way is important for C/N sensing/responsiveness, the
null mutants of the G protein under various C/N con-
dition were assayed. The leaf area differences between
the mutants of rgs1-2, gpa1-3 and agb1-2 is not uni-
form under different C/N conditions (Additional file 13:
S13). For example, agb1-2 mutants grown under limited Data analysis is performed by software SAS8.0. Single factor analysis (n = 12). Different capital letters indicated significant differences among six glucose
treatment (p < 0.05) and different lowercase letters indicated significant
differences among five nitrogen treatment (p < 0.05) Data analysis is performed by software SAS8.0. Single factor analysis (n = 12). Different capital letters indicated significant differences among six glucose
treatment (p < 0.05) and different lowercase letters indicated significant
differences among five nitrogen treatment (p < 0.05) Fig. 4 The regulatory pathway of G protein signaling in the C/N sensing evaluated by RGB value. a Proportion of green leaves of the G protein
mutants in response to nitrogen under 6% glucose. To distinguish from green and not green leaves, a threshold of was established (>15 ng/
mm2 = green) Experiments were repeated twice with 48 seedlings each. The curves were created through global curve fitting (sigmoid equation
with four parameters) with SigmaPlot 12.5. b The chlorophyll content estimated by RGB value under moderate C/N stress (4% glucose and 6 mM
nitrate, n = 40). For the box plot, solid line indicates the median and the dotted line indicates the mean value. Different lowercase letters indicate the
significant differences among six genotypes (p < 0.05) Fig. 4 The regulatory pathway of G protein signaling in the C/N sensing evaluated by RGB value. a Proportion of green leaves of the G protein
mutants in response to nitrogen under 6% glucose. To distinguish from green and not green leaves, a threshold of was established (>15 ng/
mm2 = green) Experiments were repeated twice with 48 seedlings each. The curves were created through global curve fitting (sigmoid equation
with four parameters) with SigmaPlot 12.5. b The chlorophyll content estimated by RGB value under moderate C/N stress (4% glucose and 6 mM
nitrate, n = 40). For the box plot, solid line indicates the median and the dotted line indicates the mean value. Proof of utility: chlorophyll content of Arabidopsis
seedlings grown under different C/N ratios Single factor analysis (n = 12). Different capital letters indicated significant differences among six glucose
treatment (p < 0.05) and different lowercase letters indicated significant
differences among five nitrogen treatment (p < 0.05)
Glucose
(w/v) (%)
Nitrogen concentration (mM)
0.1
0.3
0.5
2
6
0
49.10 a B
38.27 a C
49.93 a C
53.85 a C
42.71 a D
1
31.82 d C
81.49 c AB
112.26 ab A
120.08 a B
98.58 b C
2
37.31 d BC
70.00 c B
72.02 c BC
125.64 a AB
105.37 b BC
4
49.36 c B
84.27 b AB
89.73 b AB
134.22 a AB
140.03 a A
5
71.66 c A
96.63 b A
104.63 b A
138.28 a A
133.65 a AB
6
44.74 b BC
99.69 a A
111.85 a A
131.07 a AB
111.33 a ABC Table 2 Chlorophyll content (ng/mm2) in response to glu-
cose and nitrogen treatment Authors’ contributions YL designed experiments, collected the data, prepared the figures, and wrote
the manuscript. DU designed the experiments and edited the manuscript. KLL designed and wrote the Matlab code and fitted the model. TH designed
and wrote the java code for the plugin. AMJ and YG edited the manuscript. All
authors read and approved the final manuscript. Function of G protein signaling pathway in C/N sensing Different lowercase letters indicate the
significant differences among six genotypes (p < 0.05) Fig. 4 The regulatory pathway of G protein signaling in the C/N sensing evaluated by RGB value. a Proportion of green leaves of the G protein
mutants in response to nitrogen under 6% glucose. To distinguish from green and not green leaves, a threshold of was established (>15 ng/
mm2 = green) Experiments were repeated twice with 48 seedlings each. The curves were created through global curve fitting (sigmoid equation
with four parameters) with SigmaPlot 12.5. b The chlorophyll content estimated by RGB value under moderate C/N stress (4% glucose and 6 mM
nitrate, n = 40). For the box plot, solid line indicates the median and the dotted line indicates the mean value. Different lowercase letters indicate the
significant differences among six genotypes (p < 0.05) Liang et al. Plant Methods (2017) 13:26 Page 9 of 10 provides a good estimate of the relative chlorophyll
content for the Arabidopsis. nitrogen (0.1 mM) are slightly larger than the wild type
under 1 and 4% glucose but no difference under 2% glu-
cose; when the glucose increased to 5 and 6%, the plant
size of agb1-2 is smaller than wild type. Conclusion This method offer a non-destructive, sensitive, and
quantitative way to estimate the chlorophyll content
of Arabidopsis small seedlings grown on agar plates,
and it is an effective way to evaluate the growth condi-
tion of the plants. This method provides a quantitative
alternative to the qualitative ‘green’ versus not ‘green’
seedling assay [26, 27]. The use of the X-rite Color-
Checker chart eliminates differences between cameras
and environment, however, caution should be applied
in comparing images from highly different environ-
ments. Although this method is dependent on the
area of the seedlings, rather than the volume, it still Abbreviations
RGB
d RGB: red, green and blue channel of the color; r, g, b: the values of the red,
green and blue channels; r′, g′, b′: the correction value of the red, green and
blue channels; C/N: carbon to nitrogen ratio; MS medium: Murashige and
Skoog medium; MES: 2-(N-morpholino) ethanesulfonic acid. Additional files g
yp
We estimated the chlorophyll content in the G
protein mutants. The optimal concentration for the
growth of Arabidopsis grown on agar is 1–2% glucose
(Additional file 12: S12B). The G protein complex is
involve in glucose sensing [25] and AtRGS1 is a com-
ponent of a glucose sensor [11, 12, 18]. Previous stud-
ies showed that the rgs1-2 null mutant is tolerant to
high glucose levels [7], however that report used a
semi-quantitative method, the so-called “green-seed-
ling” assay. In order to compare the present quantita-
tive results to the published semi-quantitative results,
we established a threshold value of 15 ng/mm2 to
distinguish yellow seedlings from green seedlings
(Additional file 12: S12C; the threshold value was
from inner fence of agb1-2 mutants’ box plot). Tak-
ing rgs1-2 mutants as an example (Fig. 4a), the pro-
portion of green leaves was fourfold greater than wild
type when the nitrogen concentration was 0.1 mM
(0.22 and 0.05 for rgs1-2 and Col, respectively). When
the nitrogen concentration was increased to 1 mM,
the proportion of green leaves increased to 0.98 while
the wild type was 0.83. Consistent with the results of
Chen and Jones [7], the rgs1-2 mutants showed more
than 90% green seedlings versus wild type seedlings
which scored less than 40%. In addition, as previously
observed, the agb1-2 mutants contain less chloro-
phyll than wild type at high glucose and low nitrogen
growth conditions. Additional file 1: S1. Chlorophyll_imager.java. This is the .java compiler
for creating the .class file plug-in for imageJ to be used for the chlorophyll
reads. Additional file 2: S2. Chlorophyll_imager.class. This is the plug-in for
imageJ to be used for the chlorophyll reads. Additional file 3: S3. RGB_Measure.java This is the .java compiler for
creating the .class file plug-in for imageJ to be used for the the RGB reads. Additional file 4: S4. RGB_Measure.class. This is the plug-in for imageJ
used for the RGB reads. Additional file 5: S5. The chlorophyll content variation due to the posi‑
tion of the subject. Additional file 6: S6. Cleanbackground.jpg. An example of the cleaned
background JPG image. Additional file 7: S7. Greyscaleimage.jpg. The output greyscale images
created by imageJ indicated the chlorophyll content. Additional file 8: S8. The chlorophyll content estimated by RGB value for
Additional file 5: S5. Additional file 9: S9. EXAMPLE.xlsx. An example of the data set format
for the least square equation. Additional files Additional file 10: S10. Leastsquareequation.m Script written for Matlab
to apply the least square equation. Additional file 11: S11. Raw data for fitting. The original data used for
the chlorophyll content calculation with chemical extraction and RGB
value, including 4 independent experiment data of the plates grown
seedlings and 1 experiment for the plants grown in the soil. Additional file 11: S11. Raw data for fitting. The original data used for
the chlorophyll content calculation with chemical extraction and RGB
value, including 4 independent experiment data of the plates grown
seedlings and 1 experiment for the plants grown in the soil. Additional file 12: S12. The effect of different C/N ratios on Arabidopsis
seedlings growth and chlorophyll content. a Box plot shows plant sizes in
response to C/N ratio. Solid line indicates the median and the dotted line
indicates the mean value. b The plant leave area of 1 mM nitrogen under
different glucose concentrations. c The chlorophyll content of G protein
mutants under 0.2 mM nitrogen and 6% glucose. Additional file 12: S12. The effect of different C/N ratios on Arabidopsis
seedlings growth and chlorophyll content. a Box plot shows plant sizes in
response to C/N ratio. Solid line indicates the median and the dotted line
indicates the mean value. b The plant leave area of 1 mM nitrogen under
different glucose concentrations. c The chlorophyll content of G protein
mutants under 0.2 mM nitrogen and 6% glucose. We also examined the chlorophyll content of the G
protein mutants under moderate glucose and high nitro-
gen (4% glucose, 6 mM nitrogen) stress. Interestingly, as
shown in Fig. 4b, the chlorophyll content of the xlg and
agb1 mutants were significantly higher compared to the
wild type under slight stress, whereas the rgs1-2 mutant
was not significantly different from wild type. Additional file 13: S13. The effect of different C/N ratios on G protein
mutants. Growth condition and treatments are as described for Fig. 3. Data analysis was performed by software SAS8.0. Single factor analysis
(n = 48). Different capital letters indicate the similarity groups among four
genotypes (p < 0.05) and lowercase letters indicated the similarity groups
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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://crea‑
tivecommons.org/publicdomain/zero/1.0/) applies to the data made available
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in Arabidopsis. J Exp Bot. 2015;66:2763–71. • We accept pre-submission inquiries
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https://openalex.org/W4223417312
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http://pure-oai.bham.ac.uk/ws/files/168825682/juppa2022evidence.pdf
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Evidence for the encounter complex in frustrated Lewis pair chemistry
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Dalton transactions
| 2,022
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cc-by
| 8,221
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University of Birmingham University of Birmingham Evidence for the encounter complex in frustrated
Lewis pair chemistry
Jupp, Andrew R. DOI:
10.1039/D2DT00655C
License:
Creative Commons: Attribution (CC BY) Document Version
Publisher's PDF, also known as Version of record Citation for published version (Harvard):
Jupp, AR 2022, 'Evidence for the encounter complex in frustrated Lewis pair chemistry', Dalton Transactions,
vol. 51, no. 28, pp. 10681-10689. https://doi.org/10.1039/D2DT00655C Link to publication on Research at Birmingham portal Download date: 24. Oct. 2024 General rights
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Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the
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permitted by law. eely distribute the URL that is used to identify this publication. •Users may freely distribute the URL that is used to identify this publication. Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private
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Transactions View Article Online
View Journal Evidence for the encounter complex in frustrated
Lewis pair chemistry Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Andrew R. Jupp Frustrated Lewis Pairs (FLPs) are combinations of bulky Lewis acids and bases that can carry out small-
molecule activation and catalysis. Mechanistically, the reaction of the acid, base and substrate involves the
collision of three distinct molecules, and so the pre-association of the acid and base to form an encoun-
ter complex has been proposed. This article will examine the evidence for the formation of this encounter
complex, focusing on the archetypal main-group combinations P(tBu)3/B(C6F5)3 and PMes3/B(C6F5)3 (Mes
= mesityl), and includes quantum chemical calculations, molecular dynamics simulations, NMR spectro-
scopic measurements and neutron scattering. Furthermore, the recent discovery that the associated acid
and base can absorb a photon to promote single-electron transfer has enabled the encounter complex
to also be studied by UV-Vis spectroscopy, EPR spectroscopy, transient absorption spectroscopy, and
resonance Raman spectroscopy. These data all support the notion that the encounter complex is only
weakly held together and in low concentration in solution. The insights that these studies provide under-
pin the exciting transformations that can be promoted by FLPs. Finally, some observations and unan-
swered questions are provided to prompt further study in this field. Take down policy down policy
the University of Birmingham exercises care and attention in making items available there are rare occasions when an it
ded in error or has been deemed to be commercially or otherwise sensitive. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access
ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we
the work immediately and investigate. Download date: 24. Oct. 2024 School of Chemistry, University of Birmingham, Edgbaston, Birmingham, West
Midlands, B15 2TT, UK. E-mail: a.jupp@bham.ac.uk This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The Lewis base can theoretically interact with the substrate
(typified by H2 in Scheme 1A), and phosphine⋯H2 interactions
have been postulated in an argon matrix.26 However, for the
case of P(tBu)3 with H2, the interaction is computed to be
repulsive in the chemically relevant range.27 For certain small-
molecule substrates like CO2, the initial base⋯substrate inter-
action is more of a possibility, and strong Lewis bases such as
N-heterocyclic carbenes and imidazolin-2-ylidenamino-phos-
phines have been shown to bind CO2.28–31 Scheme 1B shows
the next alternative, where the substrate is initially bound to
the Lewis acid. This pathway is well established for certain
substrates; for example, the B(C6F5)3-catalysed hydrosilylation
of aromatic ketones, aldehydes and esters by Piers and co-
workers,32 where the borane activates the silane moiety,33 is
regarded as an early example of FLP chemistry.34 Furthermore,
an alkene⋯borane adduct has been observed at low tempera-
ture as an intermediate in FLP activation,35 and the zwitter-
ionic adduct of B(C6F5)3 with an alkyne was structurally charac-
terised recently.36 Regarding dihydrogen as the substrate, H2
has been shown to bind to BH3 in an argon matrix;37 HB
(C6F5)2 can undergo σ-bond metathesis with H2;38 and antiaro- It is worth noting at this stage that there are alternative
strategies for enhancing the pre-organisation of the acid and
base moieties in FLPs. The most widely employed approach is
tethering the two components with a covalent linker, known as
an
intramolecular
FLP. Erker’s
ethylene-bridged
FLP,
Mes2PCH2CH2B(C6F5)2, is a pioneering example of such a
system.41 Many different covalent linkers have been employed
in the ensuing years, and selected examples of intramolecular
FLPs include a simple methylene-bridged P/B system;42 a di-
methylxanthene-bridged system that enables the reversible
capture of N2O;43 a phenylene bridged N/B system that could
catalyse the selective Z-reduction of alkynes;44 a geminal S/B
species that could be activated by light;45 and even chiral
systems for asymmetric catalysis.46–48 An alternative approach
is for the acid and base to interact in a classical manner; there
are a number of systems that are capable of FLP-type reactivity
where the Lewis acid and base interact via a dative bond. Dalton Transactions Dalton Transactions Dalton Transactions Dalton Transactions matic boroles have been shown to activate H2.39,40 However,
calculations suggest that the interaction of H2 with B(C6F5)3 is
unfavourable due to Pauli repulsion.27 Dalton Trans. This journal is © The Royal Society of Chemistry 2022 Mechanism for FLP reactivity The mechanism for the FLP activation of small molecules like
dihydrogen has been widely discussed.22,23 The splitting of H2
by the cooperative action of a Lewis acid and Lewis base is a
three-component reaction. Although termolecular reactivity is
known in the literature,24,25 the probability of the acid, base
and substrate colliding at the same time in the correct orien-
tation for activation is low, and as such the pre-association of
two of the three components has been proposed to explain the
facile and rapid reactivity of FLPs (Scheme 1). Therefore, particularly for the activation of H2 with the
prototypical FLP combinations of P(tBu)3/B(C6F5)3 or PMes3/B
(C6F5)3 (Mes = mesityl), the prevailing theory is that depicted
in Scheme 1C, where there is a pre-association of the bulky
acid and base. This adduct is commonly referred to as the
encounter complex, and is held together by weak van der
Waals interactions between the substituents on the phosphine
and borane. Understanding the nature of the encounter
complex and the factors that affect its formation are critical to
rationalising and optimising subsequent FLP reactivity, and a
great deal of work has gone into studying this ephemeral
species. The fact that the adduct is only weakly held together
limited the studies of the encounter complex in the early days
of FLP chemistry to computational investigations, but recent
developments have gleaned key information using a range of
experimental techniques, including NMR spectroscopy, UV-Vis
spectroscopy, EPR spectroscopy, transient absorption spec-
troscopy, resonance Raman spectroscopy, and neutron scatter-
ing. These experimental breakthroughs are the focus of this
Frontier article. Introduction Combinations of a Lewis acid and a Lewis base typically form
adducts via a dative bond, as described by Gilbert Lewis in
his seminal work almost 100 years ago.1 Several exceptions to
this general rule have been found in the intervening years. In
1942, Brown and co-workers observed that 2,6-lutidine and
BMe3 do not form a Lewis adduct due to “steric interference”,2
and there are subsequent examples of non-quenching pairs
of acids and bases reacting with unsaturated substrates.3,4
However, it was the discovery by Stephan and co-workers in
2006 of a molecule containing discrete Lewis acidic and basic
sites that could reversibly activate dihydrogen that demon-
strated the true potential of these systems.5 The following
year, the term “Frustrated Lewis Pair” (FLP) was coined6 to
describe this general concept of a combination of a bulky
Lewis acid and Lewis base that is sterically precluded from
forming a Lewis adduct.7 The unquenched reactive acidic and
basic sites in FLPs have been exploited for a wide range of
small-molecule activation and catalysis, and this concept has
inspired research groups around the world to explore metal-
free approaches to reactions that were once considered the
preserve of transition metal complexes.8–13 These reactions
include the capture of environmentally relevant small mole-
cules such as CO2, N2O, and SO2;14–17 the catalytic hydrogen-
ation
of
unsaturated
organic
substrates;18–20
and
C–H
activation.21 School of Chemistry, University of Birmingham, Edgbaston, Birmingham, West
Midlands, B15 2TT, UK. E-mail: a.jupp@bham.ac.uk Andrew R. Jupp
Andy
obtained
his
Ph.D. (2012–2016) from the University
of Oxford under the supervision
of Prof. Jose Goicoechea, working
on phosphorus analogues of the
cyanate anion and urea. He sub-
sequently carried out a Banting
Postdoctoral
Fellowship
with
Prof. Doug
Stephan
at
the
University
of
Toronto
(2016–2018),
working
on
fru-
strated Lewis pairs (FLPs) and
the functionalisation of CO2. In
2018, he became an NWO VENI
laureate at the University of Amsterdam, working with Prof. Chris
Slootweg on the formation of main-group radicals in FLP systems. In 2020, he launched his independent career as a Birmingham
Fellow at the University of Birmingham (UK), working on small-
molecule activation and molecular photo-switches. He started a
Royal Society University Research Fellowship in January 2021. Andrew R. Jupp Andrew R. Jupp Andrew R. Jupp Andrew R. Jupp This journal is © The Royal Society of Chemistry 2022 Dalton Trans. View Article Online Frontier Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Examples of these systems include combinations of the Lewis
acid B(C6F5)3 with 2,6-lutidine,49 Et2O,50 1,4-dioxane,51 or the
proazaphosphatrane P(N(Me)CH2CH2)3N,52 where the classical
adduct is in equilibrium with the dissociated acid and base. Scheme 1
Mechanisms for the splitting of H2 by an FLP comprising a
bulky phosphine and borane as alternatives to true termolecular reactiv-
ity. A: pre-association of the phosphine and H2; B: pre-association of the
borane and H2; and C: pre-association of the phosphine and borane as
an encounter complex. However, this article will focus on the computational and
experimental evidence for the presence of the encounter
complex between discrete Lewis acids and bases to explain the
termolecular reactivity of FLPs. There are a very large number
of possible Lewis acids and bases; there have been interesting
studies
looking
at
N-heterocyclic
carbene/borane
combi-
nations, although a large proportion of these systems either
form a normal Lewis adduct or undergo a range of decompo-
sition pathways via C–H or C–F activation, which limits the
possibility of studying the encounter complexes in these
systems experimentally.53–55 Recently, the trioxatriangulenium
ion was explored as a carbon-centred Lewis acid in FLP chem- Scheme 1
Mechanisms for the splitting of H2 by an FLP comprising a
bulky phosphine and borane as alternatives to true termolecular reactiv-
ity. A: pre-association of the phosphine and H2; B: pre-association of the
borane and H2; and C: pre-association of the phosphine and borane as
an encounter complex. This journal is © The Royal Society of Chemistry 2022 Dalton Trans. View Article Online Dalton Transactions Frontier Fig. 1
The phosphines and borane in the prototypical encounter com-
plexes that are the focus of this article. explored the activation strain model-energy decomposition
analysis as a tool to probe reactivity in FLPs.64,65 Crucial to
these theories is the formation of the pre-organised encounter
complex stabilised by weak dispersion interactions with a reac-
tive “pocket” available for the small-molecule substrate to be
activated. A further computational study by Vankova and co-workers
corroborated the formation of the encounter complex is ener-
getically favourable, with an average association energy across
a range of systems of ΔEassoc = −10 kcal mol−1.66 Incorporating
solvent effects (toluene) using a polarisable continuum model
led to only small changes in the association energy (less than
1 kcal mol−1). However, the favourable electronic interactions
in the encounter complex are counterbalanced by the entropic
cost of adduct formation. Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1
The phosphines and borane in the prototypical encounter com-
plexes that are the focus of this article. istry, and the association with different phosphines was
probed.56 To focus the discussion and explore the evidence for
the encounter complexes in more detail, the FLPs in this
article will be limited to P(tBu)3/B(C6F5)3 or PMes3/B(C6F5)3
(Fig. 1), as these are the most commonly used FLPs and have
significance in a wide range of catalytic applications. To move beyond the static computational models used in
quantum chemical calculations, Pápai and co-workers used
molecular dynamics (MD) simulations to probe the encounter
complex of P(tBu)3/B(C6F5)3 in toluene.67 The model system
comprised one borane, one phosphine, and 1011 toluene
molecules in a periodically repeated cell, which represents a
relatively dilute solution compared to a typical experimental
set-up. The Helmholtz free energy curve (see F(r) in Fig. 2)
showed that the formation of the encounter complex is dis-
favoured; the structures with a P⋯B distance in the chemically
useful range of 4.2–5.6 Å were approximately 1.2 kcal mol−1
higher in energy than the dissociation limit. The probability of
finding a configuration with a P⋯B distance of less than 6 Å
(see C(r) curve in Fig. 2 for cumulative probability of P(r)) was
calculated to be roughly 2%, and those configurations featur-
ing the optimal P⋯B distance of 4.5 Å were only present 0.5%
of the time. This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Entropy is the dominant factor at
room temperature, and the formation of the encounter
complex is endergonic (ΔGassoc = +5 ± 2 kcal mol−1), which is
consistent with the difficulty in observing these species in the
laboratory.66 Computational models of the
encounter complex The original research into the encounter complex was compu-
tational in nature. A seminal report from Pápai and co-workers
first suggested the formation of the encounter complex in FLP
chemistry.27 They identified a weakly associated [P(tBu)3]⋯[B
(C6F5)3] adduct as a minimum on the potential energy surface,
using both B3LYP and SCS-MP2 methods. There was no evi-
dence of charge transfer from the phosphine lone pair into the
vacant orbital on boron, as shown by the planarity of the
central BC3 unit in B(C6F5)3. Furthermore, the P⋯B distance at
the energetic minimum was 4.2 Å (for the SCS-MP2 calcu-
lation); for comparison, the sum of the covalent radii would
predict a P–B bond length of 1.96 Å in a Lewis adduct.57
Instead, the adduct is stabilised by a combination of multiple
C–H⋯F non-covalent interactions, and in the absence of any
solvent modelling the association energy was calculated as
ΔEassoc = −11.5 kcal mol−1. The nature of the encounter
complex being stabilised by many weak dispersion interactions
means that there is a degree of structural flexibility, and it was
shown that large changes in the P⋯B distance can be achieved
at a relatively small energetic cost. Fig. 2
Free energy curve, F(r), and probability distribution, P(r), com-
puted from MD simulations of P(tBu)3/B(C6F5)3 in toluene. Reproduced
from ref. 67 with permission from the Royal Society of Chemistry. The significance of dispersion interactions to stabilise the
encounter complex was also supported by a number of other
studies.55,58,59 Note that there are contrasting theories for the
mechanism of the activation of dihydrogen by the FLP.60 Pápai
proposed an electron transfer mechanism based on synergistic
interactions of the donor and acceptor orbitals on the base
and acid with the acceptor and donor orbitals on dihydrogen,
respectively,27,61 whereas Grimme has proposed an electric
field model, where the H–H bond is polarised by a strong elec-
tric field generated between the donor/acceptor atoms.58,62 A
more recent publication has sought to unify these two mecha-
nisms,63 and very recently, Fernández and co-workers have Fig. 2
Free energy curve, F(r), and probability distribution, P(r), com-
puted from MD simulations of P(tBu)3/B(C6F5)3 in toluene. Reproduced
from ref. 67 with permission from the Royal Society of Chemistry. This journal is © The Royal Society of Chemistry 2022 Dalton Trans. Dalton Trans. Fig. 3
19F,1H HOESY NMR spectrum of PMes3/B(C6F5)3 in benzene-d6,
showing cross-peaks arising from all fluorine and proton environments. Computational models of the
encounter complex Adapted with permission from ref. 68. Copyright 2014 American
Chemical Society. Dalton Transactions
View Article Online View Article Online Dalton Transactions Frontier Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3
19F,1H HOESY NMR spectrum of PMes3/B(C6F5)3 in benzene-d6,
showing cross-peaks arising from all fluorine and proton environments. Adapted with permission from ref. 68. Copyright 2014 American
Chemical Society. A further complication for exploring the encounter complex
experimentally is that for one of the FLP combinations that we
are discussing, P(tBu)3/B(C6F5)3, there is a competing side-reac-
tion between the two components to form the ion pair [HP
(tBu)3][FB(C6F5)3] and the intramolecular species (tBu)2P(C6F4)
B(C6F5)2
with
elimination
of
isobutylene
(Scheme
2).70
However, despite these hurdles, there have been some recent
breakthroughs
in
the
characterisation
of
the
encounter
complex that will be explored below. any preferred directionality. The kinetics of NOE build-up and
a comparison of the relative strengths of the NOEs within
PMes3/B(C6F5)3 with computational predictions strongly indi-
cate that the two components have a random relative orien-
tation. This result suggests that the aggregation of the acid
and base in solution is dominated by intramolecular H/F inter-
actions, and not due to donation of the phosphine lone pair
into the vacant p orbital on the borane. This hypothesis was
corroborated with computations that revealed there was only
1 kcal mol−1 between the two limiting structures of PMes3/B
(C6F5)3 shown in Fig. 4, and the two extremes were roughly
equally likely to exist in solution. Grimme and co-workers
carried out a comprehensive computational investigation to
explore this further using state-of-the-art quantum chemistry
methods, building on the NMR spectroscopic data, and
extended the study to P(tBu)3/B(C6F5)3.71 The authors high-
lighted the importance of accurately modelling the dispersion
interactions, and showed that for PMes3/B(C6F5)3 there is little
energetic difference between the two extreme orientations
depicted in Fig. 4 across a range of different methods, in agree-
ment with experiment. However, for P(tBu)3/B(C6F5)3, the
orientation labelled geometry a in Fig. 4 is energetically
favoured by 3–5 kcal mol−1 (or 1–2 kcal mol−1 in free energies)
compared to geometry b, indicating that the association is less Initial observation attempts The weak stabilisation of the encounter complex in solution
hampered early attempts to observe this species experi-
mentally. It was reported that equimolar benzene or toluene
solutions of P(tBu)3/B(C6F5)3 or PMes3/B(C6F5)3 gave no indi-
cation of any interactions by 1H, 11B, 19F, or 31P NMR spec-
troscopy, and in each case the NMR spectra for the FLP looked
the
same
as
those
of
the
individual
components.7,68
Furthermore, isothermal reaction calorimetry performed by
Houghton and Autrey revealed that no appreciable heat was
produced
on
mixing
PMes3
and
B(C6F5)3
in
dichloro-
methane.69 This detailed calorimetric study concluded that
the activation of H2 by this FLP is best modelled as a termole-
cular reaction with a rate-determining step of assembling the
reactants into the solvent cage in the correct configuration. This journal is © The Royal Society of Chemistry 2022 Neutron scattering More recently, Swadźba-Kwaśny and co-workers carried out
further NMR experiments on the FLP combination P(tBu)3/B
(C6F5)3.73 Interestingly, their data show a clear change in the
chemical shifts of the 19F NMR resonances of free B(C6F5)3 in
benzene-d6 versus the same resonances in a 1 : 1 mixture of
P(tBu)3/B(C6F5)3 in benzene-d6, consistent with a small amount
of P⋯B interaction. In a bid to determine whether ionic
liquids can increase the concentration of the encounter
complex in solution, they also carried out the same analysis of
P(tBu)3/B(C6F5)3 using the ionic liquid [C10mim][NTf2] as the
solvent (Fig. 5). The 19F NMR chemical shifts of free B(C6F5)3
are significantly different when dissolved in [C10mim][NTf2]
compared to benzene-d6, consistent with interaction of the
Lewis acidic borane with one of the components of the ionic
liquid. There are undoubtedly new 19F and 31P NMR reso-
nances that appear when P(tBu)3/B(C6F5)3 is dissolved in
[C10mim][NTf2]
compared
to
the
individual
components;
according to the study, 24% of the B(C6F5)3 is no longer “free”
(and this 24% is split across at least three different environ-
ments), while 78% of the P(tBu)3 is also in a new environment. The authors state that although it is not possible to make
definitive assignments for these new resonances, they could be
attributed to the interaction between the FLP components in
the encounter complex, which is stabilised to a greater extent
in the ionic liquid compared to benzene-d6. This notion was
explored computationally in a further MD study by Liu and co-
workers, comparing the association of P(tBu)3/B(C6F5)3 in
toluene and a range of ionic liquids.74 They showed that in
general the ionic liquids led to an enhanced probability of the
phosphine and borane being associated with each other; the
computed probability of finding P(tBu)3/B(C6F5)3 with a P⋯B
distance of ≤6 Å in toluene was 2.32% (similar to the pre-
viously discussed value of 2% (ref. 67)), whereas this prob-
ability increased to 4.75–5.15% in the majority of the ionic
liquids probed. For one of the ionic liquids, specifically Swadźba-Kwaśny and co-workers also explored the nature of
the association of P(tBu)3/B(C6F5)3 using neutron scattering
experiments.73 Neutron diffraction has previously been used to
study structure and solvation,75 and when combined with
empirical potential structure refinement (EPSR) models can
provide insight into complex systems. NMR spectroscopy The first report with unequivocal experimental evidence in
support of the encounter complex was a comprehensive NMR
spectroscopic study by Rocchigiani et al.68 They performed
19F,1H
HOESY
(Heteronuclear
Overhauser
Enhancement
Spectroscopy) experiments on concentrated (220–230 mM)
samples of P(tBu)3/B(C6F5)3 or PMes3/B(C6F5)3 in benzene or
toluene, and clear cross-peaks corresponding to H/F inter-
actions could be observed (Fig. 3). Furthermore, addition of a
substoichiometric amount of B(C6F5)3 to a concentrated solu-
tion of PMes3 (relative ratio phosphine : borane of 57 : 1)
resulted in marginal shifts of the 19F resonances relative to the
free borane, and substantial line-broadening of the para-F. The
temperature-dependent broadening of the
19F NMR reso-
nances of B(C6F5)3 in the presence of excess PMes3 at low
temperature but not as the free borane is also consistent with
aggregation of the phosphine and borane in solution. The study also explored the relative orientation of the phos-
phine and borane in this aggregate to ascertain if there was Fig. 4
The two limiting geometries for the association of a phosphine
with B(C6F5)3, with the P lone pair oriented “towards” or “away” from the
p orbital on B. Scheme 2
Possible side-reaction between P(tBu)3 and B(C6F5)3. Fig. 4
The two limiting geometries for the association of a phosphine
with B(C6F5)3, with the P lone pair oriented “towards” or “away” from the
p orbital on B. Scheme 2
Possible side-reaction between P(tBu)3 and B(C6F5)3. This journal is © The Royal Society of Chemistry 2022 Dalton Trans. View Article Online View Article Online Dalton Transactions Frontier [C6mim][CTf3], the same probability actually decreased to
1.08%, reflecting a decreased stability of the encounter
complex in this case, so careful consideration of the nature of
the ionic liquid is required. The authors propose that the ionic
liquids pack together and leave large cavities that the encoun-
ter complex can accommodate, whereas toluene molecules
move in between and separate the acid and base. These
articles highlight the potential of ionic liquids as a tool to
better study the encounter complex in FLP chemistry, although
further work is required to unambiguously identify the
encounter complex in these systems. driven by dispersion interactions, and that there is a small
amount of P⋯B bonding present. Neutron scattering The measurements were
carried out on equimolar solutions in benzene at 160 mmol
concentration, which is relatively low for standard neutron
scattering experiments, and resulted in poor resolution for the
specific P⋯B interactions and significant variability between
different
refinement
runs. Nevertheless,
data
could
be
obtained that provided evidence for association of the phos-
phine and borane molecules in solution. There was <1%
chance of finding the P(tBu)3/B(C6F5)3 with a P⋯B distance of
5.7 Å, but this increased to 4.9% chance at P⋯B separations of
<8 Å. These data are consistent with the weak association of
the acid and base in solution, and highlight the utility of
neutron
diffraction
for
directly
observing
the
encounter
complex. NMR spectroscopy This non-negligible P⋯B
interaction in P(tBu)3/B(C6F5)3 has previously been discussed
in terms of the frontier orbitals.72 Rocchigiani and co-workers quantified the propensity of
PMes3/B(C6F5)3 to associate in solution using diffusion 19F and
1H NMR spectroscopy; the average association constant was
determined to be K = 0.5 ± 0.2 M−1, meaning that formation of
the encounter complex is slightly endergonic (ΔG° = +0.4 ±
0.2 kcal mol−1). These results are consistent with the pre-
viously
discussed
data
from
molecular
dynamics
simulations.67 This journal is © The Royal Society of Chemistry 2022 Equivalence with the electron donor–acceptor complex Recent experimental evidence for the encounter complex has
arisen
from
the
blossoming
field
of
frustrated
radical
pairs.76–79 The single-electron transfer (SET) from the Lewis
base to the Lewis acid to form the radical pair was first postu-
lated by Piers and co-workers, although it was discounted as a
viable mechanism in FLP chemistry due to the large discre-
pancy in redox potentials between P(tBu)3 and B(C6F5)3.80 A
breakthrough from Stephan and co-workers was published in
2017, where they studied the FLP combination PMes3/B(C6F5)3
and saw evidence of the radical cation [PMes3]•+, and postu-
lated that the radical ion pair is in thermal equilibrium with
the phosphine and borane.76 The [PMes3]•+ could be observed
by EPR spectroscopy, although the corresponding [B(C6F5)3]•−
radical was not observed, and this was attributed to the known
and rapid decomposition of this radical anion via solvolytic
pathways.81,82 Fig. 5
Structure of ionic liquid [C10mim][NTf2]. In light of the contradictory evidence above, Slootweg and
co-workers sought to better understand the SET process in
FLPs.83,84 Mulliken theory describes the interaction of an elec-
tron-rich donor (D) and an electron-poor acceptor (A) to form
an electron donor–acceptor complex [D,A], which can sub-
sequently absorb a photon of the correct energy to promote Fig. 5
Structure of ionic liquid [C10mim][NTf2]. This journal is © The Royal Society of Chemistry 2022 Dalton Trans. Dalton Trans. View Article Online Dalton Transactions Frontier Dalton Transactions Frontier Fig. 6
Mulliken theory for electron transfer in electron donor–acceptor
adducts, with analogous terms relevant to FLP chemistry highlighted in
bold. Fig. 6
Mulliken theory for electron transfer in electron donor–acceptor
adducts, with analogous terms relevant to FLP chemistry highlighted in
bold. mation was obtained by EPR spectroscopy and transient
absorption spectroscopy (Fig. 7b and c). Irradiation of the
sample at the appropriate wavelength gave characteristic
signals of the radical pair, while the same signals were not
present in the analogous experiments performed in the dark. The back-electron transfer to regenerate the neutral phosphine
and borane was rapid, and the lifetime of this radical pair was
only 237 ps. These same analyses were also carried out on the
FLP P(tBu)3/B(C6F5)3, which is pale yellow in toluene, and
showed the presence of a new absorption band at λ = 372 nm. Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6
Mulliken theory for electron transfer in electron donor–acceptor
adducts, with analogous terms relevant to FLP chemistry highlighted in
bold. Fig. 6
Mulliken theory for electron transfer in electron donor–acceptor
adducts, with analogous terms relevant to FLP chemistry highlighted in
bold. Fig. 6
Mulliken theory for electron transfer in electron donor–acceptor
adducts, with analogous terms relevant to FLP chemistry highlighted in
bold. SET and afford the charge-transfer state [D•+,A•−] (Fig. 6).85–87
Relating these concepts to FLPs, Lewis acids are acceptors, and
Lewis bases are electron donors, and therefore the encounter
complex that we have been discussing is simply another name
for the electron donor–acceptor complex. SET and afford the charge-transfer state [D•+,A•−] (Fig. 6).85–87
Relating these concepts to FLPs, Lewis acids are acceptors, and
Lewis bases are electron donors, and therefore the encounter
complex that we have been discussing is simply another name
for the electron donor–acceptor complex. The charge transfer in PMes3/B(C6F5)3 was further studied
by resonance Raman spectroscopy by Ando and co-workers.88
Resonance Raman spectroscopy can provide information on
vibrational modes that are associated with a particular elec-
tronic transition. The authors showed that the Raman spec-
trum of the FLP was the same as the superposition of the
spectra of the individual components, consistent with the pre-
vious spectroscopic evidence that there is very limited inter-
action between the acid and base in solution. The resonance
Raman spectrum of the FLP in CH2Cl2 (irradiated at λ =
457 nm) did show some enhancement of certain bands com-
pared to the normal Raman spectrum, and as these vibrational
modes were associated with both the borane and the phos-
phine, it was concluded that there must be some association
of the two components in solution. A toluene solution of PMes3/B(C6F5)3 is violet in colour,
despite the individual components each being colourless in
solution. This was noted in 2007 by Stephan, where they postu-
lated that the colour arose from π-stacking of the aryl rings on
the phosphine and borane.7 Then in 2017, it was postulated
that the colour is due to a low concentration of the [PMes3]•+
radical cation in equilibrium with the FLP.76 In 2020, Slootweg
and co-workers proposed the violet colour is actually due to a
charge-transfer
band,
where
the
electron
donor–acceptor
complex (i.e. Equivalence with the electron donor–acceptor complex The P(tBu)3/B(C6F5)3 samples were always freshly prepared and
quickly analysed to mitigate the complication in this particular
FLP system of the previously discussed side-reaction that
occurs between the acid and base as much as possible
(Scheme 2); Piers and co-workers have shown that the (tBu)2P
(C6F4)B(C6F5)2 product is also yellow.70 Irradiation of this new
absorption band also showed diagnostic signals corresponding
to the radical pair in the EPR spectrum, and transient absorp-
tion spectroscopy revealed a lifetime of only 6 ps. Acknowledgements The first unambiguous evidence for the encounter complex
in solution came from NMR measurements,68 and showed that
the association of PMes3/B(C6F5)3 had no preferred orien-
tation, which is consistent with the association being driven
by dispersion interactions. An experimental value for the
association constant of this FLP combination was obtained
from the data (K = 0.5 ± 0.2 M−1), which supported the ender-
gonic nature of the encounter complex. The concept of using
ionic liquids as the solvent to better stabilise the encounter
complex has been explored, which could open up new avenues
for aiding characterisation of the encounter complex and pro-
moting FLP reactivity.73 Neutron scattering measurements
have also provided direct evidence for the association of
P(tBu)3/B(C6F5)3 in benzene that is consistent with previous
studies.73 A. R. J. would like to thank his mentors and colleagues for all
their support in his career so far, and particularly to Prof. Doug Stephan, Dr Tim Johnstone, and Prof. Chris Slootweg for
the stimulating discussions about FLPs and their mode of
reactivity that have prompted this article. A. R. J. would also
like to thank the Royal Society (URF\R1\201636) and the
School of Chemistry at the University of Birmingham for
funding. Dalton Transactions Frontier • Can we find an experimental probe to measure the con-
centration of “active” encounter complex in solution, i.e. those
combinations that are oriented for small-molecule activation,
instead of including the non-directional and non-productive
orientations? Lewis acid. These analytical methods all provide direct evi-
dence of the encounter complex in solution, as SET is only
possible when the acid and base are in close proximity with a
suitable orbital arrangement. This finding could unlock more
ways for researchers to probe and understand the encounter
complex in the future. • Can the substituents around the Lewis acidic and Lewis
basic centres be tuned to increase the concentration of the
encounter complex in solution? Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. • Can we correlate concentration of encounter complex
with catalytic activity for different FLP combinations, and
therefore design FLP systems that are more active? The encounter complex is a key concept for rationalising the
three-component reactivity of FLPs with substrates such as
dihydrogen. The initial studies were limited to computational
models,27,58,60,66,67,71,72 which showed that the association of
the bulky Lewis acid and base is driven by dispersion inter-
actions between the large substituents on each molecule. For
example, the encounter complexes of PMes3/B(C6F5)3 and
P(tBu)3/B(C6F5)3
are
stabilised
by
multiple
C–H⋯F
non-
covalent interactions, although some studies have also shown
a small P⋯B interaction in the latter. The electronic stabilis-
ation of the encounter complex is opposed by the decrease in
entropy, leading to the formation of this adduct being
unfavourable
according
to
free
energy
calculations. The
encounter complex therefore has a low concentration in solu-
tion, which hampered early attempts to characterise this
species experimentally. The encounter complex is a key concept for rationalising the
three-component reactivity of FLPs with substrates such as
dihydrogen. The initial studies were limited to computational
models,27,58,60,66,67,71,72 which showed that the association of FLPs have unlocked
reactivity and
catalysis that
was
unthinkable by main-group compounds only twenty years ago,
and will undoubtedly continue to provide new and sustainable
routes to fundamental transformations. A thorough under-
standing of how the Lewis acids and bases associate and inter-
act with small molecules in solution will be essential to
driving this area of chemistry forward. FLPs have unlocked
reactivity and
catalysis that
was
unthinkable by main-group compounds only twenty years ago,
and will undoubtedly continue to provide new and sustainable
routes to fundamental transformations. A thorough under-
standing of how the Lewis acids and bases associate and inter-
act with small molecules in solution will be essential to
driving this area of chemistry forward. Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. encounter complex) can absorb a photon in the
visible region to promote SET from the phosphine to the
borane.83 This theory was supported computationally, as time-
dependent density functional theory on the PMes3/B(C6F5)3
encounter complex revealed a band corresponding to this elec-
tronic transition. UV-Vis spectroscopy confirmed the presence
of an absorption band at λ = 534 nm (Fig. 7a). Experimental
verification that absorption of this band led to radical for- These spectroscopic measurements, namely UV-Vis, EPR,
transient absorption, and resonance Raman spectroscopy, all
arise from the charge transfer between the Lewis base and the Fig. 7
Evidence for encounter complex of PMes3/B(C6F5)3 due to SET processes (B(C6F5)3 is abbreviated as BCF in the figure above, as this is how it
appears in the original article): (a) UV-Vis spectrum of toluene solution of PMes3/B(C6F5)3 compared to spectra of individual components; (b) experi-
mental EPR spectrum of toluene solution of PMes3/B(C6F5)3 measured at 30 K during irradiation with visible light (390–500 nm) and simulated
spectra of [PMes3]•+ and [B(C6F5)3]•−; (c) transient absorption spectra measured after pulsed excitation of PMes3/B(C6F5)3 with 530 nm light. Figure from ref. 83 used with permission from John Wiley and Sons. Copyright 2020 Wiley. Fig. 7
Evidence for encounter complex of PMes3/B(C6F5)3 due to SET processes (B(C6F5)3 is abbreviated as BCF in the figure above, as this is how it
appears in the original article): (a) UV-Vis spectrum of toluene solution of PMes3/B(C6F5)3 compared to spectra of individual components; (b) experi-
mental EPR spectrum of toluene solution of PMes3/B(C6F5)3 measured at 30 K during irradiation with visible light (390–500 nm) and simulated
spectra of [PMes3]•+ and [B(C6F5)3]•−; (c) transient absorption spectra measured after pulsed excitation of PMes3/B(C6F5)3 with 530 nm light. Figure from ref. 83 used with permission from John Wiley and Sons. Copyright 2020 Wiley. This journal is © The Royal Society of Chemistry 2022 Dalton Trans. View Article Online This journal is © The Royal Society of Chemistry 2022 Conflicts of interest There are no conflicts to declare. Open Access Article. Published on 06 April 2022. Downloaded on 5/3/2022 3:36:10 PM.
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522–525. Dalton Trans.
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Optical frequency combs for low phase noise microwave generation
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1. Introduction Combining a femtosecond laser optical frequency comb (OFC) with the high quality factor (Q) available in
optical cavities allows for the possibility of microwave signals with phase noise below state-of-the-art electronic
oscillators. The low loss of optical Fabry-Perot (FP) resonators leads to Q values exceeding 1011, greater than
microwave dielectric resonator oscillators at either room (Q ~105) or cryogenic temperatures (Q ~109). When an
optical frequency comb is phase-locked to an optical reference, the comb can be thought of as a high fidelity
frequency divider, transferring the stability of the optical reference to the microwave domain [1-3]. The division in
frequency leads to a division in phase noise, where the phase noise power spectral density scales as
⁄
Combining a femtosecond laser optical frequency comb (OFC) with the high quality factor (Q) available in
optical cavities allows for the possibility of microwave signals with phase noise below state-of-the-art electronic
oscillators. The low loss of optical Fabry-Perot (FP) resonators leads to Q values exceeding 1011, greater than
microwave dielectric resonator oscillators at either room (Q ~105) or cryogenic temperatures (Q ~109). When an
optical frequency comb is phase-locked to an optical reference, the comb can be thought of as a high fidelity
frequency divider, transferring the stability of the optical reference to the microwave domain [1-3]. The division in
frequency leads to a division in phase noise, where the phase noise power spectral density scales as
⁄
. Here, is the phase noise and
is the optical-to-microwave division ratio. Optical references with a
fractional frequency stability of <3·10-16 (at 1second) have been demonstrated [4], ideally generating a 10 GHz
signal with phase noise of <-110 f -3 dBc/Hz. At 1 Hz offset from the carrier, this represents a 40 dB improvement
over the best room temperature 10 GHz electronic microwave oscillators [5], and is below what has been
demonstrated with cryogenic 10 GHz oscillators [6, 7]. Work with Er:fiber-based frequency combs has shown a
residual phase noise in optical-to-microwave conversion of -118 dBc/Hz at 1 Hz offset from a 11.55 GHz carrier [2]. Recently, we demonstrated absolute phase noise below -104 dBc/Hz at 1 Hz offset from a 10 GHz carrier by
comparing two independent systems that employ 1 GHz mode-locked Ti:sapphire laser OFCs [3]. Abstract An optical frequency comb locked to a stable optical reference can serve as a source for microwave signals having
very low close-to-carrier phase noise. This has recently been confirmed by comparing two independent systems,
yielding an absolute phase noise of -104 dBc/Hz at 1 Hz offset from a 10 GHz carrier. The corresponding timing
jitter is 760 attoseconds, integrated from 1 Hz to 1 MHz. Here we describe the system architecture, as well as
technical and fundamental noise limitations. 1. Introduction This represents
one of the lowest close-to-carrier phase noise measurements yet reported on a 10 GHz source. The ability to
optically synthesize pure tones or even waveforms with low phase noise in the microwave, millimeter-wave or even
terahertz domains benefits a number of scientific and signal processing applications, including high speed analog-to-
digital conversion, remote synchronization, local oscillators for fountain clocks, coherent control over quantum
mechanical processes, and photonically enabled generation and processing of ultrabroadband radiofrequency
electrical signals. National Institute of Standards and Technology, 325 Broadway, Boulder CO 80305, Ph. 303-497-3295, Fax: 303-
497-6461 National Institute of Standards and Technology, 325 Broadway, Boulder CO 80305, Ph. 303-497-3295, Fax: 303-
497-6461 U.S. Government work not protected by U.S. copyright 2Current address: East China Normal University, Shanghai, 200062, China 1fquinlan@boulder.nist.gov, 3sdiddams@boulder.nist.gov 2. Optical Frequency Comb as Optical Frequency Divider The OFC in time and frequency domains is shown in Fig. 1(a), and a schematic of the optical frequency
divider architecture is shown in Fig. 1(b). For low noise microwave generation, an optical mode of the OFC is
phase-locked to a laser that is stabilized to the FP cavity. Pulse formation via passive mode-locking enforces a
constant phase among the laser modes. As a result, stabilizing one mode to the optical reference will transfer its
stability to every optical mode of the comb [8]. The equation describing the frequency of the comb lines then
becomes U.S. Government work not protected by U.S. copyright (1)
·
(1) where
is the frequency of the optical reference, is the combline spacing (or pulse repetition rate),
is an
offset frequency common to all the modes of the OFC, and is the difference or beat frequency between the optical
reference and a comb mode. We may therefore write as
⁄
(2) (2) Thus represents the frequency-divided optical reference (most directly seen in the special case of
and
equal to zero). Accessing is achieved by photodetecting the optical pulse train to generate a corresponding
electrical pulse train. In the frequency domain, this train of electrical pulses is a series of discrete tones at harmonics
of the pulse repetition rate. Any harmonic of within the photodetector bandwidth can then be selected as a low
noise microwave source. Figure1 (a) The optical frequency comb in time and frequency domains. Photodetection of the optical pulse train results in a train
of electrical pulses. The electrical pulse spectrum is an array of discrete tones at harmonics of the pulse repetition rate. (b)
Architecture for microwave generation from an optical reference. When locked to an optical reference, the OFC acts as a high
fidelity frequency divider. Figure1 (a) The optical frequency comb in time and frequency domains. Photodetection of the optical pulse train results in a train
of electrical pulses. The electrical pulse spectrum is an array of discrete tones at harmonics of the pulse repetition rate. (b)
Architecture for microwave generation from an optical reference. When locked to an optical reference, the OFC acts as a high
fidelity frequency divider. 3. Low Phase Noise Microwaves Although we may consider the microwave signal as the frequency-divided optical reference, the noise of the
resulting microwave signal is not from the optical reference alone. Also contributing are residual noise on the phase
lock between the comb and the optical reference, phase noise on the comb offset frequency, phase noise added by
the link between the OFC and photodetector, and phase noise added during photodetection. As shown in Fig. 2, from
low offset frequency to high offset frequency, the noise is in turn dominated by the optical reference, an unstabilized fiber link, the OFC, and photodetection noise. For comparison, the phase noise, scaled to a 10 GHz carrier, of an
optical reference at 518 THz is also shown in Fig. 2. This would be the microwave phase noise level if it were
determined only by the optical reference. fiber link, the OFC, and photodetection noise. For comparison, the phase noise, scaled to a 10 GHz carrier, of an
optical reference at 518 THz is also shown in Fig. 2. This would be the microwave phase noise level if it were
determined only by the optical reference. Fig. 2 Phase noise on an optically derived 10 GHz signal and contributing noise sources. Black solid line: measured absolute
phase noise at 10 GHz. The corresponding timing jitter is 760 attoseconds (1 Hz – 1 MHz integration). Red dotted curve: phase
noise of an optical reference, scaled to a 10 GHz carrier. Fig. 2 Phase noise on an optically derived 10 GHz signal and contributing noise sources. Black solid line: measured absolute
phase noise at 10 GHz. The corresponding timing jitter is 760 attoseconds (1 Hz – 1 MHz integration). Red dotted curve: phase
noise of an optical reference, scaled to a 10 GHz carrier. References [1]
J. J. McFerran, et al., "Low noise synthesis of microwave signals from an optical source," IEEE Electron. Lett., vol. 41,
2005. W. Zhang, et al., "Sub-100 attoseconds stability optics-to-microwave synchronization," Applied Physics Lett
96, May 2010. [3]
T. M. Fortier, et al., "Photonic Generation of Ultrastable 10 GHz Microwave Signals," submitted for publication, 2011. [4]
Y. Y. Jiang, et al., "Making optical atomic clocks more stable with 10-16-level laser stabilization," Nat Photon, vol. 5,
pp. 158-161, 2011. pp
E. N. Ivanov and M. E. Tobar, "Low Phase-Noise Sapphire Crystal Microwave Oscillators: Current Status,
Transactions on Ultrasonics Ferroelectrics and Frequency Control, vol. 56, pp. 263-269, Feb 2009. [6]
S. Grop, P. Y. Bourgeois, R. Boudot, Y. Kersale, E. Rubiola, and V. Giordano, "10 GHz cryocooled sapphire oscillator
with extremely low phase noise," Electronics Letters, vol. 46, pp. 420-422, Mar 2010. y
p
,
,
, pp
,
[7]
A. G. Mann, C. Sheng, and A. N. Luiten, "Cryogenic sapphire oscillator with exceptionally high frequency stability,"
IEEE Transactions on Instrumentation and Measurement, vol. 50, pp. 519-521, Apr 2001. y
p
pp
[7]
A. G. Mann, C. Sheng, and A. N. Luiten, "Cryogenic sapphire oscillator with exceptionally high frequency stability,"
IEEE Transactions on Instrumentation and Measurement vol 50 pp 519 521 Apr 2001 [7]
A. G. Mann, C. Sheng, and A. N. Luiten, "Cryogenic sapphire oscillator with exceptionally high A. G. Mann, C. Sheng, and A. N. Luiten, "Cryogenic sapphire oscillator with exceptionally high frequency
IEEE Transactions on Instrumentation and Measurement, vol. 50, pp. 519-521, Apr 2001. [ ]
,
g,
,
y g
pp
p
y
g
IEEE Transactions on Instrumentation and Measurement, vol. 50, pp. 519-521, Apr 2001. [8]
A. Bartels, C. W. Oates, L. Hollberg, and S. A. Diddams, "Stabilization of femtosecond laser frequency combs with
subhertz residual linewidths," Optics Letters, vol. 29, pp. 1081-1083, May 2004.
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Figurative Terracotta Revetments in Etruria and Latium in the VI. and V. Centuries B.C. By E. Douglas Van Buren. Pp. 74, 32 Plates. London: John Murray, 1921. 16<i>s</i>. net.
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NOTICES OF BOOKS NOTICES OF BOOKS 289 Aristotelis Meteorologicorum Libri Quattuor. Recensuit Indicem Verborum
Addidit F. H. FOBES. Cantabrigiae Massachusettensium e Typographeo Academiae
Harvardianae. MDCCCCXVini. 15s. net. What Mr. Fobes on his title-page professeB to have done he has done so well and so
thoroughly that we cannot help regretting that he has not done, nor apparently contem-
plated doing, a little more. The contents of Aristotle's Meteorologiea are so interesting
in themselves, and make so strong an impression of the author's wide knowledge, wide
research, and wider curiosity, that a few notes from a scholar so competent as Mr. Fobes
would have been very welcome, at least in those places where his emendations of the text
imply an alteration in the meaning. His discussion in the Classical Review, 1916, of a
difficult passage in the second book shows how valuable a commentary he could have
made in a small space; but when we turn to the passage we find nothing but a brief
intimation in a foot-note that the text has been changed. And surely a diagram might
have been inserted at the two or three places where the author employed one. Mr. Fobes retains not only Bekker's division into chapters, but also his paging, so
that comparison is easy. He has also given us a list of all the passages in which he has
made any considerable alteration in Bekker's text. It will be found that he is chary
of suggestion; for example, in 371 o, 4, he rejects vuperHv ZVTWV in favour of ranittS
ixovrmv without any hint as to the meaning of the unusual word thus restored to the
text. In another passage, 376 6, 23, where Bekker's TS>V Si irpos TTJ 717 (rrfip^oine
is not very satisfying, he does indeed hint in a note at a possible solution, but contents
himself with printing in the text the unmeaning and improbable MS. word •Kpo<nrTepi£oi>. A peculiarity of the volume is that idyvviu, IUKT6S, ^U{H are always spelt lulywiu,
ILfiKT&s, jueT{«. If I understand Mr. Fobes aright, he regards this unusual spellin
as merely a freak of the scribe of his favourite MS., and if so, one hardly sees why the
familiar forms should not be retained. Mr. Fobes gives a very clear and very full account
of the many MSS. NOTICES OF BOOKS We feel also that the usefulness of the book
would have been increased by even a short discussion of these terra-cottas on a chrono-
logical basis, to justify the bald statement of dates, e.g. ' VI. century,' ' VI.—V. centuries,'
etc., given without further explanation, which may puzzle readers who are naturally less
familiar with the material. Certain other omissions can hardly pass without comment:
(1) references to the Plates at the end should have been inserted in the text as well as in
the elaborate table on p. ix. f.;
(2) the scale of the illustrations is not given; (3) the
dimensions of all fragments, not merely of a selection from acroteria and friezes, should
have been furnished. Scarcely less serious, and perhaps more irritating, is the inadequacy
of the press-correction. Misprints occur rather too frequently for a book of reference of
only 74 pages. We note antifixae (p. 3, twice), satyr sand Pans (p. 25), Straticum (for
Satricum, p. 36), and Keldewey and Loescheke (pp. 57, 69, 71) among authorities cited;
PI. XXXI. represents Type V., not VI., of the friezes. The foot-notes seem to have been
inexcusably neglected, as witness the four citations of the excavations at Gordion by the
brothers Korte : p. 35 (note 8): G. A. Korte, Jahrb. d. Jnst., Erganzunsheft, v (1904), p
p. 57 (note 2): G. u. A. Korte, Jb. d. Inst. Erganzungsheft, v (1903), p. 65 (note 1): Korte, Jb. d. Inst, Erganzungsheft, v (1904), p. 66 (note 2): G. u. A. Korte, Jb. d. Inst. Enganzungsheft, v (1904). We hope that the descriptions and references have been checked with more care
than this inaccuracy and inconsistency indicates. The descriptions given are usually
clear and ample, though ' height, cm. 8 by 10'5 ' (p. 16, note 3) is a rather Thucydidean
construction, and the ' lateral akroterion of a horse' (p. 59) is mystifying without the
context. It has not been possible to check the completeness of the catalogue, but surprise
may be expressed at the omission of the large series of architectural terra-cottas from
Lanuvium presented by the late Lerd Savile to the British Museum; in fact the antefix
' Division IV., Type XX.' ( = B. M. Terracottas, B 605, of which there is another slightly
different example in the Museum at Leeds, unknown to the authoress), is almost the only
type figured from this site. NOTICES OF BOOKS he has examined, and a most valuable ' notitia litteraria' containing
a list of commentaries on the Meteorologiea, ancient and modern. There is also an index
verborum, the more valuable because the vocabulary of the fourth book in particular is
extraordinarily rich. Altogether he has given us in a beautifully-printed and very port-
able volume a most satisfactory edition of a most remarkable book. Figurative Terracotta Revetments in Etruria and Latium in the VI. and
V. Centuries B.C. By E. DOUGLAS VAN BTJKEN. Pp. 74, 32 Plates. London :
John Murray, 1921. 16s. net. This attractive volume will be welcomed on many grounds, and especially by those readers
whose appetites were whetted by the articles on Italian architectural terra-cottas by
Mrs. Strong and Mrs. Van Buren in Vol. IV. of the Journal of Roman Studies. The
authoress expresses, almost too modestly, the hope that ' a simple catalogue of the figura-
tive terra-cotta revetments from Etruria and Latium in the earliest periods may be found
useful,' for this is much more than a simple catalogue and will prove not only useful but
indispensable. In scale and sumptuousness it does not, naturally, rival Koch's Dachterra-
kotten aus Campanien—a pre-war publication—but it. provides a handy and lucid collection
of similar material from Etruria and Latium, collating duplicate examples of types,
quoting helpful parallels, and revealing an extensive acquaintance with a wide range of
material. Thirty-two plates of good photographs—many of which reproduce several pieces—
are a generous but not excessive allowance for the seventy-four pages of text, for so little
of this material is easily accessible to students in this country, and it is somewhat of a
revelation to see how many museums have been drawn upon for the purpose. The catalogue is divided into three sections—Antefixae, Acroteria (which includes a
variety of other architectural members), and Friezes—and each is prefaced by a short NOTICES OF BOOKS 290 introduction. When we observe that on pp. 31-35 there comes a brief, but clear and
scholarly discussion of the ancient authorities for the fictile decoration of Italian temples,
we realise that the book is an accretion of three articles, which might with advantage
have been rearranged so that all the introductory matter preceded the catalogue proper
under its three headings; indeed the miscellany appended to the Acroteria might well
have formed a fourth and separate section. NOTICES OF BOOKS But perhaps the other pieces would not come under the title
' Figurative,' of which the reviewer unfortunately does not know the literal meaning. And after all, even this rather formidable list of minor blemishes, mostly easy of remedy
in a subsequent edition, does not seriously impair the value of this attractive book, and
we offer congratulations to the authoress on the successful completion of a laborious
but clearly congenial task. Byzantinisch-Neugriechische
Jahrbiicher. Internationales vrissenschaftlic
Organ unter Mitwirkung zahlreicher Fachgenossen. Herausgegeben von Dr. Phil. NIKOS A. BEES (BeV). Berlin-Wilmersdorf, Weimarische Strasse, 19: Verlag der
Byzantinisch-Neugriechischen Jahrbiicher. This new periodical, of which the first volume was published in 1920, and the first half
of the second in September 1921, deserves a hearty welcome. An introduction by Dr. Bees
lays down the lines which it is to follow. The war put an end to several periodicals on
Byzantine matters; thus Byzantis and the Neos Hellenomnemon and the two Russian
journals, the Vizantijslcij Vremennik and the Journal of the Russian Archaeological
Institute at Constantinople have all disappeared, and if Byzantine studies are not to fall
behind, their place must be filled. It is remarkable that neither in this list nor in any
part of the introduction is any mention made of the most important of all these periodicals,
|
https://openalex.org/W2003641432
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0030336&type=printable
|
English
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Factors Associated with Physician Agreement on Verbal Autopsy of over 11500 Injury Deaths in India
|
PloS one
| 2,012
|
cc-by
| 7,349
|
Abstract Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: marvin.my.hsiao@gmail.com Citation: Hsiao M, Morris SK, Bassani DG, Montgomery AL, Thakur JS, et al. (2012) Factors Associated with Physician Agreement on Verbal Autopsy of over 11500
Injury Deaths in India. PLoS ONE 7(1): e30336. doi:10.1371/journal.pone.0030336
Editor: Thomas Behrens University of Bochum Germany Citation: Hsiao M, Morris SK, Bassani DG, Montgomery AL, Thakur JS, et al. (2012) Factors Associated with Physician Agreement on Verbal Autopsy of over 11500
Injury Deaths in India. PLoS ONE 7(1): e30336. doi:10.1371/journal.pone.0030336 Editor: Thomas Behrens, University of Bochum, Germany
Received August 30, 2011; Accepted December 14, 2011; Published January 17, 2012
Copyright: 2012 Hsiao 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: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: marvin.my.hsiao@gmail.com Received August 30, 2011; Accepted December 14, 2011; Published January 17, 2012 Copyright: 2012 Hsiao 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. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: marvin.my.hsiao@gmail.com Marvin Hsiao1,2*, Shaun K. Morris1,3, Diego G. Bassani1,4,5, Ann L. Montgomery1, J. S. Thakur1,6, Prabhat
Jha1,4 1 Centre for Global Health Research, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, Canada, 2 Department of Surgery, St. Michael’s
Hospital, University of Toronto, Toronto, Canada, 3 Division of Infectious Diseases, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto,
Canada, 4 Dalla Lana School of Public Health, University of Toronto, Toronto, Canada, 5 Child Health and Evaluative Sciences, Hospital for Sick Children, Toronto, Canada,
6 Department of Community Medicine, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 Abstract Introduction: Worldwide, injuries account for 9.8% of all deaths. The majority of these deaths occur in low- and middle-
income countries where vital registration systems are often inadequate. Verbal autopsy (VA) is a tool used to ascertain cause
of death in such settings. Validation studies for VA using hospital diagnosed causes of death as comparisons have shown
that injury deaths can be reliably diagnosed by VA. However, no study has assessed the factors that may affect physicians’
abilities to code specific causes of injury death using VA. Method/Principal Findings: This study used data from over 11 500 verbal autopsies of injury deaths from the Million Death
Study (MDS) in which 6.3 million people in India were monitored from 2001–2003 for vital events. Deaths that occurred in
the MDS were coded by two independent physicians. This study focused on whether physician agreement on the
classification of injury deaths was affected by characteristics of the deceased and respondent. Agreement was analyzed
using three primary methods: 1) kappa statistic; 2) sensitivity and specificity analysis using the final VA diagnosed category
of injury death as gold standard; and 3) multivariate logistic regression using a conceptual hierarchical model. The overall
agreement for all injury deaths was 77.9% with a kappa of 0.74 (99% CI 0.74–0.75). Deaths in the injury categories of
‘‘transport’’, ‘‘falls’’, ‘‘drowning’’ and ‘‘other unintentional injury’’ occurring outside the home were associated with greater
physician agreement than those occurring at home. In contrast, self-inflicted injury deaths that occurred outside the home
were associated with lower physician agreement. Conclusions/Significance: With few exceptions, most characteristics of the deceased and the respondent did not influence
physician agreement on the classification of injury deaths. Physician training and continued adaptation of the VA tool
should focus on the reasons these factors influenced physician agreement. should focus on the reasons these factors influenced physician agreement. Citation: Hsiao M, Morris SK, Bassani DG, Montgomery AL, Thakur JS, et al. (2012) Factors Associated with Physician Agreement on Verbal Autopsy of over 1150
Injury Deaths in India. PLoS ONE 7(1): e30336. doi:10.1371/journal.pone.0030336
Editor: Thomas Behrens, University of Bochum, Germany
Received August 30, 2011; Accepted December 14, 2011; Published January 17, 2012
Copyright: 2012 Hsiao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permit
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Physician Agreement on Verbal Autopsy [11,12,14]. However, no study has assessed the factors that may
influence the ability of physicians to distinguish between different
injuries using VA. We used physician agreement as a metric in our
studies to measure the performance characteristics of VA. Although no inference can be made on accuracy or external
validity, higher physician agreement should be correlated with
better reliability of VA assigned cause of death. We previously
reported that younger age and female gender were associated with
lower agreement between physicians on the cause of childhood
deaths in India [15]. The purpose of our current study is to
investigate whether characteristics of the respondent and deceased
are associated with physician agreement on the classification of
injury deaths. and keywords. If they agreed in this second reconciliation stage,
the final cause of death was assigned. However, if there was still
disagreement after the reconciliation stage, a third, senior
physician adjudicated and assigned the final cause of death
based on the VA questionnaire along with the ICD-10 codes and
keywords provided by the first two physicians. A web-based
system managed the coding process with more than 130,000
deaths coded by 130 physicians. y
p y
We used MDS data from 2001–2003 for this study. All deaths
due to injury were grouped into 9 broad categories: transport
injuries; falls; fire; drowning; venomous snakes, animals and
plants; poisonings and other unintentional injuries; self-inflicted
injuries; war, violence and other intentional injuries; and injuries
of undetermined intent. The ICD-10 codes of these 9 injury
categories are listed in Table 1, along with the number of deaths
in each category stratified by age groups: children (#14 years),
adults(15 to 69 years), and elderly ($70 years). For this study,
physician agreement was defined as agreement of the first ICD-
10 codes from the two physicians (i.e. the same injury category for
both physicians at the first coding stage prior to the second
reconciliation stage). The extent of agreement between two
physicians beyond chance was assessed using the kappa statistic
[17]. The 99% confidence interval for the kappa statistic was
calculated
using
the
bootstrap
method
for
polychotomous
variables with bias correction [18]. The Landis and Koch
classification of inter-rater reliability was used to interpret the
kappa statistic: #0.4 – poor to fair agreement; .0.4 to #0.6 –
moderate agreement; .0.6 to #0.8 – substantial agreement;
.0.8 – high agreement [17]. Physician Agreement on Verbal Autopsy Analysis was conducted on the
entire dataset and also stratified by age. Sensitivity and specificity
of each physician’s initial cause of death diagnosis was performed
using the final RHIME cause of death diagnosis as the gold
standard. This analysis should only be interpreted with the
understanding that the gold standard (final cause of death
diagnosis) was not independent of the test itself (initial physician
cause of death diagnosis). The methodology used in this study to
assess physician agreement and factors affecting agreement has
been described in detail previously [15]. Introduction accurately reported by the deceased’s family or acquaintances
during a standardized interview. Worldwide, injuries account for about 10% of all deaths and
about 12% of the total burden of diseases measured in disability-
adjusted life years [1]. The majority of these injuries occur in low
and middle-income countries [1]. In such settings with limited
resources, deaths often occur outside the formal healthcare system
and are not recorded in the country’s vital registration system. The
lack of accurate vital statistical data hampers public health action,
policy
making,
and
the
implementation
of
evidence-based
interventions. Various methodologies exist to determine a cause of death
based
on
the
signs
and
symptoms
collected
on
the
VA
questionnaire [3]. These include data- or expert-derived algo-
rithms [4], symptom pattern methodology [5], and probabilistic
models [6,7]. Another common methodology utilizes two or more
trained physicians to review the VA form to determine a cause of
death [2]. Studies using VA with physician coders have been externally
validated by comparing the VA cause of death to hospital-based
registries or death certificates [8–13]. However, in low-resource
settings, this method of validation may be biased because people
with access to healthcare facilities, death certificates, or death
registration are often not representative of the entire population. Verbal autopsy (VA) is a cost-effective tool for ascertaining
cause of death in low-resource settings with incomplete vital
registration [2]. The VA relies on the assumption that each cause
of death has a unique set of signs and symptoms that can be used
to distinguish between different causes of death. The VA also
assumes that the signs and symptoms leading up to a death can be Injury as a broad category of death can be diagnosed with high
sensitivity, specificity, and positive predictive value by VA 1 January 2012 | Volume 7 | Issue 1 | e30336 Physician Agreement on Verbal Autopsy Results There were 11 543 injury deaths in the MDS from 2001–2003. Of these, 11 509 deaths (99.7%) were coded by two physicians and
were included in this study. The process of determining the final
cause of death is shown in Figure 1. The summary of the kappa statistic analysis, stratified by age
group, is shown in Table 2. For all age groups, non-overlapping
99% confidence intervals between one or more sub-categories of a
predictor variable suggest statistical significance and require
multivariate logistic regression modeling to rule out confounding
effects. The overall agreement for all injury deaths was 77.9% with
a kappa of 0.74 (99% CI 0.74–0.75). For children (#14 years),
there was 84.1% agreement and a kappa of 0.81 (99% CI 0.79–
0.82). In this group, the 99% confidence intervals for kappa did
not overlap between one or more sub-categories of the following
variables: whether the respondent lived with the deceased,
religion,
language, deceased’s
gender, deceased’s
education,
location of death, and whether the death was registered. For
adults (15–69 years), there was 80.9% agreement overall and a
kappa of 0.77 (99% CI 0.76–0.78). In this group, the 99%
confidence intervals for kappa of all the variables did not overlap,
except for respondent education and death registration. For the
elderly ($70 years), there was 58.2% agreement overall and a
kappa of 0.44 (99% CI 0.40–0.45). In this age group, the variables
where the 99% confidence interval for kappa did not overlap were
whether the respondent lived with the deceased, respondent
education, respondent relationship to the deceased, religion,
language, deceased gender, location of death, and EAGA. Ethics
approval
for
the
MDS
was
obtained
from
the
Postgraduate Institute of Medical Education and Research,
Chandigarh, India and St. Michael’s Hospital, Toronto, Ontario,
Canada. Enrollment in the MDS was on a voluntary basis and the
study posed no or minimal risks to enrolled subjects. Families were
free to withdraw from the study. Verbal consent was obtained. All
personal identifiers present in the raw data are anonymized before
analysis. MDS data storage and usage protocols were described in
detail elsewhere [2]. The sensitivity and specificity of the initial physician coded
cause of death was calculated using the final cause of death as gold
standard (Table 3). There were 3 200 child, 16 346 adult, and
3 506 elderly initial physician coded cause of death (two codes per
death). Physician Agreement on Verbal Autopsy deceased and the respondent. The model was constructed using
an a priori hierarchical conceptual framework that grouped
variables into three blocks: distal, middle, and proximal [19]. The
distal block included geographic factors (Empowered Action
Group region including Assam (EAGA), geographic region, and
urban or rural setting). The middle block included socio-
demographic factors of the respondent (whether the respondent
lived with the deceased, respondent’s gender, education, religion,
language, and relation to the deceased). The proximal block
included characteristics of the deceased (deceased’s gender,
education, location of death, and whether the death was
registered). Variable selection was conducted using backward
elimination. A variable was retained in the final model if its
likelihood
ratio test resulted in
p-value
lower than
0.20. Respondents’ education was predefined to remain in the final
model regardless of p-value from the likelihood ratio test because
of its expected influence on the quality of VA questionnaire. The
final model for adults was also adjusted for the deceased’s age as a
continuous variable given the finding that increasing age was
correlated linearly with lower physician agreement between
injury death categories. Adjustment for age was not necessary in
the children and elderly groups because the correlation between
age and physician agreement within the group was not found to
be significant. A factor was considered to be significantly
associated with physician agreement if the likelihood ratio test
resulted in p,0.05. All statistical analysis was performed using
Stata SE version 10.0 [20]. Methods This study uses data from the Million Death Study (MDS); the
methodology of the MDS is published in detail elsewhere [2]. In
brief, the MDS monitored the vital statistics of 6.3 million people
in 1.1 million nationally representative households in India from
2001–2003. A total of 6671 sampling units, each with an average
of 150 households, were selected by stratified random sampling
based on urban or rural setting, village or city population size,
and female literacy rate according to the 1991 national census. For every death occurring in these households, a verbal autopsy
(VA) questionnaire called RHIME (Routine, Reliable, Repre-
sentative and Re-sampled Household Investigation of Mortality
with Medical Evaluation; available at http://www.cghr.org/
index.php/training/verbal-autopsy-forms-2/) was completed by
a trained fieldworker based on a structured interview with a
family member or close acquaintance of the deceased. RHIME
includes both preset close-ended questions and an open-ended
narrative. Each completed VA questionnaire was independently
reviewed by two physicians trained to use the VA questionnaire
to determine cause of death in World Health Organization
International Classification of Disease 10 (ICD-10) code [16]. The physicians also provided a set of keywords that were
important in determining the cause of death. If the two
physicians initially agreed on the same cause of death, the
corresponding ICD-10 code was assigned as the final cause of
death. If the two physicians initially disagreed, they were
required to anonymously reconcile by exchanging ICD-10 codes We used a multivariate logistic regression model to study the
association between physician agreement and the geographic,
socioeconomic, demographic, and other characteristics of the ble 1. Injury death category ICD-10 codes and numbers by age groups. Table 1. Injury death category ICD-10 codes and numbers by age groups. Table 1. Injury death category ICD-10 codes and numbers by age groups. Injury Category
ICD-10 Codes
Number of Deaths
Children
Adults
Elderly
(#14 years)
(15–69 years)
($70 years)
Transport Injuries
V01-V99,Y85
239
1948
185
Falls
W00-W19
202
824
986
Fire
X00-X09
59
278
36
Drowning
W65-W74
456
406
42
Venomous Snakes, Animals and Plants
X20-X29,W57,W60
208
395
40
Other Unintentional Injuries
X40-X44,X46-X49, W20-W56,W58-W59,W64,W75-W99,
X10-X19,X30-X39,X50-X52,X57-X59,Y40-Y84,Y86,Y88-Y89
323
1064
324
Self-Inflicted Injuries
X60-X84
53
2585
102
War, Violence and Other
Intentional Injuries
X85-Y09,Y35-Y36,Y87
45
549
26
Injuries of Undetermined Intent
Y10-Y14,Y16-Y34,Y96-Y98
17
127
13
doi:10.1371/journal.pone.0030336.t001
PLoS ONE | www.plosone.org
2
January 2012 | Volume 7 | Issue 1 | e30336 PLoS ONE | www.plosone.org 2 Physician Agreement on Verbal Autopsy Results Excluding injuries of undetermined intent, the sensitivity of
initial physician coding for all injury categories was above 82.0%
in children, above 81.3% in adults, and above 72.7% in the
elderly. Sensitivity for injuries of undetermined intent was the Figure 1. Flow diagram of Million Death Study injury deaths. doi:10.1371/journal.pone.0030336.g001
PLoS ONE | www.plosone.org
3
January 2012 | Volume 7 | Issue 1 | e30336 Figure 1. Flow diagram of Million Death Study injury deaths. doi:10.1371/journal.pone.0030336.g001 Figure 1. Flow diagram of Million Death Study injury deaths. doi:10.1371/journal.pone.0030336.g001 January 2012 | Volume 7 | Issue 1 | e30336 January 2012 | Volume 7 | Issue 1 | e30336 PLoS ONE | www.plosone.org 3 Physician Agreement on Verbal Autopsy Table 2. Kappa statistic analysis of physician agreement for injury deaths by age group. Results Children (#14 years)
Adults (15–69 years)
Elderly ($70 years)
Variable
Sub-Categories
n
% Agree Kappa 99% CI
n
% Agree Kappa 99% CI
n
% Agree Kappa 99% CI
Overall
1595 84.1
0.81
0.79–0.82
8162
80.9
0.77
0.76–0.78
1752
58.2
0.44
0.40–0.45
Live with Status
Yes
1081 82.6
0.79
0.79–0.81* 4666
78.7
0.74
0.74–0.75*
1290
56.5
0.40
0.36–0.40*
No
371
86.5
0.83
0.83–0.86* 2911
84.1
0.80
0.79–0.81*
349
61.0
0.51
0.50–0.53*
Respondent Gender
Male
894
83.8
0.81
0.79–0.84
4241
80.2
0.76
0.75–0.76*
950
58.4
0.44
0.41–0.45
Female
665
84.5
0.81
0.80–0.82
3832
81.5
0.77
0.76–0.78*
788
57.7
0.43
0.41–0.47
Respondent Education
Less than Primary
908
83.5
0.80
0.78–0.82
3912
80.5
0.77
0.75–0.78
737
59.7
0.47
0.44–0.53*
Primary
191
85.3
0.82
0.77–0.84
982
79.8
0.75
0.73–0.77
214
57.0
0.44
0.41–0.52*
.Primary
466
84.6
0.82
0.80–0.83
3074
81.3
0.77
0.76–0.78
766
56.9
0.39
0.36–0.41*
Respondent
Relationship
Related
1457 84.4
0.81
0.80–0.82
7346
80.6
0.77
0.76–0.77*
1603
57.4
0.42
0.40–0.45*
Unrelated
95
85.3
0.82
0.80–0.85
699
83.6
0.79
0.77–0.79*
132
67.4
0.58
0.48–0.64*
Religion
Hindu
1271 84.1
0.81
0.80–0.82* 6786
81.2
0.77
0.76–0.78*
1427
58.7
0.44
0.44–0.47*
Muslim
237
83.1
0.79
0.77–0.80* 675
80.4
0.77
0.73–0.80*
152
50.7
0.31
0.22–0.40*
Other
83
86.8
0.84
0.78–0.90* 678
78.2
0.73
0.71–0.76*
166
59.6
0.46
0.39–0.52*
Language
Hindi
863
82.0
0.79
0.76–0.79* 2979
76.2
0.72
0.71–0.73*
646
54.3
0.37
0.33–0.39*
English
68
95.6
0.95
0.90–0.98* 649
82.3
0.78
0.77–0.81*
98
67.4
0.58
0.53–0.70*
Other
674
85.8
0.83
0.82–0.84* 4534
83.8
0.80
0.79–0.80*
1008
59.7
0.46
0.42–0.78*
Deceased Gender
Male
916
86.0
0.83
0.82–0.88* 5637
81.1
0.77
0.77–0.78*
901
59.4
0.48
0.47–0.50*
Female
679
81.6
0.78
0.77–0.80* 2524
80.4
0.75
0.75–0.76*
851
56.9
0.38
0.37–0.40*
Deceased Education
Age-Appropriate
1337 84.7
0.82
0.81–0.82* -
-
-
-
-
-
-
-
Below Age-
Appropriate
201
89.1
0.87
0.85–0.91* -
-
-
-
-
-
-
-
Less than Primary
-
-
-
-
3964
78.6
0.75
0.74–0.75*
1437
57.8
0.43
0.41–0.45
Primary
-
-
-
-
1099
82.0
0.78
0.77–0.78*
133
60.2
0.48
0.40–0.52
.Primary
-
-
-
-
2968
83.4
0.79
0.78–0.81*
156
58.3
0.42
0.39–0.50
Death Place
Home
685
80.7
0.77
0.76–0.78* 3188
77.2
0.70
0.70–0.72*
1348
53.0
0.33
0.31–0.35*
Health Facility
248
81.1
0.78
0.76–0.81* 1652
80.8
0.77
0.76–0.77*
167
73.7
0.66
0.58–0.66*
Other
578
89.6
0.86
0.84–0.87* 3041
84.9
0.81
0.80–0.81*
184
79.4
0.75
0.71–0.79*
Death Registration
Yes
401
86.8
0.84
0.82–0.85* 3409
81.3
0.77
0.76–0.79
693
56.1
0.39
0.36–0.40
No
595
81.9
0.78
0.77–0.80* 1641
80.0
0.76
0.74–0.78
411
53.5
0.39
0.35–0.45
Rural/Urban
Rural
1429 84.3
0.81
0.79–0.82
6751
81.3
0.77
0.77–0.77*
1403
58.5
0.45
0.43–0.46
Urban
166
82.5
0.79
0.69–0.86
1411
79.2
0.74
0.72–0.75*
349
56.7
0.37
0.31–0.46
EAG+Assam
vs Non-EAG
EAGA
858
81.7
0.78
0.78–0.82
2840
77.5
0.74
0.73–0.75*
626
52.1
0.36
0.33–0.40*
Non-EAGA
737
87.0
0.84
0.81–0.86
5322
82.7
0.78
0.78–0.79*
1126
61.6
0.48
0.45–0.52*
Footnote: * indicates that the 99% confidence intervals do not overlap between one or more sub categories of the variable. PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 Results EAGA = Empowered Action Group including
Assam. doi:10.1371/journal.pone.0030336.t002 lowest in all three age groups – 61.8% in children, 59.7% in adults,
and 53.8% in the elderly. The specificity of initial physician coding
for all age groups was greater than 98.3% for all injury causes. category, death location outside of home was associated with
greater physician agreement only for deaths due to transport
injury, drowning, and other unintentional injury (Table 5). In
adult transport injury deaths, the odds ratio for physician
agreement was 5.41 (95% CI 3.18–9.22) for deaths occurring at
a health facility and 5.06 (95% CI 3.23–7.92) for deaths at a
location outside of home or health facility. For both drowning
and other unintentional injury deaths among adults, physician
agreement was greater only for deaths that occurred at a location
outside of the home or health facility (drowning - OR 2.07, 95%
CI 1.08–3.99; other unintentional injury - OR 1.76, 95% CI
1.21–2.56) compared to deaths that occurred at home. In this age
group, there was lower physician agreement for deaths due to The results of the multivariate logistic regression on factors
influencing physician agreement are summarized in Table 4. For
adults, physicians were more likely to agree on the classification of
injury deaths if deaths occurred at a health facility (OR 1.19, 95%
CI 1.01–1.39) and at a location outside of home or health facility
(OR 1.50, 95% CI 1.30–1.73) compared to deaths at home. We
postulated that injury deaths that are more likely to occur outside
of home or health facility, such as transport injuries and drowning,
may be easier to classify and would therefore have greater
physician agreement. When we stratified the analysis by injury PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 January 2012 | Volume 7 | Issue 1 | e30336 4 Physician Agreement on Verbal Autopsy Table 3. Sensitivity and specificity of initial physician coded cause of death versus final RHIME cause. Results Age Category
Cause of Death Category
n
Sensitivity
(99% CI)
Specificity
(99% CI)
Children
Specific Causes
#14 years
Drownings
911
96.8
(95.0–98.1)
99.1
(98.5–99.5)
Transport Injuries
478
96.2
(93.4–98.1)
99.9
(99.5–100)
Venomous Snakes, Animals, Plants
416
94.0
(90.3–96.6)
99.9
(99.5–100)
Falls
403
89.1
(84.5–92.7)
99.1
(98.5–99.5)
Fires
118
90.7
(81.6–96.2)
99.9
(99.6–100)
Self-inflicted Injuries
106
89.6
(79.7–95.8)
99.6
(99.3–99.9)
Non-Specific Causes
Other Unintentional Injuries
645
82.5
(78.3–86.2)
98.4
(97.6–99.0)
War, Violence, and Other Intentional Injuries
89
82.0
(69.4–91.1)
99.8
(99.5–100)
Injuries of Undetermined Intent
34
61.8
(38.5–81.7)
99.3
(98.9–99.6)
Total
3200
Adults
Specific Causes
15–69 years
Self-inflicted Injuries
5169
92.9
(92.0–93.8)
99.5
(99.2–99.6)
Transport Injuries
3895
94.9
(93.9–95.8)
99.5
(99.3–99.6)
Falls
1648
82.0
(79.5–84.4)
99.2
(99.0–99.4)
Drowning
812
90.1
(87.2–92.7)
99.7
(99.5–99.8)
Venomous Snakes, Animals, Plants
790
93.3
(90.7–95.4)
99.9
(99.9–100)
Fire
555
89.2
(85.4–92.3)
99.6
(99.5–99.7)
Non-Specific Causes
Other Unintentional Injuries
2127
81.3
(79.1–83.5)
98.3
(98.1–98.6)
War, Violence, and Other Intentional Injuries
1097
87.4
(84.6–89.9)
99.5
(99.3–99.6)
Injuries of Undetermined Intent
253
59.7
(51.4–67.6)
99.1
(98.9–99.3)
Total
16346
Elderly
Specific Causes
$70 years
Falls
1971
75.3
(72.7–77.8)
98.5
(97.5–99.2)
Transport Injuries
370
88.4
(83.4–92.3)
99.8
(99.5–99.9)
Self-inflicted Injuries
204
94.6
(89.2–97.9)
99.9
(99.7–100)
Drowning
84
83.3
(70.5–92.3)
100
(99.8–100)
Venomous Snakes, Animals, Plants
80
86.3
(73.6–94.4)
99.9
(99.6–100)
Fire
71
81.7
(67.2–91.8)
99.9
(99.6–100)
Non-Specific Causes
Other Unintentional Injuries
648
72.7
(67.9–77.1)
97.9
(97.1–98.6)
War, Violence, and Other Intentional Injuries
52
76.9
(58.9–89.9)
99.6
(99.3–99.8)
Injuries of Undetermined Intent
26
53.8
(28.1–78.2)
99.8
(99.6–100)
Total
3506
doi:10.1371/journal.pone.0030336.t003 Age Category
Cause of Death Category doi:10.1371/journal.pone.0030336.t003 fall, and other unintentional injury deaths (Table 5). In transport
injury deaths, physicians were more likely to agree for deaths that
occurred at a health facility (OR 4.92, 95% CI 1.55–15.69) and at
a location outside of the home or health facility (OR 4.18, 95% CI
1.51–11.58) compared to deaths that occurred at the home. Similarly in elderly deaths due to falls, physicians were more likely
to agree for deaths that occurred at a health facility (OR 3.12,
95% CI 1.73–5.63) and at a location outside of the home or health
facility (OR 2.42, 95% CI 1.13–5.15) compared to at the home. For other unintentional injury deaths in this age group, physician
agreement was greater only for deaths that occurred outside of the
home or health facility (OR 5.96, 95% CI 1.90–18.69) compared
to at the home. PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 Results Physicians were also more likely to agree on cause
of deaths among the elderly if the respondent was a neighbor self-inflicted injury that occurred at locations outside of the home
or health facility (OR 0.60; 95% CI 0.44–0.81) compared to self-
inflicted deaths at home. Also for adults, physicians were more likely to agree on the
classification of injury deaths if the respondent did not live with the
deceased (OR 1.33; 95% CI 1.18–1.51) compared to those who
did live with the deceased. This association was similar in all injury
categories (data not shown). For the elderly, physician agreement was greater for deaths that
occurred outside the home, whether at a health facility (OR 2.31,
95% CI 1.58–3.36) or at a location other than the home or health
facility (OR 3.28, 95% CI 2.22–4.83). When stratified by injury
category, greater physician agreement for elderly deaths that
occurred outside the home was apparent only for transport injury, PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 January 2012 | Volume 7 | Issue 1 | e30336 5 Physician Agreement on Verbal Autopsy Table 4. Multivariate logistic regression analysis of variables affecting physician agreement by age category. Results doi:10.1371/journal.pone.0030336.t004 Footnote: Results are adjusted for region, EAGA, urban/rural and age. doi:10.1371/journal.pone.0030336.t004 rather than a relative (OR 1.52, 95% CI 1.03–2.23). This
association was similar among all injury categories. hospital records and death certificates [11,12,14]. However, to
the best of our knowledge, no studies have assessed the factors that
influence physician’s ability to further classify injury deaths into
specific categories using VA. From a public health injury
prevention perspective, differentiating between injury categories
is essential to priority setting and formulating targeted interven-
tions. Using kappa statistic, sensitivity and specificity analysis, and
multivariate logistic regression, this study specifically assessed
factors that may influence physician agreement in injury deaths. For children, the only variable significantly associated with
physician
agreement
was
the
location
of
death. Physician
agreement was more common for deaths that occurred outside
of the home or health facility (OR 2.18, 95% CI 1.40–3.40),
regardless of the injury category. Results g
g
y
g p y
g
y
g
g
y
Children (#14 years)
Adults (15–69 years)
Elderly ($70 years)
Variable
Sub-Categories
n
OR
(95% CI)
p
n
OR
(95% CI)
p
n
OR
(95% CI)
p
Live with
Yes
1089
1.00
0.1073
4676
1.00
0.0000
1290
1.00
0.7086
No
372
1.32
(0.94–1.85)
2922
1.33
(1.18–1.51)
350
1.05
(0.80–1.39)
Respondent Gender
Male
900
1.00
0.9581
4254
1.00
0.3577
952
1.00
0.2137
Female
669
1.01
(0.74–1.37)
3841
1.06
(0.94–1.20)
788
0.88
(0.71–1.08)
Respondent Education
Less than Primary
914
1.00
0.9796
3926
1.00
0.2256
738
1.00
0.1191
Primary
191
0.99
(0.62–1.58)
983
0.90
(0.75–1.08)
214
0.77
(0.56–1.06)
.Primary
495
0.96
(0.58–1.58)
3222
1.06
(0.93–1.20)
794
0.82
(0.66–1.02)
Respondent
Relationship
Parent
895
1.00
0.2454
-
-
-
-
-
-
-
-
Other Relative
571
1.28
(0.93–1.76)
-
-
-
-
-
-
-
-
Neighbour
96
0.89
(0.47–1.70)
-
-
-
-
-
-
-
-
Relative
-
-
-
-
7366
1.00
0.6258
1604
1.00
0.0312
Neighbour
-
-
-
-
701
1.06
(0.84–1.34)
132
1.52
(1.03–2.23)
Religion
Hindu
1280
1.00
0.7238
6804
1.00
0.3863
1429
1.00
0.1411
Muslim
237
0.93
(0.62–1.40)
678
1.00
(0.81–1.23)
152
0.70
(0.49–1.00)
Other
84
1.30
(0.59–2.88)
679
0.86
(0.69–1.07)
166
0.93
(0.65–1.32)
Language
Hindi
863
1.00
0.0899
3001
1.00
0.1040
648
1.00
0.8252
English
68
3.46
(0.98–12.2)
649
1.03
(0.77–1.37)
98
0.92
(0.51–1.66)
Other
674
1.27
(0.76–2.12)
4534
1.21
(0.98–1.48)
1008
0.88
(0.59–1.32)
Deceased Gender
Male
924
1.00
0.4518
5654
1.00
0.8721
903
1.00
0.7541
Female
681
0.87
(0.61–1.25)
2529
1.01
(0.88–1.16)
851
0.96
(0.74–1.24)
Deceased Education
Age-appropriate
1346
1.00
0.8820
-
-
-
-
-
-
-
-
Below Age-appropriate 201
1.05
(0.57–1.92)
-
-
-
-
-
-
-
-
Less than Primary
-
-
-
-
3976
1.00
0.4979
1438
1.00
0.9613
Primary
-
-
-
-
1100
1.00
(0.83–1.22)
133
0.94
(0.58–1.51)
.Primary
-
-
-
-
2975
1.09
(0.94–1.27)
157
0.97
(0.60–1.57)
Death Place
Home
692
1.00
0.0009
3195
1.00
0.0000
1350
1.00
0.0000
Health Facility
250
0.98
(0.59–1.61)
1654
1.19
(1.01–1.39)
167
2.31
(1.58–3.36)
Other
579
2.18
(1.40–3.40)
3053
1.50
(1.30–1.73)
184
3.28
(2.22–4.83)
Death Registration
No
595
1.00
0.1859
1650
1.00
0.7483
411
1.00
0.5202
Yes
401
1.39
(0.85–2.28)
3419
0.97
(0.80–1.18)
695
0.90
(0.64–1.25)
Footnote: Results are adjusted for region, EAGA, urban/rural and age. Footnote: Results are adjusted for region, EAGA, urban/rural and age. PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 Discussion Using childhood deaths (age #14) of all causes, we previously
reported that physician agreement was not affected by features of
the death itself or by most geographic, socioeconomic, or
demographic characteristics of the respondent and/or deceased. The exceptions were with the gender and age of the deceased [15]. This study of adult and child injury deaths also suggests that, with
few exceptions, physician agreement on category of injury death
was not affected by these factors. Specifically, we did not find
consistent significant differences in physician agreement based on The purpose of this analysis was to assess factors that may
influence physicians’ ability to correctly classify injury deaths based
on VA questionnaires. We used physician agreement as the metric
in our analysis under the assumption that poor agreement is
correlated with difficulty in classifying injury deaths. Several
validation studies have shown that VA tools perform well for
injury deaths as one broad category with high sensitivity,
specificity, and positive predictive value when compared to PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 January 2012 | Volume 7 | Issue 1 | e30336 6 Physician Agreement on Verbal Autopsy Table 5. Place of death analysis of physician agreement stratified by injury category. Discussion Adults (15–69 years)
Elderly ($70 years)
Injury Category
Place of Death
n
OR
(95% CI)
n
OR
(95% CI)
Transport Injuries
Home
208
1
77
1
Health Facility
452
5.41*
(3.18–9.22)
43
4.92*
(1.55–15.69)
Other
1231
5.06*
(3.23–7.92)
59
4.18*
(1.51–11.58)
Falls
Home
490
1
852
1
Health Facility
155
1.20
(0.77–1.86)
72
3.12*
(1.73–5.63)
Other
151
1.49
(0.90–2.46)
36
2.42*
(1.13–5.15)
Fire
Home
96
1
24
1
Health Facility
137
1.39
(0.65–2.97)
7
-
-
Other
39
1.29
(0.46–3.56)
4
-
-
Drowning
Home
104
1
17
1
Health Facility
15
3.74
(0.71–19.8)
0
-
-
Other
266
2.07*
(1.08–3.99)
24
-
-
Venomous Snakes, Animals and Plants
Home
196
1
30
1
Health Facility
91
1.43
(0.57–3.56)
7
-
-
Other
100
0.99
(0.44–2.22)
2
-
-
Other Unintentional Injuries
Home
483
1
260
1
Health Facility
213
0.79
(0.55–1.14)
25
0.58
(0.21–1.55)
Other
333
1.76*
(1.21–2.56)
28
5.96*
(1.90–18.69)
Self-Inflicted Injuries
Home
1440
1
61
1
Health Facility
491
0.75
(0.53–1.05)
11
-
-
Other
581
0.60*
(0.44–0.81)
24
-
-
War, Violence and Other Intentional Injuries
Home
135
1
19
1
Health Facility
72
0.66
(0.32–1.35)
1
-
-
Other
301
1.02
(0.59–1.77)
6
-
-
Injuries of Undetermined Intent
Home
43
1
10
1
Health Facility
28
1.65
(0.34–8.00)
1
-
-
Other
51
1.61
(0.43–6.08)
1
-
-
Footnote: * indicates statistical significance. - indicates insufficient number of deaths for regression analysis. doi:10.1371/journal.pone.0030336.t005 Footnote: * indicates statistical significance. - indicates insufficient number of deaths for regression analysis. doi:10.1371/journal.pone.0030336.t005 Footnote: * indicates statistical significance. - indicates insufficient number of deaths for regression analysis. doi:10.1371/journal.pone.0030336.t005 longer time lag between injury and death (for example, an elderly
person becomes injured in a fall or car accident, undergoes
treatment in hospital for several months, is eventually discharged
and dies at home shortly after). When there is a longer lag time
and opportunity for more intervening events between initial injury
and death, as we hypothesize for injury deaths at home, physicians
may have more difficulty determining cause of death. In contrast
to these four categories, however, deaths due to self-inflicted injury
were associated with lower physician agreement if the death
occurred outside of home or health facility. Discussion One possible
explanation for this finding is that the circumstances around a
self-inflicted injury death is likely not as well known or reported by
the respondent if the death occurred outside of home or health
facility. On the other hand, detailed information on self-inflicted
deaths may be more available if the death occurred at home or at
health facilities. Additional studies exploring the open-ended
narrative section of the VA may provide support for these
hypotheses and generate additional explanations for the associa-
tion between location of death and physician agreement. geographic factors, respondents’ gender, education, religion,
language, or on the gender, education, and death registration of
the deceased. The similar level of agreement across all these
variables is reassuring to all VA based studies. Injury deaths are prevalent in the elderly population in India
(manuscript in preparation). While the ability of VA to yield a
broad classification of the underlying cause of death diminishes for
deaths that occur over age 70 [2], we have shown that, even in the
elderly population, VA performed reasonably well in distinguish-
ing between different injury categories. PLoS ONE | www.plosone.org Physician Agreement on Verbal Autopsy to identify associations with particular injury categories. We
believe that the association in children may be explained by the
same rationale as adults, but the association will need to be
assessed with a larger sample. gathering and identification of details to arrive at a more specific
category of injury death may help improve the VA tool. The degree of physician agreement is contingent on the number
of injury categories used in the analysis. Fewer categories would
result in higher physician agreement and vice versa. We decided
on the 9 injury categories based on a public health injury
prevention perspective. Ideally, VA should be able to distinguish
between injury categories such that the final output can be
informative for public health priority setting, strategic planning,
and disease monitoring. A limitation to our categorization is that
no inference can be made on physician ability to diagnose specific
causes of injury death within each injury category. Using transport
injury deaths as example, the study cannot comment on whether
VA is specific enough to determine the deceased’s mode of
transportation and the mechanism of collision (i.e. three character
ICD-10 ‘‘V’’ codes). This limitation is even more important for the
more heterogeneous injury categories of ‘‘other unintentional
injuries’’ and ‘‘injuries of undetermined intent’’. Limitations Although we were able to assess the association between
physician agreement and various characteristics of the respondent
and deceased, we did not analyze the impact of factors related to
the trained physician coders. Our analysis was performed under
the assumption that the clinical experience, knowledge, medical
specialty, and other factors that may affect the physician’s ability
to determine the cause of death based on VA were uniform among
all physician coders. This assumption was likely not true. Nevertheless, we believe it is unlikely that this limitation would
have led to a systematic bias in the results as the deaths were
randomly assigned to the physician coders. Future studies should
be performed to assess physician characteristics that affect their
ability to determine cause of death using a VA instrument. Acknowledgments We thank Brendon Pezzack for help with graphics and Ansely Wong for
editing support. The sensitivity and specificity analysis of the cause of death
determined in the first stage by physicians must be interpreted with
the
understanding
that
the
gold
standard
used
(RHIME-
determined cause of death) was not independent from the initial
physician assigned cause of death. Nonetheless, this analysis
uncovered that physicians have more difficulty arriving at a
diagnosis for deaths caused by injury of undetermined intent. Further interviewer and physician training to improve both the Whether Respondent Lived with Deceased & Respondent
Relationship For adult deaths, physicians were more likely to agree on the
classification of injury deaths if the respondent did not live with the
deceased. Similarly for elderly deaths, physicians were more likely
to agree if the respondent was a neighbour rather than a relative. We believe that this association may be reflective of the less
detailed narratives given by the respondents who did not live with
the deceased. For injury deaths, paradoxically, physicians may find
narratives with fewer details easier to determine the cause of death. The less detailed narratives often comprise of only a few key words
indicative of the cause of death such as ‘‘traffic accident’’ or ‘‘fall’’. Detailed narratives, however, may describe multiple events during
a longer intervening time gap between injury and death that, as
hypothesized above for deaths occurring at home, may make
determining cause of death more difficult. In summary, physician agreement on the injury category cause
of death was not affected by most characteristics of the deceased or
respondent, with the exceptions of location of death, respondent
relationship, and whether the respondent lived with the deceased. Specifically, for transport injury, fall, drowning, and other
unintentional injury deaths, the location of death outside of the
home was associated with greater physician agreement. In
contrast, self-inflicted injury deaths that occurred outside of the
home were associated with lower physician agreement. In
addition, physicians were more likely to be in agreement if the
respondent did not live with the deceased or if the respondent was
a neighbour instead of a relative. Recognition of these factors that
influence the physician’s ability to determine cause of injury death
is essential for continued adaptation and improvement of the VA
tool. Author Contributions Conceived and designed the experiments: MH SKM DGB ALM JST PJ. Performed the experiments: MH SKM DGB. Analyzed the data: MH
SKM DGB ALM JST PJ. Contributed reagents/materials/analysis tools:
MH SKM DGB ALM JST PJ. Wrote the paper: MH SKM DGB ALM
JST PJ. Conceived and designed the experiments: MH SKM DGB ALM JST PJ. Performed the experiments: MH SKM DGB. Analyzed the data: MH
SKM DGB ALM JST PJ. Contributed reagents/materials/analysis tools:
MH SKM DGB ALM JST PJ. Wrote the paper: MH SKM DGB ALM
JST PJ. Further interviewer and physician training to improve both the Location of Death For adult and elderly deaths in the categories of transport
injury, other unintentional injury, fall (elderly only), and drowning
(adults only), the likelihood of physician agreement increased if the
location of death was outside of home. This association suggests
that location of death provides an important clue for physicians in
determining the cause of death for these four injury categories. One hypothesis is that for these categories, deaths occurring
outside of home may be due to injuries of higher severity and
acuity, resulting in a more immediate death that may be easier to
classify. On the contrary, deaths occurring at home may have In children, the association between increased physician
agreement and death outside of home or health facility was also
present. However, due to the smaller sample size we were unable PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 January 2012 | Volume 7 | Issue 1 | e30336 7 Physician Agreement on Verbal Autopsy ( )
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34(1): 26–31. 5. Murray CJ, Lopez AD, Feehan DM, Peter ST, Yang G (2007) Validation of the
symptom pattern method for analyzing verbal autopsy data. PLoS Med 4(11):
e327. 19. Victora CG, Huttly SR, Fuchs SC, Olinto MT (1997) The role of conceptual
frameworks in epidemiological analysis: A hierarchical approach. Int J Epidemiol
26(1): 224–227. 17. Landis JR, Koch GG (1977) The measurement of observer agreement for
categorical data. Biometrics 33(1): 159–174.
18. Reichenheim ME (2004) Confidence intervals for the kappa statistic. Stata
Journal 4(4): 421–428. ( )
20. StataCorp (2007) Stata statistical software: Release 10. g
( )
18. Reichenheim ME (2004) Confidence intervals for the kappa statistic. Stata
Journal 4(4): 421–428. 17. Landis JR, Koch GG (1977) The measurement of observer agreement for
categorical data. Biometrics 33(1): 159–174. 19. Victora CG, Huttly SR, Fuchs SC, Olinto MT (1997) The role of conceptual
frameworks in epidemiological analysis: A hierarchical approach. Int J Epidemiol
26(1): 224–227.
20. StataCorp (2007) Stata statistical software: Release 10. Physician Agreement on Verbal Autopsy References World Health Organization, editor (2007) International statistical classification
of diseases and related health problems, 10th revision. 8. Gajalakshmi V, Peto R (2004) Verbal autopsy of 80,000 adult deaths in
Tamilnadu, south India. BMC Public Health 4: 47. PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 January 2012 | Volume 7 | Issue 1 | e30336 8 Physician Agreement on Verbal Autopsy PLoS ONE | www.plosone.org January 2012 | Volume 7 | Issue 1 | e30336 9
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Evaluation of Flavored Cigar Products As They Relate To Questions of Public Health
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Evaluation of Flavored Cigar Products As They
Relate To Questions of Public Health Andrew Joyce ( andrew.joyce@consiliumsciences.com )
Consilium Sciences, LLC Andrew Joyce ( andrew.joyce@consiliumsciences.com )
Consilium Sciences, LLC Research Article Keywords: Cigars, Flavors, Use Patterns, Public Health
Posted Date: November 15th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-1025233/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Evaluation of flavored cigar products as they relate to questions
1
public health
2
3
Short title: Flavored cigars and questions of public health
4
5
Andrew R Joyce1*
6
1Consilium Sciences, LLC, 7400 Beaufont Springs Drive, Suite 300, N. Chesterfield, VA
7
8
* Author for correspondence
9
E-mail: andrew.joyce@venebio.com
10 Evaluation of flavored cigar products as they relate to questions of
1
public health
2 1 1 Abstract
11
Background: Flavors in tobacco products is a subject of public health debate and in
12
regulatory attention. There is interest in gaining an in-depth understanding of flavor
13
smoking prevalence and behaviors to address the use of flavors in cigars and questio
14
health. 15
Methods: Seven publicly available data resources that assess flavored cigar use wer
16
Two focus on youth tobacco use (NYTS, MTF), four focus on adult tobacco use (HI
17
NATS, TPRPS, TUS-CPS), and one on both groups (PATH). Available data (2011-
18
analyzed to assess usage trends over time. In addition, longitudinal analysis of PAT
19
examined whether flavored cigar use was associated with future use of cigarettes or
20
use of cigars. 21
Results: Youth past 30-day estimates of cigar use ranged from 2%-10% for both fla
22
non-flavored cigars, slightly higher in high school vs. middle school age subpopulat
23
estimates have been stable or declined across all survey years within the respective s
24
Consistent trends were observed regarding frequency of use; most youth using cigar
25
days per month. 26
Similar findings were observed for adult cigar users, with five surveys indicating les
27
currently use cigars. Flavored cigar use is at less than 5% across all data sources. T
28
overarching use estimates were essentially flat over time. Frequency of youth cigar
29
remained consistent over time, with most youth reporting cigar use on 1-2 days per m
30
addition, multivariable modeling of PATH adult data did not identify an association
31
flavored cigar use and future use of cigarettes or increased use of cigars. 32
Conclusions: No evidence was found of increased use or different usage patterns, a
33
youth or adults, of flavored cigars vs. non-flavored cigars. While these trends shoul
34
be monitored, there is no indication of existing or emerging public health concerns r
35 Abstract
11 Background: Flavors in tobacco products is a subject of public health debate and increasing
12
regulatory attention. There is interest in gaining an in-depth understanding of flavored cigar
13
smoking prevalence and behaviors to address the use of flavors in cigars and questions of public
14
health. 15 Methods: Seven publicly available data resources that assess flavored cigar use were analyzed. 16
Two focus on youth tobacco use (NYTS, MTF), four focus on adult tobacco use (HINTS-FDA,
17
NATS, TPRPS, TUS-CPS), and one on both groups (PATH). Available data (2011-2019) were
18
analyzed to assess usage trends over time. In addition, longitudinal analysis of PATH adult data
19
examined whether flavored cigar use was associated with future use of cigarettes or increased
20
use of cigars. 21 Results: Youth past 30-day estimates of cigar use ranged from 2%-10% for both flavored and
22
non-flavored cigars, slightly higher in high school vs. middle school age subpopulations. These
23
estimates have been stable or declined across all survey years within the respective surveys. 24
Consistent trends were observed regarding frequency of use; most youth using cigars do so 1-2
25
days per month. 26 Results: Youth past 30-day estimates of cigar use ranged from 2%-10% for both flavored and
22
non-flavored cigars, slightly higher in high school vs. middle school age subpopulations. These
23
estimates have been stable or declined across all survey years within the respective surveys. 24
Consistent trends were observed regarding frequency of use; most youth using cigars do so 1-2
25
days per month. 26 estimates have been stable or declined across all survey years within the respective surveys. 24
Consistent trends were observed regarding frequency of use; most youth using cigars do so 1-2
25
days per month. 26
Similar findings were observed for adult cigar users, with five surveys indicating less than 10%
27
currently use cigars. Flavored cigar use is at less than 5% across all data sources. These
28
overarching use estimates were essentially flat over time. Frequency of youth cigar use
29
remained consistent over time, with most youth reporting cigar use on 1-2 days per month. In
30
addition, multivariable modeling of PATH adult data did not identify an association between
31
flavored cigar use and future use of cigarettes or increased use of cigars. Abstract
11 32
Conclusions: No evidence was found of increased use or different usage patterns, among either
33
youth or adults, of flavored cigars vs. non-flavored cigars. While these trends should continue to
34
be monitored, there is no indication of existing or emerging public health concerns related to
35
flavored cigars within the seven large, nationally representative, US government-funded
36
epidemiologic databases examined. 37 Similar findings were observed for adult cigar users, with five surveys indicating less than 10%
27
currently use cigars. Flavored cigar use is at less than 5% across all data sources. These
28
overarching use estimates were essentially flat over time. Frequency of youth cigar use
29
remained consistent over time, with most youth reporting cigar use on 1-2 days per month. In
30
addition, multivariable modeling of PATH adult data did not identify an association between
31
flavored cigar use and future use of cigarettes or increased use of cigars. 32 Conclusions: No evidence was found of increased use or different usage patterns, among either
33
youth or adults, of flavored cigars vs. non-flavored cigars. While these trends should continue to
34
be monitored, there is no indication of existing or emerging public health concerns related to
35
flavored cigars within the seven large, nationally representative, US government-funded
36
epidemiologic databases examined. 37 2 2 38 Cigars, Flavors, Use Patterns, Public Health
39 Background
40 Background
40
The issue of flavors in tobacco products has been the subject of intense public health debate and
41
increasing regulatory attention. FDA has expressed concern about flavors in tobacco products and
42
their potential effects on public health and, in March 2018, issued an Advance Notice of Proposed
43
Rulemaking on this issue [1]. This sentiment was communicated again in their Draft Guidance
44
Document from March 2019 that proposed modifying the August 2017 Compliance Policy for
45
flavors in cigars [2]; however, ultimately FDA determined to not take enhanced enforcement
46
action against flavored cigars at that time. Most recently, FDA announced an intention to release
47
a Proposed Product Standard to Prohibit Characterizing Flavors in Cigars [3]. Due to this
48
continuing regulatory interest, it is important to gain an in-depth understanding of flavored cigar
49
smoking behaviors, prevalence, and use trajectories, as they relate to questions of public health. 50
Much of the recent published literature on flavored cigar use focuses on the impact of bans on
51
flavored tobacco products, imposed at national [4] and local levels [5-7] over the past several
52
years. Other studies have assessed potential abuse liability of flavored cigars [8, 9], and/or
53
examined various associations and impacts of flavored non-cigarette tobacco product use in
54
general (i.e., aggregating the use of flavored e-cigarettes, smokeless tobacco, hookah, pipe, and
55
cigar products into a singular construct) [10-12]. Some studies continue to cite sometimes decade
56
or older trends [13-15] and relatively few studies have reported on recent prevalence and use of
57
cigars in general or flavored cigar products specifically. A 2019 systematic review of the literature
58
on cigar research on youth echoed this sentiment in identifying flavored cigar research as an
59
understudied domain [16]. 60 The issue of flavors in tobacco products has been the subject of intense public health debate and
41
increasing regulatory attention. FDA has expressed concern about flavors in tobacco products and
42
their potential effects on public health and, in March 2018, issued an Advance Notice of Proposed
43
Rulemaking on this issue [1]. This sentiment was communicated again in their Draft Guidance
44
Document from March 2019 that proposed modifying the August 2017 Compliance Policy for
45
flavors in cigars [2]; however, ultimately FDA determined to not take enhanced enforcement
46
action against flavored cigars at that time. Keywords Cigars, Flavors, Use Patterns, Public Health
39 3 Background
40 Most recently, FDA announced an intention to release
47
a Proposed Product Standard to Prohibit Characterizing Flavors in Cigars [3]. Due to this
48
continuing regulatory interest, it is important to gain an in-depth understanding of flavored cigar
49
smoking behaviors, prevalence, and use trajectories, as they relate to questions of public health. 50 Much of the recent published literature on flavored cigar use focuses on the impact of bans on
51
flavored tobacco products, imposed at national [4] and local levels [5-7] over the past several
52
years. Other studies have assessed potential abuse liability of flavored cigars [8, 9], and/or
53
examined various associations and impacts of flavored non-cigarette tobacco product use in
54
general (i.e., aggregating the use of flavored e-cigarettes, smokeless tobacco, hookah, pipe, and
55
cigar products into a singular construct) [10-12]. Some studies continue to cite sometimes decade
56
or older trends [13-15] and relatively few studies have reported on recent prevalence and use of
57
cigars in general or flavored cigar products specifically. A 2019 systematic review of the literature
58
on cigar research on youth echoed this sentiment in identifying flavored cigar research as an
59
understudied domain [16]. 60 To this end, we conducted a thorough investigation of federally funded, national databases focused
61
on tobacco product use to assess cigar use, including flavored cigar use, prevalence and frequency
62 of use in U.S. youth and adults. The National Youth Tobacco Survey (NYTS), Population
63
Assessment of Tobacco and Health Study (PATH), Health Information National Trends Survey
64
(HINTS), Monitoring the Future (MTF), National Adult Tobacco Survey (NATS), Tobacco
65
Product and Risk Perception Survey (TPRPS), and Tobacco Use Supplement to Current Population
66
Survey (TUS-CPS) were identified as resources that assess not only cigar use overall, but also
67
flavored cigar use specifically, in U.S. youth and/or adults. Here, we present findings from our
68
analyses of these resources to identify similarities and differences in flavored versus non-flavored
69
cigars as they relate to youth and adult use prevalence, smoking behaviors, and use trajectories
70
including potential transition to combustible cigarette smoking or increased use frequency. 71 of use in U.S. youth and adults. Data Sources
73 Several nationally representative epidemiologic surveys and studies were identified based on a
74
review of medical and public health literature as well as online resources including the Inter-
75
University Consortium for Political and Social Research (ICPSR), the Georgia State University
76
Tobacco Portal; the Centers for Disease Control Office on Smoking and Health website, and a
77
broad web-based search using key search terms related to cigar products and flavors. The seven
78
identified publicly available data sources included cross-sectional collections of survey data and
79
questions related to use of flavored cigar products. Table 1 provides a brief description of the
80
population assessed and overarching content of each dataset. 81 Two of the identified resources focus on youth tobacco use (NYTS [17] and MTF [18]), four focus
82
on adult tobacco use (HINTS-FDA [19, 20], NATS [21], TPRPS [22], and TUS-CPS [23]), and
83
one collects survey results on both youth and adult tobacco users (PATH [24]). Notably, the Youth
84
Risk Behavior Surveillance System (YRBSS [25]), a long-established data resource on youth
85
tobacco use, was not included in this analysis because it does not include questions that specifically
86
address the use of flavored tobacco. 87 Two of the identified resources focus on youth tobacco use (NYTS [17] and MTF [18]), four focus
82
on adult tobacco use (HINTS-FDA [19, 20], NATS [21], TPRPS [22], and TUS-CPS [23]), and
83
one collects survey results on both youth and adult tobacco users (PATH [24]). Notably, the Youth
84
Risk Behavior Surveillance System (YRBSS [25]), a long-established data resource on youth
85
tobacco use, was not included in this analysis because it does not include questions that specifically
86
address the use of flavored tobacco. 87 Datasets that included questions related to use of flavored cigars were acquired from each of the
88
seven identified resources. Table 2 specifies the datasets, the dates that surveys were administered,
89
and brief descriptions of data elements that were extracted for analysis. For all but the TRPRS,
90
public use files were downloaded directly from the respective websites that provide access. The
91
TRPRS data files were acquired via direct download after formally requesting access and entering
92 Datasets that included questions related to use of flavored cigars were acquired from each of the
88
seven identified resources. Background
40 The National Youth Tobacco Survey (NYTS), Population
63
Assessment of Tobacco and Health Study (PATH), Health Information National Trends Survey
64
(HINTS), Monitoring the Future (MTF), National Adult Tobacco Survey (NATS), Tobacco
65
Product and Risk Perception Survey (TPRPS), and Tobacco Use Supplement to Current Population
66
Survey (TUS-CPS) were identified as resources that assess not only cigar use overall, but also
67
flavored cigar use specifically, in U.S. youth and/or adults. Here, we present findings from our
68
analyses of these resources to identify similarities and differences in flavored versus non-flavored
69
cigars as they relate to youth and adult use prevalence, smoking behaviors, and use trajectories
70
including potential transition to combustible cigarette smoking or increased use frequency. 71 5 5 Data Sources
73 Table 2 specifies the datasets, the dates that surveys were administered,
89
and brief descriptions of data elements that were extracted for analysis. For all but the TRPRS,
90
public use files were downloaded directly from the respective websites that provide access. The
91
TRPRS data files were acquired via direct download after formally requesting access and entering
92 6 6 into a Data Sharing Agreement for the survey data and related materials from the Georgia State
93
University Tobacco Center of Regulatory Science (TCORS). 94 into a Data Sharing Agreement for the survey data and related materials from the Georgia State
93
University Tobacco Center of Regulatory Science (TCORS). 94 into a Data Sharing Agreement for the survey data and related materials from the Georgia State
93
University Tobacco Center of Regulatory Science (TCORS). 94 In order to adjust appropriately for complex study design characteristics, such as stratification,
95
clustering within primary sampling units, and oversampling, population and replicate weights (as
96
available) were used in calculating all estimates presented herein. The weights also adjust for
97
nonresponse as appropriate. Use of these weights is required to produce results that are nationally
98
representative of the U.S. civilian, noninstitutionalized population. 99 In order to adjust appropriately for complex study design characteristics, such as stratification,
95
clustering within primary sampling units, and oversampling, population and replicate weights (as
96
available) were used in calculating all estimates presented herein. The weights also adjust for
97
nonresponse as appropriate. Use of these weights is required to produce results that are nationally
98
representative of the U.S. civilian, noninstitutionalized population. 99 Measures
100 Cigar types. The cigar category is diverse, with cigars varying in size, shape, and weight, being
101
filtered or unfiltered, and considered premium hand-rolled, or more economical machine-made
102
cigars. The various surveys refer to cigars, and therefore collect data on their use, in somewhat
103
different manners. Most surveys do not specify the category of cigar with extensive granularity in
104
their questioning and tend to ask about cigar usage in a more general manner. Certain surveys,
105
however, offer sets of questions specific to larger cigars versus smaller cigars. For example, PATH
106
Study participants report separately whether they used filtered cigars, cigarillos, and/or traditional
107
cigars (i.e., large cigars), with the PATH questionnaire displaying a photo of example products,
108
describing their physical characteristics, and listing popular brands. Researchers have also
109
attempted to stratify the large cigar category into premium and non-premium subtypes using price
110
per unit and/or respondent-provided brand name information [26, 27]. Doing so, however,
111
introduces potential complications related to recall error/inconsistency that can raise questions
112
regarding estimate accuracy and validity. 113 Cigar types. The cigar category is diverse, with cigars varying in size, shape, and weight, being
101
filtered or unfiltered, and considered premium hand-rolled, or more economical machine-made
102
cigars. The various surveys refer to cigars, and therefore collect data on their use, in somewhat
103
different manners. Most surveys do not specify the category of cigar with extensive granularity in
104
their questioning and tend to ask about cigar usage in a more general manner. Certain surveys,
105
however, offer sets of questions specific to larger cigars versus smaller cigars. For example, PATH
106
Study participants report separately whether they used filtered cigars, cigarillos, and/or traditional
107
cigars (i.e., large cigars), with the PATH questionnaire displaying a photo of example products,
108
describing their physical characteristics, and listing popular brands. Researchers have also
109
attempted to stratify the large cigar category into premium and non-premium subtypes using price
110
per unit and/or respondent-provided brand name information [26, 27]. Doing so, however,
111
introduces potential complications related to recall error/inconsistency that can raise questions
112
regarding estimate accuracy and validity. 113 7 7 7 For simplicity, the analyses generally consider cigar usage in the overall sense, without
114
stratification into subtypes. Measures
100 One exception to this paradigm is in the MTF survey, which asks
115
questions about use of flavored little cigars and cigarillos separately from use of “regular” little
116
cigars and cigarillos. A third large cigar category is also considered. This three-group
117
stratification presents challenges in separating flavored cigar use from cigar use in general. As
118
such, the three categories are summarized separately. In all other surveys, use of flavored cigars
119
is queried separately from use of cigars in general. This split enables investigation of possible
120
differences in overall cigar usage versus usage of flavored cigars, without further complicating
121
matters by introducing product subtypes. 122 One additional instance in which cigar usage data were not aggregated is represented in the
123
longitudinal analyses of PATH. Here, data were retained in their standard form, stratified into the
124
three PATH cigar categories (cigarillos, filtered cigars, traditional cigars) due to challenges related
125
to combining validly results from the relevant survey questions required to conduct the
126
corresponding analyses. 127 Cigar use. Cigar use is defined in various ways in the data sources considered. Current use by
128
adults is defined in PATH as those who currently used the cigar type “every day” or “some
129
days”. Other surveys report on use “now” or in the past 30 days. Current use for youth tends
130
to be defined differently and requires only past 30-day use of cigars. In all cases, the respective
131
Results subsections and corresponding figure and table descriptions use precise terminology
132
to align with the language used in the survey. 133 Cigar use. Cigar use is defined in various ways in the data sources considered. Current use by
128
adults is defined in PATH as those who currently used the cigar type “every day” or “some
129
days”. Other surveys report on use “now” or in the past 30 days. Current use for youth tends
130
to be defined differently and requires only past 30-day use of cigars. In all cases, the respective
131
Results subsections and corresponding figure and table descriptions use precise terminology
132
to align with the language used in the survey. 133 Cigarette use. Current adult users of cigarettes are identified in the PATH Study as currently using
134
cigarettes “every day” or “some days.” A minimum level of lifetime use (≥100 cigarettes) is also
135 Cigarette use. Measures
100 Current adult users of cigarettes are identified in the PATH Study as currently using
134
cigarettes “every day” or “some days.” A minimum level of lifetime use (≥100 cigarettes) is also
135 8 8 required for current established cigarette users. Current use of cigarettes by youth is defined
136
somewhat differently in PATH by requiring only past 30-day use of any frequency. 137
Analyses
138
Analyses included adult and youth current users of cigars (typically in aggregate, although in some
139
circumstances stratified into broad subtypes). Analyses of each cigar type were not mutually
140
exclusive in that participants could be users of multiple cigar types. 141
We conducted analyses separately for each survey using R 3.4.0 [28] and the survey package [29,
142
30] to generate weighted estimates to represent the US civilian, non-institutionalized adult (18+)
143
and youth (12-17) populations. Where appropriate (e.g., PATH), estimated standard errors and
144
95% confidence intervals (CIs) were calculated using the balanced repeated replication method
145
[31] with Fay’s adjustment set to 0.3 to increase estimate stability [32] as recommended in survey
146
data user guide documentation. 147 required for current established cigarette users. Current use of cigarettes by youth is defined
136
somewhat differently in PATH by requiring only past 30-day use of any frequency. 137 required for current established cigarette users. Current use of cigarettes by youth is defined
136
somewhat differently in PATH by requiring only past 30-day use of any frequency. 137 required for current established cigarette users. Current use of cigarettes by youth is defined
136
somewhat differently in PATH by requiring only past 30-day use of any frequency. 137 Analyses included adult and youth current users of cigars (typically in aggregate, although in some
139
circumstances stratified into broad subtypes). Analyses of each cigar type were not mutually
140
exclusive in that participants could be users of multiple cigar types. 141 Analyses included adult and youth current users of cigars (typically in aggregate, although in some
139
circumstances stratified into broad subtypes). Analyses of each cigar type were not mutually
140
exclusive in that participants could be users of multiple cigar types. 141 We conducted analyses separately for each survey using R 3.4.0 [28] and the survey package [29,
142
30] to generate weighted estimates to represent the US civilian, non-institutionalized adult (18+)
143
and youth (12-17) populations. Measures
100 Where appropriate (e.g., PATH), estimated standard errors and
144
95% confidence intervals (CIs) were calculated using the balanced repeated replication method
145
[31] with Fay’s adjustment set to 0.3 to increase estimate stability [32] as recommended in survey
146
data user guide documentation. 147 We conducted analyses separately for each survey using R 3.4.0 [28] and the survey package [29,
142
30] to generate weighted estimates to represent the US civilian, non-institutionalized adult (18+)
143
and youth (12-17) populations. Where appropriate (e.g., PATH), estimated standard errors and
144
95% confidence intervals (CIs) were calculated using the balanced repeated replication method
145
[31] with Fay’s adjustment set to 0.3 to increase estimate stability [32] as recommended in survey
146
data user guide documentation. 147 For PATH, cross-sectional analyses used weights corresponding to the appropriate wave and
148
weight type (i.e., single wave weights for Waves 1-3 and cross-sectional weights for Wave 4). 149
Longitudinal analyses used the predicted wave weights in accordance with PATH Study
150
recommendations and multivariable logistic regression to examine associations between flavored
151
cigar use and future product use behavior. Adjusted analyses controlled for sex (male, female),
152
age, race/ethnicity (White, non-Hispanic; Black, non-Hispanic; Other, non-Hispanic; Hispanic),
153
and education (less than high school, General Educational Development [GED], high school, some
154
college or associate’s degree, bachelor’s degree or more advanced degree). This approach follows
155
that used by Persoskie and colleagues in their study of cigar package quantity and pricing on
156
smoking behavior [27]. Missing data on these variables were not imputed. So-called “imputed”
157 For PATH, cross-sectional analyses used weights corresponding to the appropriate wave and
148
weight type (i.e., single wave weights for Waves 1-3 and cross-sectional weights for Wave 4). 149
Longitudinal analyses used the predicted wave weights in accordance with PATH Study
150
recommendations and multivariable logistic regression to examine associations between flavored
151
cigar use and future product use behavior. Adjusted analyses controlled for sex (male, female),
152
age, race/ethnicity (White, non-Hispanic; Black, non-Hispanic; Other, non-Hispanic; Hispanic),
153
and education (less than high school, General Educational Development [GED], high school, some
154
college or associate’s degree, bachelor’s degree or more advanced degree). This approach follows
155
that used by Persoskie and colleagues in their study of cigar package quantity and pricing on
156
smoking behavior [27]. Missing data on these variables were not imputed. versions of demographic variables were used where available. These variables typically make
158
reasonable inferences based on other PATH Survey documentation to make assignments for
159
otherwise missing sex, age, or race/ethnicity variables, for example.
160 Measures
100 So-called “imputed”
157 9 9 versions of demographic variables were used where available. These variables typically make
158
reasonable inferences based on other PATH Survey documentation to make assignments for
159
otherwise missing sex, age, or race/ethnicity variables, for example. 160 10 10 Results
161
Youth Cigar Use
162
Current youth use trends over time. Cigar usage estimates for the United States youth populat
163
are generally similar based on NYTS, PATH, and MTF data. Past 30-day estimates of cigar
164
overall tend to be approximately 2%-10% overall or for flavored cigars specifically, slightly hig
165
in older relative to younger subpopulations. These estimates have remained generally stable
166
declined across all survey years within the respective surveys. 167
Figure 1 shows youth usage of cigars overall and for flavored cigars specifically across all analy
168
survey years for NYTS and PATH. NYTS estimates of past 30-day use (on at least one d
169
between 2011 and 2013 were consistent indicating that approximately 8% of the United St
170
youth population used cigar products at least once in the past 30 days. A notable shift occurre
171
2014 when estimates dropped to approximately 5% with estimates remaining essentially stabl
172
that level through the most recently available survey year in 2019. This trend appears to be dri
173
largely by the high school subpopulation (approximately 12% past 30-day use from 2011-20
174
dropping to approximately 7.5% from 2014-2019), whereas middle school estimates w
175
essentially flat or slightly declining throughout (approximately 3.5% in 2011 to approxima
176
2.5% in 2019). 177 Current youth use trends over time. Cigar usage estimates for the United States youth population
163
are generally similar based on NYTS, PATH, and MTF data. Past 30-day estimates of cigar use
164
overall tend to be approximately 2%-10% overall or for flavored cigars specifically, slightly higher
165
in older relative to younger subpopulations. These estimates have remained generally stable or
166
declined across all survey years within the respective surveys. 167 Current youth use trends over time. Cigar usage estimates for the United States youth population
163
are generally similar based on NYTS, PATH, and MTF data. Past 30-day estimates of cigar use
164
overall tend to be approximately 2%-10% overall or for flavored cigars specifically, slightly higher
165
in older relative to younger subpopulations. Measures
100 These estimates have remained generally stable or
166
declined across all survey years within the respective surveys. 167 Figure 1 shows youth usage of cigars overall and for flavored cigars specifically across all analyzed
168
survey years for NYTS and PATH. NYTS estimates of past 30-day use (on at least one day)
169
between 2011 and 2013 were consistent indicating that approximately 8% of the United States
170
youth population used cigar products at least once in the past 30 days. A notable shift occurred in
171
2014 when estimates dropped to approximately 5% with estimates remaining essentially stable at
172
that level through the most recently available survey year in 2019. This trend appears to be driven
173
largely by the high school subpopulation (approximately 12% past 30-day use from 2011-2013,
174
dropping to approximately 7.5% from 2014-2019), whereas middle school estimates were
175
essentially flat or slightly declining throughout (approximately 3.5% in 2011 to approximately
176
2.5% in 2019). 177 The NYTS trend over time for past 30-day flavored cigar use is more volatile, largely due to
178
changes to the corresponding question between survey years. In survey years 2011 and 2013,
179
respondents were asked only about use of flavored little cigars, and in 2012, respondents were
180
asked only about use of flavored tobacco products in general. It should also be noted that the
181
flavored cigar use question was not asked only of individuals indicating past 30-day cigar use, but
182 11 11 instead of all survey participants. Past 30-day flavored cigar users, therefore, should not be
183
interpreted as reflecting a true subset of NYTS participants indicating past 30-day cigar use. 184
Recognizing some estimate instability early in the time course, estimates for past 30-day use of
185
flavored cigars among United States youth overall have declined steadily from a high of
186
approximately 5% in 2015 to approximately 3% in 2019. This trend again appears to be driven by
187
the high school population (declining from a high of over 7.5% in 2015 to just over 4% in 2019),
188
with middle school estimates remaining generally stable at approximately 1.5% from 2011-2019. 189
190
Youth usage of any cigars based on PATH data from Wave 1 (collected 2013/14) through Wave
191
4 (collected late 2016 through early 2018) followed a similar trend to NYTS (Figure 1). Measures
100 192
Estimates of past 30-day cigar use in youths overall declined from Wave 1 (approximately 2.5%)
193
through Wave 3 and remained stable at approximately 1.5% through Wave 4. Use of flavored
194
cigars has followed a similar trend falling from approximately 2% in Wave 1 to approximately
195
0.5% in Wave 4. As observed in NYTS, these trends appear driven by the high school age 15–
196
17-year subpopulation that tracks the overall trend with slightly higher estimates, with the middle
197
school age 12–14-year subpopulation estimates remaining at consistent low levels across Waves
198
1 through Wave 4 (approximately 0.5% overall and approximately 0.25% for flavored cigar use
199
specifically). 200
MTF structures its cigar-related questions differently and does not assess flavored cigars
201 instead of all survey participants. Past 30-day flavored cigar users, therefore, should not be
183
interpreted as reflecting a true subset of NYTS participants indicating past 30-day cigar use. 184 instead of all survey participants. Past 30-day flavored cigar users, therefore, should not be
183
interpreted as reflecting a true subset of NYTS participants indicating past 30-day cigar use. 184 Recognizing some estimate instability early in the time course, estimates for past 30-day use of
185
flavored cigars among United States youth overall have declined steadily from a high of
186
approximately 5% in 2015 to approximately 3% in 2019. This trend again appears to be driven by
187
the high school population (declining from a high of over 7.5% in 2015 to just over 4% in 2019),
188
with middle school estimates remaining generally stable at approximately 1.5% from 2011-2019. 189
190 Recognizing some estimate instability early in the time course, estimates for past 30-day use of
185
flavored cigars among United States youth overall have declined steadily from a high of
186
approximately 5% in 2015 to approximately 3% in 2019. This trend again appears to be driven by
187
the high school population (declining from a high of over 7.5% in 2015 to just over 4% in 2019),
188
with middle school estimates remaining generally stable at approximately 1.5% from 2011-2019. 189
190 Youth usage of any cigars based on PATH data from Wave 1 (collected 2013/14) through Wave
191
4 (collected late 2016 through early 2018) followed a similar trend to NYTS (Figure 1). Measures
100 192
Estimates of past 30-day cigar use in youths overall declined from Wave 1 (approximately 2.5%)
193
through Wave 3 and remained stable at approximately 1.5% through Wave 4. Use of flavored
194
cigars has followed a similar trend falling from approximately 2% in Wave 1 to approximately
195
0.5% in Wave 4. As observed in NYTS, these trends appear driven by the high school age 15–
196
17-year subpopulation that tracks the overall trend with slightly higher estimates, with the middle
197
school age 12–14-year subpopulation estimates remaining at consistent low levels across Waves
198
1 through Wave 4 (approximately 0.5% overall and approximately 0.25% for flavored cigar use
199
specifically). 200 MTF structures its cigar-related questions differently and does not assess flavored cigars
201
generally. Rather, MTF asks more targeted questions focused on recent use of flavored little
202
cigars or cigarillos, regular (i.e., non-flavored) little cigars or cigarillos, and large cigars,
203
separately. The three usage groups are not mutually exclusive, and some respondents are
204 MTF structures its cigar-related questions differently and does not assess flavored cigars
201
generally. Rather, MTF asks more targeted questions focused on recent use of flavored little
202
cigars or cigarillos, regular (i.e., non-flavored) little cigars or cigarillos, and large cigars,
203
separately. The three usage groups are not mutually exclusive, and some respondents are
204 12 represented in multiple subgroups. Estimates of youth use of flavored little cigars or cigarillos in
205
MTF have declined somewhat for 12th graders from approximately 12% in 2014 to less than 9%
206
in 2018 (Figure 2). A more modest declining trend was similarly observed for 10th and 8th
207
graders. Declining trends were likewise observed for all MTF youth subgroups for regular little
208
cigars or cigarillos and large cigars. 209 Frequency of youth use Current cigar usage in these surveys only requires only affirmative
210
indication of any use in the past 30 days; in other words, use on one day is counted the same as
211
use on every day in the past month. To better understand usage patterns, the frequency of use in
212
the past month was examined for NYTS and MTF among past 30-day cigar users overall and when
213
limited to flavored cigar users only, respectively. Measures
100 Consistent trends were again observed with the
214
majority of past 30-day youth users limiting cigar use to 1-2 days per month with another
215
approximately 20% of past 30-day youth users limiting cigar use to 3-5 days. Further, overall youth
216
estimates are generally similar to those from both high school and middle school past 30-day cigar
217
using subpopulations. These trends hold across all survey years examined for overall or flavored
218
cigar use in NYTS (Figure 3) and across the three cigar product categories assessed in MTF (Figure
219
4). 220 Current adult use trends over time. Cigar usage estimates for the United States adult population
222
are generally similar based on the five epidemiologic data sources analyzed. Despite slight
223
differences in how current cigar use is defined across the respective surveys, all surveys indicate
224
that less than 10% of adults currently use cigars of any type. Flavored cigar use estimates are
225
similarly consistent at less than 5% across all data sources examined. Further, while estimates
226 Current adult use trends over time. Cigar usage estimates for the United States adult population
222
are generally similar based on the five epidemiologic data sources analyzed. Despite slight
223
differences in how current cigar use is defined across the respective surveys, all surveys indicate
224
that less than 10% of adults currently use cigars of any type. Flavored cigar use estimates are
225
similarly consistent at less than 5% across all data sources examined. Further, while estimates
226 13 vary somewhat between data collections within respective surveys, these overarching use
227
estimates are essentially flat over time. 228 vary somewhat between data collections within respective surveys, these overarching use
227
estimates are essentially flat over time. 228 Figure 5 shows adult usage of cigars overall and for flavored cigars specifically, across all analyzed
229
survey years for NATS, PATH, and the TPRPS. NATS estimates for the adults who use cigars
230
rarely, some days or every day were essentially unchanged from 2009/10 through 2013/14
231
(approximately 7% of United States adults overall). Estimates of past 30-day flavored cigar use
232
are similarly consistent over time at approximately 2.5% from 2009/10 through 2013/14. PATH
233
estimates were similar for current established adult users of cigars in the United States with Wave
234
1 (collected 2013/14) through Wave 4 (collected 2016/18) estimates generally stable at
235
approximately 3%. Measures
100 255 cigar users were essentially flat at 5% (the survey questions were structured such that current non-
250
flavored cigar users who use other non-cigar, flavored tobacco products could be counted among
251
this subgroup). TUS-CPS estimates of current adult use (every day or some days) of regular cigars,
252
or cigarillos, or little filtered cigars were stable at approximately 2% based on data collected in
253
2014-2015 and 2018. A similar pattern for current users of cigars that usually use flavored cigar
254
products was observed with estimates stable at less than 0.5%. 255 Frequency of adult use. Consistent trends were likewise observed when more closely examining
256
the frequency of use among adult cigar users. A relatively small proportion are every day cigar
257
users and the most commonly reported use pattern is 1-2 days per month for surveys with
258
sufficiently granular information on use frequency. These trends have held stable across several
259
data sources and over time. 260 Frequency of adult use. Consistent trends were likewise observed when more closely examining
256
the frequency of use among adult cigar users. A relatively small proportion are every day cigar
257
users and the most commonly reported use pattern is 1-2 days per month for surveys with
258
sufficiently granular information on use frequency. These trends have held stable across several
259
data sources and over time. 260 TUS-CPS and TPRPS past 30-day frequency of use was examined for cigar users overall and when
261
limited to flavored cigar users only (Figure 6). Consistent trends were observed across data
262
collections with the majority of cigar users indicating use on 1-2 days or 3-5 days. Less than 5%
263
of users indicated use on 20+ days or more per month in the TUS-CPS, whereas TPRPS estimates
264
were somewhat higher at approximately 15-20% in all data collection years except for 2015. These
265
trends hold for both overall and flavored cigar use only. 266 Measures
100 Past 30-day flavored cigar use among adults in the United States were also
236
consistent at approximately 2% across Waves 2 through 4 (Wave 1 did not include specific
237
flavored cigar use questions). TPRPS estimates of past 30-day adult use (even one or two puffs)
238
of traditional cigars, cigarillos, filtered or little cigars varied between 4% in 2014 to nearly 7
239
percent in 2017 without a clear direction in trend. A similar pattern for past 30-day use of flavored
240
cigars was observed between 2015 and 2017 (the 2014 survey did not ask specifically about past
241
30-day flavored cigar use). Only respondents indicating recent use of little cigars, cigarillos, or
242
filtered cigars were asked about flavored cigar use, with estimates varying between approximately
243
2% in 2015 and approximately 2.5% in 2017 also without a clear discernible direction in trend. 244 The two respective sets of data analyzed for HINTS-FDA and TUS-CPS yielded similar findings
245
(Supplementary Figure 1). HINTS-FDA current (every day or some days) cigar use estimates
246
among American adults showed a somewhat declining trend, from approximately 5% in 2015 to
247
approximately 4% in 2017, although with largely overlapping confidence intervals. HINTS-FDA
248
estimates of past 30-day use of flavored tobacco products among current (every day or some day)
249 The two respective sets of data analyzed for HINTS-FDA and TUS-CPS yielded similar findings
245
(Supplementary Figure 1). HINTS-FDA current (every day or some days) cigar use estimates
246
among American adults showed a somewhat declining trend, from approximately 5% in 2015 to
247
approximately 4% in 2017, although with largely overlapping confidence intervals. HINTS-FDA
248
estimates of past 30-day use of flavored tobacco products among current (every day or some day)
249 14 cigar users were essentially flat at 5% (the survey questions were structured such that current non-
250
flavored cigar users who use other non-cigar, flavored tobacco products could be counted among
251
this subgroup). TUS-CPS estimates of current adult use (every day or some days) of regular cigars,
252
or cigarillos, or little filtered cigars were stable at approximately 2% based on data collected in
253
2014-2015 and 2018. A similar pattern for current users of cigars that usually use flavored cigar
254
products was observed with estimates stable at less than 0.5%. Flavored Cigar Use and Other Tobacco Use Behavior
267 Flavored Cigar Use and Subsequent Cigarette Use. In addition to the cross-sectional analyses of
268
PATH data described previously, longitudinal analyses were conducted using weighted logistic
269
regressions to test whether prior use of flavored cigars predicted becoming an established cigarette
270
smoker in adult cigar users who did not smoke cigarettes in the prior wave. Analyses considered
271 15 15 cigarillo, filtered cigar, and traditional cigar categories separately as defined and coded in PATH. 272
Flavored cigar use was established based on the respective survey questions that inquired about
273
past 30-day use of such products. Valid responses were “Yes”, “No”, and “I don’t know”, and the
274
primary question of interest was whether those individuals responding “Yes” were more or less
275
likely than individuals responding “No” to become established cigarette users in the subsequent
276
wave. 277 Table 3 reports results from analyses that assessed subsequent wave cigarette smoking status
278
among prior wave non-cigarette smoking cigar using respondents who indicated whether or not
279
there was past 30-day flavored cigar use. A similar Wave 1/Wave 2 analysis was not conducted
280
because past 30-day use of flavored cigars was not assessed in Wave 1. 281 Table 3 reports results from analyses that assessed subsequent wave cigarette smoking status
278
among prior wave non-cigarette smoking cigar using respondents who indicated whether or not
279
there was past 30-day flavored cigar use. A similar Wave 1/Wave 2 analysis was not conducted
280
because past 30-day use of flavored cigars was not assessed in Wave 1. 281 Table 3 reports results from analyses that assessed subsequent wave cigarette smoking status
278
among prior wave non-cigarette smoking cigar using respondents who indicated whether or not
279
there was past 30-day flavored cigar use. A similar Wave 1/Wave 2 analysis was not conducted
280
because past 30-day use of flavored cigars was not assessed in Wave 1. 281 The prevalence of Wave 3 established cigarette smokers ranged from approximately 5% to
282
approximately 25% across Wave 2 cigar type and flavored subgroups (top Table 3). Filtered
283
cigar users reporting past 30-day flavored filtered cigar use had the highest prevalence with
284
cigarillo and traditional cigar users having similar, lower prevalence of Wave 3 established
285
cigarette smoking. A Wave 3/Wave 4 analysis yielded similar findings (bottom Table 3). Flavored Cigar Use and Other Tobacco Use Behavior
267 286 The prevalence of Wave 3 established cigarette smokers ranged from approximately 5% to
282
approximately 25% across Wave 2 cigar type and flavored subgroups (top Table 3). Filtered
283
cigar users reporting past 30-day flavored filtered cigar use had the highest prevalence with
284
cigarillo and traditional cigar users having similar, lower prevalence of Wave 3 established
285
cigarette smoking. A Wave 3/Wave 4 analysis yielded similar findings (bottom Table 3). 286 No statistically significant results were obtained from comparisons designed to determine if prior
287
wave past 30-day flavored cigar use among non-cigarette smokers influenced established
288
cigarette smoking status in the subsequent wave. The consistent lack of statistically significant
289
findings suggests that flavored cigar use among non-cigarette smokers was not found to be
290
associated with becoming an established cigarette smoker in the future. It should be noted,
291
however, that the PATH data indicate that such a transition is a rare event among cigar users
292
with a limited number of records available for analysis (see Record Count column in Table 3). 293 No statistically significant results were obtained from comparisons designed to determine if prior
287
wave past 30-day flavored cigar use among non-cigarette smokers influenced established
288
cigarette smoking status in the subsequent wave. The consistent lack of statistically significant
289
findings suggests that flavored cigar use among non-cigarette smokers was not found to be
290
associated with becoming an established cigarette smoker in the future. It should be noted,
291
however, that the PATH data indicate that such a transition is a rare event among cigar users
292
with a limited number of records available for analysis (see Record Count column in Table 3). 293 16 As such, this pattern of product use should continue to be monitored with future data collections
294
and estimates based on ten or fewer records interpreted cautiously. 295 As such, this pattern of product use should continue to be monitored with future data collections
294
and estimates based on ten or fewer records interpreted cautiously. 295 Flavored Cigar Use and Frequency of Subsequent Use. In a separate set of longitudinal analyses,
296
the transition to increased frequency of use was assessed. Flavored Cigar Use and Other Tobacco Use Behavior
267 Specifically, weighted logistic
297
regressions also were used to evaluate whether prior flavored cigar use predicted progression from
298
regular but non-daily use to daily use among established adult cigar users (i.e., some day to every
299
day use). In addition, a conceptually similar analysis assessed whether flavored cigar use predicted
300
progression from regular but non-daily use to cessation (i.e., some day to non-current use). As in
301
the prior section, analyses considered cigarillo, filtered cigar, and traditional cigar categories
302
separately as defined and coded in PATH. Also similar to the previously discussed longitudinal
303
analysis, flavored cigar use was established based on the respective survey questions that inquired
304
about past 30-day use of such products. Valid responses were “Yes”, “No”, and “I don’t know”,
305
and the primary question of interest was whether those some day cigar users responding “Yes”
306
were more or less likely than those responding “No” to become an every day cigar user in the
307
subsequent wave, or, alternatively, a non-current cigar user in the subsequent wave. 308 Table 4 reports analytic results focused on the transition from regular but nondaily cigar use to
309
daily cigar use between Waves 2 and 3. Among Wave 2 non-daily established users of all cigar
310
types and prior flavored cigar use subgroups, progression to daily cigar use was infrequent. 311
Approximately 13% of filtered cigar users progressed to daily use, with cigarillo and traditional
312
cigar users doing so to a lesser extent (see Prevalence column of Table 4). Prevalence estimates
313
were generally similar whether or not prior use of flavored cigars was indicated with no statistically
314
significant findings identified. As in the analysis of transition to established cigarette use,
315
however, the PATH data indicate that transition to everyday cigar use is a rare event with a limited
316 Table 4 reports analytic results focused on the transition from regular but nondaily cigar use to
309
daily cigar use between Waves 2 and 3. Among Wave 2 non-daily established users of all cigar
310
types and prior flavored cigar use subgroups, progression to daily cigar use was infrequent. 311
Approximately 13% of filtered cigar users progressed to daily use, with cigarillo and traditional
312
cigar users doing so to a lesser extent (see Prevalence column of Table 4). Flavored Cigar Use and Other Tobacco Use Behavior
267 Prevalence estimates
313
were generally similar whether or not prior use of flavored cigars was indicated with no statistically
314
significant findings identified. As in the analysis of transition to established cigarette use,
315
however, the PATH data indicate that transition to everyday cigar use is a rare event with a limited
316 17 number of records available for analysis (see Record Count column in Table 4). As such, this
317
pattern of product use should continue to be monitored with future data collections and estimates
318
based on ten or fewer records interpreted cautiously. 319 number of records available for analysis (see Record Count column in Table 4). As such, this
317
pattern of product use should continue to be monitored with future data collections and estimates
318
based on ten or fewer records interpreted cautiously. 319 The transition from some day cigar use in Wave 2 to non-current use in Wave 3, (i.e., cessation)
320
was more common. Approximately 50% of Wave 2 cigarillo and filtered cigar users no longer
321
indicated established cigar use in Wave 3, with essentially no difference identified between
322
Wave 2 flavored versus non-flavored cigar users. Cessation among traditional cigar users was
323
less common with approximately 28% and 14% of Wave 2 flavored and non-flavored cigar
324
users, respectively, no longer indicating established use in Wave 3. Interestingly, a statistically
325
significant difference was identified in an unadjusted analysis indicating Wave 2 flavored cigar
326
users were more likely to cease use of traditional cigar products than Wave 2 users of non-
327
flavored cigars (OR: 2.35, 95%CI:1.30-4.27). The statistically significant difference is not
328
retained in a similar multivariable analysis adjusting for sex, age, race/ethnicity, and education
329
(AOR: 1.28, 95%CI: 0.58-2.80), with apparent confounders of increased age and more advanced
330
education associated with lower likelihood of cessation in Wave 3. 331 The transition from some day cigar use in Wave 2 to non-current use in Wave 3, (i.e., cessation)
320
was more common. Approximately 50% of Wave 2 cigarillo and filtered cigar users no longer
321
indicated established cigar use in Wave 3, with essentially no difference identified between
322
Wave 2 flavored versus non-flavored cigar users. Flavored Cigar Use and Other Tobacco Use Behavior
267 Cessation among traditional cigar users was
323
less common with approximately 28% and 14% of Wave 2 flavored and non-flavored cigar
324
users, respectively, no longer indicating established use in Wave 3. Interestingly, a statistically
325
significant difference was identified in an unadjusted analysis indicating Wave 2 flavored cigar
326
users were more likely to cease use of traditional cigar products than Wave 2 users of non-
327
flavored cigars (OR: 2.35, 95%CI:1.30-4.27). The statistically significant difference is not
328
retained in a similar multivariable analysis adjusting for sex, age, race/ethnicity, and education
329
(AOR: 1.28, 95%CI: 0.58-2.80), with apparent confounders of increased age and more advanced
330
education associated with lower likelihood of cessation in Wave 3. 331 Results from a conceptually equivalent analysis of transition in cigar use frequency between Wave
332
3 and Wave 4 are shown in Table 5. Findings were consistent with those from the Wave 2/Wave
333
3 analysis. Progression to daily use was infrequent (approximately 10% for filtered cigar,
334
somewhat less frequent for cigarillo, and less still for traditional cigar users) and no statistically
335
significant differences were identified based on prior use of flavored cigars. In addition, transition
336
from some day use in Wave 3 to non-current use in Wave 4 was similarly more common. A
337
statistically significant difference was again observed in an unadjusted analysis of Wave 4
338
traditional cigar users indicating increased likelihood of cessation for individuals using flavored
339 Results from a conceptually equivalent analysis of transition in cigar use frequency between Wave
332
3 and Wave 4 are shown in Table 5. Findings were consistent with those from the Wave 2/Wave
333
3 analysis. Progression to daily use was infrequent (approximately 10% for filtered cigar,
334
somewhat less frequent for cigarillo, and less still for traditional cigar users) and no statistically
335
significant differences were identified based on prior use of flavored cigars. In addition, transition
336
from some day use in Wave 3 to non-current use in Wave 4 was similarly more common. A
337
statistically significant difference was again observed in an unadjusted analysis of Wave 4
338
traditional cigar users indicating increased likelihood of cessation for individuals using flavored
339 18 traditional cigar products in the prior Wave 3 (OR: 2.06, 95%CI: 1.08-3.96). Flavored Cigar Use and Other Tobacco Use Behavior
267 However, the
340
statistically significant finding again was not retained after adjusting for sex, age, race/ethnicity,
341
and education in a multivariable model (AOR: 1.28, 95%CI: 0.55-2.96). Education level attained
342
appears to be the primary confounder in this model, with more advanced education associated with
343
lower likelihood of cessation in Wave 4. 344 Flavored Cigar Use and Youth Cigarette Use. The PATH youth data also enables longitudinal
345
analysis in which tobacco use behavior can be assessed over time. For example, the likelihood
346
of cigarette initiation based on prior use of flavored cigars can be assessed by integrating
347
information for individuals across PATH waves (similar to that previously described in the prior
348
subsections using PATH adult data). To do so, however, requires sufficient data records across
349
waves. 350 Flavored Cigar Use and Youth Cigarette Use. The PATH youth data also enables longitudinal
345
analysis in which tobacco use behavior can be assessed over time. For example, the likelihood
346
of cigarette initiation based on prior use of flavored cigars can be assessed by integrating
347
information for individuals across PATH waves (similar to that previously described in the prior
348
subsections using PATH adult data). To do so, however, requires sufficient data records across
349
waves. 350 As detailed in Table 6, with the exception of Wave 1 cigarillo use, data records on youth use of
351
cigars are limited, thus restricting the types of analyses that can be conducted to assess whether
352
use of flavored cigars is associated with subsequent initiation of cigarette use or other public
353
health-relevant behaviors. Simple assessments of prevalence within individual waves are
354
possible, but more complex cross-wave analyses will not yield robust results due to data
355
limitations. Under these circumstances, even prevalence assessments are of limited value. The
356
wide confidence intervals for dual use of cigarettes and flavored or unflavored cigars presented
357
in Table 6 illustrate this point. The wide, overlapping confidence intervals, coupled with often
358
unstable point estimates, complicate interpretation and preclude the development of firm
359
conclusions. As such, data on these tobacco use behaviors should continue to be monitored, but
360
conclusive findings await collection of considerably more information. 361 19 19 Discussion
362 After analyses of several outcomes across the various data sources, the results do not
363
demonstrate an association between use of flavored cigar products and differential behaviors
364
related to public health. Our findings with these large representative datasets were notably
365
consistent among both youth and adult populations. Cigar usage estimates for the United States
366
youth population are generally similar based on NYTS, MTF, and PATH with past 30-day
367
estimates of cigar use between approximately 2%-10% overall or for flavored cigar products
368
specifically. These estimates have remained generally stable or declined across all data
369
collection years within the respective surveys. Consistent trends were likewise observed when
370
looking at the frequency of use during the past 30 days. The majority of past 30-day youth users
371
limit cigar use to 1-2 days per month with another approximately 20% of past 30-day youth users
372
limiting cigar use to 3-5 days. 373 After analyses of several outcomes across the various data sources, the results do not
363
demonstrate an association between use of flavored cigar products and differential behaviors
364
related to public health. Our findings with these large representative datasets were notably
365
consistent among both youth and adult populations. Cigar usage estimates for the United States
366
youth population are generally similar based on NYTS, MTF, and PATH with past 30-day
367
estimates of cigar use between approximately 2%-10% overall or for flavored cigar products
368
specifically. These estimates have remained generally stable or declined across all data
369
collection years within the respective surveys. Consistent trends were likewise observed when
370
looking at the frequency of use during the past 30 days. The majority of past 30-day youth users
371
limit cigar use to 1-2 days per month with another approximately 20% of past 30-day youth users
372
limiting cigar use to 3-5 days. 373 Similarly, analysis of NATS, HINTS, PATH, TUS-CPS, and TPRPS indicate that less than 10%
374
of adults currently use cigars, and less than 5% currently use flavored cigars, indicating that
375
flavored cigars continue to be an important product category to adult cigar users but with stable
376
use prevalence over time. Discussion
362 Also similar to the youth cigar use estimates, frequency of use per
377
month estimates remained consistent across data sources and over time with the most commonly
378
reported use pattern being 1-2 days per month, and only a relatively small proportion using
379
cigars every day. In addition, longitudinal analysis of PATH adult data found that flavored cigar
380
use was not differentially associated with future established use of traditional cigarettes or
381
increased future use of cigars among regular but non-daily users. Data limitations precluded
382
conducting the same analysis using PATH youth data. 383 Similarly, analysis of NATS, HINTS, PATH, TUS-CPS, and TPRPS indicate that less than 10%
374
of adults currently use cigars, and less than 5% currently use flavored cigars, indicating that
375
flavored cigars continue to be an important product category to adult cigar users but with stable
376
use prevalence over time. Also similar to the youth cigar use estimates, frequency of use per
377
month estimates remained consistent across data sources and over time with the most commonly
378
reported use pattern being 1-2 days per month, and only a relatively small proportion using
379
cigars every day. In addition, longitudinal analysis of PATH adult data found that flavored cigar
380
use was not differentially associated with future established use of traditional cigarettes or
381
increased future use of cigars among regular but non-daily users. Data limitations precluded
382
conducting the same analysis using PATH youth data. 383 20 20 Our findings related to current use metrics and corresponding daily use frequency echo those in a
384
recent publication on tobacco product prevalence. Sánchez-Romero and colleagues detail
385
variability in current use prevalence estimates in the context of various daily use thresholds (1+,
386
10+, and 25+ days in the past 30 days) for various tobacco products across two of the surveys
387
considered in this study (PATH and TUS-CPS) as well as the National Health Interview Survey
388
(NHIS) [33]. They found differences in prevalence to vary least for cigarette and smokeless
389
tobacco use across the daily use thresholds. In contrast, current use prevalence of other
390
combustibles (cigars were grouped with hookah and pipe use) varied the most with increasing
391
daily use thresholds and e-cigarette current use prevalence variation falling in between. Discussion
362 The
392
authors note that the large variation for other combustibles likely reflects their less stable usage
393
pattern among primarily intermittent or social users. Further they note that a common definition
394
of “current” use (e.g., use on one or more of the past 30 days) may not be an adequate single
395
solution to assess/address tobacco use patterns and corresponding public health considerations. 396
Our work also touches on another potential issue with treating flavored tobacco products as
397
monolithic. While our analyses did not identify a link between flavored cigar use and
398
subsequent use behaviors of concern (i.e., cigarette smoking or increased cigar use), other studies
399
have reported such associations [11, 34, 35]. Those studies, however, aggregated across flavored
400
products such that flavored e-cigarette, flavored smokeless tobacco, and/or flavored cigar use are
401
combined in a singular predictor of tobacco use behavior of concern. In contrast, a study of
402
young adult college students that more specifically targeted use of flavored cigars found the
403
number of days of cigar use and current use of other tobacco products were not associated with
404
use of flavored cigars [36]. 405 21 21 21 As such, while broad flavor bans may effectively restrict sales in the target localities [5],
406
potential unintended consequences with public health relevance should be considered. For
407
example, some evidence is emerging that cigarette smoking increased following the imposition
408
of the flavored tobacco product ban in San Francisco [7, 37]. Additional unintended public
409
health consequences could also include increasing use of after-market addition of unregulated
410
and potentially unsafe flavor additives. In a study of e-cigarette users, the majority of
411
participants reported their intent to circumvent potential FDA flavor restrictions via illicit
412
sources or self-manufacture [38]. 413 ch, while broad flavor bans may effectively restrict sales in the target localities [5], As such, while broad flavor bans may effectively restrict sales in the target localities [5],
406
potential unintended consequences with public health relevance should be considered. For
407
example, some evidence is emerging that cigarette smoking increased following the imposition
408
of the flavored tobacco product ban in San Francisco [7, 37]. Additional unintended public
409
health consequences could also include increasing use of after-market addition of unregulated
410
and potentially unsafe flavor additives. Discussion
362 In a study of e-cigarette users, the majority of
411
participants reported their intent to circumvent potential FDA flavor restrictions via illicit
412
sources or self-manufacture [38]. 413 There are opportunities for future research to explore these and related topics. Many of these
414
data resources collect a wide array of information on risk perception, reasons for smoking, and
415
other potentially relevant public health information. Our analysis focused on current use overall,
416
frequency of use, and trends over time with respect to those parameters. Other analyses are
417
possible using a similar framework. In addition, data from many of these surveys (e.g., NYTS,
418
MTF, PATH) will continue to be collected and released for analysis on an ongoing basis. As
419
such, the analyses presented herein can be replicated with future data releases to continue to
420
monitor these and potentially other relevant issues on an ongoing basis. 421 There are opportunities for future research to explore these and related topics. Many of these
414
data resources collect a wide array of information on risk perception, reasons for smoking, and
415
other potentially relevant public health information. Our analysis focused on current use overall,
416
frequency of use, and trends over time with respect to those parameters. Other analyses are
417
possible using a similar framework. In addition, data from many of these surveys (e.g., NYTS,
418
MTF, PATH) will continue to be collected and released for analysis on an ongoing basis. As
419
such, the analyses presented herein can be replicated with future data releases to continue to
420
monitor these and potentially other relevant issues on an ongoing basis. 421 Several important considerations and potential limitations should be kept in mind when
422
interpreting these findings. First, cigar misclassification is a well-recognized issue, even with
423
surveys such as PATH [26, 27]. The photos presented to survey participants and detailed
424
descriptions of each cigar type are designed to limit such problems, but some participants may still
425
misclassify, for example, traditional/premium cigars and cigarillos, or vice versa. Further, brand
426
misclassification [26, 27]has also been observed, with respondents occasionally reporting well
427
known cigarette brands in response to questions about cigar use. 428 Several important considerations and potential limitations should be kept in mind when
422
interpreting these findings. First, cigar misclassification is a well-recognized issue, even with
423
surveys such as PATH [26, 27]. Discussion
362 The photos presented to survey participants and detailed
424
descriptions of each cigar type are designed to limit such problems, but some participants may still
425
misclassify, for example, traditional/premium cigars and cigarillos, or vice versa. Further, brand
426
misclassification [26, 27]has also been observed, with respondents occasionally reporting well
427
known cigarette brands in response to questions about cigar use. 428 22 In addition, the weighted estimates are not intended to represent exact values. All estimates should
429
be viewed in the context of their 95% confidence intervals and the number of individual survey
430
subjects/records that underlie each estimate. While there is no firm guideline on the minimum
431
number of records needed to provide a robust estimate, a common rule of thumb is for any
432
estimates based on the low tens of records to be treated cautiously. This consideration is
433
particularly relevant should additional stratification or statistical adjustment be required. Data
434
limitations of this type can, and in some cases should, preclude the use of certain analytic methods. 435
The longitudinal analyses of PATH should be interpreted carefully as several estimates were based
436
on few records and corresponding estimates may be unreliable. 437 In addition, the weighted estimates are not intended to represent exact values. All estimates should
429
be viewed in the context of their 95% confidence intervals and the number of individual survey
430
subjects/records that underlie each estimate. While there is no firm guideline on the minimum
431
number of records needed to provide a robust estimate, a common rule of thumb is for any
432
estimates based on the low tens of records to be treated cautiously. This consideration is
433
particularly relevant should additional stratification or statistical adjustment be required. Data
434
limitations of this type can, and in some cases should, preclude the use of certain analytic methods. 435
The longitudinal analyses of PATH should be interpreted carefully as several estimates were based
436
on few records and corresponding estimates may be unreliable. 437 23 23 Conclusions
438 In conclusion, after assessing the most current, publicly available US public health datasets on
439
tobacco use, we did not identify any indication that flavored cigar product use raises different
440
questions of public health. The proportion of current users has remained low and stable or
441
declining over time, and the pattern of use (rarely every day, and most often on very few days
442
per month) has likewise remained stable in both youth and adult cigar using populations. No
443
evidence was found of increased use or different usage patterns, either among youth or adults, of
444
flavored relative to unflavored cigar products. The frequency of daily usage of cigars in general
445
and flavored products specifically, has not increased during the study period, in either adults or
446
youth. Further, no differences in prevalence of reported use were found specifically for flavored
447
products. That is, neither adult nor youth users of any cigars, showed increases in use of flavored
448
cigars concomitant with decreases in unflavored products. In addition, longitudinal analysis of
449
PATH adult data failed to identify associations between flavored cigar use and subsequent use
450
behaviors of potential concern (i.e., cigarette use initiation or increased cigar use). 451 In conclusion, after assessing the most current, publicly available US public health datasets on
439
tobacco use, we did not identify any indication that flavored cigar product use raises different
440
questions of public health. The proportion of current users has remained low and stable or
441
declining over time, and the pattern of use (rarely every day, and most often on very few days
442
per month) has likewise remained stable in both youth and adult cigar using populations. No
443
evidence was found of increased use or different usage patterns, either among youth or adults, of
444
flavored relative to unflavored cigar products. The frequency of daily usage of cigars in general
445
and flavored products specifically, has not increased during the study period, in either adults or
446
youth. Further, no differences in prevalence of reported use were found specifically for flavored
447
products. That is, neither adult nor youth users of any cigars, showed increases in use of flavored
448
cigars concomitant with decreases in unflavored products. Declarations
458 Declarations
458
Ethics approval and consent to participate
459
This study used public-use, de-identified, secondary data and, as such, is not considered human
460
subjects research under 45 CFR 46.102; therefore, IRB approval was not required. The study was
461
conducted and reported according to STROBE guidelines, and, as applicable, in accordance with
462
the Declaration of Helsinki. 463
Consent for publication
464
Not applicable
465
Availability of data and materials
466
All data analyzed during this study are publicly available with data access details provided in Table
467
2. 468
Competing interests
469
ARJ is a principal with Consilium Sciences, a company that provides consulting services on
470
tobacco and nicotine, among other products, on a project basis to companies involved in tobacco
471
harm reduction. 472
Funding
473
The independent conduct of this study was funded by the Cigar Association of America, Inc.,
474
Altria Client Services LLC, ITG Brands LLC, Swedish Match North America and Swisher
475
International Inc. 476
Authors’ contributions
477
ARJ determined the methodological approach and conducted and checked the analyses reported. 478
Initial and final drafts of the paper were prepared by ARJ. 479 Ethics approval and consent to participate
459 This study used public-use, de-identified, secondary data and, as such, is not considered human
460
subjects research under 45 CFR 46.102; therefore, IRB approval was not required. The study was
461
conducted and reported according to STROBE guidelines, and, as applicable, in accordance with
462
the Declaration of Helsinki. 463 Conclusions
438 In addition, longitudinal analysis of
449
PATH adult data failed to identify associations between flavored cigar use and subsequent use
450
behaviors of potential concern (i.e., cigarette use initiation or increased cigar use). 451 Therefore, while these trends should continue to be monitored on an ongoing basis, there is no
452
indication of existing or emerging public health concerns related specifically to flavored cigar
453
products within the seven large, nationally representative epidemiologic databases examined for
454
this study. 455 24 List of abbreviations
456
AOR
Adjusted Odds Ratio
CI
Confidence Interval
CTP
Center for Tobacco Products
FDA
Food and Drug Administration
GSU
Georgia State University
HINTS
Health Information National Trends Survey
ICPSR
Inter-University Consortium for Political and Social Research
MTF
Monitoring the Future
NATS
National Adult Tobacco Survey
NHIS
National Health Interview Survey
NIH
National Institutes of Health
NYTS
National Youth Tobacco Survey
OR
Odds Ratio
PATH
Population Assessment of Tobacco and Health Study
TCORS
Tobacco Center of Regulatory Science
TPRPS
Tobacco Product and Risk Perception Survey
TUS-CPS
Tobacco Use Supplement to Current Population Survey
YRBSS
Youth Risk Behavior Surveillance System
457 457 25 25 Consent for publication
464 Not applicable
465
Availability of data and materials
466
All data analyzed during this study are publicly available with data access details provided in Table
467
2. 468
Competing interests
469
ARJ is a principal with Consilium Sciences, a company that provides consulting services on
470
tobacco and nicotine, among other products, on a project basis to companies involved in tobacco
471
harm reduction. 472
Funding
473
The independent conduct of this study was funded by the Cigar Association of America, Inc.,
474
Altria Client Services LLC, ITG Brands LLC, Swedish Match North America and Swisher
475
International Inc. 476
Authors’ contributions
477
ARJ d t
i
d th
th d l
i
l
h
d
d
t d
d h
k d th
l
t d
478 Authors’ contributions
477 ARJ determined the methodological approach and conducted and checked the analyses reported. 478
Initial and final drafts of the paper were prepared by ARJ. 479 ARJ determined the methodological approach and conducted and checked the analyses reported. 478
Initial and final drafts of the paper were prepared by ARJ. 479 ARJ determined the methodological approach and conducted and checked the analyses reported. 478
Initial and final drafts of the paper were prepared by ARJ. 479 26 26 Acknowledgements
480 Kephart L, Setodji C, Pane J, Shadel W, Song G, Robertson J, Harding N, Henley P,
506
Ursprung WWS: Evaluating tobacco retailer experience and compliance with a
507
flavoured tobacco product restriction in Boston, Massachusetts: impact on product
508
availability, advertisement and consumer demand. Tob Control 2020, 29(e1):e71-e77
509
6. Rogers T, Feld A, Gammon DG, Coats EM, Brown EM, Olson LT, Nonnemaker JM,
510
Engstrom M, McCrae T, Holder-Hayes E et al: Changes in cigar sales following
511
implementation of a local policy restricting sales of flavoured non-cigarette tobacco
512
products. Tob Control 2020, 29(4):412-419. 513
7. Yang Y, Lindblom EN, Salloum RG, Ward KD: The impact of a comprehensive
514
tobacco product flavor ban in San Francisco among young adults. Addict Behav Rep
515
2020, 11:100273. 516
8. Bono RS, Cobb CO, Wall CS, Lester RC, Hoetger C, Lipato T, Guy MC, Eissenberg T,
517
Bickel WK, Barnes AJ: Behavioral economic assessment of abuse liability for Black
518
& Mild cigar flavors among young adults. Exp Clin Psychopharmacol 2020. 519
9. Wall CS, Bono RS, Lester RC, Hoetger C, Lipato T, Guy MC, Eissenberg TE, Bickel
520
WK, Barnes AJ, Cobb CO: Triangulating abuse liability assessment for flavoured
521
cigar products using physiological, behavioural economic and subjective
522
assessments: a within-subjects clinical laboratory protocol. BMJ Open 2018,
523
8(10):e023850. 524
10. Dai H, Hao J: Flavored Tobacco Use Among U.S. Adults by Age Group: 2013-2014. 525
Subst Use Misuse 2019, 54(2):315-323. 526
11. Farley SM, Seoh H, Sacks R, Johns M: Teen use of flavored tobacco products in new
527
york city. Nicotine Tob Res 2014, 16(11):1518-1521. 528
12. Villanti AC, Johnson AL, Ambrose BK, Cummings KM, Stanton CA, Rose SW, Feirman
529
SP, Tworek C, Glasser AM, Pearson JL et al: Flavored Tobacco Product Use in Youth
530
and Adults: Findings From the First Wave of the PATH Study (2013-2014). Am J
531
Prev Med 2017, 53(2):139-151. 532 References
490 References
490
1. Advance Notice of Proposed Rulemaking, Regulation of Flavors in Tobacco
491
Products, 83 Fed. Reg. 12,294 (March 21, 2018). 492
[https://www.federalregister.gov/documents/2018/03/21/2018-05655/regulation-of-
493
flavors-in-tobacco-products]
494
2. Modifications to Compliance Policy for Certain Deemed Tobacco Products; Draft
495
Guidance for Industry, 84 Fed. Reg. 9345
496
[https://www.federalregister.gov/documents/2019/03/14/2019-04765/modifications-to-
497
compliance-policy-for-certain-deemed-tobacco-products-draft-guidance-for-industry]
498
3. FDA Commits to Evidence-Based Actions Aimed at Saving Lives and Preventing
499
Future Generations of Smokers [https://www.fda.gov/news-events/press-
500
announcements/fda-commits-evidence-based-actions-aimed-saving-lives-and-preventing
501
future-generations-smokers]
502
4. Chaiton MO, Schwartz R, Tremblay G, Nugent R: Association of flavoured cigar
503
regulations with wholesale tobacco volumes in Canada: an interrupted time series
504
analysis. Tob Control 2019, 28(4):457-461. 505
5. Kephart L, Setodji C, Pane J, Shadel W, Song G, Robertson J, Harding N, Henley P,
506
Ursprung WWS: Evaluating tobacco retailer experience and compliance with a
507
flavoured tobacco product restriction in Boston, Massachusetts: impact on product
508
availability, advertisement and consumer demand. Tob Control 2020, 29(e1):e71-e77
509
6. Rogers T, Feld A, Gammon DG, Coats EM, Brown EM, Olson LT, Nonnemaker JM,
510
Engstrom M, McCrae T, Holder-Hayes E et al: Changes in cigar sales following
511
implementation of a local policy restricting sales of flavoured non-cigarette tobacco
512
products. Tob Control 2020, 29(4):412-419. 513
7. Yang Y, Lindblom EN, Salloum RG, Ward KD: The impact of a comprehensive
514
tobacco product flavor ban in San Francisco among young adults. Addict Behav Rep
515
2020, 11:100273. 516
8. Bono RS, Cobb CO, Wall CS, Lester RC, Hoetger C, Lipato T, Guy MC, Eissenberg T,
517
Bickel WK, Barnes AJ: Behavioral economic assessment of abuse liability for Black
518
& Mild cigar flavors among young adults. Exp Clin Psychopharmacol 2020. 519
9. Wall CS, Bono RS, Lester RC, Hoetger C, Lipato T, Guy MC, Eissenberg TE, Bickel
520
WK, Barnes AJ, Cobb CO: Triangulating abuse liability assessment for flavoured
521
cigar products using physiological, behavioural economic and subjective
522
assessments: a within-subjects clinical laboratory protocol. BMJ Open 2018,
523
8(10):e023850. 524
10. Dai H, Hao J: Flavored Tobacco Use Among U.S. Adults by Age Group: 2013-2014. 525
Subst Use Misuse 2019, 54(2):315-323. 526
11. Farley SM, Seoh H, Sacks R, Johns M: Teen use of flavored tobacco products in new
527
york city. Nicotine Tob Res 2014, 16(11):1518-1521. 528
12. Acknowledgements
480 Acknowledgements
480
We thank our colleague at Consilium Sciences, Rachael Posey for assistance with literature search
481
and screening as well as for preparation of the References list. We also thank the Cigar Association
482
of America, Inc. and certain member and supporting companies for financial support for the work
483
conducted, and other members of the Consilium Sciences team, particularly Lenn Murrelle, who
484
provided useful comments at various stages. We also thank Georgia State University, the GSU
485
Tobacco Center of Regulatory Sciences research team for making the TPRPS data available to us
486
for this study. However, we should note that information presented in this paper does not
487
necessarily represent the official views of the Georgia State University, the GSU Tobacco Center
488
of Regulatory Science research team, the NIH or the Food and Drug Administration. 489 We thank our colleague at Consilium Sciences, Rachael Posey for assistance with literature search
481
and screening as well as for preparation of the References list. We also thank the Cigar Association
482
of America, Inc. and certain member and supporting companies for financial support for the work
483
conducted, and other members of the Consilium Sciences team, particularly Lenn Murrelle, who
484
provided useful comments at various stages. We also thank Georgia State University, the GSU
485
Tobacco Center of Regulatory Sciences research team for making the TPRPS data available to us
486
for this study. However, we should note that information presented in this paper does not
487
necessarily represent the official views of the Georgia State University, the GSU Tobacco Center
488
of Regulatory Science research team, the NIH or the Food and Drug Administration. 489 27 27 References
490
1. Advance Notice of Proposed Rulemaking, Regulation of Flavors in Tobacco
491
Products, 83 Fed. Reg. 12,294 (March 21, 2018). 492
[https://www.federalregister.gov/documents/2018/03/21/2018-05655/regulation-of-
493
flavors-in-tobacco-products]
494
2. Modifications to Compliance Policy for Certain Deemed Tobacco Products; Draft
495
Guidance for Industry, 84 Fed. Reg. 9345
496
[https://www.federalregister.gov/documents/2019/03/14/2019-04765/modifications-to-
497
compliance-policy-for-certain-deemed-tobacco-products-draft-guidance-for-industry]
498
3. FDA Commits to Evidence-Based Actions Aimed at Saving Lives and Preventing
499
Future Generations of Smokers [https://www.fda.gov/news-events/press-
500
announcements/fda-commits-evidence-based-actions-aimed-saving-lives-and-preventing
501
future-generations-smokers]
502
4. Chaiton MO, Schwartz R, Tremblay G, Nugent R: Association of flavoured cigar
503
regulations with wholesale tobacco volumes in Canada: an interrupted time series
504
analysis. Tob Control 2019, 28(4):457-461. 505
5. References
490 Hyland A, Ambrose BK, Conway KP, Borek N, Lambert E, Carusi C, Taylor K, Crosse
570
S, Fong GT, Cummings KM et al: Design and methods of the Population Assessment
571
of Tobacco and Health (PATH) Study. Tob Control 2017, 26(4):371-378. 572
25. Brener ND, Kann L, Shanklin S, Kinchen S, Eaton DK, Hawkins J, Flint KH:
573
Methodology of the Youth Risk Behavior Surveillance System--2013. MMWR
574
Recomm Rep 2013, 62(Rr-1):1-20. 575
26. Corey CG, Holder-Hayes E, Nguyen AB, Delnevo CD, Rostron BL, Bansal-Travers M,
576
Kimmel HL Koblitz A Lambert E Pearson JL et al: US Adult Cigar Smoking
577 13. Dunn DS, Johnson AL, Sterling KL, Cohn AM: Differences in reasons for little
533
cigar/cigarillo use across white and black/African American young adult users. 534
Addict Behav 2021, 118:106884. 535 14. Lawyer GR, Jackson M, Prinz M, Lamb T, Wang Q, Muthumalage T, Rahman I:
536
Classification of flavors in cigarillos and little cigars and their variable cellular and
537
acellular oxidative and cytotoxic responses. PLoS One 2019, 14(12):e0226066. 538 15. Trapl ES, Yoder LD, Frank JL, Borawski EA, Sattar A: Individual, Parental, and
539
Environmental Correlates of Cigar, Cigarillo, and Little Cigar Use Among Middle
540
School Adolescents. Nicotine Tob Res 2016, 18(5):834-841. 541 16. Kong G, Creamer MR, Simon P, Cavallo DA, Ross JC, Hinds JT, Fishbein H, Gutierrez
542
K: Systematic review of cigars, cigarillos, and little cigars among adolescents:
543
Setting research agenda to inform tobacco control policy. Addict Behav 2019, 96:192-
544
197. 545 17. Office on Smoking and Health: Methodology Report. In: 2020 National Youth Tobacco
546
Survey. Atlanta, GA: U.S. Department of Health and Human Services, Centers for
547
Disease Control and Prevention, National Center for Chronic Disease Prevention and
548
Health Promotion, Office on Smoking and Health; 2020. 549 18. Johnston LD, Miech RA, O'Malley PM, Bachman JG, Schulenberg JE, Patrick ME: 2020
550
Overview, Key Findings on Adolescent Drug Use. In: Monitoring the Future National
551
Survey Results on Drug Use, 1975-2020. Ann Arbor, Michigan: Institute for Social
552
Research, University of Michigan; 2021. 553 19. Nelson DE, Kreps GL, Hesse BW, Croyle RT, Willis G, Arora NK, Rimer BK,
554
Viswanath KV, Weinstein N, Alden S: The Health Information National Trends
555
Survey (HINTS): development, design, and dissemination. J Health Commun 2004,
556
9(5):443-460; discussion 481-444. 557 20. References
490 Villanti AC, Johnson AL, Ambrose BK, Cummings KM, Stanton CA, Rose SW, Feirman
529
SP, Tworek C, Glasser AM, Pearson JL et al: Flavored Tobacco Product Use in Youth
530
and Adults: Findings From the First Wave of the PATH Study (2013-2014). Am J
531
Prev Med 2017, 53(2):139-151. 532 p
p
y
p
g
y
3. FDA Commits to Evidence-Based Actions Aimed at Saving Lives and Preventing
499
Future Generations of Smokers [https://www.fda.gov/news-events/press-
500
announcements/fda-commits-evidence-based-actions-aimed-saving-lives-and-preventing-
501
future-generations-smokers]
502 4. Chaiton MO, Schwartz R, Tremblay G, Nugent R: Association of flavoured cigar
503
regulations with wholesale tobacco volumes in Canada: an interrupted time series
504
analysis. Tob Control 2019, 28(4):457-461. 505 7. Yang Y, Lindblom EN, Salloum RG, Ward KD: The impact of a comprehensive
514
tobacco product flavor ban in San Francisco among young adults. Addict Behav Rep
515
2020, 11:100273. 516 8. Bono RS, Cobb CO, Wall CS, Lester RC, Hoetger C, Lipato T, Guy MC, Eissenberg T,
517
Bickel WK, Barnes AJ: Behavioral economic assessment of abuse liability for Black
518
& Mild cigar flavors among young adults. Exp Clin Psychopharmacol 2020. 519 g
g y
g
p
y
p
9. Wall CS, Bono RS, Lester RC, Hoetger C, Lipato T, Guy MC, Eissenberg TE, Bickel
520
WK, Barnes AJ, Cobb CO: Triangulating abuse liability assessment for flavoured
521
cigar products using physiological, behavioural economic and subjective
522
assessments: a within-subjects clinical laboratory protocol. BMJ Open 2018,
523
8(10):e023850. 524 10. Dai H, Hao J: Flavored Tobacco Use Among U.S. Adults by Age Group: 2013-2014. 525
Subst Use Misuse 2019, 54(2):315-323. 526 11. Farley SM, Seoh H, Sacks R, Johns M: Teen use of flavored tobacco products in new
527
york city. Nicotine Tob Res 2014, 16(11):1518-1521. 528 12. Villanti AC, Johnson AL, Ambrose BK, Cummings KM, Stanton CA, Rose SW, Feirman
529
SP, Tworek C, Glasser AM, Pearson JL et al: Flavored Tobacco Product Use in Youth
530
and Adults: Findings From the First Wave of the PATH Study (2013-2014). Am J
531
Prev Med 2017, 53(2):139-151. 532 28 13. Dunn DS, Johnson AL, Sterling KL, Cohn AM: Differences in reasons for little
533
cigar/cigarillo use across white and black/African American young adult users. 534
Addict Behav 2021, 118:106884. 535
14. References
490 Lawyer GR, Jackson M, Prinz M, Lamb T, Wang Q, Muthumalage T, Rahman I:
536
Classification of flavors in cigarillos and little cigars and their variable cellular and
537
acellular oxidative and cytotoxic responses. PLoS One 2019, 14(12):e0226066. 538
15. Trapl ES, Yoder LD, Frank JL, Borawski EA, Sattar A: Individual, Parental, and
539
Environmental Correlates of Cigar, Cigarillo, and Little Cigar Use Among Middle
540
School Adolescents. Nicotine Tob Res 2016, 18(5):834-841. 541
16. Kong G, Creamer MR, Simon P, Cavallo DA, Ross JC, Hinds JT, Fishbein H, Gutierrez
542
K: Systematic review of cigars, cigarillos, and little cigars among adolescents:
543
Setting research agenda to inform tobacco control policy. Addict Behav 2019, 96:192-
544
197. 545
17. Office on Smoking and Health: Methodology Report. In: 2020 National Youth Tobacco
546
Survey. Atlanta, GA: U.S. Department of Health and Human Services, Centers for
547
Disease Control and Prevention, National Center for Chronic Disease Prevention and
548
Health Promotion, Office on Smoking and Health; 2020. 549
18. Johnston LD, Miech RA, O'Malley PM, Bachman JG, Schulenberg JE, Patrick ME: 2020
550
Overview, Key Findings on Adolescent Drug Use. In: Monitoring the Future National
551
Survey Results on Drug Use, 1975-2020. Ann Arbor, Michigan: Institute for Social
552
Research, University of Michigan; 2021. 553
19. Nelson DE, Kreps GL, Hesse BW, Croyle RT, Willis G, Arora NK, Rimer BK,
554
Viswanath KV, Weinstein N, Alden S: The Health Information National Trends
555
Survey (HINTS): development, design, and dissemination. J Health Commun 2004,
556
9(5):443-460; discussion 481-444. 557
20. Blake KD, Portnoy DB, Kaufman AR, Lin CJ, Lo SC, Backlund E, Cantor D, Hicks L,
558
Lin A, Caporaso A et al: Rationale, Procedures, and Response Rates for the 2015
559
Administration of NCI's Health Information National Trends Survey: HINTS-FDA
560
2015. J Health Commun 2016, 21(12):1269-1275. 561
21. Agaku IT, King BA, Husten CG, Bunnell R, Ambrose BK, Hu SS, Holder-Hayes E, Day
562
HR: Tobacco product use among adults--United States, 2012-2013. MMWR Morb
563
Mortal Wkly Rep 2014, 63(25):542-547. 564
22. Weaver SR, Majeed BA, Pechacek TF, Nyman AL, Gregory KR, Eriksen MP: Use of
565
electronic nicotine delivery systems and other tobacco products among USA adults,
566
2014: results from a national survey. Int J Public Health 2016, 61(2):177-188. 567
23. Tobacco Use Supplement to the Current Population Survey Harmonized Data,
568
1992-2019 [https://cancercontrol.cancer.gov/brp/tcrb/tus-cps/]
569
24. References
490 Blake KD, Portnoy DB, Kaufman AR, Lin CJ, Lo SC, Backlund E, Cantor D, Hicks L,
558
Lin A, Caporaso A et al: Rationale, Procedures, and Response Rates for the 2015
559
Administration of NCI's Health Information National Trends Survey: HINTS-FDA
560
2015. J Health Commun 2016, 21(12):1269-1275. 561 21. Agaku IT, King BA, Husten CG, Bunnell R, Ambrose BK, Hu SS, Holder-Hayes E, Day
562
HR: Tobacco product use among adults--United States, 2012-2013. MMWR Morb
563
Mortal Wkly Rep 2014, 63(25):542-547. 564 22. Weaver SR, Majeed BA, Pechacek TF, Nyman AL, Gregory KR, Eriksen MP: Use of
565
electronic nicotine delivery systems and other tobacco products among USA adults,
566
2014: results from a national survey. Int J Public Health 2016, 61(2):177-188. 567 y
23. Tobacco Use Supplement to the Current Population Survey Harmonized Data,
68
1992 2019 obacco Use Supplement to the Current Population Survey H
992-2019 [https://cancercontrol.cancer.gov/brp/tcrb/tus-cps/] y
Brener ND, Kann L, Shanklin S, Kinchen S, Eaton DK, Hawkins J, Flint KH: Methodology of the Youth Risk Behavior Surveillance System--2013. MMWR
74
Recomm Rep 2013, 62(Rr-1):1-20. 75 29 Findings From the Population Assessment of Tobacco and Health (PATH) Study,
579
2013-2014. Nicotine Tob Res 2018, 20(12):1457-1466. 580 27. Persoskie A, O'Brien EK, Donaldson EA, Pearson J, Choi K, Kaufman A, Stanton CA,
581
Delnevo CD: Cigar package quantity and smoking behavior. BMC Public Health
582
2019, 19(1):868. 583 28. R: A language and environment for statistical computing. R Foundation for
584
Statistical Computing, Vienna, Austria [https://www.R-project.org]
585 31. McCarthy PJ: Pseudoreplication: further evaluation and applications of the balanced
589
half-sample technique. Vital Health Stat 2 1969(31):1-24. 590 32. Judkins DR: Fay's method for variance estimation. Journal of Official Statistics 1990,
591
6(3):223-239. 592 33. Sánchez-Romero LM, Cadham CJ, Hirschtick JL, Mattingly DT, Cho B, Fleischer NL,
593
Brouwer A, Mistry R, Land SR, Jeon J et al: A comparison of tobacco product
594
prevalence by different frequency of use thresholds across three US surveys. BMC
595
Public Health 2021, 21(1):1203. 596 34. Mantey DS, Omega-Njemnobi O, Montgomery L: Flavored tobacco use is associated
597
with dual and poly tobacco use among adolescents. Addict Behav 2019, 93:269-273. 598 35. Smith DM, Bansal-Travers M, Huang J, Barker D, Hyland AJ, Chaloupka F: Association
599
between use of flavoured tobacco products and quit behaviours: findings from a
600
cross-sectional survey of US adult tobacco users. References
490 Tob Control 2016, 25(Suppl 2):ii73-
601
ii80. 602 36. Hinds JT, 3rd, Li X, Loukas A, Pasch KE, Perry CL: Flavored Cigars Appeal to
603
Younger, Female, and Racial/Ethnic Minority College Students. Nicotine Tob Res
604
2018, 20(3):347-354. 605 37. Friedman AS: A Difference-in-Differences Analysis of Youth Smoking and a Ban on
606
Sales of Flavored Tobacco Products in San Francisco, California. JAMA Pediatr
607
2021. 608 38. Du P, Bascom R, Fan T, Sinharoy A, Yingst J, Mondal P, Foulds J: Changes in Flavor
609
Preference in a Cohort of Long-Term Electronic Cigarette Users. Ann Am Thorac
610
Soc 2020, 17(5):573-581. 611 30 30 613 Figures
614 614 615 616
617
Figure 1. Youth past 30-day use (on one or more days) of cigars overall (left) and flavored (right) based
618
on (A) NYTS and (B) PATH. Note that respondents to NYTS flavor-related questions were not limited to
619
those who responded affirmatively to indicate use of cigar products generally in the past 30 days. Also
620
note that the 2012 survey asked only if respondents used flavored tobacco products. In addition, the
621
2011 and 2013 survey asked only if respondents used flavored little cigars. This variation in question
622
content/format likely explains the estimate instability between 2011-2013. 623
B
A A 616 B B 617 Figure 1. Youth past 30-day use (on one or more days) of cigars overall (left) and flavored (right) based
618
on (A) NYTS and (B) PATH. Note that respondents to NYTS flavor-related questions were not limited to
619
those who responded affirmatively to indicate use of cigar products generally in the past 30 days. Also
620
note that the 2012 survey asked only if respondents used flavored tobacco products. In addition, the
621
2011 and 2013 survey asked only if respondents used flavored little cigars. This variation in question
622
content/format likely explains the estimate instability between 2011-2013. 623 31 624
Figure 2. Past 30 day use of cigars based on youth respondents in MTF. Flavored little cigars/cigarillos
625
(top), “regular” little cigars/cigarillos (middle) and “large” cigars (bottom) are considered separately in
626
MTF. Note that the three usage groups are not mutually exclusive, and some respondents are
627
represented in multiple subgroups. 628 624 624 Figure 2. Past 30 day use of cigars based on youth respondents in MTF. References
490 Flavored little cigars/cigarillos
625
(top), “regular” little cigars/cigarillos (middle) and “large” cigars (bottom) are considered separately in
626
MTF. Note that the three usage groups are not mutually exclusive, and some respondents are
627
represented in multiple subgroups. 628 32 29
Figure 3. Frequency of monthly use based on NYTS respondents (A) reporting cigar use overall in the
30
past 30 days and (B) reporting flavored cigar use in the past 30 days. 31
B
A A A 29
B B 629 Figure 3. Frequency of monthly use based on NYTS respondents (A) reporting cigar use overall in the
630
past 30 days and (B) reporting flavored cigar use in the past 30 days. 631 33 Figure 4. Frequency of monthly youth cigar use based on MTF respondents reporting cigar use in the
past 30 days. The plot is organized such that cigar type appears in individual columns (left, large cigars;
middle, regular little cigars or cigarillos; right, flavored little cigars or cigarillos) and survey year are
consistent per row (2014 at top through 2018 at bottom). 632 Figure 4. Frequency of monthly youth cigar use based on MTF respondents reporting cigar use in the
633
past 30 days. The plot is organized such that cigar type appears in individual columns (left, large cigars;
634
middle, regular little cigars or cigarillos; right, flavored little cigars or cigarillos) and survey year are
635
consistent per row (2014 at top through 2018 at bottom). 636 34 637
638
639
Figure 5. Adult cigar use overall (left) and flavored cigar use specifically (right) based on (A) NATS (every
640
day, some days, or rarely in the past 30 days), (B) PATH (current established users), and (C) TPRPS (past
641
30-day use on one or more days). PATH Wave 1 and TPRPS 2014 did not ask specifically about current
642
flavored cigar use. 643
B
A
C 7
A A A 637 B B 638 9
C 639 Figure 5. Adult cigar use overall (left) and flavored cigar use specifically (right) based on (A) NATS (every
640
day, some days, or rarely in the past 30 days), (B) PATH (current established users), and (C) TPRPS (past
641
30-day use on one or more days). PATH Wave 1 and TPRPS 2014 did not ask specifically about current
642
flavored cigar use. 643 35 A Figure 6. References
490 Frequency of adult cigar usage patterns based on (A) TUS-CPS (2014/15 – 2018) and (B) TPRPS
(2014 – 2017). B
A A 4
B B Figure 6. Frequency of adult cigar usage patterns based on (A) TUS-CPS (2014/15 – 2018) and (B) TPRPS
645
(2014 – 2017). 646 Figure 6. Frequency of adult cigar usage patterns based on (A) TUS-CPS (2014/15 – 2018) and (B) TPRPS
645
(2014 – 2017). 646 36 36 Table 1. Brief description of nationally representative surveys/studies with applicability to the Research
648
Question
649
Survey/study name
Years conducted
Description
Health Information National
Trends Survey (HINTS)
2003-present
HINTS is a biennial, cross-sectional survey of a nationally
representative sample of American adults that is used to assess the
impact of the health information environment. Specifically, HINTS
measures how people access and use health information; how people
use information technology to manage health and health information;
and the degree to which people are engaged in healthy behaviors. In
addition, several items in recent HINTS cycles that were administered
in conjunction with FDA have a specific focus on tobacco use and risk
perception. Survey results from these cycles are typically referred to
as HINTS-FDA. Monitoring the Future (MTF)
1975-present
MTF collects data annually on behaviors, attitudes and values of
American youth and young adults. Each year, a nationally
representative sample of 8th, 10th, and 12th graders are surveyed, and
a sample of participants receive follow-up surveys into young
adulthood. National Adult Tobacco
Survey (NATS)
2009-present
The NATS provides data about tobacco use among adults, as well as
the factors promoting, and impeding tobacco use. National Youth Tobacco
Survey (NYTS)
1999-present
The NYTS provides data about tobacco-related beliefs, attitudes,
behaviors, and exposure to pro- and anti-tobacco influences from
middle and high school youth. Population Assessment of
Tobacco and Health Study
(PATH)
2013-present
The PATH Study is a national longitudinal study of tobacco use and
health that surveys participants aged 12 and older. Tobacco Product and Risk
Perception Survey (TPRPS)
2014-present
The TPRPS assesses tobacco use, consumer reaction to tobacco
product marketing, and individual perception of the risk of tobacco
products, specifically novel and alternative products. The most recent
iteration of the survey was completed in 2017. Tobacco Use Supplement
to Current Population
Survey (TUS-CPS)
1992-present
The TUS-CPS is a survey of tobacco use that is administered every 3
to 4 years. References
490 Data are available from adults in all waves and from youth
aged 15-17 from years 1992-2006. 650 650 37 Table 2. Individual datasets acquired for each nationally representative survey/study identified for
analysis. Survey/study name
Years conducted
Dataset Name
Data Access/URL
Data on Youth Users
Monitoring the Future
(MTF)
1975-present
2014 – 2018 Annual Survey of 8th
and 10th Graders
2014 – 2018 Annual Survey of 12th
Graders
https://www.icpsr.umich.edu/web/N
AHDAP/series/35
National Youth Tobacco
Survey (NYTS)
1999-present
2011 – 2019 Annual Survey of
Middle and High School Students
https://www.cdc.gov/tobacco/data_s
tatistics/surveys/nyts/data/index.htm
l
Population Assessment
of Tobacco and Health
Study (PATH)
2013-present
Wave 1 (09/2013 – 12/2014) Youth
Wave 2 (10/2014 – 10/2015) Youth
Wave 3 (10/2015 – 10/2016) Youth
Wave 4 (12/2016 – 01/2018) Youth
https://www.icpsr.umich.edu/web/N
AHDAP/studies/36498
Data on Adult Users
Health Information
National Trends Survey
(HINTS)
2015, 2017
HINTS FDA 2015
HINTS FDA Cycle 2 2017
https://hints.cancer.gov/data/downlo
ad-data.aspx
National Adult Tobacco
Survey (NATS)
2009-2014
2009/10 NATS Survey
2012/13 NATS Survey
2013/14 NATS Survey
https://www.cdc.gov/tobacco/data_s
tatistics/surveys/nats/index.htm
Population Assessment
of Tobacco and Health
Study (PATH)
2013-present
Wave 1 (09/2013 – 12/2014) Adult
Wave 2 (10/2014 – 10/2015) Adult
Wave 3 (10/2015 – 10/2016) Adult
Wave 4 (12/2016 – 01/2018) Adult
https://www.icpsr.umich.edu/web/N
AHDAP/studies/36498
Tobacco Product and
Risk Perception Survey
(TPRPS)
2014-2017
2014 GSU TCORS TPRPS Survey
2015 GSU TCORS TPRPS Survey
2016 GSU TCORS TPRPS Survey
2017 GSU TCORS TPRPS Survey
via Data Sharing Agreement with
the Georgia State University
Tobacco Center of Regulatory
Science (TCORS)
Tobacco Use
Supplement to Current
Population Survey (TUS-
CPS)
2010-2018
2014/2015 TUS-CPS Survey
July 2018 TUS-CPS Survey
https://cancercontrol.cancer.gov/brp
/tcrb/tus-cps/questionnaires-data 651
652 653 38 Table 3. Prior wave flavored adult cigar use predicting established use of cigarettes in subsequent wave
in PATH. Cigar Type
Flavored Cigar
Use in Wave 2? breviations: AOR=adjusted odds ratio, CI=confidence interval, NA=Not Applicable, OR=odds ratio.
lid dd
i
b
l
l
d
h
l
d
i
l d d i
h d breviations: AOR=adjusted odds ratio, CI=confidence interval, NA=Not Applicable, OR=odds ratio.
alid odds ratios cannot be calculated when zero relevant records are included in the data. j
,
f
,
pp
,
d odds ratios cannot be calculated when zero relevant records are included in the data. References
490 Record
Count (n)
Prevalence (95% CI)
OR (95% CI)
AOR (95% CI)
Wave 3 Cigarette Use by Wave 2 Past 30 day Flavored Cigar Use in Wave 2 Cigarette Nonsmokers†
Cigarillos
No
10
9.424 (3.14, 15.71)
Reference
Reference
Yes
12
10.61 (4.658, 16.56)
1.14 (0.394, 3.3)
0.914 (0.23, 3.63)
I don't know
1
13.73 (0, 41.51)
1.53 (0.0886, 26.4)
1.37 (0.0517, 36.4)
Filtered
Cigars
No
8
25.08 (4.777, 32.56)
Reference
Reference
Yes
15
23.63 (12.25, 30.93)
0.924 (0.3, 2.85)
0.806 (0.172, 3.77)
I don't know
1
37.6 (0, 43.48)
1.8 (0.0533, 60.8)
4.44 (0.109, 181)
Traditional
Cigars
No
8
4.747 (1.237, 8.257)
Reference
Reference
Yes
6
10.27 (1.141, 19.41)
2.3 (0.572, 9.23)
0.636 (0.083, 4.88)
I don't know
1
13.02 (0, 40.63)
3 (0.199, 45.2)
3.49 (0.0213, 572)
Wave 4 Cigarette Use by Wave3 Past 30 day Flavored Cigar Use in Wave 3 Cigarette Nonsmokers‡
Cigarillos
No
14
10.31 (4.073, 16.55)
Reference
Reference
Yes
14
15 (7.288, 22.72)
1.54 (0.598, 3.94)
1.46 (0.505, 4.24)
I don't know
0
0 (0, 0)
NA*
NA*
Filtered
Cigars
No
8
15.85 (3.736, 25.96)
Reference
Reference
Yes
11
19.81 (9.185, 31.88)
1.31 (0.36, 4.78)
1.48 (0.233, 9.35)
I don't know
1
20.84 (-10.39, 28.91)
1.4 (0.089, 22)
0.922 (0.00724, 118)
Traditional
Cigars
No
8
2.868 (0.6246, 5.111)
Reference
Reference
Yes
3
5.827 (0, 12.14)
2.1 (0.417, 10.5)
0.948 (0.0913, 9.86)
I don't know
0
0 (0, 0)
NA*
NA*
Abbreviations: AOR=adjusted odds ratio, CI=confidence interval, NA=Not Applicable, OR=odds ratio. *Valid odds ratios cannot be calculated when zero relevant records are included in the data. †Wave 2 non-cigarette smoking cigarillo, filtered cigar, and traditional cigar users were represented by 273, 115,
d 308 t t l
d
ti
l ( f
hi h 134 69
d 79 l
i di
t d
f fl
d i
) 654
655 654
655 †Wave 2 non-cigarette smoking cigarillo, filtered cigar, and traditional cigar users were represented by 273, 115,
and 308 total records, respectively (of which 134, 69, and 79 also indicated use of flavored cigars). ‡Wave 3 non-cigarette smoking cigarillo, filtered cigar, and traditional cigar users were represented by 255, 115,
and 320 total records, respectively (of which 107, 60, and 56 also indicated use of flavored cigars). 656 39 39 Table 4. breviations: AOR=adjusted odds ratio, CI=confidence interval, OR=odds ratio.
atistically significant finding indicated in bold. References
490 Wave 2 flavored cigar use predicting transitions to daily or non-current Wave 3 cigar use
657
among wave 2 regular but not daily (i.e., “some day”) adults in PATH. 658 657
658 Table 4. Wave 2 flavored cigar use predicting transitions to daily or non-current Wave 3 cigar use
among wave 2 regular but not daily (i.e., “some day”) adults in PATH. Wave 2
Cigar Type
Flavored
Cigar Use
in Wave
2? Wave 3
Cigar Use
Frequency
Record
Count
(n)
Prevalence (95% CI)
OR (95% CI)
AOR (95% CI)
Cigarillos*
No
Every day
11
5.284 (1.773, 8.795)
Reference
Reference
Some day
75
41.88 (34.08, 49.68)
--
--
Non-current
95
52.83 (44.75, 60.91)
Reference
Reference
Yes
Every day
16
8.552 (3.56, 13.54)
1.68 (0.606, 4.63)
1.89 (0.557, 6.4)
Some day
104
40.75 (32.98, 48.52)
--
--
Non-current
125
50.7 (42.69, 58.71)
0.918 (0.586, 1.44)
0.796 (0.472, 1.34)
I don't
know
Every day
1
14.33 (-13.82, 42.48)
3 (0.182, 49.5)
3.83 (0, Inf)
Some day
1
9.826 (-12.12, 31.77)
--
--
Non-current
7
75.84 (42.97, 100)
2.8 (0.326, 24.1)
2.15 (0.227, 20.3)
Filtered
Cigars†
No
Every day
10
12.82 (3.782, 21.85)
Reference
Reference
Some day
34
40.19 (29.13, 51.24)
--
--
Non-current
32
46.99 (35.49, 58.5)
Reference
Reference
Yes
Every day
16
13.6 (6.79, 20.41)
1.07 (0.392, 2.92)
1.26 (0.337, 4.7)
Some day
48
36.62 (25.24, 48)
Non-current
60
49.78 (38.58, 60.97)
1.12 (0.582, 2.15)
1.28 (0.588, 2.8)
I don't
know
Every day
1
24.19 (-28.49, 76.86)
2.17 (0.0669, 70.4)
9.09 (0.0505, 1,635)
Some day
1
31.89 (-4.706, 68.48)
Non-current
2
43.93 (6.031, 81.82)
0.884 (0.144, 5.4)
1.05 (0.168, 6.56)
Traditional
Cigars‡
No
Every day
11
3.815 (1.445, 6.185)
Reference
Reference
Some day
179
81.74 (76.34, 87.13)
--
--
Non-current
41
14.45 (9.515, 19.38)
Reference
Reference
Yes
Every day
4
5.362 (-0.788, 11.51)
1.43 (0.276, 7.39)
0.751 (0.0952, 5.93)
Some day
47
66.21 (56.17, 76.24)
--
--
Non-current
25
28.43 (17.59, 39.28)
2.35 (1.3, 4.27)
1.28 (0.584, 2.8)
I don't
know
Every day
2
13.02 (-9.024, 35.07)
3.78 (0.43, 33.2)
4.41 (0.365, 53.3)
Some day
3
35.11 (-3.182, 73.4)
--
--
Non-current
3
51.87 (8.842, 94.9)
6.38 (0.859, 47.4)
4.89 (0.26, 91.9)
Abbreviations: AOR=adjusted odds ratio, CI=confidence interval, OR=odds ratio. Statistically significant finding indicated in bold. 659 40 660
661 among Wave 3 regular but not daily (i.e., some day ) adult users in PATH. y
g f
f
g
alid odds ratios cannot be calculated when zero relevant records are included in the data. References
490 Wave 3
Cigar Type
Flavored
Cigar
Use in
Wave 3? Wave 4
Cigar Use
Frequency
Record
Count
(n)
Prevalence (95% CI)
OR (95% CI)
AOR (95% CI)
Cigarillos†
No
Every day
16
9.353 (3.742, 14.96)
Reference
Reference
Some day
69
48.74 (40.53, 56.96)
--
--
Non-current
64
41.91 (33.76, 50.05)
Reference
Reference
Yes
Every day
10
4.313 (0.9084, 7.718)
0.437 (0.152, 1.26)
0.372 (0.126, 1.1)
Some day
99
52.41 (44.15, 60.68)
--
--
Non-current
88
43.27 (34.91, 51.63)
1.06 (0.65, 1.72)
0.946 (0.538, 1.66)
I don't
know
Every day
3
9.771 (0, 20.35)
1.05 (0.228, 4.82)
1.1 (0.127, 9.53)
Some day
7
36.58 (13.63, 59.52)
--
--
Non-current
9
53.65 (27.4, 79.91)
1.6 (0.514, 5.01)
1.36 (0.418, 4.41)
Filtered
Cigars‡
No
Every day
10
12.37 (3.086, 21.65)
Reference
Reference
Some day
29
46.52 (28.66, 64.38)
--
--
Non-current
36
41.11 (26.96, 55.26)
Reference
Reference
Yes
Every day
7
7.422 (1.117, 13.73)
0.568 (0.147, 2.19)
0.479 (0.062, 3.69)
Some day
33
38.94 (25.93, 51.95)
--
--
Non-current
50
53.64 (40.92, 66.35)
1.66 (0.812, 3.38)
1.71 (0.69, 4.24)
I don't
know
Every day
0
--
NA*
NA*
Some day
3
47.87 (0 100)
--
--
Non-current
3
52.13 (0, 100)
1.56 (0.134, 18.1)
0.789 (0.105, 5.95)
Traditional
Cigars§
No
Every day
10
6.449 (0.5351, 12.36)
Reference
Reference
Some day
164
72.81 (65.25, 80.38)
--
--
Non-current
48
20.74 (14.54, 26.93)
Reference
Reference
Yes
Every day
5
3.632 (0, 8.695)
0.547 (0.0761,
3.93)
0.575 (0.0931, 3.55)
Some day
41
61.33 (48.4, 74.27)
--
--
Non-current
28
35.03 (23.64, 46.43)
2.06 (1.07, 3.96)
1.28 (0.552, 2.96)
I don't
know
Every day
0
--
NA*
NA*
Some day
7
31.4 (0, 70.15)
--
--
Non-current
5
68.6 (29.85, 100)
8.35 (1.45, 48.2)
7.73 (0.918, 65.1)
Abbreviations: AOR=adjusted odds ratio, CI=confidence interval, NA=Not Applicable, OR=odds ratio. St ti ti
ll
i
ifi
t fi di
i di
t d i b ld Abbreviations: AOR=adjusted odds ratio, CI=confidence interval, NA=Not Applicable, OR=odds ratio. Statistically significant finding indicated in bold. 662 41 Table 6. Prevalence of current cigarette smoking in youth cigar users of flavored or unflavored products
663
in PATH. 664 Table 6. Prevalence of current cigarette smoking in youth cigar users of flavored or unflavored products
663
in PATH. 664 Table 6. Prevalence of current cigarette smoking in youth cigar users of flavored or unflavored products
in PATH. Flavored
Cigar
Use? References
490 PATH
Wave
Cigarillos
Filtered Cigars
Traditional Cigars
Record
Count
(n)
Prevalence (95%CI)
Record
Count
(n)
Prevalence (95%CI)
Record
Count
(n)
Prevalence (95%CI)
Yes†
1
131
58.42 (50.9, 65.93)
34
68.51 (51.73, 85.29)
31
56.48 (39.96, 73.01)
2
34
51.06 (36.38, 65.74)
17
61.49 (38.53, 84.44)
19
58.39 (40.87, 75.91)
3
34
64.01 (48.53, 79.49)
17
63.04 (36.11, 89.97)
10
55.89 (27.03, 84.75)
4
41
61.95 (48.83, 75.08)
13
68.4 (47.77, 89.02)
9
73.11 (45.65, 100)
No‡
1
36
49.83 (37.71, 61.95)
14
76.8 (53.27, 100)
21
46.83 (28.86, 64.8)
2
25
56.44 (40.71, 72.17)
15
87.19 (66.75, 100)
20
49.63 (29.62, 69.65)
3
13
57.09 (36.04, 78.14)
14
92.12 (76.28, 100)
7
52.85 (23, 82.7)
4
26
55.2 (41.28, 69.12)
10
55.11 (27.4, 82.82)
13
53.49 (30.24, 76.74)
I don't
know§
1*
--
--
--
2
6
42.55 (14.39, 70.7)
5
79.65 (48.35, 100)
2
29.69 (-7.742, 67.11)
3
7
62.59 (31.5, 93.69)
3
70.17 (20.87, 100)
2
68.33 (12.16, 100)
4
10
39.76 (19.2, 60.32)
2
31.91 (-10.16, 73.99)
4
29.81 (2.997, 56.62)
*The response of “I don’t know” to flavored cigar use questions was not enabled as a third possible option in Wave 1
d t
ll
ti 663
664 663
664 *The response of “I don’t know” to flavored cigar use questions was not enabled as a third possible option in Wave 1
data collection. respectively. ‡Unflavored cigarillo users were represented by 72, 43, 24, and 50 total records in Waves 1 through 4, respectively. Unflavored filtered cigar users were represented by 18, 17, 15, and 17 total records in Waves 1 through 4, respectively. Unflavored traditional cigar users were represented by 43, 35, 13, and 24 total records in Waves 1 through 4,
respectively. §Cigarillo users unsure whether product was flavored were represented by 16, 12, and 27 total records in Waves 2
through 4, respectively. Filtered cigar users unsure whether product was flavored were represented by 7, 4, and 7 total
d
h
h
l
d
l
h
h
d
fl
d p
y
‡Unflavored cigarillo users were represented by 72, 43, 24, and 50 total records in Waves 1 through 4, respectively. Unflavored filtered cigar users were represented by 18, 17, 15, and 17 total records in Waves 1 through 4, respectively. Unflavored traditional cigar users were represented by 43, 35, 13, and 24 total records in Waves 1 through 4,
respectively. *The response of “I don’t know” to flavored cigar use questions was not enabled as a third possible option in Wave 1
data collection. References
490 §Cigarillo users unsure whether product was flavored were represented by 16, 12, and 27 total records in Waves 2
through 4, respectively. Filtered cigar users unsure whether product was flavored were represented by 7, 4, and 7 total
records in Waves 2 through 4, respectively. Traditional cigar users unsure whether product was flavored were
represented by 8, 3, and 10 total records in Waves 2 through 4, respectively. 665 666 42 Additional Files Additional Files Additional Files 667 Additional Files
667
668
669
Supplementary Figure 1. Adult cigar use overall (left) and flavored cigar use specifically (right) based on
670
(A) HINTS-FDA (current every day or some day users) and (B) TUS-CPS (past 30-day use on one or more
671
days). Note that these HINTS-FDA estimates reflect use of any flavored tobacco product (including e-
672
cigarette/vaping products whose popularity grew considerably during this timespan). The HINTS-FDA
673
survey question does not ask specifically about use of flavored cigars. 674
B
A 68
A 668 69
B 9
B B 669 Supplementary Figure 1. Adult cigar use overall (left) and flavored cigar use specifically (right) based on
670
(A) HINTS-FDA (current every day or some day users) and (B) TUS-CPS (past 30-day use on one or more
671
days). Note that these HINTS-FDA estimates reflect use of any flavored tobacco product (including e-
672
cigarette/vaping products whose popularity grew considerably during this timespan). The HINTS-FDA
673
survey question does not ask specifically about use of flavored cigars. 674 43
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https://openalex.org/W2171455789
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https://europepmc.org/articles/pmc4155041?pdf=render
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English
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A turn-on fluorescent solid-sensor for Hg(II) detection
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Nanoscale research letters
| 2,014
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cc-by
| 7,147
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NANO EXPRESS Open Access Abstract A rhodamine organosilane derivative (Rh-UTES) has been obtained by one-pot synthesis. The chemical structure of
Rh-UTES was confirmed by nuclear magnetic resonance (NMR) and infrared (FTIR) techniques. To obtain an
inorganic-organic hybrid sensor, Rh-UTES was covalently immobilized on a porous silicon microcavity (PSiMc) via
triethoxysilane groups. The attachment of the organic derivative into PSiMc was confirmed by FTIR, specular
reflectance, and scanning electron microscopy (SEM). The optical performance of Rh-UTES receptor for Hg2+
detection was investigated by fluorescent spectroscopy and microscopy. Upon the addition of increasing amounts
of Hg2+ ions, a remarkable enhancement in emission intensity was produced in both systems. In the solid phase, an
increase of integrated fluorescent emission of 0.12- and 0.15-fold after Hg2+ receptor coordination was observed. The light harvesting capability of PSiMc devices allowed obtaining an enhanced fluorescent emission after Rh-UTES
immobilization (277-fold). The fluorescence microscopy of hybrid PSiMc sensor provided an optical qualitative test
for Hg2+ detection. Keywords: Chemosensor; Porous silicon; Rhodamine derivative; Fluorescence; Heavy metal particularly attractive due to their simplicity, high sensitiv-
ity, high selectivity, and instantaneous response [10]. Fluorescent chemosensors based on xanthenes and related
derivatives for the Hg2+ ions detection have been increas-
ing due to the low cost and high applicability in industrial
and biological processes [11]. During recent years, novel
inorganic-rhodamine hybrid sensors have been published. The rhodamine derivatives have been immobilized into the
different inorganic receptors. Huang et al. reported fluores-
cent gold nanoparticle sensors for detection of Hg2+ ions
[12]. Since gold nanoparticles (AuNPs) are highly efficient
fluorescence quenchers, the rhodamine derivative had to
be released from the AuNPs to restore the rhodamine
fluorescence. Lee et al. and Zhou's group developed a
covalently bonded mesoporous silica rhodamine derivative
[13,14]. Childress and co-workers reported dye-doped
polymer nanoparticles that are able to detect mercury ions. The nanoparticles were prepared by precipitation of highly
fluorescent conjugated polymers and doped with rhoda-
mine derivatives [15]. Recently, Wang and Gao designed a
mercury sensor using β-NaYF4:Yb3+/Eu3+ nanorods as
the excitation source and a rhodamine derivative as a
probe [16]. In this proposal, our research group has © 2014 De la Cruz-Guzman et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. A turn-on fluorescent solid-sensor for Hg(II)
detection Mayela De la Cruz-Guzman1, Angelica Aguilar-Aguilar1, Luis Hernandez-Adame1, Alan Bañuelos-Frias3,
Francisco J Medellín-Rodríguez2 and Gabriela Palestino1* De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 * Correspondence: palestinogabriela@fcq.uaslp.mx
1Biopolymers and Nanostructures Laboratory, Faculty of Chemical Sciences,
Universidad Autónoma de San Luis Potosí, Av. Manuel Nava No. 6, San Luis
Potosí, San Luis Potosí 78210, México
Full list of author information is available at the end of the article Synthesis of porous silicon designed a new functional rhodamine derivative (Rh-
UTES) that acts as a receptor of heavy metal ions. The
Rh-UTES derivative was covalently bonded to porous
silicon microcavity (PSiMc) to develop a hybrid sensor. The main advantage of the proposed method is the sim-
plicity of the system and the fact that the hybrid sensor
should be easy to carry for field applications. The PSiMc
has proven to be a suitable material with unique optical
properties for the development of this kind of fluores-
cent sensor [17]. Our previous approaches in this field
have shown that the detection of fluorescent molecules
is possible using the optical properties of specific PSi
structure (mirror or microcavity) [18]. Increased excitation
and enhanced emission, both driven by the efficient reflec-
tion of light and resonance effects within the PSi micro-
cavities, allowed the enhancement of the fluorescent
response of the Rh-UTES derivative even at low molecular
concentration. Hence, the variation of this method was
used here to produce detection of low concentrations of
heavy metals by forming metallic complexes within the
pores that turn on the luminescence emission. PSi samples were prepared by the wet electrochemical etch-
ing process using high-doped p-type (boron-doped) silicon
wafers (thickness 500 to 550 μm) with 0.001 to 0.005 Ω cm
resistivity, and with the crystallographic orientation of
(100), purchased from WRS Materials (San Jose, CA, USA). The electrolyte consisted of hydrofluoric acid (48 wt%) and
ethanol in the volumetric ratio of 3:7. The anodization
time and current density were controlled by a computer-
interfaced electronic circuit. The samples were fabricated at
room temperature, and freshly etched samples were washed
with ethanol and dried with pentane. To perform this work,
we have selected a PSiMc, mainly due to its optical features
in the reflectance spectra that allows the detection of infil-
trated material into the porous structure. PSiMc configur-
ation consists of an active porous layer embedded between
two multilayered mirrors (Bragg reflectors). The PSiMc was
produced by alternating layers of high porosity (H; refract-
ive index, n = 1.14395) and low porosity (L; n = 1.25865),
with current densities of 70 and 30 mA/cm2. Anodization
times of 6.35 and 10.67 s for H and for L, respectively, were
used for the fabrication of the corresponding dielectric
Bragg mirrors. Synthesis of porous silicon The PSiMc structures were fabricated with
the configuration of (HL) × 5 HH (LH) × 5, where (HL) × 5
corresponded to the first Bragg reflector, HH to the cavity
and (LH) × 5 to the second Bragg reflector. The PSiMc
samples were thermally oxidized at 600°C for 30 min in
O2 atmosphere to stabilize and protect them against en-
vironmental contaminants and/or natural aging [19]. Synthesis of rhodamine fluorescent derivative Synthesis of rhodamine fluorescent derivative
Herein, we synthesize a new rhodamine fluorescent de-
rivative Rh-UTES bearing urea groups. To obtain this
compound, several steps were needed. For the synthesis
of Rh-amine derivative (1), following the procedure in
the literature [20], rhodamine base (4.0 g, 8.3 mmol) and
ethylendiamine (4.2 g, 70 mmol) were dissolved in EtOH
(210 mL) and refluxed for 18 h. The solvent was removed
by evaporation, and the residue was dissolved in an
aqueous HCl solution (1 M, 333 mL). An aqueous NaOH
solution (1 M) was added carefully to the solution with
magnetic stirring. The precipitate was recovered by filtra-
tion, washed thoroughly with water, and then dried under
vacuum, yielding (1) as a pink fluffy powder (3.21 g, 80%);
1H NMR (CDCl3): δ (ppm) 7.85 (d, 1H, J = 2.5 Hz), 7.44 (t,
2H, J = 6.7 Hz), 7.06 (s, 1H), 6.42 to 6.37 (m, 6H), 3.33 (q,
10H, J = 7.1 Hz), 2.91 (t, 2H, J = 6.7 Hz), 1.16 (t, 12H, J =
6.7 Hz); 13C NMR (CDCl3): δ (ppm) 170.5, 153.7, 153.3,
149.1, 133.2, 130.0, 128.4, 128.3, 123.9, 123.2, 108.6, 103.6,
97.8, 66.4, 44.4, 41.1, 39.5, 12.66. Figure 1 shows the syn-
thesis to obtain derivative (1). Background g
The toxicity of mercury (Hg) and its complex forms on
ecosystems and human health is well known. The need
to create new sensitive and practical analytical methods
to detect the mercury ions in different sources has in-
creased. Recently, ion-selective sensors have attracted
attention due to their diverse potential applications as
tools for the quantitative and qualitative monitoring of
metal ions in many biological and environmental pro-
cesses [1-6]. Ion-selective sensors could find applicability
in monitoring metal ion concentrations and can be prac-
tical solutions to monitor industrial waste effluent streams
and potable water. Emphasis has been placed on com-
pound development that selectively responds to the pres-
ence of specific metal ions through a change in one or
more properties of the system, such as redox potentials [7],
absorption [8], or fluorescence spectra [9]. Such sensors
based on ion-induced changes in fluorescence appear to be * Correspondence: palestinogabriela@fcq.uaslp.mx
1Biopolymers and Nanostructures Laboratory, Faculty of Chemical Sciences,
Universidad Autónoma de San Luis Potosí, Av. Manuel Nava No. 6, San Luis
Potosí, San Luis Potosí 78210, México
Full list of author information is available at the end of the article Page 2 of 9 Page 2 of 9 De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 designed a new functional rhodamine derivative (Rh-
UTES) that acts as a receptor of heavy metal ions. The
Rh-UTES derivative was covalently bonded to porous
silicon microcavity (PSiMc) to develop a hybrid sensor. The main advantage of the proposed method is the sim-
plicity of the system and the fact that the hybrid sensor
should be easy to carry for field applications. The PSiMc
has proven to be a suitable material with unique optical
properties for the development of this kind of fluores-
cent sensor [17]. Our previous approaches in this field
have shown that the detection of fluorescent molecules
is possible using the optical properties of specific PSi
structure (mirror or microcavity) [18]. Increased excitation
and enhanced emission, both driven by the efficient reflec-
tion of light and resonance effects within the PSi micro-
cavities, allowed the enhancement of the fluorescent
response of the Rh-UTES derivative even at low molecular
concentration. Hence, the variation of this method was
used here to produce detection of low concentrations of
heavy metals by forming metallic complexes within the
pores that turn on the luminescence emission. Methods Rhodamine base, ethylenediamine, m-xylenediisocyanate,
3-aminopropyltriethoxysilane (APTES), hydrochloric acid,
hydrofluoric acid, nitric acid, sodium hydroxide, and mer-
cury nitrate were purchased from Sigma-Aldrich (St. Louis,
MO, USA). All solvents were analytical reagent grade and
used as received. Instruments and spectroscopy measurements
Th
fl
i i
d d i
A Instruments and spectroscopy measurements
The reflectivity spectra were recorded in an Agilent Cary
60 UV-Vis spectrophotometer (Agilent Technologies, Sta. Clara, CA, USA) coupled with a 30° specular reflection
unit. PSi samples were illuminated with the xenon source,
and the reflected beam was detected with the silicon diode
detector. The resulting spectra were captured in the range
from 500 to 900 nm. The fluorescence images of PSiMc/
Rh-UTES sensor were recorded in a Nikon Optiphot-2
fluorescence microscope (super high pressure mercury
lamp power supply; Nikon, Tokyo, Japan). The Fourier
transform infrared spectra (FTIR) were recorded in a
Bruker Tensor 27 spectrophotometer (Bruker Corporation,
Billerica, MA, USA), with 128 scans and 4-cm−1 reso-
lution, coupled with a diamond crystal attenuated total
reflectance unit (ATR). Nuclear magnetic resonance
(NMR) measurements of 1H and 13C were carried out in
a Bruker 500 MHz spectrometer. Scanning electron mi-
croscopy (SEM) was performed using a UHR dual-beam
FEI Helios Nanolab 600 field emission scanning electron
microscope (FEI Company, Hillsboro, OR, USA). Samples
were mounted on a conductive carbon tape. Images were
captured at magnifications of × 20,000 and × 25,000. The Rh-UTES derivative was obtained by following
the next procedure (Figure 2): In a 10-mL round-bottom
flask fitted with magnetic stirrer, m-xylenediisocyanate De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 Page 3 of 9 Figure 1 Synthesis to obtain derivative (1). Figure 1 Synthesis to obtain derivative (1). (0.05 g, 0.26 mmol) and 3-aminopropyltriethoxysilane
(APTES) (0.04 g, 0.18 mmol) were refluxed in 5 mL of
toluene under N2 for 12 h. Derivative (2) was used without
isolation, the Rh-amine derivative (1) was added (0.1 g,
0.21 mmol) under N2, and the reaction was refluxed for
3 h. The solvent was evaporated under reduced pressure
to give a beige powder (0.22 g, 96%); 13C NMR (DMSO-
d6): δ (ppm) 168.0, 158.1, 154.2, 153.0, 148.1, 141.0, 133.2,
130.5, 128.6, 128.5, 126.2, 126.1, 126.0, 125.9, 125.7, 124.0,
122.8, 108.3, 105.3, 97.8, 64.6, 60.2, 44.1, 43.4, 40.6, 38.4,
21.2, 15.1, 14.5, 12.8; IR data: νmax (cm−1): 3331, 2970 to
2890, 1695, 1624, 1574, 1513, 1082, 962, 771. method through silane chemistry by reacting the meth-
oxy groups (-OCH3)3 of the fluorescent molecule with
the siloxane (-Si-O) groups of the thermally oxidized PSi
surface [18]. Metal capture Once obtained, the PSiMc/Rh-UTES sensors were ex-
posed to 2.0 mL of mercury aqueous solutions. To as-
sure the presence of the free Hg2+ ions, the solutions
were adjusted at pH 3.0 using HNO3 0.1 M (based in
the Hg speciation diagram). The complexation reactions Instruments and spectroscopy measurements
Th
fl
i i
d d i
A Briefly, the PSi samples were dipped in
2 mL of Rh-UTES derivative solution (1.16 μM in ACN)
at room temperature, and all of the reaction system was
kept under inert atmosphere with magnetic stirring. The
reaction time was fixed at 3 h to obtain the final PSiMc/
Rh-UTES sensors. Results and discussion Rh-UTES derivative was successfully synthesized from a
rhodamine base in a relatively good yield. To evaluate
the metal ion binding capability of this new compound,
a colorimetric evaluation was performed in a liquid phase. Figure 3 shows the optical behavior of the fluorescent che-
mosensor in solution (1.16 μM in ACN). It was observed
that after the Hg2+ addition, the colorless solution immedi-
ately becomes pink. It is interesting to notice that the color
intensity of the solution is linearly dependent on the metal
concentration. The color change in the chemosensor solu-
tion after Hg2+ addition is attributed to the chelator-metal
binding. Thus, the colorimetric change produced during
Hg2+ capture can be used as ‘naked-eye’ detection of this
metallic contaminant in solution. Figure 4 Fluorescence response of Rh-UTES derivative in liquid
phase at different metal concentration. Fluorescence response
of Rh-UTES derivative in liquid phase (1 mM in ACN) upon addition
of different concentrations of Hg2+ ions (0.166 to 27.0 μM). λexc =
485 nm. The inset shows the fluorescence intensity of the Rh-UTES-
Hg2+ complex as a function of [Hg2+]/[Rh-UTES] ratio. The photoluminescent properties of Rh-UTES deriva-
tive in solution were investigated toward the metal ion
complexation. Figure 4a shows the excitation and emis-
sion spectra of Rh-UTES derivative with peaks centered
at 513 and 583 nm, respectively. In the figure we can no-
tice that the organic receptor exhibited a slight fluores-
cence emission. Upon the addition of increasing amount
of Hg2+ ions (0.166 to 27.0 μM) to the solution of Rh-
UTES receptor, a remarkable enhancement in the emis-
sion intensity was observed. This fluorescent enhance-
ment is attributed to the formation of the Rh-UTES-Hg2+
complex. Thus, it is clear that the addition of Hg2+ ions
‘turns-on’ the fluorescence whereby the colorless weak
fluorescent derivative changed to a colored highly fluores-
cent complex, as was also shown in Figure 3. Additionally,
we found that the Rh-UTES-Hg2+ complex presents a
maximum emission at 11.9 μM Hg2+ concentration,
after which a fluorescent quenching phenomenon was
observed. The fluorescent intensity is reduced since
some molecules of the complex act as a quencher (be-
cause the high concentration of the complex may induce a self-absorption process) which in turn decreases the num-
ber of molecules that can emit. PSi device functionalization The binding of Rh-UTES derivative within the PSi nano-
structured devices was performed following one-step Figure 2 Synthesis of Rh-UTES (3). Figure 2 Synthesis of Rh-UTES (3). Page 4 of 9 De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 Figure 4 Fluorescence response of Rh-UTES derivative in liquid
phase at different metal concentration. Fluorescence response
of Rh-UTES derivative in liquid phase (1 mM in ACN) upon addition
of different concentrations of Hg2+ ions (0.166 to 27.0 μM). λexc =
485 nm. The inset shows the fluorescence intensity of the Rh-UTES-
Hg2+ complex as a function of [Hg2+]/[Rh-UTES] ratio. were carried out at room temperature for 12 h under
magnetic stirring. Results and discussion Finally, after addition of
24.2 μM Hg2+ concentration, the fluorescent emission of
complex remains constant, which is attributed to the de-
pletion of Rh-UTES derivative. The fluorophore selectivity was also investigated by
measuring the changes in the fluorescent emission pro-
duced by the addition of the following metal ions: Ag+,
Hg2+, Ca2+, Pb2+, Li2+, Zn2+, Fe2+, Ni2+, K+, Cu2+, Na+, and
Mn2+ to various solutions of Rh-UTES. The results are dis-
played in Figure 5; it is clear that the presence of these ions
led to increases in the fluorescence intensity to varying de-
grees. It was observed that only Li2+ ions promote small
fluorescence intensity changes, while the other metal ions
did not cause any significant changes under identical con-
ditions. The fluorescent emission intensity observed for
Hg2+ over the other ions is remarkably high pointing out
the high selectivity of Rh-UTES toward Hg2+. Reflectance spectra
h
fl Figure 3 Colorimetric changes in the Rh-UTES derivative
solutions. (a) Before Hg2+ addition and after Rh-UTES-Hg2+ complex
formation at the following molar ratios: (b) 1:1, (c) 1:6, and (d) 1:10,
respectively. Rh-UTES concentration remained fixed at 1.16 μM in
ACN solution. The reflectance spectra of the PSiMc were recorded after
each modification step using the UV-vis spectrophotom-
eter. Figure 6 compares reflectance spectra taken before
and after PSiMc functionalization and a metal capture. It
is observed that Rh-UTES derivative binding produces a
red shift (12 nm) in the PSiMc reflectance spectrum; we
also found that this process is repeatable showing a stand-
ard deviation (SD) of ±2.12 nm. The red shift can be at-
tributed to the effective refractive index (ȵ) changes after
infiltration of the fluorescent molecule into the PSi pores
[18]. After exposition of PSiMc/Rh-UTES sensor to Hg2+
solution, surprisingly and contrary to the expectation, a
blue shift was observed in the specular reflectance Figure 3 Colorimetric changes in the Rh-UTES derivative
solutions. (a) Before Hg2+ addition and after Rh-UTES-Hg2+ complex
formation at the following molar ratios: (b) 1:1, (c) 1:6, and (d) 1:10,
respectively. Rh-UTES concentration remained fixed at 1.16 μM in
ACN solution. Page 5 of 9 De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 Figure 5 Maximum fluorescence emission of Rh-UTES after
metal capture. Maximum fluorescence emission of Rh-UTES (10 μM
in ACN) derivative upon addition of 100 μM of Ag+, Hg2+, Ca2+,
Pb2+, Li2+, Zn2+, Fe2+, Ni2+, K+, Cu2+, Na+, and Mn2+, respectively. The emission spectra were recorded under identical experimental
conditions at excitation wavelength of 485 nm. produces a decrease of ȵ. Nevertheless, to have a better
understanding of the metal-ligand-substrate interac-
tions and their effect on the optical properties of the
PSiMc structure, more studies are being conducted in
our research group. Thus, the capture of the metal ions
for the PSi/Rh-UTES sensor was confirmed using com-
plementary analytical techniques. Monitoring molecular infiltration PSi nanostructured devices were analyzed by FTIR be-
fore and after derivative functionalization and the metal
capture. Riikonen and co-workers reported the typical
strong absorptions of oxidized PSi (OxPSi) [22]. Bands
characteristic of the stretching mode of silanol groups
ν(SiO-H) were observed at around 3400 cm−1, the δ(SiO-
H) bending mode at 1640 cm−1, and the ν(Si-OH) stretch-
ing modes at 950 and 887 cm−1. An intense broad peak at
around 1085 cm−1 was also seen, which may be due to the
ν(Si-O) stretching mode for surface silicon-hydroxyl spe-
cies. All of these bands are consistent with FTIR spectrum
of our thermally (OxPSi) device [19]. The immobilization
of Rh-UTES derivative into the PSiMc surface was carried
out and confirmed by FTIR spectroscopy (Figure 7a); the
hybrid sensor owns the next characteristics bands: ν(N-H)
stretching modes at 3344 cm−1, ν(C = O) stretching modes
at 2924 cm−1, δ(N-H) bending mode at 1571 cm−1 of
secondary amide, ν(C-H) stretching modes of methylene Figure 5 Maximum fluorescence emission of Rh-UTES after
metal capture. Maximum fluorescence emission of Rh-UTES (10 μM
in ACN) derivative upon addition of 100 μM of Ag+, Hg2+, Ca2+,
Pb2+, Li2+, Zn2+, Fe2+, Ni2+, K+, Cu2+, Na+, and Mn2+, respectively. The emission spectra were recorded under identical experimental
conditions at excitation wavelength of 485 nm. spectrum (9 nm, SD ± 3.35 nm). Normally, this drift in
signal (blue shifts) can be associated to the degradation
(or oxidation) of PSi [21]. However, in this work, the ob-
served negative shift is attributed to the derivative-
metal binding. This was confirmed by the negative con-
trols that were carried out to ensure the specificity of
the linking chemistry. These results showed a negligible
drift in the PSi sensor reflectance spectrum over the
same incubation periods used to collect data in the per-
formed experiments. It seems that the metal capture Figure 7 Infrared spectra. (a) Functionalized PSiMc/Rh-UTES
device and (b) pure Rh-UTES derivative. Figure 7 Infrared spectra. (a) Functionalized PSiMc/Rh-UTES
device and (b) pure Rh-UTES derivative. Figure 6 Specular reflectance spectra of PSiMc devices. (a)
Thermally oxidized sample (black line), (b) after Rh-UTES
immobilization (red line), and (c) after metal coordination (blue
line). [Hg2+] = 3.48 μM. Figure 7 Infrared spectra. (a) Functionalized PSiMc/Rh-UTES
device and (b) pure Rh-UTES derivative. Figure 6 Specular reflectance spectra of PSiMc devices. Photoluminescence properties In solid phase, photoluminescence (PL) measurements
were used to characterize the performance of the fluor-
escent sensor under λexc = 490 nm. Figure 9 shows the
fluorescent emission of (a) thermally oxidized PSiMc, (b)
PSiMc/Rh-UTES functionalized device [1.16 μM of de-
rivative (3)], and (c, d) PSiMc/Rh-UTES sensors after ex-
posure to solutions contaminated with Hg2+ (3.45 and
6.95 μM, respectively). The amount of infiltrated derivative
into the PSi pores was obtained by calculating the concen-
tration of the residual supernatant (recovered after the ex-
posure time of the sample was completed) and making a
mass balance. The obtained concentration value was of
1.4058 ± 0.35 nmol of Rh-UTES/cm2 of etched area, which
corresponds at approximately 20% of the initial solution
concentration (1.16 μM) [19]. By comparing the optical
features of bare PSiMc with that obtained after device
functionalization, it is clear that the emission spectra show
important optical changes. The most remarkable is the
well-defined emission curve in the 525 to 625-nm range at-
tributed to the fluorescent emission of Rh-UTES derivative,
which confirms the attachment of the derivative molecule
on the PSi surface. Exposure of PSiMc/Rh-UTES sensor at
a heavy metal solution produced two new changes: first, an
increase in the integrated emission intensity of 0.13-fold
and secondly, a 16-nm red shift (552 to 568 nm) of the Monitoring molecular infiltration (a)
Thermally oxidized sample (black line), (b) after Rh-UTES
immobilization (red line), and (c) after metal coordination (blue
line). [Hg2+] = 3.48 μM. Figure 6 Specular reflectance spectra of PSiMc devices. (a)
Thermally oxidized sample (black line), (b) after Rh-UTES
immobilization (red line), and (c) after metal coordination (blue
line). [Hg2+] = 3.48 μM. Figure 6 Specular reflectance spectra of PSiMc devices. (a)
Thermally oxidized sample (black line), (b) after Rh-UTES
immobilization (red line), and (c) after metal coordination (blue
line). [Hg2+] = 3.48 μM. Figure 6 Specular reflectance spectra of PSiMc devices. (a)
Thermally oxidized sample (black line), (b) after Rh-UTES
immobilization (red line), and (c) after metal coordination (blue
line). [Hg2+] = 3.48 μM. De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 Page 6 of 9 (black line) layers together with the defect layer (cen-
tered in the middle of the structure). The morphology of
the PSiMc structures after chemical modification is shown
in Figure 8b, and we observed a homogeneous layer of
organic derivative covering the first layers of the PSi struc-
ture, which confirms the infiltration of Rh-UTES deriva-
tive into the porous device. groups at 3008 to 2861 cm−1, and mainly the siloxane
(Si-O) bands of OxPSi at 1054 cm−1. These bands are
similar to those belonging to the pure Rh-UTES deriva-
tive reported in the ‘Methods’ section (Figure 7b), thus
confirming that incorporation of Rh-UTES into the
PSiMc was successful. The hybrid sensor was then ex-
posed in a Hg2+ solution (1.16 μM) for 12 h, and the FTIR
analysis of the PSiMc/Rh-UTES-Hg2+ sample showed no
significant changes in the infrared bands (not shown)
compared with the reference spectrum of Figure 7b. groups at 3008 to 2861 cm−1, and mainly the siloxane
(Si-O) bands of OxPSi at 1054 cm−1. These bands are
similar to those belonging to the pure Rh-UTES deriva-
tive reported in the ‘Methods’ section (Figure 7b), thus
confirming that incorporation of Rh-UTES into the
PSiMc was successful. The hybrid sensor was then ex-
posed in a Hg2+ solution (1.16 μM) for 12 h, and the FTIR
analysis of the PSiMc/Rh-UTES-Hg2+ sample showed no
significant changes in the infrared bands (not shown)
compared with the reference spectrum of Figure 7b. Morphological analysis In
agreement with those contributions, we believe that the
enhanced emission observed when the PSiMc/Rh-UTES
sensor captured the Hg2+ ions is produced by the forma-
tion of metal-ligand coordination bonds, which in turn in-
duces the spirolactam ring opening [23]. Thus, based on
this coordination mechanism, the red shift in the fluores-
cent emission may be attributed to the electronic interac-
tions of PSiMc/Rh-UTES-Hg2+ complex (Figure 9c). A
similar optical behavior was found in the liquid phase che-
mosensor; however, our solid device presents several ad-
vantages that are related with (i) the easy operation of the
device, (ii) special solvents that are not needed, (iii) the
higher stability of the fluorescent derivative when immobi-
lized in the solid support, and (iv) the possibility of port-
ability. Then, by comparing spectra (c) and (d) which
correspond at the sensing of two different Hg2+ ion con-
centrations (3.45 and 6.95 μM, respectively), a 6-nm red
shift (from 568 to 574 nm) and a fluorescent emission en-
hancement of 0.12-fold was observed. In this case, the red
shift may be attributed to PSi-derivative-Hg2+ interaction
processes produced in the reduced space of PSi pores. Our
hypothesis is that after increasing the metal ion concentra-
tion, the derivative Rh-UTES receptor changed its chem-
ical structure, provoking a molecular reorganization inside
the pore. According to Tu and co-workers [24], the chem-
ical change can reduce the distances between neighboring
molecules limiting their free stretching movement and
leading to their self-interaction, which may reduce their ex-
cited state energy and produce the red shift in the spectra. Figure 10 shows a proposed mechanism of the coordin-
ation mode of Hg2+ ions. Several proposed binding modes
have been reported on which oxygen, sulfur, and nitrogen
atoms have provided higher affinity toward Hg2+ [11]. In
our study and as the FTIR spectra have showed, two
carbonyl oxygen atoms as well as the amide oxygen can
provide a binding pocket for Hg2+. To confirm the pro-
posed mechanism, further studies need to be completed
(X-ray diffraction). An analysis using fluorescence microscopy was also
carried out to characterize the emission intensity over the
entire surface of the hybrid sensor. The samples were ex-
cited using a mercury lamp with 510 to 560-nm filter in a
Nikon Optiphot-2 (G2-A) microscope coupled with 3CCD
MTI 8-bit camera. The emission intensities are shown in
the Figure 11. Morphological analysis Figure 8 shows cross-sectional SEM images of PSiMc
devices before (a) and after (b) functionalization with
Rh-UTES derivative. The top view of unmodified PSiMc
device (image not shown) shows a high porosity struc-
ture composed of well-defined pores with an average
size distribution of 19.25 ± 4 nm. In these PSi structures,
the pore sizes were big enough to allow the molecular
infiltration as demonstrated by specular reflectance spec-
trometry. The lateral view of the unmodified sample
(Figure 8a) shows the high (white line) and low porosity Figure 8 Cross-sectional SEM micrographs of PSiMc before and
after derivative immobilization. (a) Thermally oxidized sample. (b) PSiMc/Rh-UTES hybrid device. Figure 9 Emission spectra of PSiMc devices (λexc = 490 nm)
before and after chemical functionalization and metal device
recognition. (a) Thermally oxidized sample, (b) PSiMc/Rh-UTES
sensor (derivative (3) concentration = 1.16 μM), and (c and d) PSiMc/
Rh-UTES-Hg2+ complexes (3.45 and 6.95 μM respectively). Figure 9 Emission spectra of PSiMc devices (λexc = 490 nm)
before and after chemical functionalization and metal device
recognition. (a) Thermally oxidized sample, (b) PSiMc/Rh-UTES
sensor (derivative (3) concentration = 1.16 μM), and (c and d) PSiMc/
Rh-UTES-Hg2+ complexes (3.45 and 6.95 μM respectively). Figure 8 Cross-sectional SEM micrographs of PSiMc before and
after derivative immobilization. (a) Thermally oxidized sample. (b) PSiMc/Rh-UTES hybrid device. Page 7 of 9 De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 Page 7 of 9 main peak position. As we mentioned before, some studies
have demonstrated that the spirolactam-rhodamine deriva-
tives can be used to develop liquid phase OFF-ON metal
ion-fluorescent chemosensors, mainly because their chem-
ical structure may change in the presence of metal ions. In
agreement with those contributions, we believe that the
enhanced emission observed when the PSiMc/Rh-UTES
sensor captured the Hg2+ ions is produced by the forma-
tion of metal-ligand coordination bonds, which in turn in-
duces the spirolactam ring opening [23]. Thus, based on
this coordination mechanism, the red shift in the fluores-
cent emission may be attributed to the electronic interac-
tions of PSiMc/Rh-UTES-Hg2+ complex (Figure 9c). Morphological analysis A
similar optical behavior was found in the liquid phase che-
mosensor; however, our solid device presents several ad-
vantages that are related with (i) the easy operation of the
device, (ii) special solvents that are not needed, (iii) the
higher stability of the fluorescent derivative when immobi-
lized in the solid support, and (iv) the possibility of port-
ability. Then, by comparing spectra (c) and (d) which
correspond at the sensing of two different Hg2+ ion con-
centrations (3.45 and 6.95 μM, respectively), a 6-nm red
shift (from 568 to 574 nm) and a fluorescent emission en-
hancement of 0.12-fold was observed. In this case, the red
shift may be attributed to PSi-derivative-Hg2+ interaction
processes produced in the reduced space of PSi pores. Our
hypothesis is that after increasing the metal ion concentra-
tion, the derivative Rh-UTES receptor changed its chem-
ical structure, provoking a molecular reorganization inside
the pore. According to Tu and co-workers [24], the chem-
ical change can reduce the distances between neighboring
molecules limiting their free stretching movement and
leading to their self-interaction, which may reduce their ex-
cited state energy and produce the red shift in the spectra. On other hand, the enhancement of the emission intensity
observed when the PSiMc/Rh-UTES device coordinates
higher amount of Hg2+ ions confirms that the fluorescent
intensity of the PSiMc hybrid device is metal concentration
dependent [25,26]. This is important to notice if the sensing
principle of the PSiMc/Rh-UTES sensor is based on the
fluorescence spectroscopy measurements. Finally, to inves-
tigate the optical contribution of PSi devices in the fluores-
cence response, we compared the fluorescence emission of
Rh-UTES derivative in liquid (ACN) and immobilized on
PSi structures. We observed a 277-fold fluorescence in-
crease in the case of PSi/Rh-UTES nanostructure, and it is
important to keep in mind that the derivative concentration
in the solid device is three orders of magnitude lower than
in the solution (1.4058 ± 0.35 nmol cm−2 compared with
1.16 μM). Therefore, these results highlight the benefits of
use PSi optical device as support of the organic receptor. main peak position. As we mentioned before, some studies
have demonstrated that the spirolactam-rhodamine deriva-
tives can be used to develop liquid phase OFF-ON metal
ion-fluorescent chemosensors, mainly because their chem-
ical structure may change in the presence of metal ions. Acknowledgements This work was supported by the National Council for Science and
Technology of Mexico (CONACYT), Project No. CB-153161. We thank
CONACYT for the following student scholarships: MDG No. 237466, LHA No. 270040, ABF No. 229949, and AA postdoctoral scholarship 2013 (3). We
would like to thank the University of Guanajuato for NMR support via the
CONACYT-UGTO National Laboratory (Grant 123732). We acknowledge to I.Q. Olga Dávalos Montoya for her technical support during FTIR studies and Dr. Jaime Ruiz Garcia (Physics Institute-UASLP) for the facilities given for use the
fluorescence microscope. Morphological analysis The image in the Figure 11a is presenting a
real view of the PSiMc/Rh-UTES hybrid sensor and its Figure 10 Proposed mechanism of the coordination mode of Hg2+ ions. Figure 10 Proposed mechanism of the coordination mode of Hg2+ ions. Figure 10 Proposed mechanism of the coordination mode of Hg2+ ions. De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 De la Cruz-Guzman et al. Nanoscale Research Letters 2014, 9:431
http://www.nanoscalereslett.com/content/9/1/431 Page 8 of 9 Figure 11 Fluorescence emission of PSiMc sensor and its tridimensional profile before and after metal detection. (a) PSiMc/Rh-UTES
(Rh-UTES = 1.16 μM) and (b) PSiMc/Rh-UTES-Hg2+ (Hg2+ = 6.95 μM). Figure 11 Fluorescence emission of PSiMc sensor and its tridimensional profile before and after metal detection. (a) PSiMc/Rh-UTES
(Rh-UTES = 1.16 μM) and (b) PSiMc/Rh-UTES-Hg2+ (Hg2+ = 6.95 μM). uorescence emission of PSiMc sensor and its tridimensional profile before and after metal detection. (a) PSiMc/Rh-U
16 μM) and (b) PSiMc/Rh-UTES-Hg2+ (Hg2+ = 6.95 μM). receptor. This work may open the door to the develop-
ment of optical fluorescence-based sensors that can be
easily used in field without the need of complicated instru-
mentation, allowing the fast diagnosis of the quality of
natural water sources or water from the industrial waste. corresponding tridimensional fluorescence profile over the
entire surface, on which we can see the emission intensity
produced for the immobilized Rh-UTES derivative. After
metal sensor exposure, the hybrid sensor showed a strong
brilliant red light (Figure 11b), and the fluorescence en-
hancement was 0.22-fold (integrated emission). This value
coincided well with the fluorescent enhancement observed
on the fluorescent spectroscopy analysis (0.25-fold for the
same metal concentration). Authors' contributions GP designed the project, coordinated, reviewed and drafted the manuscript. MDC carried out the main experimental work, and performed the
characterizations of interferometry, Infrared, fluorescent spectroscopy,
fluorescent microscopy and SEM, and wrote the in liquid phase discussion of
fluorescence spectroscopy. AA carried out the organic synthesis, NMR
experiments, FTIR and NMR discussion, organized and drafted the
manuscript. LHA participated in the PL characterization and results
discussion, analysis data, and in drafting the manuscript. ABF performed the
fluorescence microscopy analysis and made the tridimensional emission
profile through computing data processing. FJMR participated in infrared
measurements. All the authors read and approved the manuscript. Abbreviations
ACN
l ACN: acetonitrile; APTES: 3-aminopropyltriethoxysilane; ATR: attenuated total
reflectance; au: arbitrary unities; AuNPs: gold nanoparticles; C: carbon;
CDCl3: deuterated chloroform; d: double; DMSO: dimethyl sulfoxide;
FTIR: Fourier transform infrared; H: high current density; H: proton; IR: infrared
spectroscopy; L: low current density; m: multiplet; NMR: nuclear magnetic
resonance; PL: photoluminescence; ppm: parts per million; Psi: porous silicon;
PSiMc: porous silicon microcavity; q: quartet; Rh-UTES: rhodamine
organosilane derivative (3); SD: standard deviation; SEM: scanning electron
microscopy; s: singlet; t: triplet. Conclusions
h
k In this work we have proposed a novel method for detec-
tion of Hg2+ ions using rhodamine fluorescent derivative
as the recognizing element. We studied the fluorescent
performance of the derivative receptor in liquid and solid
phases. In solution, after the Hg2+ addition to the Rh-
UTES receptor, it was observed that the colorless solution
becomes colored (pink) and a remarkable enhancement in
the emission intensity. We found that both the color in-
tensity and the fluorescent intensity of the solution are
linearly dependent on the metal concentration. This dis-
tinct color and fluorescent change due to the spirolactam
ring opening makes this derivative valuable for sensing
ions through fluorescent or naked-eye detection. Add-
itionally, a new sensing strategy was evaluated by immo-
bilizing the Rh-UTES derivative on porous silicon devices. We found that after immobilization procedure, the Rh-
UTES derivate maintained its fluorescent properties. PSi/
Rh-UTES' sensing capabilities for Hg2+ detection were
studied. It was observed that metal-hybrid sensor coordin-
ation produces a 0.25-fold enhancement in the integrated
fluorescent emission at 6.95 μM Hg2+ ion concentration. By comparing the fluorescence response of Rh-UTES de-
rivative in liquid and solid phases, we found that the
immobilization procedure produced a 277-fold integrated
fluorescence increasing which highlights the benefits of
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How to objectively assess and observe maladaptive pain behaviors in clinical rehabilitation: a systematic search and review
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Naye et al. Archives of Physiotherapy
(2021) 11:15
https://doi.org/10.1186/s40945-021-00109-y
REVIEW
Open Access
How to objectively assess and observe
maladaptive pain behaviors in clinical
rehabilitation: a systematic search and
review
Florian Naye1, Chloé Cachinho2, Annie-Pier Tremblay1, Maude Saint-Germain Lavoie1, Gabriel Lepage1,
Emma Larochelle1, Lorijane Labrecque1 and Yannick Tousignant-Laflamme1,3*
Abstract
Background: Cognitive-affective factors influence the perception of pain and disability. These factors can lead to
pain behaviors (PB) that can persist and become maladaptive. These maladaptive PB will further increase the risk of
chronicity or persistence of symptoms and disability. Thus, clinicians must be prepared to recognize maladaptive PB
in a clinical context. To date, in the context of assessment in a rehabilitation setting, PB in clinical settings are
poorly documented. The main objective of this study was to identify direct observation methods and critically
appraise them in order to propose recommendations for practice. As a secondary objective, we explored and
extracted the different observable PB that patients could exhibit and that clinicians could observe.
Methods: We conducted a comprehensive review on four databases with a generic search strategy in order to
obtain the largest range of PB. For the first objective, a two-step critical appraisal used clinical criteria (from
qualitative studies on barriers to implement routine measures) and psychometric criteria (from Brink and Louw
critical appraisal tool) to determine which observation methods could be recommended for clinical practice. For
the second objective, we extracted PB found in the literature to list potential PB that patients could exhibit, and
clinicians could observe.
Results: From the 3362 retrieved studies, 47 met the inclusion criteria for the first objective. The clinical criteria
allowed us to select three observation methods. After the psychometric step, two observation methods were
retained and recommended for clinical practice: the Behavioral Avoidance Test-Back Pain (BAT-Back) and the Pain
Behaviour Scale (PaBS). For the second objective, 107 studies met the inclusion criteria. The extraction of the PB
allowed us to list a large range of PB and classify the data in 7 categories of PB.
* Correspondence: yannick.tousignant-laflamme@usherbrooke.ca
1
School of Rehabilitation, Faculty of Medicine and Health Sciences, Université
de Sherbrooke, Sherbrooke, QC, Canada
3
Research Center of the Centre Hospitalier Universitaire de Sherbrooke,
Sherbrooke, QC, Canada
Full list of author information is available at the end of the article
© The Author(s). 2021 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/.
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.
Naye et al. Archives of Physiotherapy
(2021) 11:15
Page 2 of 14
Conclusion: Our results allowed us to recommend two observation methods for clinical practice. However, these
methods have limitations and are validated only in chronic low back pain populations. With the extraction of PB
presented in the literature, we contribute to better prepare clinicians to recognize PB in all patients who are
experiencing pain.
Keywords: Pain behavior, Assessment, Protective behavior, Endurance behavior, Avoidance behavior, Musculoskeletal
pain
Introduction
The biopsychosocial model of pain strongly supports that
in addition to biological and social factors, cognitiveemotional factors drive the experience of pain and disability [1–4]. According to a systematic review of the best
practice care for musculoskeletal pain, the authors conclude that the assessment of psychosocial factors should
be an essential part of the evaluation process [5]. This suggests that the evaluation of maladaptive cognitions and
emotions should specifically be assessed by rehabilitation
professionals.
According to the Fear-Avoidance Model, maladaptive
cognitions (e.g., Pain catastrophizing) and maladaptive
emotions (e.g., Fear of movement) may contribute to the
development of avoidance-related pain behaviors (PB) [6].
In addition to the avoidance patterns, the EnduranceAvoidance Model proposes that thought suppression or
distraction may lead to endurance-related pain behaviors
[7], namely, the opposite of the avoidance behaviors. The
persistence of these PB may lead to poor outcomes and
are known risk factors for the recurrence of pain and
chronicity [8–10].
PB are defined as “the behavioral alterations observed
in individuals experiencing pain” [11] and consist of two
main categories. The first category includes protective
PBs, which is defined as “any action primarily aimed at
minimizing the experience of pain, promoting recovery
from injury, or reducing the probability of further injury”
[11] (e.g. avoiding a threatening task). The second category includes communicative PBs, which is defined as
“observable behaviors meant to communicate to others
that one is experiencing pain” [12] (e.g. touching the
painful area after task performance). Some could argue
that protective PB may also serve as a communicative
function when they are viewed by others, and that communicative behaviors may also serve to seek support or
assistance from the patient’s social environment [11]. By
definition, these categories are only applicable to avoidance behaviors.
However, since the definition of PBs covers a large
range of behaviors such as vocalizations, sighing, rubbing, posture modification, and movement modification,
the interpretation of the PB as adaptive or maladaptive
is often difficult. A specific PB may be adaptive in the
short term (e.g. relative rest after injury), but may
become maladaptive if it persists [9] or becomes more
frequent [13]. A PB may have negative outcomes in the
short term but may have a positive outcome in a longterm (e.g. a return to physical activity leads to an increase in pain in the short term, but a decrease in pain
in the long term) [14, 15]. Moreover, the contextual and
social environment can also modify the manifestations
of the PB [16, 17].
As maladaptive pain behaviors can be expressed in
many different ways [18], clinicians can often struggle to
detect relevant findings in a clinical environment. Knowledge of maladaptive behaviors is critical in understanding, assessing, and treating persistent pain [19]. Yet, to
our knowledge, there is no review documenting the observation methods to objectively assess PB in patients
with musculoskeletal pain. The main objective of this
study was to identify direct observation methods and
critically appraise them in order to propose recommendations for practice. As a secondary objective, we explored and extracted the different observable PB that
patients could exhibit and that clinicians could observe.
Methods
Design
We chose a systematic search and review to answer our
main objective. This design combines the strengths of a
critical review with a comprehensive search process that
typically addresses broad questions to produce a synthesis
of best evidence [20]. We aimed to answer this specific research question: What are the direct observation methods,
adapted to clinical settings, to assess PBs in an adult population (≥18 years-old) experiencing musculoskeletal pain.
For our second objective, we chose a narrative review to
present the PBs identified from the literature.
This review was registered with the PROSPERO database: CRD42018093102.
Identification and selection of studies
For both objectives, four (4) databases (CINAHL,
PubMed, PsycInfo, Scopus) were explored. Literature addressing observable pain behavior was examined using
the most generic search strategy: (“pain behavior” OR
“pain behaviour” OR “avoidance behavior” OR “endurance behavior” OR “avoidance behaviour” OR “endurance behaviour”) NOT (animal OR animals OR mice OR
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mouse OR rat OR rats OR dog OR dogs OR rodent OR
rodents OR murine OR adolescent OR adolescents OR
child OR children OR pediatr* OR “cognitive impaired”
OR “cognitive impairment”) with title filter. The choice
of a generic search strategy was based on the intention
to target the largest range of studies on pain behavior.
Also, we used title filter to focus on the literature that
the purpose is specific to PB. Only literature published
in English and French was included. This search was
performed in March 2020 thus the search period was
from inception to March 2020.
After removing duplicates, the screening of the records
was made by two independent evaluators (CC, and FN)
who screened the study titles and abstracts to identify eligible articles for the full-text review. For this first step, the
selection was based on common criteria for the two objectives. To be included, the potential studies had to present
content meeting three inclusion criteria. Because of the
abundance of literature on the specific topics of pain in
people with cognitive and communicative impairments,
we decided to add two exclusion criteria (detailed inclusion/exclusion criteria are presented in Table 1).
The assessment for eligibility of the full-text articles
was made by the same two independent evaluators
(CC, and FN). This assessment presented specific criteria for each objective. For the first objective, two inclusion criteria were applied (Table 1). For the
second objective, one inclusion criterion was applied
(Table 1). Because pain is not specific to a condition,
we broadened the selection of studies to include all
populations for objective 2. The final decision on article inclusion was made by consensus. In case of disagreement, a third reviewer (YTL) was available to
make the final decision.
Table 1 Selection criteria
RECORDS SCREENING
For the two objectives
Inclusion
criteria
1) Observable behaviors related to the experience of
pain
2) Human participants
3) Adult participants (> 18 years-old)
Exclusion
criteria
1) Participants with cognitive or communicative
impairments
2) Studies in other language than English and French
FULL-TEXT ASSESSMENT FOR ELIGIBILITY
For the first objective: Observation methods to assess pain behaviors
Inclusion
criteria
1) Participants with musculoskeletal pain
2) Use of a direct observation method with enough
details to reproduce it
For the second objective: Pain behaviors present in the selected literature
Inclusion
criterion
1) behavior that can be directly observed by clinicians
Page 3 of 14
Critical appraisal of assessment tools (1st objective)
Because one of the aims of a systematic search and review is to make recommendations for practice, we developed a triage process to further refine the selection
before extracting the data. The triage process was based
on clinical and psychometric aspects. We used the reported barriers to implement outcome measures from
qualitative data to determine relevant clinical criteria
[21, 22]. To be included, the tool had to meet each of
these clinical criteria:
– The time to complete the observation method had
to be equal or less than 10 min. In case of
observation during a more comprehensive
assessment (clinicians obtain more information than
PB alone), this procedure had to be equal or less
than 30 min.
– The scoring method had to be made without the use
of videotaping.
– An interpretation of the score to help clinicians in
their care plan had to be inherent to the tool.
– The tasks performed during the observation method
had to require no special equipment and had to be
made with commonly-used equipment (if required).
Afterwards, the studies that met the clinical criteria
were methodologically appraised for their measurement
(psychometric) properties based on the Critical Appraisal Tool (CAT) developed by Brink and Louw [23].
The CAT consists of a 13-item checklist to assess the
validity and reliability of clinical instruments. We removed items three, seven, nine, and eleven as they were
specific to concurrent validity and not relevant to the
nature of our analysis. As other items were conditional,
some items could be rated as not applicable. To estimate
the study quality (based on the CAT), we used the ratio
(percentage) between the number of items with a positive answer (yes) and the total number of relevant items
[24]. We used a cut-off of 60%, where a given tool was
rated > 60%, it was deemed acceptable and retained for
further analysis [25]. All appraisal-related procedures
were made by two independent evaluators (CC, FN). In
case of disagreement, a third evaluator (YTL) was available to make the final decision.
Data extraction and data analysis
Two independent evaluators (CC, FN) extracted the data
from the retained observation methods. A third evaluator (YTL) verified the extraction.
For the first objective, we extracted the data regarding:
the aim of the observation method, its clinical administration, the observed PB, the scoring and its interpretation, the clinical benefits of the method, the result of
the statistical analysis of validity and reliability, and the
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Page 4 of 14
Fig. 1 Flow chart
target population. A narrative synthesis was made to inform clinicians about the characteristics of each recommended observation method. For the second objective,
all observable PB were extracted and regrouped into
homogenous categories.
screening (1694 excluded with 112 abstracts not available), we obtained a pool of 180 articles. From this pool,
28 articles were not available and, 105 articles failed to
meet our inclusion parameters, which left 47 articles for
dedicated assessment (see Fig. 1 for the flow chart
diagram).
Results
Selection of the studies
Critical appraisal
For the first objective, 3360 relevant articles were found
in the various databases consulted. Two more articles
were included after an exploratory hand search. After
the removal of duplicates (1488 excluded), title/abstract
From the 47 articles, we found 14 different observation
methods. From the 14 initial methods, 9 used videotaped
sequences to determine the number of different PBs
during task execution which considerably limit their use
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Page 5 of 14
in the clinic setting. Also, 11 observation methods did
not propose an interpretation of the score which made it
difficult to use these data to determine or adapt the
treatment plan. Table 2 presents the extracted data for
the assessment of the clinical criteria. Only three observation methods met all clinical criteria: 1) the Behavioral
Table 2 First step of the critical appraise: clinical criteria appraisal
Method
Time to complete
Scoring method
≤ 10 min OR ≤ 30 min if part Not videotaped
of a comprehensive
assessment
Interpretation
Does the method propose an
interpretation of the score
(severity, …)?
No special
equipment
required
Decision
(retain
or
reject)
Reject
Aung’s method [26]
We can assume < 10 min
Behavioral Avoidance
Test-Back Pain (BAT-Back)
[27]
Retain
< 10 min
Specific rating
according to the level
of avoidance
Range of possible scores is 0 to 60
(0 = no avoidance, 60 = every
movement is avoided)
Butler and Kozey’s
method [28]
Reject
We can assume < 10 min
Cinciripini’s method [29]
Reject
We can assume < 10 min
Cold pressure method
[30]
Keefe and Block [31]
(K&B) and modified K&B
[32]
Reject
2-min
Reject
10-min
Reject
Keefe’s walk method [33]
We can assume < 10 min
Koho’s method [34]
Reject
We can assume > 10 min
Moores’ method [35]
Reject
We can assume > 10 min
Pain Behaviour Scale
(PaBS) [36]
Frequencies of PB
Retain
Part of a physical performance Frequencies of PB
test of 10 to 15 min
A total score of severity can be
determined from 0 to 15
Reject
Prkachin’s method [18]
Number of PB
Test Instrument for Profile
of Physical Ability (TIPPA)
[37]
Part of a comprehensive
assessment but we can
assume < 30 min
Retain
Presence of PB
A severity scale is proposed based
on the number of activities with
PBs
Reject
Thieme’s method [38]
8-min
Reject
Watson’s method [39]
We can assume > 10 min
: unclear
: no
: yes
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Avoidance Test-Back Pain (BAT-Back) [27], 2) the Pain
Behavior Scale (PaBS) [36], and 3) the Test Instrument
for Profile of Physical Ability (TIPPA) [37].
The methodology of the three selected observation
methods was then evaluated using the CAT. The CAT
was modified for the BAT-Back and the TIPPA, as these
two methods only analyzed the inter-rater reliability.
Items 5 (Raters blindness in intra-rater reliability), 6
(Variation of the order of examination), and 8 (Stability
of variable) were not applicable. The BAT-Back obtained
a percentage of 66.7%, the PaBS obtained a percentage
of 77.8%, and the TIPPA obtained a percentage of 16.7%.
Given the score below the a priori threshold of 60%, the
TIPPA was not retained for further analysis. Table 3 presents the completed CAT scores for the 3 instruments.
The clinically relevant characteristics of the recommended
observation methods
Table 4 presents the main characteristics of the two observation methods retained: the BAT-Back and the
PaBS.
Listing of the observable PB identified from the literature
For the second objective, 3360 relevant articles were
found in the various databases consulted. Two more articles were included after an exploratory research. After
the removal of duplicates (1488 excluded), title/abstract
screening (1694 excluded with 112 abstracts not available), we obtained a pool of 180 articles. From this pool,
28 articles were not available, 45 were excluded for failure to meet inclusion criteria, which left 107 articles for
review. See Fig. 1 for the flow chart diagram.
Page 6 of 14
Based on the extracted data from the 107 studies, 21
different groups of PB were identified. We grouped together similar PB and we classified these groups into 7
categories: (1) verbal and non-verbal communication, (2)
sounds, (3) posture and movements, (4) inconsistent
findings during clinical examination, (5) physical activities, (6) social and occupational activities, and (7) inappropriate use of. The complete listing and categories
are presented in Table 5.
Discussion
To our knowledge, this study is the first exhaustive and
comprehensive review to critically appraise observation
methods to assess PB considering clinical and psychometric criteria, identify, and categorize PB described in
the literature that patients can exhibit. Concerning the
assessment tools, our review shows that observation
methods easily applied in clinical practice are scarce. We
extracted from the literature, a large spectrum of possible PB that may be observed, from subtle behaviors
(e.g. drink water to delay task) to more obvious (e.g.
avoidance of the painful task). Clinicians may benefit
from awareness of the different PB clinical presentations
to detect maladaptive behaviors in people with musculoskeletal pain, which often suggest the presence of
cognitive-emotional factors that may interfere with the
rehabilitation process.
The clinical criteria of our triage process allowed us to
select 3 observation methods, but the psychometric assessment suggests that the TIPPA presented low methodological quality. Thus, we recommend the use of the
PaBS or the BAT-Back in clinical practice since both of
Table 3 Second step of the critical appraisal: psychometric criteria according to the Critical Appraisal Tool [23]
Item from the CAT
BATBack
PaBS
TIPPA
1
If human subjects were used, did the authors give a detailed description of the sample of subjects used to perform
the (index) test?
Yes
Yes
No
2
Did the authors clarify the qualification, or competence of the rater(s) who performed the (index) test?
No
Yes
No
4
If interrater reliability was tested, were raters blinded to the findings of other raters?
No
Yes
No
5
If intrarater reliability was tested, were raters blinded to their own prior findings of the test under evaluation?
n/a
Yes
n/a
6
Was the order of examination varied?
n/a
No
n/a
8
Was the stability (or theoretical stability) of the variable being measured taken into account when determining the
suitability of the time interval between repeated measures?
n/a
No
n/a
10 Was the execution of the (index) test described in sufficient detail to permit replication of the test?
Yes
Yes
No
12 Were withdrawals from the study explained?
Yes
Yes
Yes
13 Were the statistical methods appropriate for the purpose of the study?
Yes
Yes
No
Ratio between the number of items with a positive answer and the total number of “applicable” items
4/6
7/9
1/6
Percentage
66.7%
77.8%
16.7%
Decision before extraction
Retain
Retain Reject
No: no information or insufficient information
Yes: sufficient information
n/a: not applicable
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Table 4 Recommended observation methods suitable for utilization in clinical settings
Tool
Behavioral avoidance test (BAT-BACK) [27]
What does the tool
measure?
Measures observable avoidance behaviors. May be used to plan graded Measures observable pain behaviors.
exposure for patients with chronic lumbar pain or as a tool to measure
therapeutic success.
Pain Behaviour Scale [36]
How is the tool
administered?
The patient must approach the feared stimulus in a standardized
environment to induce fear and avoidance reactions
1. Instructions are given to the patient
2. Demonstration of movements (bending forward, lifting a box ~ 8 kg,
rotation) by the evaluator
3. Movements are executed by the patient (10 repetitions)
4. Assessment of behavior (according to 3 categories)
The patient performs a standardized
sequence of physical performance tests
1. Repeated trunk flexion
2. Repeated sit to stand
3. Timed up and go
4. Loaded reach
5. 50-ft walk
Observed PB
Category 1: The movement is carried out as demonstrated by the
evaluator. No avoidance or protective behavior.
Category 2: The movement is carried out with protective behaviors
(bended knees, keep the back straight by lifting or bending, move feet
while turning, deep breaths, taking medication before the task, drinking
water, seeking support, asking for help).
Category 3: The patient avoids making the movement. If less than 10
repetitions, missing repetitions are scored as avoided.
The specific pain behaviors assessed are:
- Sighing
- Breath-holding,
- Grimacing
- Guarding
- Rubbing
- Antalgic gait
Scoring and interpretation Each repetition is scored as follows:
of the observation
Category 1 = 0 point
method
Category 2 = 1 point
Category 3 = 2 points
Thus, a score of 0 means that the patient avoided no movement or did
not engage in a protective movement, and a score of 60 means that
the patient avoided all movements.
For each task, the intensity and severity of
PB are rated as below:
a) Intensity
Presence or absence of each PB
b) Severity
For each task, determination of PB severity
with a 4-point scale:
0. None
1. Mild
2. Moderate
3. Severe
Finally, a total severity score (0–15) is
obtained with the sum of the 5-task PB severity score.
Clinical benefits
• Easy to administer and interpret
• Short (approx. 5 min)
• Requires little to no material
• Easy to administer
• Short (10–15 min) and assess physical
performance at the same time
• Requires little to no material
• Also informs on physical performance
Validity and Reliability
• The BAT-Back is a reliable and valid measure of pain avoidance
behavior
Inter-rater reliability: good to excellent
• Internal consistency: excellent
• Convergent validity and divergent validity were determined
• Cross-cultural validity (Turkish) [40]
• The PaBS is a reliable and valid measure to
assess the presence and severity of PB.
• Inter-rater reliability: excellent
• Intra-rater reliability: excellent
• Agreement for each PB in each task
between 95 and 100%
• Perfect consistency for the absence/
presence of PB
• Acceptable construct validity
Target population
People with chronic low back pain. (CLBP)
Participants in the validity study were between 18 and 65 years old.
People with chronic low back pain
Participants in this study were between 21
and 65 years old.
these scored well during the psychometric assessment.
However, these two observation methods are only validated for people with chronic low back pain which limits
the objective assessment of maladaptive PB of other
musculoskeletal conditions. It is worth noting that these
two observation methods present some differences and
limitations. The first main difference involves instrument
scoring. The BAT-Back proposes a score of avoidance,
and more precisely physical avoidance (protective PBs)
[27], as opposed to the PaBS, which proposes a severity
score within a range of protective or communicative PBs
[36]. As a result, the PaBS allowed clinicians to evaluate
a larger diversity of PB.
Clinicians must use caution when evaluating communicative PBs, as they are not always related to pain severity and pain-related disability [28]. Moreover, observers
give more weight to communicative PBs than protective
behaviors [28]. This overinterpretation and its consequences on clinicians’ attitudes towards the patient may
lead to reinforce these communicative PBs [28]. Thus,
despite the lack of diversity in the PBs it assesses, the
BAT-Back’s focus on protective PBs may avoid this
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Table 5 Listing of observable pain behaviors found in the systematic search and review
Does the patient exhibit this PB?
If yes, with the integration of the patient’s context, beliefs, the
frequency of this PB, etc., the clinician can interpret if the PB is
adaptive or not and interferes with the rehabilitation process
Verbal and nonverbal communication
Pain-related behaviors that can be verbalized or executed by the patient to communicate with the therapist about his/her pain
• The patient stays focused on pain communication (e.g., always refers
to his/her pain during conversation) [28, 29, 35, 41–56]
• The patient stays focused on disability or impairments despite clinical
improvements [43, 52, 54, 57–60]
• The patient verbalizes hesitation or questions about his/her capacity
to perform feasible tasks [42, 54, 55, 58, 61]
• The patient asks for help for tasks he/she can perform independently
(alone) [27, 44–46, 49, 55, 60, 62–65]
Touching/rubbing the painful area after task accomplishment [11, 18, 28,
29, 31–36, 39, 42, 45, 51–56, 58, 61, 66–111]
Sounds
Pain-related behaviors that can be heard by the therapist when the patient performs tasks or activities
• Groaning, Moaning, Whining, Whimpering, Crying, Screaming
[11, 18, 27–30, 34, 35, 37, 39, 42, 44–47, 49–51, 53–59, 61–63, 67, 68, 70–
74, 88, 97, 103–105, 107, 109, 111–114]
Sighing, Holding their breath, Taking a deep breath [11, 18, 27–36, 39, 42,
44, 50, 53–57, 61, 66, 68, 69, 71–74, 76–82, 85–87, 89–94, 96, 97, 100–103,
106, 107, 109–111, 115, 116]
Posture and movements
Pain-related behaviors that can be seen by the therapist when the patient moves or remains in a static position
• Overcautious/overprotective during movements
○ Self-limiting range of motion
○ Stiff or rigid movements
○ Abnormally slow movements
[11, 18, 26, 28, 29, 31–38, 42, 44–46, 49, 52, 53, 55, 57–59, 61–63, 66–69,
71, 72, 74–82, 85–87, 89, 90, 92–104, 106, 107, 109–111, 116–122]
• Strategies to minimize the threat and/or the load on the painful area
during movement
○ Avoids or minimizes lifting, bending
○ Bending knees, kneeling, keeping the back straight
○ Moving the feet while rotating
○ Imbalance on the distribution of body weight
[11, 18, 27, 28, 30, 32, 33, 37, 42, 45, 50, 52–59, 61–63, 66, 67, 69–71, 74–89,
92–103, 105–110, 119, 120, 123, 124]
• Keeps distorted gait despite clinical improvements
○ Limps
○ Drag one’s leg
[11, 29, 33–36, 39, 41, 45, 46, 48, 49, 52, 54–56, 58, 62–64, 73, 74, 83, 84,
88, 91, 105, 113, 115]
• Delays activity execution
○ Drinks water between the order and the performance of a requested
task/movement
○ Latency to initiate a requested task/movement
○ Misses therapy sessions if not reminded
[27, 55, 58, 117, 120]
• Excessive rest [34, 35, 43, 44, 51, 54, 55, 60, 62, 63, 70, 74, 84, 88, 105,
118, 119, 124–128]
Inconsistent findings during clinical examination
Pain-related behaviors that can be provoked during the clinical examination
• Discrepancies between:
○ clinical findings and observed functional capacity or incapacity
(dressing, …)
○ the demonstrated range of motion during clinical examination and
during distraction tasks
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Table 5 Listing of observable pain behaviors found in the systematic search and review (Continued)
Does the patient exhibit this PB?
If yes, with the integration of the patient’s context, beliefs, the
frequency of this PB, etc., the clinician can interpret if the PB is
adaptive or not and interferes with the rehabilitation process
[42, 129, 135]
• Overreaction during examination [42, 129, 135]
Physical activities
Pain-related behaviors that can be mentioned by the patient while talking about physical activities or performing tasks
• Avoid or minimize:
○ leisure activities
○ housework
○ sports
○ sexual intercourse
[11, 33, 37, 43, 50, 51, 54–56, 60, 65, 67, 84, 125, 128, 130, 136]
• Undertakes nothing outside therapy time despite therapist’s
encouragement [55]
Social and occupational activities
Pain-related behaviors that can be mentioned by the patient while talking about social or occupational activities
• Avoids or minimizes spending time with people [50, 51, 55, 56, 62, 63,
74, 125]
• Repeated work absences [51, 56]
Inappropriate use of
Pain-related behaviors that can be mentioned by the patient while talking about or seen by the therapist
• Medication (prescribed or not) [27, 29, 45, 46, 51, 54–56, 58, 60, 67, 88,
105, 118, 119, 126, 127]
• Healthcare system
○ Asking for further specialized medical treatment [51, 55]
• Non-prescribed equipment
○ TENS
○ Cane or crutch
○ Brace
[27, 29, 37, 39, 44, 46, 49, 52, 54, 55, 58, 62, 63, 67, 70, 86–88, 105, 119, 127]
reinforcement, while providing information about pain
severity and pain-related disability [28]. The difference
in the type of PBs assessed does not seem to be a
limitation.
The BAT-Back scoring can be confusing as it is based
on a sequence of 3 movements. If a patient stops the sequence during the first movement, the remaining two
are not performed, but scored as avoided movements,
which can lead to an overestimation of avoidance [40].
Furthermore, as the first movement is bending forward,
its scoring can be influenced (biased) by physical consequences of underactivity such as stiffness, shortness of
muscles, among many other factors [40]. For example, if
the patient bends to the knees or keeps his back straight,
the BAT-Back considers that the patient engages in
safety behaviors. Physical limitations, such as less flexible
hamstrings may lead to an overestimation of patient
avoidance. The score based on a sequence and the rating
that can be influenced by physical or cognitive consequences of the patient’s life are the main limitations of
the BAT-Back. Another limitation of the BAT-Back is
the tasks that are performed. Even if the 3 movements
of the BAT-Back are known to be fearful tasks for patients with low back pain, it is also well known that a patient can avoid certain tasks, but can perform others
without avoidance [66]. The PaBS uses tasks from the
physical performance assessment to evaluate PBs. With
this strategy, the PaBS increases the number of tasks that
are performed. However, all the tasks performed for the
PaBS are in a sagittal plan whereas the BAT-Back uses
movements in the sagittal and horizontal plans.
Our results also highlights the ubiquitous of the avoidance behaviors reported in the literature, as the types of
most PB found in the literature were either protective or
communicative. This discrepancy could be explained by
the fact that the Fear-Avoidance Model was conceptually
proposed in 2000 [6], whereas the Endurance-Avoidance
Model was conceptually proposed 10 years later [9]; not
surprisingly, much more literature is based on the FearAvoidance Model. Another reason relates to the behaviors themselves. Contrary to the avoidance response,
pattern that is characterized by pain-related fear, catastrophizing, and behavioral avoidance [6], the endurance
response pattern is characterized by thought
Naye et al. Archives of Physiotherapy
(2021) 11:15
suppression, anxiety/depression, and task persistence
(endurance behaviors) [7]. Thus, even if avoidance behaviors are subtle, they still remain observable [67]. On
the other hand, as “endurers” carry out the task to the
end despite a significant increase in pain [131], endurance behaviors seem less “observable” and could be better captured by a questionnaire. If a clinician suspects
that his or her patient presents endurance behaviors
when performing a task, it would seem more appropriate
to use a questionnaire such as the Avoidance Endurance
Questionnaire to assess the patient’s PB [125, 132–134].
The assessment of behavioral components is an integral part of a biopsychosocial approach. However, clinicians can feel uncomfortable in the assessment of
psychosocial factors [26] and want the support of simple
screening tools [29]. Also, because PB are dynamic
(adapted in the short term and can turn into maladaptive behavior), it is essential to have the possibility of a
rapid screening. With a screening perspective, we summarized the different observable PB found in the literature. Yet, when available, clinicians must also objectively
document these with a proper tool. In this case, the
PaBS or the BAT-Back can be used.
Our systematic search and review present some limitations. The first one concerns the clinical criteria used to
select the observation methods. As no specific tools were
available, we had to create our own grid based on data
from the literature on the clinical integration of outcome
measures in rehabilitation. The second main limitation
concerns the clinical assessment tool (CAT) developed by
Brink and Louw. Although cited in several studies (n =
40), this CAT is not validated for this type of analysis.
Conclusion
This is the first review to identify and critically appraise
observation methods to assess pain behaviors in patients
with musculoskeletal pain in clinical setting. The critical
appraisal process allowed us to recommend two observation methods that are rapid to complete, with few equipment, and using tasks perceived as threatening by
patients. These methods are the PaBS and the BAT-Back.
However, these two tools are only validated for people
with chronic low back pain. In order to help clinicians in
the detection of possible maladaptive PBs in patients with
various musculoskeletal conditions, we extracted the different PBs present in the literature and that patients can
exhibit. This extraction allowed us to propose 7 categories
of PBs. With that, clinicians can perform a screening of
PBs, but not an objective assessment. Also, this review
shows the ubiquitous of the avoidance behaviors in the literature. Thus, clinicians may use a questionnaire like the
Avoidance Endurance Questionnaire to perform a global
evaluation of behaviors that can be part of the two models
of transition to chronicity.
Page 10 of 14
Acknowledgements
Not applicable.
Authors’ contributions
FN contributed to the conception, design, acquisition, analysis, interpretation
of data and drafted the manuscript. CC contributed to the acquisition,
analysis and interpretation of data. YTL contributed to the conception,
design, interpretation of the data, and substantively revised the manuscript.
APT, MSGL, GL, EL, and LL contributed to the conception, design, and
substantively revised the manuscript. The authors read and approved the
final manuscript.
Funding
No funding was obtained to support this study.
Availability of data and materials
The datasets used and/or analysed during the current study are available
from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
School of Rehabilitation, Faculty of Medicine and Health Sciences, Université
de Sherbrooke, Sherbrooke, QC, Canada. 262 Rue de la Rondonnerie, 45120
Corquilleroy, France. 3Research Center of the Centre Hospitalier Universitaire
de Sherbrooke, Sherbrooke, QC, Canada.
Received: 10 December 2020 Accepted: 6 May 2021
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The development of mathematical HOTS questions based on banten culture
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1. INTRODUCTION 1. INTRODUCTION One of the toughest challenges in the world of 21st century education is the rapid development of human civilization around
the world. Indonesian children must be able to compete in various aspects. Competition in the era of globalization requires
Indonesian human resources to be supported by competence, skills, and sensitivity to rapid changes. These human resources
will be born through transformation in the field of education. One of the efforts is to improve high order thinking skills,
namely cognitive processes that require students to process various ideas and information with certain mechanisms that
can provide the latest meanings and consequences (Istiqomah, 2018). Students must have the ability to carry out
mathematical activities called mathematical abilities to achieve higher order thinking skills. Mathematical ability according
to NCTM (in Maulyda, 2020) is the ability that is used to deal with various problems, both in mathematics or in daily
activities. The Organization for Economic Cooperation and Development (OECD) conducted a survey using the Program for
International Student Assessment (PISA) 2018 and it was found that out of 78 countries, Indonesian students ranked 72nd
on the math ability test. The average score obtained by Indonesian students is 379, while the average score deter-mined by
the OECD is 487. Referring to the statement submitted by the Ministry of Education and Culture (2014: 2) that the
achievement of Indonesian children is low because most of the material studies presented in PISA are not available in
learning in Indonesia. One of the elements in PISA is the HOTS-based questions in the 2013 curriculum. The problem that
arises is that the 2013 curriculum has been implemented, but the questions tested are not able to train students' high-er-
order thinking skills (Farihah, et al, 2018). g
(
,
,
)
The results of research conducted by Supriadi (Supriadi, et al, 2016) produced data that most (80%) students did not un-
derstand the culture presented when learning mathematics activities were carried out. The facts on the ground state that
learning mathematics in schools often does not integrate cul-ture into learning. Meanwhile Bishop (in Supriadi 2016: 54)
stated that the problem was caused by mathematics learning activities in the education sector which were considered to be
less integrated with culture as a context in learning. The Indonesian curriculum is still eurocentric, which is in-clined to
Western civilization and is considered incompatible with the culture and personality of Indonesian students. Anis Yuliani*, Fitri Alfarisa & Tiurlina Universitas Pendidikan Indonesia, Serang, Indonesia, 42116
anisyuliani@upi.edu; alfarisa@upi.edu; p.tiurlina59@gmail.com
*Corresponding Author: anisyuliani@upi.edu Received: 20 January 2022
Revised: 19 February 2022
Accepted: 26 March 2022
Available online: 30 March 20 Received: 20 January 2022
Revised: 19 February 2022
Accepted: 26 March 2022
Available o ABSTRACT The resulted of a survey conducted by the Organization for Economic Cooperation and Develop-ment based on the 2018
Program for International Student Assessment were obtained from 78 countries, Indonesian students ranked 72nd in
math ability. The reason is that the 2013 curricu-lum that has been implemented in Indonesia is eurocentric. The
transformation sought in the field of education is the improvement of high order thinking skills and mathematical
abilities. Educa-tion and culture cannot be separated from regular daily life because culture covers all aspects of life
while education is a primary need for everyone. This study used the Research and Develop-ment (R&D) method with the
Martin Tessmer (1993) model, which is formative research. The validity of the values obtained above 0.80 means that
the questions are valid. The level of practi-cality of the questions obtained is 96.25%, very practical category. The
reliability coefficient of 0.891, reliable. The level of difficulty is known to be 80% of the items in the medium category,
20% of the items in the difficult category. The discriminatory power of the question is known to all items (100%) in good
category, meaning that good questions are used to classify the level of stu-dents thinking abilities. Keywords: Development; Banten; Culture; Mathematical The development of mathematical HOTS questions based on
B
t
lt Anis Yuliani*, Fitri Alfarisa & Tiurlina International Journal of Trends in Mathematics Education Research
Vol. 5, No. 1 (2022), pp. 44-52
ISSN 2621-8488 (online)
DOI: https://doi.org/10.33122/ijtmer.v5i1.112 DOI: https://doi.org/10.33122/ijtmer.v5i1.112 Research Article 2. RESEARCH METHOD The method used is Research and Development (R&D) re-ferring to the Martin Tessmer (1993) model, namely formative
research including the pleminary stage and formative evalu-ation stage which includes the self-evaluation stage, the pro-
totyping stage (expert review, one-to-one, small group), and field test. The preliminary stage is the beginning of the
development process. Researchers carry out evaluations on several reference materials related to the research process,
determining the location and research subjects. Then there is the prototyping stage which includes the expert review, one-
to-one, and small group stages. The instrument validity test was carried out to 3 experts (validators) which included 1
mathematics teacher at SD Negeri Karawaci Baru 3 and 2 mathematics lecturers, then an experiment was conducted on 3
students to deal with HOTS questions and then provide comments or reactions at the one-to-one stage. Based on the results
of the reaction or response in the one-to-one stage, the next question instrument is a small group trial, namely on 6 students. Based on the questionnaire that has been analyzed from the small group stage, the question instrument can be forwarded
to the next research stage. The Field Test stage, is a field test to determine the ability of students to correctly answer the HOTS questions based
on Banten culture, the value of reliability, the level of difficulty, and the distinguishing power of the items that have been
made. The test subjects are fifth grade students in the 2021/2022 odd semester at SD Negeri Karawaci Baru 3, Tangerang
City. The research instruments were interview guidelines, validation, students’ questionnaire sheets, and question
instruments. 1. INTRODUCTION De-veloped
countries have used their own culture to study mathematics for a long time. So that their country can progress in all fields. According to Kurumeh, the success of Japan and China in learning mathematics is due to the use of ethno-mathematics in
their mathematics learning (Supriadi, 2016). Education and culture cannot be avoided from real life. Because culture is integral and holistic in society. In this regard,
education is a primary need for every individual in society. Besides that, one of the cultural expressions is education (Zulfah,
2018). Banten is a province located at the western tip of Java Island, Indonesia. Banten culture has a plural potential. 44 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 Yuliani et.al Culture can be a variety of mathematical ideas that can be researched and studied in every cultural activity carried out so
that it can become a source of contextual mathematics teaching and learning activities (Sutrimo, et al, 2019). Culture can be a variety of mathematical ideas that can be researched and studied in every cultural activity carried out so
that it can become a source of contextual mathematics teaching and learning activities (Sutrimo, et al, 2019). The results of observations before the study obtained in-formation that so far the teacher had carried out routine
evaluations at the end of each lesson by giving questions to class V semester 1 students according to the material that had
been studied. Some of the questions presented include the criteria for HOTS questions obtained from various sources such
as mathematics textbooks, Student Worksheets (LKS), the internet, and other sources. However, the HOTS questions
presented are not based on Banten culture even though the geographical location of SD is in the province of Banten. In fact,
by incorporating Banten cultural content, it can produce meaningful learning in all activities carried out, so that it has a
function as a reference for contextual teaching and learning activities in mathematics. (Kusmaryono, 2012). Research and
development has the aim of knowing how to develop questions, knowing the feasibility of questions and the character-istics
of HOTS questions based on Banten culture. 2. RESEARCH METHOD 2. RESEARCH METHOD Data accumulation techniques used are documentation, walkthroughs, questionnaires, interviews, and
tests.Data analysis techniques in this study are as follows: 2.1 Eligibility Test of HOTS Questions Based on Banten Culture 2.1.1 Validity Test Content validity according to Aiken were said to be valid or suitable for use with a rating of 3 and a scale of 4, so the Aiken
V index was at least 0.80 (in Azwar, 2012): Content validity according to Aiken were said to be valid or suitable for use with a rating of 3 an
V index was at least 0.80 (in Azwar, 2012): 45
V index was at least 0.80 (in Azwar, 2012):
V= Σs/[n (c-1)]
Description:
s
: r-lo
lo
: Minimum validity score
c
: Maximum validity score
r
: Score from expert
2.1.2 Practical Test of HOTS Questions Based on Banten Culture
P =
𝑅
𝑆𝑀 x 100%
(Source : Zainal Arifin, 2006:50)
Description:
P
: Practical value
R
: Earned value
SM : Maximum value
Table 1. Practical Score Category
Practical Score (%)
Category
85 ≤ P ≤ 100
Very Practical
75 ≤ P ≤ 85
Practical
60 ≤ P ≤ 75
Enough Practical
55 ≤ P ≤ 60
Less Practical
0≤ P ≤ 55
Not Practical 45
V index was at least 0.80 (in Azwar, 2012):
V= Σs/[n (c-1)]
Description:
s
: r-lo
lo
: Minimum validity score
c
: Maximum validity score
r
: Score from expert
2.1.2 Practical Test of HOTS Questions Based on Banten Culture
P =
𝑅
𝑆𝑀 x 100%
(Source : Zainal Arifin, 2006:50)
Description:
P
: Practical value
R
: Earned value
SM : Maximum value
Table 1. Practical Score Category
Practical Score (%)
Category
85 ≤ P ≤ 100
Very Practical
75 ≤ P ≤ 85
Practical
60 ≤ P ≤ 75
Enough Practical
55 ≤ P ≤ 60
Less Practical
0≤ P ≤ 55
Not Practical V= Σs/[n (c-1)] ractical Test of HOTS Questions Based on Banten Culture P =
𝑅
𝑆𝑀 x 100% Category
Very Practical
Practical
Enough Practical
Less Practical
Not Practical 45 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 3.1 Development of Banten Culture-Based HOTS Questions
3.1.1 Pleminary Stage This stage is the initial stage. It begins with some reference materials related to research activities, namely the development
of Banten culture-based High Order Thinking Skills (HOTS) questions to measure the mathematical abilities of elementary
school students. From various references obtained several theories that have been submitted by experts and related to this
research. One of the references in this research is the HOTS theory according to B. S. Bloom which produces Bloom's
Taxonomy where High Order Thinking Skills (HOTS) consists of analyzing, evaluating, and creating. Based on existing
references, the location and subject of the research were determined. ,
j
The location of the trial in this research process is SD Negeri Karawaci Baru 3, Tangerang City. While the subjects in
this study were grade 5 students in semester 1 at SD Negeri Karawaci Baru 3. The study was held on October 27, 2021 -
December 24, 2021. The schedule of research implementation can be seen in the table 5. Table 5. Research schedule
Research Stage
Execution time
Preliminary Stage
October 2021
Self-Evaluation Stage
November 2021
Prototyping Stage
November-December 2021
Field Test Stage
December 2021
3.1.2 Self-Evaluation Stage Table 5. Research schedule
Research Stage
Execution time
Preliminary Stage
October 2021
Self-Evaluation Stage
November 2021
Prototyping Stage
November-December 2021
Field Test Stage
December 2021
3.1.2 Self-Evaluation Stage This stage aims to develop High Order Thinking Skills (HOTS) questions based on Banten culture based on the results of
the preliminary stage, the question instruments to be developed consist of HOTS question grids, HOTS questions, answer
keys, and scoring guidelines. At this stage there are several stages, including analysis and design. This analysis phase
consists of curriculum analysis, student analysis, and material analysis. The results of the curriculum analysis showed
learning using K-13 which has the aim of increasing students' High Order Thinking Skills (HOTS) which refers to the
assessment of attitudes, skills, and knowledge. The results of the analysis of students in class V semester 1 as the subject
of this research and development trial, it was found that the number of students was 30 students. It is also known that the
mathematical knowledge and mathematical ability of class V SD Negeri Karawaci Baru 3 varies. Based on the results of
the fifth semester homeroom interview, this is because students' enjoyment of each subject is different. 2. RESEARCH METHOD 44-52 Yuliani et.al 2.2 Characteristics of HOTS items based on Banten culture
2.2.1 Reliability Test
𝑟11 = [
𝑘
𝑘−1 ] [ 1-
∑𝜎𝑏
2
𝑉𝑡
2 ]
(Source: Sugiyono, 2013: 121)
Description:
𝑟11
: Reliability
𝑘
: Amount of questions
∑𝜎𝑏
2
: Amount of variance
𝑉𝑡
2
: Total variance
Table 2. Reliability Score Category
Reliability Score
Category
0,90 < 𝑟11 ≤ 1,00
Very high
0,70 < 𝑟11 ≤ 0,90
Tall
0,40 < 𝑟11 ≤ 0,70
Currently
0,20 < 𝑟11 ≤ 0,40
Low
𝑟11 ≤ 0,20
Very low
2.2.2. Question Difficulty Level
IK =
𝑛𝐴 + 𝑛𝐵
𝑁𝐴 + 𝑁𝐵
(Source: Lestari & Yudhanegara, 2017)
Description:
IK: difficulty index
𝑛𝐴: the number of correct answers is upper class
𝑛𝐵: the number of correct answers lower class
𝑁𝐴: amount of upper class students
𝑁𝐵: amount of lower class students
Table 3. Difficulty Level Category
IK
Category
IK = 0,00
Too Difficult
0,00 < IK < 0,30
Hard
0,30 < IK < 0,70
Currently
0,70 < IK < 1,00
Easy
IK= 1,00
Too easy
2.2.3. Power of Differing Questions
DP =
(𝑋̅𝐴− 𝑋̅𝐵)
𝑆𝑀𝐼
(Source: Lestari & Yudhanegara, 2017)
Description:
DP : power of differing questions
𝑋̅𝐴
: the average score of upper class students
𝑋̅𝐴
: the average score of lower class students
𝑆𝑀𝐼 : maximum value
Table 4. Power of Differing Category
Power of Differing
Category
0,70 > DP ≤ 1,00
Very Good
0 40 > DP ≤0 70
Good IK
IK = 0,00
0,00 < IK < 0,30
0,30 < IK < 0,70
0,70 < IK < 1,00
IK= 1,00 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 Yuliani et.al 3.1 Development of Banten Culture-Based HOTS Questions
3.1.1 Pleminary Stage The results of the material analysis obtained information that the learning material that will be used as a reference in
the research and development process on High Order Thinking Skills (HOTS) questions is based on the material in K-13 for
mathematics subjects in class V SD semester 1. The mathematics material for class V semester 1 covers the operation of
counting fractions, scale, speed and discharge material. At the design stage, the researcher designed 10 items representing
each grade 5 mathematics material in semester 1. The items were designed in such a way based on indicators of higher-
order thinking skills and indicators of mathematical reasoning integrated with Banten culture. The result of this stage is
prototype 1. 3. RESULTS AND DISCUSSION 3.1 Development of Banten Culture-Based HOTS Questions 3.1.3 Prototyping Stage
3.1.3.1 Expert Review 3.1.3 Prototyping Stage
3.1.3.1 Expert Review 3.1.3.2 One-to-one Add the fractions in the two pictures above... 3.1.3 Prototyping Stage 3.1.3.1 Expert Review Expert review or assessment by experts is used as a foundation in revising or repairing prototypes that produce prototypes
2. Instrument validation by experts is carried out by the process of providing validation sheets for HOTS question grids,
HOTS questions, answer keys, and scoring guidelines for validators, consisting of two mathematics lecturers at the
Indonesian Education University, Serang Regional Campus. Based on the expert review validation stage, the general results
are as follows: Validator 1 stated that the Banten culture-based HOTS questions were fairly good and fit to be used without
any corrections or revisions. Validator 2 stated that the Banten culture-based HOTS questions were fairly good and deserved
to be used with minor improvements or revisions. Validator 3 stated that the Banten culture-based HOTS questions were
fairly good and feasible to use with minor improvements or revisions. Experts (experts) were asked to evaluate and give an
assessment of all instruments about High Order Thinking Skills (HOTS) based on Banten culture. After the analysis
activities are completed on the validation sheet by the experts, the validity test is carried out. One of the questions before
and after the revision based on the validator can be seen in the table 6. 47 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 Yuliani et.al Table 6. Questions before and after revision
Before Revision
After Revision
2. Take a look at the two pictures of the baduy batik motifs that
have been shaded below! llustrate a fraction that is equivalent to the number of fractions in
the image above on the Baduy batik motif below by shading it…
2. Take a look at the two pictures of the baduy batik motifs that
have been shaded below! The yellow part is the shading. Add the fractions in the two pictures above... After Revision have been shaded below! The yellow part is the shading. llustrate a fraction that is equivalent to the number of fractions in
the image above on the Baduy batik motif below by shading it… Add the fractions in the two pictures above... 3.1.3.2 One-to-one 3.1.3.2 One-to-one 3.1.3.2 One-to-one At this stage a trial was carried out on several students who were in class V semester 1 of SD Negeri Karawaci Baru 3. The
question of High Order Thinking Skills (HOTS) based on Banten culture was tested on 3 students who were the subject of
research and students submitted their comments on the High Order question. Thinking Skills (HOTS) are on the student
response questionnaire given. The research subjects at this one-to-one stage consisted of students who had high, medium,
and low-level thinking skills. The results obtained from the one-to-one stage are that students feel that the material for
question number 2 is in accordance with what has been studied in class 5 semester 1 of SD Negeri Karawaci Baru 3 in
mathematics, students have difficulty understanding the questions in item number 2 which contains material for counting
fractions operations, and there are suggestions from students to add a plus sign to the images presented so that they are
easy to understand. The response becomes a reference for improvement or revision of the prototype. The results of the 0ne-
to-one stage can be seen in the table 7. g
Table 7. One-on-one results
Before Table 7 Revision
After Revision
2. Take a look at the two pictures of the baduy batik
motifs that have been shaded below! The yellow part is the shading. Add the fractions in the two pictures above... 2. Take a look at the two pictures of the baduy batik motifs
that have been shaded below! The yellow part is the shading. Add the fractions in the two pictures above... Table 7. One-on-one results ne-on-one results
After Revision
2. Take a look at the two pictures of the baduy batik motifs
that have been shaded below! The yellow part is the shading. Add the fractions in the two pictures above... b
Before Table 7 Revision
2. Take a look at the two pictures of the baduy batik
motifs that have been shaded below! The yellow part is the shading. Add the fractions in the two pictures above... The yellow part is the shading. The yellow part is the shading. Add the fractions in the two pictures above... The yellow part is the shading. Add the fractions in the two pictures above... The yellow part is the shading. Add the fractions in the two pictures above... Add the fractions in the two pictures above... 3.2. Eligibility of Banten Culture-Based HOTS Questions 3.2. Eligibility of Banten Culture-Based HOTS Question The results of the validity test showed that the HOTS questions based on Banten culture were said to be valid or suitable
for use with a rating of 3 and a scale of 4, so the Aiken V index was at least 0.80 (in Azwar, 2012). validity results in the
table 8. table 8. Table 8. Validity results
Items
Expert Review
s1
s2
s3
∑S
n(c-1)
V
Results
I
II
III
1
4
3
4
3
2
3
8
9
0.888888889
Valid
2
4
4
3
3
3
2
8
9
0.888888889
Valid
3
4
4
4
3
3
3
9
9
1
Valid
4
4
4
4
3
3
3
9
9
1
Valid
5
4
4
4
3
3
3
9
9
1
Valid
6
4
4
4
3
3
3
9
9
1
Valid
7
4
4
4
3
3
3
9
9
1
Valid
8
4
4
4
3
3
3
9
9
1
Valid
9
4
4
4
3
3
3
9
9
1
Valid
10
4
4
4
3
3
3
9
9
1
Valid
Expert validation results were obtained from 10 HOTS questions based on Banten culture, all of which had an AIken score
above 0.80. This means that the Banten culture-based High Order Thinking Skills (HOTS) test questions have valid criteria
and are ready to be used. Based on the results of the student response questionnaire there is a practicality test, the results
obtained are as follows in the table 9 Expert validation results were obtained from 10 HOTS questions based on Banten culture, all of which had an AIken score
above 0.80. This means that the Banten culture-based High Order Thinking Skills (HOTS) test questions have valid criteria
and are ready to be used. Based on the results of the student response questionnaire there is a practicality test, the results
obtained are as follows in the table 9. Expert validation results were obtained from 10 HOTS questions based on Banten culture, all of which had an AIken score
above 0.80. This means that the Banten culture-based High Order Thinking Skills (HOTS) test questions have valid criteria
and are ready to be used. Based on the results of the student response questionnaire there is a practicality test, the results
obtained are as follows in the table 9. Table 9. 3.1.3.3 Small Group An experiment was conducted in small groups with 6 students from class V in semester 1 of SD Negeri Karawaci Baru 3. The characteristics of the research subjects consisted of students with high-level, medium-level, and low-level thinking
abilities. Each characteristic consists of two students from class V. Students who are the subject of research trials at the
small group stage are assigned to work on the Banten Culture-Based HOTS questions. In order to assess the practicality of
the items and determine student responses or responses, students are assigned to fill out student response questionnaire
sheets related to the HOTS questions that have been done. The results of the small group stage are students feel that the
questions are easy to understand, the questions presented have a special attraction for students, especially in the pictures
and story texts that are presented, the items presented have varying levels of difficulty depending on the individual students 48 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-5 Yuliani et.al themselves, students like story questions. which is integrated with Banten culture, and students feel they know new things
about Banten culture after reading the questions presented. The response is used as a reference for repair or revision of the
prototype and produces prototype 3. themselves, students like story questions. which is integrated with Banten culture, and students feel they know new things
about Banten culture after reading the questions presented. The response is used as a reference for repair or revision of the
prototype and produces prototype 3. 3.1.4. Field Test Stage The prototype that has passed the validation process and is repaired or revised, is then carried out on a trial run on grade
5 students in semester 1 at SD Negeri Karawaci Baru 3 with 30 students. The Banten culture-based High Order Thinking
Skills (HOTS) test was held for 2 x 45 minutes. Students are assigned to work on the Banten culture-based High Order
Thinking Skills (HOTS) question which consists of 10 questions in the form of a description. In the implementation of the
High Order Thinking Skills (HOTS) field test activity, the researcher provided question sheets and answer sheets to
students. The process of working on or solving High Order Thinking Skills (HOTS) questions begins with the researcher
delivering guidelines for solving the Banten culture-based High Order Thinking Skills (HOTS) questions. Each of these
students completes the HOTS questions on the student answer sheets that have been given. The results obtained from the
student's answers were then analyzed to determine the value of reliability, level of difficulty, discriminating power of
questions, and the ability of students to correctly answer the High Order Thinking Skills (HOTS) question based on Banten
culture. 3.3. Characteristics of HOTS Question Items Based on Banten Culture 3.3. Characteristics of HOTS Question Items Based on Banten Cultur 3.2. Eligibility of Banten Culture-Based HOTS Questions Practical Results
Items
Students
Practicality Score
1
AM
92.5
2
AP
97.5
3
CW
97.5
4
DA
97.5
5
ML
95
6
S
97.5
Average
96.25 49 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-5 Yuliani et.al The practical value obtained is 96.25%. This means that the Banten culture-based High Order Thinking Skills (HOTS) that
has been developed is in the very practical category according to the practicality criteria according to Zainal Arifin (2016:
50). The practical value obtained is 96.25%. This means that the Banten culture-based High Order Thinking Skills (HOTS) that
has been developed is in the very practical category according to the practicality criteria according to Zainal Arifin (2016:
50). 3.3.2. Question Difficulty Level Each HOTS item based on Banten culture can be declared appropriate or good if each HOTS item meets the criteria fo
minimum level of difficulty with a value of 0.31 (Lestari & Yudhanegara, 2017). Each HOTS item based on Banten culture can be declared appropriate or good if each HOTS item meets the criteria for a
minimum level of difficulty with a value of 0.31 (Lestari & Yudhanegara, 2017). y
(
g
,
)
Table 10. Difficulty Level Results
Items
Difficulty Level
Category
1
0,542
Medium
2
0,233
Difficult
3
0,483
Medium
4
0,575
Medium
5
0,250
Difficult
6
0,592
Medium
7
0,558
Medium
8
0,525
Medium
9
0,550
Medium
10
0,500
Medium The results of the difficulty level test found that 8 items (80%) entered the level of difficulty with the "Medium" criteria. This means that there are many students whose answers are correct and whose answers are wrong, or it can be said to be
a draw. While 2 items (20%) entered the level of difficulty with the "Difficult" criteria, meaning that many students answered
incorrectly. In the research of Alfarisa, et al (2019), it was stated that the items that entered the accepted criteria were the
medium difficulty category, those that entered the revised criteria were the difficult and easy difficulty categories, the
rejected criteria were the very difficult and very easy categories. This can be interpreted that the majority of questions are
accepted, while two items that have a difficult category need to be corrected or revised. This is in line with the statement of
Amalia & Widayati (2012) on the results of the research they have done that the questions that fall into the category are
not so easy and not so difficult, or it can be said that they are good items. 3.3. Characteristics of HOTS Question Items Based on Banten Culture The reliability test of HOTS questions based on Banten culture was calculated using the Alfa Cronbach formula. The
instrument reliability value is 0.891. Then 0.70 < r_11 0.90. This means that the HOTS questions based on Banten culture
are declared to have high reliability criteria according to the reliability criteria according to Jihad and Abdul (2013: 181). 3.3.2. Question Difficulty Level 3.3.3. Power of Differing Questions 3.3.3. Power of Differing Questions HOTS question items based on Banten culture can be declared good or good if the HOTS items have a minimum
distinguishing power of 0.2 (Lestari & Yudhanegara, 2017). This states that the High Order Thinking Skills (HOTS) item
is declared good if it has sufficient discriminatory power. Different power test results in the following table 11. Tabel 11. Power of Differing Results 50
Tabel 11. Power of Differing Results
Items
Power of Differing
1
0,486
2
0,747
3
0,633
4
0,622
5
0,782
6
0,770
7
0,650
8
0,442
9
0,576
10
0,631 50 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 Yuliani et.al The results of the discriminatory power test found that 10 items (100%) were included in the discriminatory criteria, which
means that the HOTS questions are good for classifying the level of students' thinking abilities. The test results are in line
with the theory presented by Mardapi (2017) and Kartowagiran (2012) that the items are of good quality and deserve to be
accepted if the discrepancy index is more than 0.30. This means that all items of High Order Thinking Skills (HOTS) based
on Banten culture tested are of good quality and deserve to be accepted. The results of the analysis carried out on the ability
of students to correctly answer the High Order Thinking Skills (HOTS) questions based on Banten culture found that the
ability to answer questions on the HOTS C4 criteria was mostly (26%) at a sufficient score. Most of the HOTS C5 criteria
were in the sufficient and less scores (26%). And most of the HOTS C6 criteria (30%) are at a very low score. It states that
the higher the criteria for High Order Thinking Skills (HOTS) students face difficulties in working on questions. This is in
line with the purpose of Bloom's taxonomy (in Munzenmaier & Rubin, 2013) to classify cognitive levels C1-C6 which
describes the level of students' thinking abilities, the higher the cognitive level, the higher the difficulty level. 4. CONCLUSION The development of HOTS questions based on Banten cul-ture to measure the mathematical abilities of elementary school
students through several stages based on the Research and Development (R&D) method with the Martin Tessmer (1993)
model, namely formative research which includes pleminary stages and formative evaluation stages. There are no conflicts of interest declared by the author. There are no conflicts of interest declared by the author. REFERENCES 3.3.3. Power of Differing Questions The development of
the questions resulted in 10 mathe-matical description questions. The value of validity 0.80 for each item, meaning that the
item is feasible to use. The level of practicality of the questions obtained a value of 96.25% with a very practical category. The reliability of the question obtained a reliability coefficient of 0.891 and declared reliable. Based on the difficulty level
test, it was found that 8 items (80%) entered the "medium" diffi-culty level criteria. this means the number of students who
answered right and wrong, balanced. Meanwhile, 2 items (20%) are included in the criteria for "difficult" level of diffi-culty. This means that most of the students answered the questions incorrectly. Based on the discriminatory power test, it was
found that 10 items (100%) were categorized as good discriminating power. This means that the HOTS questions are good
and good for classifying the level of students' thinking abilities. Based on the results obtained from this study, it can be
stated that the following suggestions: for students in learning mathematics, they must be able to improve higher-order
thinking skills so that the dimensions of students' knowledge also increase, teachers are expected to be able to provide cul-
ture-based questions that contain the dimensions of students' high order thinking skills in learning so that students are ac-
customed to solving these questions, and to find out further whether or not the question instrument that has been devel-
oped, it is recommended for further researchers to be able to try it out on a wider trial subject. AUTHOR’S CONTRIBUTIONS The author discussed the results and contributed to from the start to final manuscript. ACKNOWLEDGEMENTS The author would like to thank all those who have helped, both morally and materially, so that this study was completed. AUTHOR’S CONTRIBUTIONS The author would like to thank all those who have helped, both morally and materially, so that this study was completed. The author would like to thank all those who have helped, both morally and materially, s International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 Rudhito, Andy, dkk. (2019). Matematika Dalam Budaya: Kumpulan Kajian Etnomatika. Yogyakarta: Garudhawaca.
Sugiyono. (2013). Statistika Untuk Penelitian. Bandung: Alfabeta. Prawiradilaga, Dewi S. (2012). Prinsip Desain Pembelajaran. Jakarta: Kencana. Prawiradilaga, Dewi S. (2012). Prinsip Desain Pembelajaran. Jakarta: Kencana. Rudhito, Andy, dkk. (2019). Matematika Dalam Budaya: Kumpulan Kajian Etnomatika. Yogyakarta: Garudhawaca.
S
i
(2013) St ti tik
U t k P
liti
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d
Alf b t Sugiyono. (2017). Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, dan R & D. Bandung : Alfabeta.
Supriadi, S., Arisetyawan, A., & Tiurlina. (2016). Mengintegrasikan Pembelajaran Matematika Berbasis Budaya Banten
Pada Pendirian SD Laboratorium UPI Kampus Serang. Jurnal Mimbar Sekolah Dasar, Vol. 3 (1), 1-18. Sugiyono. (2017). Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, dan R & D. Bandung : Alfabeta. Sutarti, Tati., dan Irawan, Edi. (2017). Kiat Sukses Meraih Hibah Penelitian Pengembangan. Yogyakarta: Deepublish. Sugiyono. (2013). Statistika Untuk Penelitian. Bandung: Alfabeta.
Sugiyono. (2017). Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, dan R & D. Bandung : Alfabeta.
Supriadi, S., Arisetyawan, A., & Tiurlina. (2016). Mengintegrasikan Pembelajaran Matematika Berbasis Budaya Banten
Pada Pendirian SD Laboratorium UPI Kampus Serang. Jurnal Mimbar Sekolah Dasar, Vol. 3 (1), 1-18.
Sutarti, Tati., dan Irawan, Edi. (2017). Kiat Sukses Meraih Hibah Penelitian Pengembangan. Yogyakarta: Deepublish. Sugiyono. (2017). Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, dan R & D. Bandung : Alfabeta.
S
i di S
A i
t
A
& Ti
li
(2016) M
i t
ik
P
b l j
M t
tik
B
b
i
B d
B
t Sutarti, Tati., dan Irawan, Edi. (2017). Kiat Sukses Meraih Hibah Penelitian Pengembangan. Y Pembelajaran. Jakarta: Kencana. REFERENCES Alfarisa, F., Chudari, I. N. M., & Robiansyah, F. (2019). Analisis Butir Soal IPS Kelas V Sekolah Dasar Menggunakan
Software ITEMAN. EduBasic Journal: Jurnal Pendidikan Dasar, 1(2), 100-106. Alfarisa, F., Chudari, I. N. M., & Robiansyah, F. (2019). Analisis Butir Soal IPS Kelas V Sekolah Dasar Menggunakan
Software ITEMAN. EduBasic Journal: Jurnal Pendidikan Dasar, 1(2), 100-106. Alfarisa, F., Chudari, I. N. M., & Robiansyah, F. (2019). Analisis Butir Soal IPS Kelas V Sekolah Dasar Menggunakan
Software ITEMAN. EduBasic Journal: Jurnal Pendidikan Dasar, 1(2), 100-106. Arikunto, Suharsimi. (2012). Prosedur Penelitian Suatu Pendekatan Praktik. Jakarta: PT Rineke Cipta. Arikunto, Suharsimi. (2012). Prosedur Penelitian Suatu Pendekatan Praktik. Jakarta: PT Rineke Cipta. Arikunto, Suharsimi. (2012). Prosedur Penelitian Suatu Pendekatan Praktik. Jakarta: PT Azwar, S. (2012). Reliabilitas dan Validitas. Yogyakarta: Pustaka Pelajar. ar, S. (2012). Reliabilitas dan Validitas. Yogyakarta: Pustaka Pelajar. Farihah, Nailul dkk. (2018). Pengembangan Soal Higher Order Thingking Skill (HOTS) pada materi Barisan dan Deret
Bilangan”. Jurnal Majamath. Vol.1 (2), 142-154. Farihah, Nailul dkk. (2018). Pengembangan Soal Higher Order Thingking Skill (HOTS) pada materi Barisan dan Deret
Bilangan”. Jurnal Majamath. Vol.1 (2), 142-154. Helmawati. (2019). Pembelajaran dan Penilaian Berbasis HOTS. Bandung: Remaja Rosdakarya Helmawati. (2019). Pembelajaran dan Penilaian Berbasis HOTS. Bandung: Remaja Rosdakarya. Jihad, A., & Abdul, H. (2013). Evaluasi Pembelajaran. Yogyakarta: Multi Presindo. Jihad, A., & Abdul, H. (2013). Evaluasi Pembelajaran. Yogyakarta: Multi Presindo. Jihad, A., & Abdul, H. (2013). Evaluasi Pembelajaran. Yogyakarta: Multi Presindo. Lestari, K. E., & Yudhanegara, M. R. (2017). Penelitian Pendidikan Matematika. Bandung: PT Mardapi, D. (2017). Pengukuran, penilaian, dan evaluasi pendidikan: Edisi 2. Yogyakarta: Para Mardapi, D. (2017). Pengukuran, penilaian, dan evaluasi pendidikan: Edisi 2. Yogyakarta: Parama Publising. Maulyda, & Archi, Mohammad. (2020). Paradigma Pembelajaran Matematika Berbasis NCTM. mad. (2020). Paradigma Pembelajaran Matematika Berbasis NCTM. Malang : CV IRDH. Munzenmaier, C., & Rubin, N. (2013). Perspectives Bloom’s Taxonomy ?: What’s Old is New Again?. Santa Rosa: The
eLearning Guild Research. Munzenmaier, C., & Rubin, N. (2013). Perspectives Bloom’s Taxonomy ?: What’s Old is New Again?. Santa Rosa: The
eLearning Guild Research. 51 International Journal of Trends in Mathematics Education Research, Vol. 5, No. 1 (2022), pp. 44-52 Yuliani et.al 52
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https://openalex.org/W4245301140
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https://www.qeios.com/read/6KZE8J/pdf
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BMP15 wt Allele
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Definitions
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cc-by
| 77
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios BMP15 wt Allele National Cancer Institute National Cancer Institute Qeios ID: 6KZE8J · https://doi.org/10.32388/6KZE8J Source National Cancer Institute. BMP15 wt Allele. NCI Thesaurus. Code C51528. Human BMP15 wild-type allele is located in the vicinity of Xp11.2 and is approximately 6
kb in length. This allele, which encodes bone morphogenetic protein 15, plays a role in
oocyte maturation and follicular development. Qeios ID: 6KZE8J · https://doi.org/10.32388/6KZE8J 1/1
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Synthesis of dandelion-like bimetallic nickel-cobalt metal-organic framework as an efficient cathode for high-performance asymmetric supercapacitors
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Research Square (Research Square)
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Synthesis of dandelion-like bimetallic nickel-cobalt
metal-organic framework as an efficient cathode for
high-performance asymmetric supercapacitors Chii-Rong Yang
National Taiwan Normal University
Yu-Chiao Li
National Taiwan Normal University
Jeng-Yu Lin
Tunghai University
Mao-Jung Huang
(
huangmj@ntu.edu.tw
)
National Taiwan University Tunghai University
Mao-Jung Huang
(
huangmj@ntu.edu.tw
)
National Taiwan University Research Article Keywords: bimetallic, metal-organic framework, pyromellitic acid, high specific surface area, cathode,
supercapacitor Posted Date: April 17th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2790800/v1 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 1/27 Page 1/27 Abstract In this current work, pyromellitic acid (PMA) was employed to synthesize a NiCo metal-organic framework
(MOF) for supercapacitors. The resultant NiCo-MOF (PMA) showed dandelion-like structure and therefore
its surface area was reached as high as 500.7 m2 g− 1. The NiCo-MOF (PMA) delivered an impressive
specific capacitance value of 918.8 F g− 1 at the current density of 1 A g− 1. Upon increasing the current
density to 10 A g− 1, it retained 61.1% of the capacitance obtained at 1 A g− 1. Moreover, the capacitance
retention of 64.3% was achieved even after being charged/discharged at 10 A g− 1 for 10,000 cycles. More
importantly, the asymmetric supercapacitor (ASC) based on the NiCo-MOF (PMA) cathode showed
specific capacitance of 83.47 F g− 1 at 0.5 A g− 1 and 80.34% of its initial capacitance was still maintained
after 5,000 cycles at 5 A g− 1. Furthermore, the ASC delivered an energy density of 23.88 Wh kg− 1 at a
power density of 750 Wkg− 1. Additionally, two ASCs in series successfully drove 120 green LEDs lighting
for 21 minutes, demonstrating its potential practical applications. 1. Introduction Supercapacitors have been regarded as novel, environmentally friendly electrical energy storage systems
due to their high power density and long cycle life. They have partially substituted the existing
rechargeable batteries for IoT-enabled devices, smart instruments, electric automobiles and motorcycles,
and wearable electronic devices. The supercapacitor is the optimal energy system for coordinating the
difference between traditional capacitors with low energy density and lithium-ion batteries with high
energy density [1]. However, as the energy density of a supercapacitor is still lower than that of batteries,
supercapacitors are currently not used in energy storage systems by themselves for long-term energy
supply. With the characteristics of small sizes, quick charge/discharge, a wide operating temperature
range, and a long life, when supercapacitors are used alongside batteries, the high-power characteristic of
supercapacitors can be demonstrated, and the service life of batteries can be prolonged to achieve the
optimal energy storage effect. Asymmetric supercapacitors (ASC) are generally made from
pseudocapacitors type cathode and electric double layer capacitor (EDLC) type anode, simultaneously
satisfying a wide voltage range and high capacitance storage capacity. The EDLC stores charge by
adsorbing electrolyte ions at the interface of the electrolyte and electrode. It provides high power density
and long cycle life. However, in organic electrolytes or aqueous solutions, the energy density of EDLC is
very low. On the other hand, the pseudocapacitor can store charge using reversible oxidation and
reduction reactions. Its specific capacitance value and energy density are almost ten times that of EDLC
[2]. However, continuous redox reactions may induce microstructure deformation of pseudocapacitor
electrode materials and lead to degradation of the loop stability of pseudocapacitors. Therefore,
developing a novel electrode material with a high specific surface area and redox site has become the key
to implementing high-performance supercapacitors [3]. The metal-organic framework (MOF) has been
proven to be an excellent material for making SCs electrodes [4–5]. Additionally, the MOF applies to gas
storage, chemosensing, drug delivery, and catalyst fields [6–13]. Page 2/27 The MOF is a porous crystalline material composed of metallic ions (Cu2 +, Zn2 +, Mn2 +, Co2 +, Mg2 +, Ni2
+, and Al3 +) and organic ligands through coordinative bonds. It is a 3D structure with the same pore size
distribution [14–17]. The MOF of monometallic components has worse electrical conductivity and
structural stability. This MOF has obvious defects in supercapacitor electrodes [18]. 1. Introduction Therefore, many
studies on MOF have used bimetallic ions Ni2+ and Co2+ for synthesis. Since Ni and Co have similar ionic
radii, their synthesis does not result in noticeable structural changes in bimetallic MOF [19]. Partial Ni2+ is
substituted by Co2+ to provide more free holes. Hence, a synergistic effect occurs [20], and the charge
transfer efficiency is increased. In addition, the bimetallic MOF has tunable electrochemical activity, high
charging capacity, and better conductivity [4–5, 21]. Wanga et al. indicated that the Ni-MOF had a high
specific surface area and high porosity, but the poor conductivity of MOF material could induce poor
specific capacitance value. Therefore, more free holes and synergistic effects were generated by Wanga
et al. doped cobalt ions to substitute partial nickel ions. The charge transfer efficiency and the
conductivity of MOF material were increased. The developed NiCo-MOF had 1300 F g− 1 specific
capacitance value under 1 A g− 1 current density [22]. The metallic ions can be combined with different
organic ligands with different coordination numbers. The structure of MOF material can be adjusted by
selecting different organic ligands, including chemical composition, particle size, morphology, and hole
feature [23]. Gao et al. [ref] indicated that the MOF had a high specific surface area, bad electrical
conductivity, unsuitable pore size for ion transmission, and poor stability in the charging/discharging
process. As a result, the MOF had a lower specific capacitance value and loop stability than transition
metal oxides. Therefore, Gao et al. used organic ligands to adjust the porosity of MOF to remedy the
defects and used trimesic acid, nickel, and cobalt metallic ions to synthesize a Ni/Co-MOF with a
dandelion structure. The developed NiCo-MOF had a 758 F g− 1 specific capacitance value under 1 A g− 1
current density and had relatively excellent rate capability under high current density. The scattered rod-
like structures on the NiCo-MOF sphere provided more effective active reaction sites and reduced ion
diffusion impedance at a higher scanning speed [24]. Additionally, the disordered structure is favorable
for enhancing electrochemical and structural stability, so Ni2+ is partially substituted by Co2+. The NiCo-
MOF forms a low crystal material and can have excellent capacitance retention and loop stability under
high current density [25]. Chen et al. 2.1 Synthesis of three kinds of NiCo-MOF This study used Ni (II) nitrate hexahydrate (Ni(NO3)2·6H2O) and Co (II) nitrate
hexahydrate(Co(NO3)2·6H2O) as metal sources. These were combined with organic ligands terephthalic
acid (TPA), trimesic acid (TMA), and pyromellitic acid (PMA) to synthesize three kinds of NiCo-MOF with
different organic ligands individually. The synthesis diagram is shown in Fig. 1. One-step hydrothermal
synthesis was used in the experiment. 0.05 M TPA, TMA, and PMA were combined with 0.05 M Ni (II)
nitrate hexahydrate and Co(II) nitrate hexahydrate separately. It was then dissolved in deionized (DI)
water, ethanol (99.5%), and dimethylformamide (DMF) solvent with a 5:2:14 ratio. The magnets stirred
the subsequent solutions at 500 rpm for 20 minutes. The containers carrying these solutions were heated
at 120℃ for 8 hours in oil. After they were cooled to room temperature, the precipitates were centrifuged
and cleaned with ethanol and DI water several times. The precipitates were dried in a vacuum at 60℃
overnight to obtain earthy yellow NiCo-MOF (TPA) powder, lavender purple NiCo-MOF (TMA) powder, and
peach purple NiCo-MOF (PMA) powder. The specification information of chemicals used in this study is
shown in Fig. 1. 2.2 Materials characterization The surface morphology and elements of the prepared three kinds of NiCo-MOF were analyzed using the
field emission scanning electron microscope (FESEM, ZEISS ∑IGMA Essential) equipped with energy-
dispersive X-ray spectroscopy (EDS) and transmission electron microscope (TEM, FEI Tecnai G2 20). The
crystal structure of NiCo-MOF was evaluated by an X-ray diffraction system (Bruker AXS GmbH New D8
DISCOVER). The functional group was measured using Fourier-transform infrared spectroscopy (FTIR,
Shimadzu IRSpirit) to ensure the synthesis quality of NiCo-MOF. Raman spectroscopy (NRS-4100, Jasco)
was used to analyze the lattice characteristic peak of NiCo-MOF. The Accelerated Surface Area and
Porosimetry System (Micromeritics ASAP2010) was used for N2 measurement adsorption/desorption of
specific surface areas and pore sizes of samples. The samples were degassed in a vacuum at 100℃ for
8 hours before measurement. To evaluate the elementary composition of NiCo-MOF, the Auger electron
microprobe (VG Scientific Microlab 350) was used for X-ray photoelectron spectroscopy (XPS)
measurements. 1. Introduction used p-phthalic acid as a ligand to synthesize cellular Ni/Co-MO, and
the maximum specific capacity was 1180.5 mC cm− 2 at 3 mA cm− 2 [26]. Habibb et al. used terephthalic
acid to synthesize rod-like Ni/Co-MOF with a specific capacitance of 1049 F g− 1 under 1 A g− 1 current
density [27]. Ye et al. used isophthalic acid to synthesize a flower-like structure, and the specific
capacitance was 833 C g− 1 under 0.5 A g− 1 current density [28]. Wang et al. connected terephthalic acid,
nickel, and cobalt ions in a molar ratio of 3:2 [29] to nickel foam (NF) to synthesize a triangular plate-like
structure. The specific capacitance value was 2200 F g− 1 under 1 A g− 1 current density. As mentioned
above, the Ni-Co MOF used for energy storage mostly uses benzene dicarboxylic and trimesic acids as
i li
d [2 22 24 30 32] Th
f
thi
t d
tili
d th l
l
d
lliti
id Page 3/27 2.3 Electrode fabrication and measurement of
electrochemical properties NiCo-MOF active electrode materials, carbon black, and polyvinylidene difluoride (PVDF), were mixed in N
Methyl-2-pyrrolidone (NMP) in the ratio of 8:1:1 to prepare the working electrode paste. This paste was
coated on a 1 cm2 nickel foam and dried overnight at 60℃ to obtain a working electrode. The
potentiostat (Metrohm Autolab PGSTAT30 & FRA) was used for cyclic voltammetry (CV), galvanostatic
charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS) measuring techniques to Page 4/27 evaluate the electrochemical properties of electrodes. The three-pole measurement system was used for
semi-electrode tests. In these tests, NiCo-MOF was used as the working electrode, Pt mesh was used as
the counter electrode, and Ag/AgCl was used as the reference electrode. The measurement was
performed in 1 M KOH electrolyte. The EIS test was performed under open-circuit voltage in the 0.01 Hz-
100 kHz frequency range. In this study, the active material mass of three kinds of NiCo-MOF electrodes
was controlled at 1 mg. According to the following Eq. (1), the specific capacitance value of three-pole measurement and ASC
was calculated using the GCD measurement result [30]: C =
I × t
m × ΔV C =
I × t
m × ΔV 1 where C (F g− 1) is the specific capacitance value, I is the discharge current (A), t is the discharge time (s),
m is the mass of active material (g), and ∆V is the voltage drop (V) in the discharge process. where C (F g− 1) is the specific capacitance value, I is the discharge current (A), t is the discharge time (s),
m is the mass of active material (g), and ∆V is the voltage drop (V) in the discharge process. After evaluating the electrode efficiency, the NiCo-MOF (PMA) was used as a cathode, and the activated
carbon was used as an anode. The cellulose filter paper (Advantec NO.5C) was used as a separator, and 1
M KOH electrolyte was filled in to make ASC. The capacitor efficiency was evaluated using bipolar
measurements. To implement the best electrochemical properties, the cathode and anode should
maintain a charge balance to satisfy the relationship of q+=q−. Hence, the optimal mass ratio of cathode
and anode should satisfy Eq. (2) [22]. 3. Result And Discussion
3.1 Material analysis and discussion about three kinds of
NiCo-MOF The surface morphology and elementary composition of three kinds of NiCo-MOF were observed through
SEM and TEM. Figure 2 shows the FESEM images of NiCo-MOF (TPA), NiCo-MOF (TMA), and NiCo-MOF
(PMA) under 5,000X and 50,000X magnifications and the TEM image under 11,500X magnification. Figures 2 (a-b) show that the NiCo-MOF (TPA) was a flower-like structure composed of microplate-like
structures. Its diameter was about 20 µm and its thickness was smaller than 100 nm. Figure 2 (c) shows
the TEM image of NiCo-MOF (TPA) in a plate-like structure. Figures 2 (d-e) show that the NiCo-MOF
(TMA) was a dandelion-like structure with a diameter of about 2 µm. The image with 50,000 X
magnification shows that each dandelion structure is composed of columns. The NiCo-MOF (TMA) was
observed through TEM. The diameter of the columnar structure was about 50 nm, as shown in Fig. 2 (f). Figures 2 (g-h) show that the NiCo-MOF (PMA) material was similar to NiCo-MOF (TMA). Both of them
were dense dandelion-like structures. The diameter of the dandelion structure of NiCo-MOF (PMA) was
about 10 µm. It was observed in the image with 50,000 X magnification that the dandelion-like structure
was also composed of columnar structures. Figure 2 (i) shows the TEM image of NiCo-MOF (PMA). The
diameter of the nano column was about 50 nm. To prove the successful synthesis of Ni and Co in NiCo-
MOF (PMA), the EDS was used for composition analysis. The result is shown in Fig. 3. It was observed in
the EDS image that the C, O, Co, and Ni elements were uniformly distributed in the image. This proves the
successful synthesis of Ni and Co in NiCo-MOF (PMA). Figure 3 (f) shows the atomic percentages of
different elements: C is 42.24%, O is 47.23%, Co is 4.14%, and Ni is 6.39%. The EDS images of NiCo-MOF
(TPA) and NiCo-MOF (TMA) are shown in Figs. S1-S2. The data were integrated into Table 1 to further
discuss the element contents in the three kinds of NiCo-MOF. There were slight differences in the total
content of transition metals (Ni and Co) in the three kinds of NiCo-MOF. The NiCo-MOF (TPA) had the
highest content of transition metals (18.01%). The NiCo-MOF (TMA) took second place (11.32%), and
NiCo-MOF (PMA) had the lowest amount of transition metals (10.53%). 2.3 Electrode fabrication and measurement of
electrochemical properties =
m+
m−
C−× ΔV−
C+ × ΔV + =
m+
m−
C−× ΔV−
C+ × ΔV + 2 where m is the mass of active material (g), C is the specific capacitance value (F g− 1), and ∆V is the
voltage drop (V) in the discharge process. The energy density and power density of ASC were calculated
using Eqs. (3) and (4) [23]: where m is the mass of active material (g), C is the specific capacitance value (F g− 1), and ∆V is the
voltage drop (V) in the discharge process. The energy density and power density of ASC were calculated
using Eqs. (3) and (4) [23]: Page 5/27
3
4
E = 0.5 × C × ΔV 2
3.6
P = E × 3600
Δt E = 0.5 × C × ΔV 2
3.6 3 Page 5/27
4
P = E × 3600
Δt 4 Page 5/27 where E is the energy density (Whkg− 1), C is the specific capacitance value (F g− 1), ∆V is the voltage drop
(V) in the discharge process, P is the power density (W kg− 1), and ∆t is the discharge time (s). 3. Result And Discussion
3.1 Material analysis and discussion about three kinds of
NiCo-MOF It was suspected that the
difference in the content of transition metals might influence the performance of NiCo-MOF. Page 6/27 Table 1
The element ratio of NiCo-MOFs. Samples
C
O
Co
Ni
Total
NiCo-MOF (TPA)
46.45%
35.54%
4.94%
13.07%
100%
NiCo-MOF (TMA)
32.93%
55.75%
4.09%
7.23%
100%
NiCo-MOF (PMA)
42.24%
47.23%
4.14%
6.39%
100% XRD measurements were performed to discuss the crystal structure of the three kinds of NiCo-MOF. As
depicted in Fig. 4, the three kinds of NiCo-MOF materials had an obvious peak within 10°. NiCo-MOF
(TPA) had obvious peaks near 8° and 17°, corresponding to the (2 0 0) and ( 0 1) planes, respectively
[22]. NiCo-MOF (TMA) had obvious peaks near 7° and 10°, corresponding to the (0 1 1) and (1 0 0) planes,
respectively [33]. In addition to NiCo-MOF (PMA), Ni-MOF (PMA) and Co-MOF (PMA) were synthesized in
this study for analysis. It was observed that Ni-MOF (PMA), Co-MOF (PMA), and NiCo-MOF (PMA) had
very coincident peaks. There was a common peak of Ni-based MOF material near 8°, corresponding to the
(1 0 0) planes [34]. According to these results, it is speculated that NiCo-MOF (PMA) was successfully
synthesized. ¯¯¯2 XRD measurements were performed to discuss the crystal structure of the three kinds of NiCo-MOF. As
depicted in Fig. 4, the three kinds of NiCo-MOF materials had an obvious peak within 10°. NiCo-MOF
(TPA) had obvious peaks near 8° and 17°, corresponding to the (2 0 0) and ( 0 1) planes, respectively
[22]. NiCo-MOF (TMA) had obvious peaks near 7° and 10°, corresponding to the (0 1 1) and (1 0 0) planes,
respectively [33]. In addition to NiCo-MOF (PMA), Ni-MOF (PMA) and Co-MOF (PMA) were synthesized in
this study for analysis. It was observed that Ni-MOF (PMA), Co-MOF (PMA), and NiCo-MOF (PMA) had
very coincident peaks. There was a common peak of Ni-based MOF material near 8°, corresponding to the
(1 0 0) planes [34]. According to these results, it is speculated that NiCo-MOF (PMA) was successfully
synthesized. ¯¯¯2 The FT-IR and Raman spectra of NiCo-MOF types are shown in Fig. 5. The FT-IR spectrum in Fig. 5 (a)
shows the absorption peak of NiCo-MOF (TPA) at 3600 cm− 1. The broad absorption peaks of NiCo-MOF
(TMA) and NiCo-MOF (PMA) near 3450 cm− 1 were related to the stretching vibration of OH, meaning that
the water molecules existed in the NiCo-MOF structure. 3. Result And Discussion
3.1 Material analysis and discussion about three kinds of
NiCo-MOF The strong absorption peaks at 1600 cm− 1 and
1400 cm− 1 represented the asymmetric and symmetrical stretching vibrations of the COO- anionic group,
respectively. The absorption peaks at 813 cm− 1 and 765 cm− 1 are the stretching vibrations of benzene
rings [22, 30, 33]. The Raman spectrum in Fig. 5 (b) shows that the three kinds of NiCo-MOF present
consistent peaks. The peak at 1600 cm− 1 represents the asymmetric stretching vibration of the COO-
anionic group, and the peak at 1400 cm− 1 represents the symmetrical stretching vibration of the COO-
anionic group. The peak nearby 800 cm− 1 represents the bending vibration of the C-H bond. The
measurement results and FT-IR show that the three kinds of NiCo-MOF had the vibration characteristics
of the COO- anionic group, coinciding with the literature [30]. The XPS measurement was performed to study the chemical composition of the three kinds of NiCo-MOF. The XPS spectra of NiCo-MOF are shown in Fig. 6(a). It was observed that these NiCo-MOF had similar
peaks in the full spectrogram. The peaks of C, O, Co, and Ni were detected in all three compositions of
NiCo-MOF, the obtained peaks were carbon corrected and deconvoluted. Further to discuss the
composition of chemical bonds of the NiCo-MOF (PMA) developed in this study, the chemical
composition of each peak was discussed, as shown in Figs. 6(b-e). Figure 6(b) shows the XPS spectrum
of C 1s, with the peak, observed at a binding energy of 284.4 eV, and the peak at 288.2 eV corresponds to
the bonds of C = C and O = C-O [35–36]. Figure 6(c) shows the XPS spectrum of O 1s, with the peak at
529.7 eV, 530.6 eV and 533.3 eV belonging to the metal-oxygen bond and the peak at 531.7 eV Page 7/27 Page 7/27 corresponding to OH- which is attributed to Ni and Co-O bonds confirming the presence of oxygen atoms,
which also confirms the low concentrated oxygen defect with the metal [35–36]. The XPS spectrum of Co
2p in Fig. 6(d) shows two spin-orbit doublets which were deconvoluted into four peaks of the Co 2p3/2
and Co 2p1/2. The corresponding peaks were observed at 781.5 and 797.1 eV belonging to Co 2p3/2 and
Co 2p1/2. respectively. 3. Result And Discussion
3.1 Material analysis and discussion about three kinds of
NiCo-MOF Samples
Specific surface area (m2 g− 1)
Pore size (nm)
NiCo-MOF(TPA)
6.91
18
NiCo-MOF(TMA)
20.66
23
NiCo-MOF(PMA)
500.70
16 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF 3. Result And Discussion
3.1 Material analysis and discussion about three kinds of
NiCo-MOF The two peaks observed at 784.0 and 803.1 eV corresponds to the satellite peak,
and the corresponding peaks at these binding energies belong to the Co2+ state [35–36]. Figure 6(e)
shows the XPS spectrum of Ni 2p, the main peaks observed at 856.3 and 874.0 eV, corresponding to Ni
2p3/2 and Ni 2p1/2 respectively. The two additional peaks observed along with the Ni peaks at 860.2 and
880.2 eV correspond to satellite peaks, respectively. These peaks were observed at 856.3 and 874.0 eV,
corresponding to the Ni + 2 oxidation state [36–37]. The satellite peaks in the XPS spectra of Co 2p and
Ni 2p might have resulted from energy loss in the valence electron excitation process [38]. This result
confirms the presence of the synthesized elements, which further confirms that there are no other
impurities were found in the sample. The three kinds of NiCo-MOF materials were degassed at 100℃ for 8 hours before the BET
measurement. The BET results are summarized in Table 2. The results show that the micro-flower
structure’s specific surface area of NiCo-MOF (TPA) was 6.91 m2 g− 1 with a pore size of 18 nm. Whereas
the specific surface area of NiCo-MOF (TMA) of the dandelion-like structure was 20.66 m2 g− 1 with a pore
size of 23 nm. The NiCo-MOF (PMA) of the dandelion-like structure developed in this study had a high
specific surface area of 500.70 m2 g− 1 and a pore size of 16 nm. It was proved with the SEM image of
NiCo-MOF (PMA) that the NiCo-MOF (PMA) of micron dandelion-like structures composed of nanorod
structures had the highest specific surface area. The cilia-like nanorod structures could effectively
increase the absorbed charges on the active material surface and enhance the capacitive character of
this active material. Page 8/27
Table 2
Specific surface area and pore size of NiCo-MOFs. Samples
Specific surface area (m2 g− 1)
Pore size (nm)
NiCo-MOF(TPA)
6.91
18
NiCo-MOF(TMA)
20.66
23
NiCo-MOF(PMA)
500.70
16
3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF
The electrochemical properties of various NiCo-MOF electrodes were measured using a conventional
three-electrode system in 1 M KOH electrolyte. The mass loading active material for each electrode was Table 2
Specific surface area and pore size of NiCo-MOFs. 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF Figure 7 (b) shows the GCD curves of various NiCo-MOF electrodes at 1 A
g− 1. Their corresponding specific capacitance values are estimated by Eq. (1). NiCo-MOF (TPA)
discharged for 500 secs (1000 F g− 1), NiCo-MOF (PMA) for 450.2 secs (918.8 F g− 1), and the NiCo-MOF
(TMA) discharged for 432 secs (864 F g− 1). Therefore, the performance of NiCo-MOF (TPA) was better
than that of NiCo-MOF (PMA) and NiCo-MOF (TMA). Figure 7 (c) shows the CV curves of NiCo-MOF
(PMA) developed in this study at a scan rate of 5 to 100 mV/s in the 0-0.6 V voltage range. The CV curve
did not deform with an increase in the scan rate. The redox peak could be observed even at a scan rate of
100 mv/, representing excellent reversible capacitive characteristics and rate performance. [39]
Additionally, as the scan rate increased, the reduction and oxidation peaks gradually moved in negative
and positive directions. This phenomenon was induced by the inherent impedance of materials (B). The
redox peak could be attributed to the following reversible Faraday redox process [40]: Additionally, as the scan rate increased, the reduction and oxidation peaks gradually moved in negative
and positive directions. This phenomenon was induced by the inherent impedance of materials (B). The
redox peak could be attributed to the following reversible Faraday redox process [40]: Ni(OH)2+OH−↔ NiO(OH) + H2O + e− (5)
Co(OH)2+OH−↔CoO(OH) + H2O + e− (6) Figure 7 (d) shows the GCD measurements of NiCo-MOF (PMA) under different current densities. The
charge/discharge platform could be observed. This means that the electrode material had the
characteristics of pseudocapacitors, coinciding with the characteristics of CV curves. The CV curves and
GCD measurements of NiCo-MOF (TPA) and NiCo-MOF (TMA) are shown in Figs. S3-S4. Figure 7 (e)
shows the specific capacitance values of NiCo-MOF types under different current densities calculated
based on GCD curves. The specific capacitance value of NiCo-MOF (TPA) under a high current density of
10 A g− 1 is 662 F g− 1. The specific capacitance value of NiCo-MOF (TMA) is 372 F g− 1, and the specific
capacitance value of NiCo-MOF (PMA) is 561.2 F g− 1. Compared to the initial capacitance under 1 A g− 1
current density, the three materials maintained the rate capability of 66.2%, 43.1%, and 61.1%,
respectively. The NiCo-MOF (TPA) had the best rate capability under high current density. 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF The electrochemical properties of various NiCo-MOF electrodes were measured using a conventional
three-electrode system in 1 M KOH electrolyte. The mass loading active material for each electrode was Page 8/27 Page 8/27 controlled at about 1 mg. As depicted in Fig. 7 (a), the resultant CV curves of various NiCo-MOF
electrodes were recorded at the scan rate of 5 mV s− 1 within the potential range of 0-0.6 V. It is observed
that both of NiCo-MOF (TPA) and NiCo-MOF (PMA) electrodes display similar CV areas, which is
apparently larger than that of NiCo-MOF (TMA) electrode. Notably, a pair of redox peaks are observed in
all CV curves, signifying that the NiCo-MOF electrode materials are with pseudocapacitive characteristics
due to Faraday redox reactions. Figure 7 (b) shows the GCD curves of various NiCo-MOF electrodes at 1 A
g− 1. Their corresponding specific capacitance values are estimated by Eq. (1). NiCo-MOF (TPA)
discharged for 500 secs (1000 F g− 1), NiCo-MOF (PMA) for 450.2 secs (918.8 F g− 1), and the NiCo-MOF
(TMA) discharged for 432 secs (864 F g− 1). Therefore, the performance of NiCo-MOF (TPA) was better
than that of NiCo-MOF (PMA) and NiCo-MOF (TMA). Figure 7 (c) shows the CV curves of NiCo-MOF
(PMA) developed in this study at a scan rate of 5 to 100 mV/s in the 0-0.6 V voltage range. The CV curve
did not deform with an increase in the scan rate. The redox peak could be observed even at a scan rate of
100 mv/, representing excellent reversible capacitive characteristics and rate performance. [39]
Additionally, as the scan rate increased, the reduction and oxidation peaks gradually moved in negative
and positive directions. This phenomenon was induced by the inherent impedance of materials (B). The
redox peak could be attributed to the following reversible Faraday redox process [40]: controlled at about 1 mg. As depicted in Fig. 7 (a), the resultant CV curves of various NiCo-MOF
electrodes were recorded at the scan rate of 5 mV s− 1 within the potential range of 0-0.6 V. It is observed
that both of NiCo-MOF (TPA) and NiCo-MOF (PMA) electrodes display similar CV areas, which is
apparently larger than that of NiCo-MOF (TMA) electrode. Notably, a pair of redox peaks are observed in
all CV curves, signifying that the NiCo-MOF electrode materials are with pseudocapacitive characteristics
due to Faraday redox reactions. 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF Figure 7 (f)
shows the Nyquist plot of NiCo-MOF types. The three Nyquist curves were composed of small semicircles
of high-frequency and slashes of low-frequency regions. The semi-circular arc curve of the high-frequency
region and the real axis intercept represented the electrode’s equivalent series resistance (Rs). It includes
the intrinsic resistance of active material, ionic resistance, and the contact resistance between the active
material and the current collector. The diameter of the semi-circular arc represented the charge transform
resistance (Rct) at the interface between the electrode and electrolyte. The oblique straight line in the low-
frequency region represented the Warburg spreading resistance (Zw) of electrolyte ions [23]. The results
show that the three materials had similar curves and close electrolyte (solution) resistance (Rs) values. The Rs values of NiCo-MOF (TPA), NiCo-MOF (TMA), and NiCo-MOF (PMA) are 1.55 Ω, 1.19 Ω, and 1.37 Page 9/27 Page 9/27 Ω, respectively. According to the enlarged view, the charge transfer resistance (Rct) values of NiCo-MOF
(TPA), NiCo-MOF (TMA), and NiCo-MOF (PMA) are 2.05 Ω, 4.17 Ω, and 3.49 Ω, respectively. Based on the
above results, the NiCo-MOF (TPA) had the smallest Rct value, the NiCo-MOF (PMA) took second place,
and the NiCo-MOF (TMA) had the largest value. Rct value positively correlated with the specific
capacitance value. According to the above findings, the NiCo-MOF (TPA) had the lowest Rct resistance
(2.05 Ω) and the highest specific capacitance value (1000 F g− 1). This might be because of the highest
transition metal content (18.01%) in EDS elemental analyses. The transition metal content (10.53%) in
NiCo-MOF (PMA) was lower than that in NiCo-MOF (TMA) (11.32%). However, the BET analysis showed
the NiCo-MOF (PMA) had a superhigh specific surface area of 500 m2 g− 1. The Rct resistance was lower
than NiCo-MOF (TMA), so the NiCo-MOF (PMA) developed in this study had a performance comparable
with the other two. The performance of NiCo-MOF types was compared with similar MOF materials in
literature and compiled in Table 3. It was observed that the specific capacitance values of the NiCo-MOF
types developed in this study were close to that in the literature, and the specific capacitance value was
not the highest. This might be because the NiCo-MOF types synthesized in this study controlled the molar
ratio of nickel ions to cobalt ions at basic 1:1. 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF In the literature, the metal ratio was usually adjusted, or the
graphene material was added to increase the specific capacitance value. For example, the NiCo-MOF/rGO
developed by Kumaraguru et al. used a nickel-cobalt molar ratio of 3:2 and rGO to achieve the specific
capacitance value of 958 F g− 1 [30]. The NiCo-MOF developed by Zhao et al. used a nickel-cobalt molar
ratio of 1:0.25 to achieve the specific capacitance value of 1067 F g− 1 [31]. Wang et al. used a nickel-
cobalt molar ratio of 1:0.02 so that the NiCo-MOF achieved the specific capacitance value of 1300 F g− 1
[22]. Ren et al. used a nickel-cobalt molar ratio of 1:3 to develop NiCo-MOF with a specific capacitance
value of 1230.3 F g− 1 [41]. Zheng et al. used a nickel-cobalt molar ratio of 5:1 so that the developed NiCo-
MOF could achieve a high specific capacitance value of 1498 F g− 1 [32]. NiCo-MOF (PMA) developed
using a nickel-cobalt molar ratio of 1:1 in this study has achieved a high specific capacitance value
comparable with the literature. It could be a promising cathode material for supercapacitors. If the nickel-
cobalt molar ratio can be adjusted, the specific capacitance value could be further improved. Page 10/27 Table 3 Specific capacitance values of MOF-based materials. Page 11/27
Specific capacitance values of MOF-based materials. 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF The rapid decline of cycle life before 1,000 cycles might be related to
the scaling of active materials. These bubbles formed on the electrode surface in the constant current
charge/discharge process. These bubbles pushed out some less adhesive active materials and scaled
off, rapidly reducing the cycle life. Electrode
materials
Organic ligand
Specific
capacitance
(F g− 1)
Current
density
(A g− 1)
Electrolyte
Reference
NiCo-
MOF(PMA)
Pyromellitic acid
918.8
1
1 M KOH
This work
*1 GNS: Graphene nanosheets
*2 rGO: Reduced graphene oxide
*3 AB: Acetylene black The long cycle durability test was performed for the NiCo-MOF (PMA) developed in this study under 10 A
g− 1 current density using constant current charge/discharge to verify its applicability to a cathode of
supercapacitors, as shown in Fig. 8. After 6,000 cycles under 10 A g− 1 current density, NiCo-MOF (PMA)
could keep 69.7% of initial capacitance. The NiCo-MOF made by Wang et al. [29] kept 68.5% of initial
capacitance after 6,000 cycles under 6 A g− 1 current density. This study’s capacitance retention after
6,000 cycles was close to the value of Wang et al.. However, the 10 A g− 1 current density selected in this
study was stricter than the 6 A g− 1 current density used in the study of Wang et al. Therefore, the NiCo-
MOF (PMA) developed in this study was theoretically better than the performances in literature during
long cycle life. After 10,000 cycles, NiCo-MOF (PMA) kept 64.3% of initial capacitance, meaning the NiCo-
MOF (PMA) had a good duty cycle. The rapid decline of cycle life before 1,000 cycles might be related to
the scaling of active materials. These bubbles formed on the electrode surface in the constant current
charge/discharge process. These bubbles pushed out some less adhesive active materials and scaled
off, rapidly reducing the cycle life. To verify that NiCo-MOF (PMA) developed in this study could be used as the electrode material of
supercapacitors, NiCo-MOF (PMA) was used as a cathode, and the active carbon (AC) was used as an
anode. A cellulose filter paper was placed between the cathode and anode as a separator to make an
ASC. The system was expressed as NiCo-MOF(PMA) // AC. The assembly diagram of this ASC is shown
in Fig. 9 (a). To make NiCo-MOF(PMA) // AC have a better matching degree, the matching degree of the
cathode and anode was calculated by Eq. 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF Electrode
materials
Organic ligand
Specific
capacitance
(F g− 1)
Current
density
(A g− 1)
Electrolyte
Reference
Ni-MOF
Terephthalic acid
552.0
1
2 M KOH
【25】
Co-MOF
4,4′-bipyridyl
446.8
1.2
1 M KOH
【42】
Co-MOF
Aminobenzoic acid
414.5
0.5
3 M KOH
【43】
Co-
MOF/GNS*1
Trimesic acid
608.2
0.25
1 M KOH
【44】
Ni/Co-MOF
Trimesic acid
758.0
1
2 M KOH
【24】
Ni/Co-MOF-
rGO*2
4,4′-Oxydibenzoic acid
860.0
1
6 M KOH
【45】
NiCo-
MOF/AB*3
Terephthalic acid
916.1
1
2 M KOH
【2】
NiCo-
MOF/rGO
Trimesic acid
958.0
1
1 M KOH
【30】
NiCo-MOF
H3BTC
639.8
1
3 M KOH
【46】
NiCo-MOF
4,4′-
biphenyldicarboxylic
acid
990.7
1
2 M KOH
【20】
NiCo-MOF
p-phthalic acid
998
1
2 M KOH
【47】
NiCo-MOF
Trimesic acid
1067
1
3 M KOH
【31】
NiCo-MOF
Terephthalic acid
1300
1
6 M KOH
【22】
NiCo-MOF
Isophthalic acid
1230.3
1
2 M KOH
【41】
NiCo-MOF
Trimesic acid
1498
1
6 M KOH
【32】
NiCo-
MOF(TPA)
Terephthalic acid
1000
1
1 M KOH
This work
NiCo-
MOF(TMA)
Trimesic acid
864.0
1
1 M KOH
This work
*1 GNS: Graphene nanosheets
*2 rGO: Reduced graphene oxide
*3 AB: Acetylene black Page 11/27 Electrode
materials
Organic ligand
Specific
capacitance
(F g− 1)
Current
density
(A g− 1)
Electrolyte
Reference
NiCo-
MOF(PMA)
Pyromellitic acid
918.8
1
1 M KOH
This work
*1 GNS: Graphene nanosheets
*2 rGO: Reduced graphene oxide
*3 AB: Acetylene black
The long cycle durability test was performed for the NiCo-MOF (PMA) developed in this study under 10 A
g− 1 current density using constant current charge/discharge to verify its applicability to a cathode of
supercapacitors, as shown in Fig. 8. After 6,000 cycles under 10 A g− 1 current density, NiCo-MOF (PMA)
could keep 69.7% of initial capacitance. The NiCo-MOF made by Wang et al. [29] kept 68.5% of initial
capacitance after 6,000 cycles under 6 A g− 1 current density. This study’s capacitance retention after
6,000 cycles was close to the value of Wang et al.. However, the 10 A g− 1 current density selected in this
study was stricter than the 6 A g− 1 current density used in the study of Wang et al. Therefore, the NiCo-
MOF (PMA) developed in this study was theoretically better than the performances in literature during
long cycle life. After 10,000 cycles, NiCo-MOF (PMA) kept 64.3% of initial capacitance, meaning the NiCo-
MOF (PMA) had a good duty cycle. 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF (2) before measurement. Then the active material mass of the
cathode of NiCo-MOF (PMA) was controlled at 1.11 mg. The active material of the activated carbon
anode was controlled at 4.56 mg, and the overall active material of NiCo-MOF(PMA) // AC was 5.67 mg. To evaluate the electrochemical properties of NiCo-MOF (PMA) and AC electrodes and a stable voltage
range, the CV curves of the two electrode materials were measured in 1 M KOH electrolyte. The voltage
range of NiCo-MOF (PMA) was from 0 to 0.6 V, and that of AC was from − 1.0 to 0 V, as shown in Fig. 9
(b). The ASC did not generate hydrogen/oxygen in the 0-1.5 V voltage range, so 1.5 V was regarded as the
optimum operating voltage of this ASC. Figure 9 (c) shows the CV curves of NiCo-MOF(PMA) // AC at
different scan rates in the 5–50 mV/s range. The pattern of the CV curve was gradually enlarged with an
increase in the scan rate. This CV curve shows that this ASC could reach the 1.5 V high voltage range. Page 12/27 Page 12/27 Figure 9 (d) shows the GCD curves of NiCo-MOF(PMA) // AC under different current densities in the range
of 0.5-5 A g− 1. This GCD diagram proves that the ASC could reach the 1.5 V voltage range. The GCD
measurement result shows that the time of discharge from 1.5 V to 0 V of ASC under 0.5 A g− 1 current
density was 250.4 secs. The specific capacitance value calculated by Eq. (1) was 83.47 F g− 1. Figure 9
(e) shows the specific capacitance values of NiCo-MOF(PMA) // AC under different current densities. The
specific capacitance value was 77.6 F g− 1 under 1A g− 1 current density. The specific capacitance value
was 38.3 F g− 1 under 5 A g− 1 current density. The Ragone plot of NiCo-MOF(PMA) // AC was drawn
based on the constant current charge/discharge curve. The ASC had an energy density of 23.88 Whkg− 1
under 750 Wkg− 1 power density. The energy density was still 11.98 Whkg− 1, under a high power density
of 3750 Wkg− 1. The value was equivalent to the performance of supercapacitors in similar
research/literature. 3.2 Measurement of electrochemical properties of three
kinds of NiCo-MOF To discuss the capabilities of the developed ASC in practical applications, a long cycle durability test was
performed under 5 A g− 1 current density using a constant current charge/discharge, as shown in Fig. 10
(a). This ASC still had 80.34% of initial capacitance after 5,000 charge/discharge cycles under 5 A g− 1
current density. The ASC proposed by Du et al. maintained 70% of initial capacitance after 2,000
charge/discharge cycles under 5 A g− 1 current density [33]. In another test, two ASCs were connected in
series for lighting tests, as shown in Fig. 10 (b). The test result shows that 120 pieces of 80 mW green
LED lamps (LS-F5UPGC-YBL) connected in parallel, which displayed a pattern of NTNU ME, were lit for 21
minutes. Conflict of Interest The authors declare that they have no conflict of interest. 4. Conclusions This study developed a NiCo-MOF (PMA) with a dandelion-like structure and a superhigh specific surface
area of 500.7 m2g− 1 using PMA as a ligand and one-step hydrothermal method. The specific surface
area of the developed structure was much higher than the common flower-like structure NiCo-MOF (TPA)
(6.91 m2g− 1) and dandelion-like structure NiCo-MOF (TMA) (20.66 m2g− 1) found in the literature. The
NiCo-MOF (PMA) had a high specific capacitance value of 918.8 F g-1 under 1 A g− 1 current density but
lower than 1000 F g− 1 of NiCo-MOF (TPA). By adjusting Ni and Co, the specific capacitance value of
NiCo-MOF (PMA) could be enhanced. NiCo-MOF (PMA) had a capacitance retention of 64.3% after 10,000
charge/discharge cycles under 10 A g− 1 current density. The ASC developed using NiCo-MOF (PMA) and
activated carbon had a specific capacitance value of 83.47 F g− 1 under 0.5 A g− 1 current density and an
energy density of 23.88 Wh kg− 1 under 750 W kg− 1 power density. Moreover, the developed ASC still had
an energy density of 11.98 Wh kg− 1 even under a high power density of 3750 W kg− 1. The ASC had
80.34% initial capacitance after 5,000 charge/discharge cycles under 5 A g− 1 current density. Finally, in
practical applications, two ASCs connected in series could keep 120 pieces of 80 mW green LED lamps
connected in parallel displaying the pattern of NTNU ME lit for 21 minutes. The above results prove that
the NiCo-MOF (PMA) developed in this study was comparable to the NiCo-MOF (TPA) developed from
terephthalic acid and the NiCo-MOF (TMA) developed from trimesic acid in the literature. The material Page 13/27 Page 13/27 exhibited a high specific capacitance value and good cycle life, proving that the NiCo-MOF (PMA) with
practical applicability developed in this study can be a promising electrode material in developing
supercapacitors. exhibited a high specific capacitance value and good cycle life, proving that the NiCo-MOF (PMA) with
practical applicability developed in this study can be a promising electrode material in developing
supercapacitors. Author Contributions Statement All authors contributed to the study conception and design. The resources and the project administration
were performed by Chii-Rong Yang. The conceptualization and methodology were performed by Chii-
Rong Yang and Yu-Chiao Li. The first draft of the manuscript was written by Yu-Chiao Li, Jeng-Yu Lin and
Mao-Jung Huang; and all authors commented on previous versions of the manuscript. All authors read
and approved the final manuscript. All authors reviewed the manuscript. Funding The authors would like to thank the Ministry of Science and Technology (MOST) of the Republic of China
(Taiwan) for financially supporting this research under Contract No. MOST 111-2221-E-003-010, and
subsidized by the National Taiwan Normal University (NTNU), Taiwan, ROC. Acknowledgements We thank the excellent technical assistant of Instrumentation Center at NTU (Taiwan) with the TEM
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Energy Storage 47:103627. https://doi.org/10.1016/j.est.2021.103627 Figures Page 18/27 Figure 1 Schematic diagram of synthesizing three kinds of NiCo-MOF materials. Schematic diagram of synthesizing three kinds of NiCo-MOF materials. Page 19/27 FESEM (left, center) and TEM (right) images of (a-c) NiCo-MOF(TPA), (d-f) NiCo-MOF(TMA), (g-i) NiCo-
MOF(PMA). Figure 2 FESEM (left, center) and TEM (right) images of (a-c) NiCo-MOF(TPA), (d-f) NiCo-MOF(TMA), (g-i) NiCo-
MOF(PMA). Page 20/27 Page 20/27 Figure 3
EDS elemental mapping images of NiCo-MOF(PMA): (a) FESEM image, (b) carbon, (c) oxygen, (d)
(e) nickel mappings, and (f) the element ratio of NiCo-MOF(PMA). Figure 3 EDS elemental mapping images of NiCo-MOF(PMA): (a) FESEM image, (b) carbon, (c) oxygen, (d) cobalt,
(e) nickel mappings, and (f) the element ratio of NiCo-MOF(PMA). Page 21/27 Page 21/27 Page 21/27 Figure 4
XRD pattern of NiCo-MOFs with different organic ligands. Figure 4
XRD pattern of NiCo-MOFs with different organic ligands. Figure 4 Figure 4 XRD pattern of NiCo-MOFs with different organic ligands. Page 22/27 Page 22/27 Page 22/27 Page 22/27 Figure 5 Figure 5 (a) FTIR spectrum and (b) Raman spectrum of NiCo-MOFs with different organic ligands. gure 6 Figure 6 XPS spectra of bimetallic NiCo-MOF(PMA). (a) Survey, (b) C 1s, (c) O 1s, (d) Co 2p, (e) Ni 2p. Page 23/27 Page 23/27 gure 7
ectrochemical performance of bimetallic NiCo-MOFs in a three-electrode configuration: Comparis
e (a) CV, (b) GCD and (c) CV curves of NiCo-MOF(PMA) at different scan rates, (d) GCD curves of
OF(PMA) at different current densities. (e) Specific capacitances at different current densities, (f)
yquist plots of NiCo-MOFs. Figure 7 Electrochemical performance of bimetallic NiCo-MOFs in a three-electrode configuration: Comparisons of
the (a) CV, (b) GCD and (c) CV curves of NiCo-MOF(PMA) at different scan rates, (d) GCD curves of NiCo-
MOF(PMA) at different current densities. (e) Specific capacitances at different current densities, (f)
Nyquist plots of NiCo-MOFs. Page 24/27 Figure 8
The cyclic stability of the NiCo-MOF(PMA) at current density of 10 A g-1 for 10000 cycles. Figure 10 (a) Cycling stability of ASCs at the current density of 5 A g-1, (b) lighting one hundred twenty LED lamps
arranged as a NTNU ME pattern. (a) Cycling stability of ASCs at the current density of 5 A g-1, (b) lighting one hundred twenty LED lamps
arranged as a NTNU ME pattern. Figure 8 The cyclic stability of the NiCo-MOF(PMA) at current density of 10 A g-1 for 10000 cycles. The cyclic stability of the NiCo-MOF(PMA) at current density of 10 A g-1 for 10000 cycles. Page 25/27 Page 25/27 Figure 9
(a) Schematic illustration of the ASCs based on the NiCo-MOF(PMA) and AC, (b) CV curves of NiC
MOF(PMA) and AC with the scan rate at 25 mV/s, (c) CV curves of the ASC with the scan Figure 9 (a) Schematic illustration of the ASCs based on the NiCo-MOF(PMA) and AC, (b) CV curves of NiCo-
MOF(PMA) and AC with the scan rate at 25 mV/s, (c) CV curves of the ASC with the scan Page 26/27 Page 26/27 Figure 10 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. ACHMsupplementarymaterials.docx Page 27/27
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English
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Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR
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Viruses
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cc-by
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Luciana Alexandra CRIVEI 1,*, Sara MOUTAILLER 2
, Gaëlle GONZALEZ 3, Steeve LOWENSKI 3,
Ioana Cristina CRIVEI 1, Daniela POREA 1, Dragos, Constantin ANITA 1
, Ioana Alexandra RATOI 1,
Stéphan ZIENTARA 3
, Luanda Elena OSLOBANU 1, Alexandru TOMAZATOS 4, Gheorghe SAVUTA 1,†
and Sylvie LECOLLINET 3,†,‡ https://doi.org/10.3390/v15010186 Keywords:
arbovirus; West Nile virus; Sindbis virus; mosquito-borne; Romania; flavivirus;
alphavirus; high-throughput microfluidic real-time PCR Academic Editor: Boris Pastorino Received: 14 December 2022
Revised: 3 January 2023
Accepted: 4 January 2023
Published: 9 January 2023 viruses viruses viruses Luciana Alexandra CRIVEI 1,*, Sara MOUTAILLER 2
, Gaëlle GONZALEZ 3, Steeve LOWENSKI 3,
Ioana Cristina CRIVEI 1, Daniela POREA 1, Dragos, Constantin ANITA 1
, Ioana Alexandra RATOI 1,
Stéphan ZIENTARA 3
, Luanda Elena OSLOBANU 1, Alexandru TOMAZATOS 4, Gheorghe SAVUTA 1,†
and Sylvie LECOLLINET 3,†,‡ 1
Regional Center of Advanced Research for Emerging Diseases, Zoonoses and Food Safety,
Ias,i University of Life Sciences, 700490 Ias,i, Romania 1
Regional Center of Advanced Research for Emerging Diseases, Zoonoses and Food Safety, g
g
g
,
y,
Ias,i University of Life Sciences, 700490 Ias,i, Romania ,
y
,
2
ANSES, INRAE, Ecole Nationale Vétérinaire d’Alfort, UMR BIPAR, Laboratoire de Santé Animale,
94700 Maisons-Alfort, France 3
ANSES, INRAE, Ecole Nationale Vétérinaire d’Alfort, UMR VIROLOGIE, Laboratoire de Santé Animale,
94700 Maisons-Alfort, France 4
Department of Arbovirology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany
*
Correspondence: criveilucianaalexandra@gmail.com †
These authors contributed equally to this work. †
These authors contributed equally to this work. ‡
Current address: CIRAD, UMR ASTRE, 97170 Petit Bourg, Guadeloupe, France. Abstract: The impact of mosquito-borne diseases on human and veterinary health is being exacer-
bated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeast-
ern Romania, we have used a combination of sampling methods in natural, urban and peri-urban
sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a
high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365
pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied
counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in
one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus
isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas
observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional
SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of
theCentral-Southeast European clade, has a wider circulation in Romania than previously known. Citation: CRIVEI, L.A.;
MOUTAILLER, S.; GONZALEZ, G.;
LOWENSKI, S.; CRIVEI, I.C.;
POREA, D.; ANITA, D.C.; RATOI,
I.A.; ZIENTARA, S.; OSLOBANU,
L.E.; et al. Detection of West Nile
Virus Lineage 2 in Eastern Romania
and First Identification of Sindbis
Virus RNA in Mosquitoes Analyzed
using High-Throughput Microfluidic
Real-Time PCR. Viruses 2023, 15, 186. viruses viruses 1. Introduction In recent decades, the impact of emerging and re-emerging infectious diseases on
human and animal health has increased, posing a major challenge for global health and
economy [1]. Roughly two thirds of human infectious diseases originate from wildlife,
and many recently emerging diseases are caused by vector-borne viruses [2]. At least
10 mosquito-borne viruses (MBV) have been detected in Europe, and some of them have
become a major concern in the last decade. The majority of these MBVs belong to the
Flaviviridae family (e.g., West Nile, Dengue or Usutu viruses) [3,4]. Other pathogenic MBVs
of the families Togaviridae (e.g., Sindbis, Chikungunya viruses) and Peribunyaviridae (Tahyna,
Inkoo and Lednice viruses) are known to have medical importance in Europe [5]. West
Nile virus (WNV, genus Flavivirus) is the most widespread encephalitic arthropod-borne
virus [6] and a member of the Japanese encephalitis virus serocomplex. Of its nine potential Detection of West Nile Virus Lineage 2 in Eastern Romania
and First Identification of Sindbis Virus RNA in Mosquitoes
Analyzed using High-Throughput Microfluidic Real-Time PCR Luciana Alexandra CRIVEI 1,*, Sara MOUTAILLER 2
, Gaëlle GONZALEZ 3, Steeve LOWENSKI 3,
Ioana Cristina CRIVEI 1, Daniela POREA 1, Dragos, Constantin ANITA 1
, Ioana Alexandra RATOI 1,
Stéphan ZIENTARA 3
, Luanda Elena OSLOBANU 1, Alexandru TOMAZATOS 4, Gheorghe SAVUTA 1,†
and Sylvie LECOLLINET 3,†,‡ Copyright:
© 2023 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/). https://www.mdpi.com/journal/viruses Viruses 2023, 15, 186. https://doi.org/10.3390/v15010186 Viruses 2023, 15, 186 2 of 14 lineages documented to date [7,8], the main pathogenic WNV strains belong to lineages
1 and 2. However, human cases of acute encephalitis have also been attributed to WNV
lineage 5 (formerly lineage 1c [9]) in India [10]. In the natural enzootic cycle, WNV is
transmitted mainly by ornithophilic Culex mosquitoes as vectors and birds as amplifying
reservoir hosts [9]. West Nile virus is one of the most important viral pathogens in Europe,
and since its initial detection in Albania in 1958, it has become an endemo-epidemic virus
on the continent [11]. Although the great majority of animal species are not susceptible to
WNV infection, humans and equids are incidental hosts which may become ill without
developing viraemia levels high enough for reinfecting a new mosquito during feeding. Most infections remain asymptomatic, while 1–4% lead to neuroinvasion and disease of
the central nervous system [12]. Mortality is usually higher in immunocompromised or
chronically ill hosts [13] and impact on public health is compounded by risks associated
with organ donation and blood transfusion [14]. The currently most affected European regions include south, southeast and central
Europe [15]. In recent years, consecutive vector seasons with high temperatures enabled
WNV to expand its range westwards and at higher latitudes [13,14]. A sharp increase in
human and equid cases has been experienced most acutely by southern and southeastern
regions of the continent as a result of the rapid emergence and spread of West Nile lineage
2 virus first reported in Hungary, in 2004 [8,15]. In Romania, the 1996 epidemic caused by a
lineage 1 WNV strain in southeastern Romania remains the largest one documented in the
country (393 hospitalized cases and 17 deaths) [16] Since then, WNV infections of humans
and equids have been recorded annually. Large outbreaks occurred also in 2010, 2016 and
2018 (277 cases), when WNV activity and impact on public health were particularly high
throughout the country [17–19]. Circulation of WNV in Romania is endemic in southern
(Romanian Plain) and southeastern parts (Constant,a, Tulcea, Danube Delta) [20]. Human
cases have also been detected in the last decade in the western parts of the country (Sibiu). p
y
Unlike WNV, the presence of Sindbis virus (SINV, genus Alphavirus) has been evi-
denced worldwide, though human outbreaks are reported almost exclusively in northern
Europe (Sweden, Finland, Russia) and rarely in South Africa, China and Oceania [21,22]. This alphavirus is the etiological agent of Sindbis fever, characterized mainly by rash,
arthralgia and fever. Although the illness is typically self-limiting, arthralgia and myalgia
can manifest for months or even years, in rare cases resulting in chronic arthritis. Widely
detected in Culex and Culiseta mosquito vectors worldwide, SINV is also [23] found in
vertebrates from Eurasia, Africa and Oceania. In Europe, SINV or anti-SINV antibodies
have been found in more than 20 European countries from all the main regions of the
continent. The presence of SINV in Romania remains doubtful, as animals were reportedly
found seropositive in the mid-1970s by Drăgănescu et al. [24] at very low frequency. Despite endemic circulation of WNV and high incidence of WNV infections in humans,
Romania still lacks an operational integrated surveillance program for arboviruses. This
is also reflected in the scarcity of genetic and serological data, hampering molecular and
genomic epidemiological studies in the region. Obtaining baseline data about arbovirus
spatial distribution, diversity, prevalence in vectors and impact on hosts are necessary steps
for pursuing regional and country-wide integrated surveillance. Therefore, the aims of this
study were to investigate the presence and diversity of MBVs in two counties from eastern
and southeastern Romania: one which is known for endemic circulation of WNV (Tulcea)
and one where sporadic WNV cases occurred in recent years (Ias,i). 2.1. Study Areas and Mosquito Sampling 2.1. Study Areas and Mosquito Sampling Mosquitoes were collected during two consecutive transmission seasons (May–September,
2018 and 2019) in two counties from eastern (Ias,i) and southeastern (Tulcea) Romania. Col-
lection sites were selected by the apparent suitability of habitats for MBV transmission,
including the abundance of vertebrate hosts. Mosquitoes were collected using a com-
bination of CDC Light Trap Miniature traps (BioQuip, Rancho Dominguez, CA, USA), Viruses 2023, 15, 186 3 of 14
ted usi
ngue 3 of 14
ted usi
nguez BG-Sentinel traps (BioGents, Regensburg, Germany), manual aspirator “Pooter” Style (Bio-
Quip, Rancho Dominguez, CA, USA) and the manual aspirator Heavy Duty Hand-Held
DC Vac (BioQuip, Rancho Dominguez, CA, USA). tyle (BioQuip, Rancho Dominguez, CA, USA) and the manual aspirator Heavy D
and-Held DC Vac (BioQuip, Rancho Dominguez, CA, USA). Sampling in Iași was conducted in three urban sites, one peri-urban site (a pri BG-Sentinel traps (BioGents, Regensburg, Germany), manual aspirator “Pooter” Style (Bio-
Quip, Rancho Dominguez, CA, USA) and the manual aspirator Heavy Duty Hand-Held
DC Vac (BioQuip, Rancho Dominguez, CA, USA). yle (BioQuip, Rancho Dominguez, CA, USA) and the manual aspirator Heavy D
and-Held DC Vac (BioQuip, Rancho Dominguez, CA, USA). Sampling in Iași was conducted in three urban sites, one peri-urban site (a pri Sampling in Ias,i was conducted in three urban sites, one peri-urban site (a private
household) and one natural site (game reserve). The urban sampling sites were highly
anthropogenic, characterized by a mix of wooded areas (campus, parks), water bodies, high
density of bird hosts and availability of artificial resting and breeding sites (car tire depots
and old buildings). The peri-urban and natural sites were chosen based on ecological
characteristics favorable to MBV transmission: wetland, density of wildlife hosts including
birds, abundance of vectors (game reserve), domestic mammal hosts and synanthropic
birds (private household) (Figure 1). ousehold) and one natural site (game reserve). The urban sampling sites were hi
nthropogenic, characterized by a mix of wooded areas (campus, parks), water bod
igh density of bird hosts and availability of artificial resting and breeding sites (car
epots and old buildings). The peri-urban and natural sites were chosen based on eco
al characteristics favorable to MBV transmission: wetland, density of wildlife host
uding birds, abundance of vectors (game reserve), domestic mammal hosts and syn
hropic birds (private household) (Figure 1). Figure 1. Mosquito sampling sites in eastern Romania used in this study (2018–2019) (I). 2.1. Study Areas and Mosquito Sampling Up
right panel shows the sampling sites in Iași county (II) and the lower-left panel indicates the
ping locations in Tulcea county (III). Purple circles represent urban sites, green circles repre
natural sites and the orange circle indicates peri-urban sampling sites. Natural site 1 = A, peri-u
site 1 = B, urban site 1 = C, urban site 2 = D, urban site 3 = E, natural site 2 = F, urban site 4 = G. Figure 1. Mosquito sampling sites in eastern Romania used in this study (2018–2019) (I). Upper-right
panel shows the sampling sites in Ias,i county (II) and the lower-left panel indicates the trapping
locations in Tulcea county (III). Purple circles represent urban sites, green circles represent natural
sites and the orange circle indicates peri-urban sampling sites. Natural site 1 = A, peri-urban site 1 = B,
urban site 1 = C, urban site 2 = D, urban site 3 = E, natural site 2 = F, urban site 4 = G. gure 1. Mosquito sampling sites in eastern Romania used in this study (2018–2019) (I). Up
ght panel shows the sampling sites in Iași county (II) and the lower-left panel indicates the
ng locations in Tulcea county (III). Purple circles represent urban sites, green circles repre
atural sites and the orange circle indicates peri-urban sampling sites. Natural site 1 = A, peri-u
te 1 = B, urban site 1 = C, urban site 2 = D, urban site 3 = E, natural site 2 = F, urban site 4 = G. Figure 1. Mosquito sampling sites in eastern Romania used in this study (2018–2019) (I). Upper-right
panel shows the sampling sites in Ias,i county (II) and the lower-left panel indicates the trapping
locations in Tulcea county (III). Purple circles represent urban sites, green circles represent natural
sites and the orange circle indicates peri-urban sampling sites. Natural site 1 = A, peri-urban site 1 = B,
urban site 1 = C, urban site 2 = D, urban site 3 = E, natural site 2 = F, urban site 4 = G. Two sites were selected in Tulcea county for opportunistic collection of mosqui
n July and September 2019. The first site was located in an urban area comprising
ential/commercial complexes. 2.1. Study Areas and Mosquito Sampling The second site was located in a natural area with a r
vely low degree of anthropization adjacent wetlands and a horse shelter
Two sites were selected in Tulcea county for opportunistic collection of mosquitoes in
July and September 2019. The first site was located in an urban area comprising residen-
tial/commercial complexes. The second site was located in a natural area with a relatively
low degree of anthropization, adjacent wetlands and a horse shelter. 2.3. RNA Extraction 2.3. RNA Extraction Mosquito pools were homogenized at 5500 rpm in 2 mL tubes containing silica beads
(0.1 mm diameter, BioSpec, Bartlesville, OK, USA) and 500 µL of DMEM with 10% fetal
calf serum on a Precellys 24 Dual homogenizer (Bertin, Montigny-le-Bretonneux, France). The homogenate was clarified by centrifugation for 2 min at 1.500 rpm and 120 µL of
the supernatant was used for RNA extractions using the MagVet Universal Isolation kit
(Lifetechnology, MA, USA) and MagMAX Express-96 Deep Well Magnetic Particle Processor
workstation (Thermo Fisher Scientific, MA, USA). The remaining homogenate was stored
at −80 ◦C for subsequent virus isolation. 2.5. High-Throughput Microfluidic Real-Time PCR Mosquito-borne viruses, their targeted genes and the corresponding primers/probe sets
were selected according to Moutailler et al. [26]. For this study, 365 field-collected mosquito
pools were screened for 37 viruses, targeting a total of 94 genes (Supplementary Table S1). The pre-amplified cDNA was subjected to high-throughput microfluidic real-time PCR
amplification using the 96.96 dynamic arrays (Starlab Biotools, Hamburg, Germany). For
the purpose of this assay, the BioMark real-time PCR system (Starlab Biotools, Hamburg,
Germany) was used. High-throughput real-time PCRs were performed using FAM and
black hole quencher (BHQ1)-labeled TaqMan probes with TaqMan Gene Expression Master
Mix in accordance with manufacturer’s instructions (Applied Biosystems, MA, USA). The
reactions were performed for 2 min at 50 ◦C and 10 min at 95 ◦C, followed by 40 cycles of
two-step amplifications of 15 s at 95 ◦C and, finally, 10 min at 60 ◦C. p
p
y
Data were acquired on the BioMark real-time PCR system and analyzed using the
Fluidigm real-time PCR Analysis software to obtain cycle threshold values, which for
positive samples ranged between 16.3 and 26.5. False positive or inconclusive (unreliable)
results were declared at Ct > 30. Primers and probes were evaluated before use for their
specificity against cDNA reference samples (see [26] for details). One negative water control
was included per chip. To determine if factors present in the sample could inhibit the PCR,
Escherichia coli strain EDL933 DNA was added to each sample as an internal inhibition
control, using primers and probes specific for the E. coli eae gene [26]. 2.4. Reverse Transcription and cDNA Pre—Amplification Reverse transcription of RNA extracts was performed using Reverse Transcriptase
Master Mix (Starlab Biotools, Hamburg, Germany). One µL extracted RNA was added
with 1 µL of Reverse Transcription Master Mix to 3 µL of RNase free water, resulting in a
total reaction volume of 5 µL. The reaction’s thermal profile consisted of one cycle of 5 min,
at 25 ◦C, one step of 30 min, at 42 ◦C, and one last cycle, at 85 ◦C, for 5 min. Pre-amplification reactions were performed using the PreAmp Master Mix Kit (Starlab
Biotools, Hamburg, Germany), following the manufacturer’s instructions. Pre-amplification
allowed a better amplification of viral cDNA relative to mosquito cDNA. The pre-amplification
reaction used 1 µL of the master mix, 1.25 µL of cDNA, 1.25 µL of pooled primers (all the
primers targeting arboviruses) and 1.5 µL distilled water. This operation was conducted
using the following thermal conditions: 95 ◦C for 2 min, 14 cycles at 95 ◦C for 15 s, and
4 min at 60 ◦C. After the pre-amplification step, cDNAs/amplicons were diluted 1:5 in
pure water and stored at −20 ◦C until further analysis. vely low degree of anthropization, adja
2.2. Mosquito Identification and Processing 2. Mosquito Identification and Processing
Mosquitoes were brought alive to the laboratory in catch nets and killed by de
eezing for 5 min, at −20 °C. Females were sorted according to gonotrophic stage (un
Mosquitoes were brought alive to the laboratory in catch nets and killed by deep-
freezing for 5 min, at −20 ◦C. Females were sorted according to gonotrophic stage (un-
fed, fed, gravid) and identified on chilled tablets using morphological keys [25]. Unfed
mosquitoes were pooled according to the taxa, sex, gonotrophic stage, collection date and
site. After identification, monospecific and monogeneric pools (of individuals which could
not be identified below the genus level), were transferred to −80 ◦C until viral screen-
ing. Blood-fed females were screened individually, while the unfed mosquito pools were
analyzed in monospecific pool, each containing 20–50 specimens (“group testing”). Viruses 2023, 15, 186 4 of 14 y
95 ◦C for 15 s and 60 ◦C for 1 min. 2.7. Calculation of Minimum Infection Rates Considering that the expected prevalences of WNV and other MBVs in mosquitoes are
lower than 0.1% in Europe and assuming that generally a single individual was infected
within a positive pool, prevalences were expressed as the minimum infection rate per
1000 tested individuals (MIR), by calculating the ratio of the positive pools to the total
mosquitoes tested. Considering that the expected prevalences of WNV and other MBVs in mosquitoes are
lower than 0.1% in Europe and assuming that generally a single individual was infected
within a positive pool, prevalences were expressed as the minimum infection rate per
1000 tested individuals (MIR), by calculating the ratio of the positive pools to the total
mosquitoes tested. 2.8. Sequencing and Phylogenetic Analysis of WNV Amplification of partial NS5 gene by conventional pan-flavivirus PCR was performed on
positive mosquito pool homogenates and virus isolates, as described by Weissenböck et al. [30]. Amplification of partial NS5 gene by conventional pan-flavivirus PCR was performed on
positive mosquito pool homogenates and virus isolates, as described by Weissenböck et al. [30]. Amplification was achieved under the following thermal cycling conditions: 30 min at
50 ◦C, 15 min at 95 ◦C, 45 cycles at 94 ◦C for 30 s, 60 ◦C for 30 s, 72 ◦C for 1 min and 10 min
at 72 ◦C. Resulting amplicons were visualized by electrophoresis (30 min at 90V) on a 1%
agarose gel in the presence of a 1kb ladder (Hyperladder, Bioline, London, UK). g
g
y
Sanger sequencing was performed in both directions and the resulting nucleotide
sequence was identified by basic alignment search tool (BLAST) in the Genbank database
(https://blast.ncbi.nlm.nih.gov/, accessed on 30 November 2022). Homologous sequences
were retrieved and aligned using the MAFFT algorithm implemented in Geneious Prime
(Biomatters, Auckland, New Zealand). To assess the phylogenetic relationship of the new
WNV isolates, a maximum likelihood phylogenetic tree rooted by WNV lineage 3 (Rabens-
burg strain) was built with MEGA 11 [31] following substitution model selection by Mega
11 and jModelTest 2.1.10 [32]. Robustness of tree nodes was assessed by 1000 bootstraps,
and the result was displayed with iTOL v5 [33]. 2.6. Validation by Virus Isolation and Partial Genome Sequencing WNV-positive pools detected by microfluidic real-time PCR assays were further
screened by WNV real-time PCR using Applied Biosystems StepOnePlus Instrument as
described by Linke et al. [27]. The quantitative duplex real-time PCR used FAM–TAMRA-
labeled probes for WNV and VIC–TAMRA for the endogenous β-actin RNA, as described
in [28] (Supplementary Table S2). An initial reverse transcription step was performed at
45 ◦C for 10 min, followed by DNA denaturation at 95 ◦C for 10 min, and 45 PCR cycles at Viruses 2023, 15, 186 5 of 14 After confirmation of WNV positive samples by duplex real-time PCR, virus isolation
was performed on freshly prepared semi-confluent monolayers of Vero cells (ATCC CCL81)
maintained in Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with 1%
L-glutamine, 1% non-essential amino acids, 1% penicillin/streptomycin, 1 mM sodium
pyruvate and 10% fetal bovine serum (FBS), at 37 ◦C and 5% CO2. T25 flasks of Vero cells
were inoculated with 1 mL inoculum, consisting of 100 µL of 0.45 µm-filtered mosquito
homogenate and 900 µL DMEM. Seven days post-infection (dpi), cell supernatants were
recovered, aliquoted and stored at −80 ◦C for further passage in cell culture, while RNA
was extracted using QIAmp Viral RNA Mini QIAcube Kit (Qiagen, Hilden, Germany) and
the QIAcube robot (Qiagen, Hilden, Germany), following the manufacturer’s instructions. g
y
g
Virus supernatants recovered from infected Vero cells were further propagated on
C6/36 cells (ATCC CRL-1660). An amount of 1 ml of supernatants was added on confluent
monolayers of C6/36 cells. Aedes albopictus-derived C6/36 cells were maintained at 28◦C in
Leibovitz’s L-15 medium, supplemented with 10% FBS, 1% L-glutamine, 1% non-essential
amino acids, 1% penicillin/streptomycin and 1 mM sodium pyruvate. The supernatant
was harvested at 7 dpi, and C6/36 RNA extracts were checked for the presence of WNV
RNA by real-time PCR. y
The positive SINV pool detected by microfluidic real-time PCR assays was further
screened by conventional SINV PCR, as described by Lundström and Pfeffer [29], and viral
isolation was attempted on Vero and C6/36 cell cultures (Supplementary Table S2). 2.7. Calculation of Minimum Infection Rates 3.1. Mosquito Trapping Between June 2018 and September 2019, we collected 17,694 female mosquitoes repre-
senting 11 taxa in four urban, one peri-urban and two natural sites from two counties in
eastern and southeastern Romania. From this collection, seven taxa which are known to
be MBV vectors (including the Aedes spp. pools) were screened using a high-throughput
microfluidic real-time PCR targeting 37 viruses from 4 families (Peribunyaviridae, Flaviviridae,
Reoviridae, Togaviridae). Viruses 2023, 15, 186 6 of 14
from
A d Aedes vexans (43.77%) and Culex pipiens s.l. (29.21%) were the most commonly detected
of the 11 mosquito taxa identified (Figure 2). The rest of the identified taxa represented
between 0.03 % and 7.34% of the mosquito collection. From the total collection, 6 taxa of the Aedes and Culex genera, along with the uniden-
tified Aedes spp. individuals, (n = 16,827, 95.1%) were pooled (n = 365) and subjected to
virus screening by high-throughput microfluidic real-time PCR. Figure 2. Number of mosquito specimens trapped in 2018 and 2019 in eastern Romania, ordered by
taxa and sampling site (Iași county in the left panel, Tulcea in the central panel, and total per taxa
on the right panel). Figure 2. Number of mosquito specimens trapped in 2018 and 2019 in eastern Romania, ordered by
taxa and sampling site (Ias,i county in the left panel, Tulcea in the central panel, and total per taxa on
the right panel). Aedes vexans (43.77%) and Culex pipiens s.l. (29.21%) were the most commonly detected
of the 11 mosquito taxa identified (Figure 2). The rest of the identified taxa represented
between 0.03 % and 7.34% of the mosquito collection. From the total collection, 6 taxa of the Aedes and Culex genera, along with the uniden-
tified Aedes spp. individuals, (n = 16,827, 95.1%) were pooled (n = 365) and subjected to
virus screening by high-throughput microfluidic real-time PCR. Figure 2. Number of mosquito specimens trapped in 2018 and 2019 in eastern Romania, ordered by
taxa and sampling site (Iași county in the left panel, Tulcea in the central panel, and total per taxa
on the right panel). Figure 2. Number of mosquito specimens trapped in 2018 and 2019 in eastern Romania, ordered by
taxa and sampling site (Ias,i county in the left panel, Tulcea in the central panel, and total per taxa on
the right panel). 3.2. Virus Detection
3.2.1. 3.1. Mosquito Trapping Detection of Viral Pathogens Using High-Throughput Microfluidic Real-Time
PCRs
The largest number of individuals was captured in the natural sites from the two coun-
ties, with 8727 specimens (49.3%) collected in Ias,i and 6863 (38.8%) in Tulcea. Consequently,
in these sites, we recorded the highest taxonomic diversity, amounting to nine taxa in the
natural site from Ias,i (natural site 1) and eight taxa in the natural site from Tulcea (natural
site 2). The largest sample in urban environments was obtained in Ias,i, in urban site 1
(1286 specimens from three taxa), followed by urban site 2 (551 specimens from three taxa),
urban site 3 (10 specimens of Culex pipiens s.l.) and urban site 4 in Tulcea (4 individuals
from three taxa) (Figure 2). The majority of the identified mosquitoes were collected in Ias,i
county (n = 10,827, 61.2%, versus 6,867 individuals from Tulcea, 38.8%). Aedes vexans and
Culex pipiens s.l. were the dominant taxa in the majority of sampling sites and the overall
collection (Figure 2). Exceptions were found in the urban sites 2 and 3 from Ias,i and urban
site 4 from Tulcea, where Culex pipiens s.l. was the main taxon and Aedes vexans were absent. F
h
l
ll
i
6
f h A d
d C l
l
i h h
id
i p p
From the total collection, 6 taxa of the Aedes and Culex genera, along with the unidenti-
fied Aedes spp. individuals, (n = 16,827, 95.1%) were pooled (n = 365) and subjected to virus
screening by high-throughput microfluidic real-time PCR. 3.2. Virus Detection
3.2.1. Detection of Viral Pathogens Using High-Throughput Microfluidic Real-Time PCRs 3.2. Virus Detection Purple circles represent urban sites, green circles represent the natural sites an
the orange circle indicates the peri–urban sampling site. Natural site 1 = A, peri-urban site 1 = B
urban site 1 = C, urban site 2 = D, urban site 3 = E, natural site 2 = F, urban site 4 = G. Figure 3. Mosquito screening results relative to the distribution of sampling sites in the two counties
in eastern Romania. Purple circles represent urban sites, green circles represent the natural sites and
the orange circle indicates the peri–urban sampling site. Natural site 1 = A, peri-urban site 1 = B,
urban site 1 = C, urban site 2 = D, urban site 3 = E, natural site 2 = F, urban site 4 = G. igure 3. Mosquito screening results relative to the distribution of sampling sites in the two counti
n eastern Romania. Purple circles represent urban sites, green circles represent the natural sites an
he orange circle indicates the peri–urban sampling site. Natural site 1 = A, peri-urban site 1 =
urban site 1 = C, urban site 2 = D, urban site 3 = E, natural site 2 = F, urban site 4 = G. Figure 3. Mosquito screening results relative to the distribution of sampling sites in the two counties
in eastern Romania. Purple circles represent urban sites, green circles represent the natural sites and
the orange circle indicates the peri–urban sampling site. Natural site 1 = A, peri-urban site 1 = B,
urban site 1 = C, urban site 2 = D, urban site 3 = E, natural site 2 = F, urban site 4 = G. able 1. Virological findings in mosquitoes collected in Iași and Tulcea counties, in 2018 and 201
he number of tested mosquitoes (Nt) and tested pools (NP) and WNV and SINV positive pools a
Table 1. Virological findings in mosquitoes collected in Ias,i and Tulcea counties, in 2018 and 2019. The
number of tested mosquitoes (Nt) and tested pools (NP) and WNV and SINV positive pools are indicated. The number of tested mosquitoes (Nt) and tested pools (NP) and WNV and SINV positive pools ar
indicated. Species
NP
Nt
WNV+
Date of
Collection
of Positive
Pool
Site
SINV +
Date of
Collection
Site
Aedes vexans
167
7745
1
9.07.2019
Natural site 2 (F)
Culex
pipiens s.l. 3.2. Virus Detection 3.2.1. Detection of Viral Pathogens Using High-Throughput Microfluidic Real-Time PCRs Through the screening of 37 MBVs using high-throughput microfluidic real-time PCRs,
a total of 11 out of 365 (3%) mosquito pools were found positive for WNV (10 pools 2.73%)
and SINV (1 pool, 0.27%) (Figure 3). Three mosquito taxa were found to be WNV positive: Culex pipiens s.l. (eight pools),
Culex modestus (one pool) and Aedes vexans (one pool) (Table 1). The majority of WNV-
positive pools originated from natural site 1 (Ias,i, 5/127), followed by natural site 2 (Tulcea,
3/127) and urban site 2 (Ias,i, 2/12). Culex pipiens s.l. was found to be WNV positive in each
month from June to September (2018) in both natural sites (1 and 2) and urban site 2. In all
these sites, the habitat consisted of wooded landscape and wetland. Culex modestus positive
for WNV was detected in August 2019 in urban site 2. Positive Aedes vexans were collected Viruses 2023, 15, 186 7 of 14
an site
d t 7 of 14
an site
d t in July 2019 at the natural site 2, from Tulcea. Sindbis virus was detected in a single pool of
Culex modestus sampled in June 2018, at the natural site 1, in Ias,i (Figure 3). collected in July 2019 at the natural site 2, from Tulcea. Sindbis virus was detected in
single pool of Culex modestus sampled in June 2018, at the natural site 1, in Iași (Figure 3 in July 2019 at the natural site 2, from Tulcea. Sindbis virus was detected in a single pool of
Culex modestus sampled in June 2018, at the natural site 1, in Ias,i (Figure 3). collected in July 2019 at the natural site 2, from Tulcea. Sindbis virus was detected in
single pool of Culex modestus sampled in June 2018, at the natural site 1, in Iași (Figure 3 in July 2019 at the natural site 2, from Tulcea. Sindbis virus was detected in a single pool of
Culex modestus sampled in June 2018, at the natural site 1, in Ias,i (Figure 3). collected in July 2019 at the natural site 2, from Tulcea. Sindbis virus was detected in
single pool of Culex modestus sampled in June 2018, at the natural site 1, in Iași (Figure 3 Figure 3. Mosquito screening results relative to the distribution of sampling sites in the two countie
in eastern Romania. 3.2. Virus Detection 116
5169
8
19.08.2018
Natural site 1 (A)
8.09.2018
Natural site 1 (A)
23.08.2018
Natural site 1 (A)
14.09.2018
Natural site 1 (A)
14.08.2019
Urban site 2 (D)
9.07.2019
Natural site 2 (F)
10.07.2019
Natural site 2 (F)
11.08.2019
Natural site 1 (A)
Culex modes-
tus
26
1300
1
30.08.2019
Urban site 2 (D)
1
14.06.2018
Natural
site 1 (A)
number of tested mosquitoes (Nt) and tested pools (NP) and WNV and SINV positive pools are indicated. Species
NP
Nt
WNV+
Date of Collection
of Positive Pool
Site
SINV +
Date of
Collection
Site
Aedes vexans
167
7745
1
9 July 2019
Natural site 2 (F)
Culex pipiens s.l. 116
5169
8
19 August 2018
Natural site 1 (A)
8 September 2018
Natural site 1 (A)
23 August 2018
Natural site 1 (A)
14 September 2018
Natural site 1 (A)
14 August 2019
Urban site 2 (D)
9 July 2019
Natural site 2 (F)
10 July 2019
Natural site 2 (F)
11 August 2019
Natural site 1 (A)
Culex modestus
26
1300
1
30.08.2019
Urban site 2 (D)
1
14 June 2018
Natural site 1 (A)
Aedes caspius
24
1044
0
Ochlerotatus
sticticus
8
400
0
Aedes spp. 23
1119
0
Aedes intrudens
1
50
0
365
16,827
10
1
Overall, the number of WNV-positive pools was similar in 2018 (n = 4) and 2019 (n = 6),
respectively. The minimum infection rate (MIR) for WNV ranged between 0.34 for Aedes
vexans to 3.54 for Culex pipiens s.l. in Ias,i in 2018. The MIR for SINV-positive Culex modestus
was 1.33 (Table 2). Overall, the number of WNV-positive pools was similar in 2018 (n = 4) and 2019 (n = 6),
respectively. The minimum infection rate (MIR) for WNV ranged between 0.34 for Aedes
vexans to 3.54 for Culex pipiens s.l. in Ias,i in 2018. The MIR for SINV-positive Culex modestus
was 1.33 (Table 2). Viruses 2023, 15, 186 8 of 14 Table 2. MIR by WNV for Culex pipiens s.l., Culex modestus and Aedes vexans mosquitoes, per time
period (2018 or 2019) and county (Ias,i or Tulcea). 2019
2018
Culex
pipiens s.l. Culex
modestus
Aedes
vexans
Culex
pipiens s.l. 3.2. Virus Detection Culex
modestus
Ias,i
Total WNV-positive pools
2
1
0
4
0
Total analyzed specimens
2576
550
4460
1128
750
MIR WNV
0.77
1.81
0.00
3.54
0.00
Total SINV-positive pools
0
0
0
0
1
Total analyzed specimens
2576
550
4460
1128
750
MIR SINV
0.00
0.00
0.00
0.00
1.33
Tulcea
Total WNV-positive pools
2
0
1
Total analyzed specimens
1465
0
2878
MIR WNV
1.36
0.00
0.34 Table 2. MIR by WNV for Culex pipiens s.l., Culex modestus and Aedes vexans mosquitoes, per time
period (2018 or 2019) and county (Ias,i or Tulcea). 3.2.2. Validation of Pathogen Detection by Monospecific Real-Time RT-PCR and Virus Isolation Confirmation of WNV infection was carried out on positive pools evidenced in high-
throughput microfluidic assays by real-time RT-PCR and by virus isolation. The real-time
RT-PCR assay targeted a different fragment of the WNV genome (e.g., the 5′ non coding
region and part of the capsid coding sequence) [27]; Ct values ranging between 20.7 and
32.6 were obtained for every WNV-positive pool (Supplementary Table S3). Successful virus isolation, as assessed by observation of cytopathic effects (CPE), was
obtained in 4 of 10 inoculated Vero cell cultures and for one sample in C6/36 cells. The Culex modestus pool found positive by real-time microfluidic SINV PCRs did not
induce CPE on Vero and C6/36 cells, nor was positive in conventional SINV RT-PCR [29]. 3.3. Phylogenetic Analysis Two partial NS5 sequences were obtained from positive pools of Culex pipiens s.l. Bioinformatic analysis of these sequences indicated that both mosquito homogenates,
originating from Culex pipiens s.l. pools collected on the 14th of September 2018 and 11th of
August 2019, respectively, were infected by WNV lineage 2 strains of the Central-Southeast
European clade (Figure 4). 9 of 14
-South- Viruses 2023, 15, 186 Figure 4. Maximum likelihood phylogenetic analysis of WNV lineage 2 partial NS5 gene. Romanian
strains are indicated in blue (previous studies) and red color (present study). Rabensburg strain 97–
103 was used as an outgroup, and bootstrap support >80% is displayed at nodes of major clades. Figure 4. Maximum likelihood phylogenetic analysis of WNV lineage 2 partial NS5 gene. Romanian
strains are indicated in blue (previous studies) and red color (present study). Rabensburg strain
97–103 was used as an outgroup, and bootstrap support >80% is displayed at nodes of major clades. Figure 4. Maximum likelihood phylogenetic analysis of WNV lineage 2 partial NS5 gene. Romanian
strains are indicated in blue (previous studies) and red color (present study). Rabensburg strain 97–
103 was used as an outgroup, and bootstrap support >80% is displayed at nodes of major clades. Figure 4. Maximum likelihood phylogenetic analysis of WNV lineage 2 partial NS5 gene. Romanian
strains are indicated in blue (previous studies) and red color (present study). Rabensburg strain
97–103 was used as an outgroup, and bootstrap support >80% is displayed at nodes of major clades. 4. Discussion The same can be observed for Aedes vexans, whose secondary
role is reflected by the lowest MIR found in the present study. The higher prevalence of
WNV observed in our study in 2018 was also reflected by the high number of human
cases reported in Ias,i, suggesting that high levels of enzootic WNV circulation resulted
in more frequent spill-over to humans during this vector season. This epidemiological
scenario was observed in 2018 by other European countries where incidence rates in
humans grew sharply along with new reports of infections with the closely related Usutu
virus [45–48]. In parallel with increasing incidence in endemic regions, during the unusually
hot seasons of 2018 and following years, WNV expanded its range into northwestern
Europe [49,50], where epizootics affecting birds and equids were followed the next season
by WNV epidemics [49]. Following the large epidemic of 1996 in southeastern Romania, the causative WNV
lineage 1 strain was replaced by WNV lineage 2 strains of the Eastern European clade (EEC,
Danube Delta, Volga Delta, Ukraine) and the Central-Southeastern European clade (CSEC). Lineage 2 was first detected in Romania in 2010, as closely related to the 2007 Volgograd
outbreak strain (EEC). The epidemiological data showed that the EEC strains dominate transmission in
Romania from 2010-2015 in mosquitoes collected in the Danube Delta [41] and Bucharest
while CSEC strains were detected from 2015 onwards and had replaced earlier EEC lineage
2 strains by 2016 [51]. Lineage 2 strains had not been evidenced previously in Ias,i county,
although WNV infections in humans are reported annually, and sustained WNV circulation
has been evidenced through prevalence studies in horses [20,42] and dogs [42,52]. Bird migration is considered one of the main mechanisms of WNV introduction and
spread [34]. For Europe, intercontinental flight along the main corridors of the Afro-
Eurasian migration network is generally accepted as a mechanism of introduction for new
WNV strains, and it may play a significant role at major migratory hubs. However, new
introductions from Africa are infrequent, and WNV relies more on short-range movements
of infected birds where local mosquito populations acquire the virus for local transmission. The presence of the virus is also associated with intensive farming, mammal richness,
urbanization and wetland coverage [53]. These environmental factors are conducive to
viral transmission as they support rich and dense communities of hosts and mosquito
vectors. 4. Discussion 4. Discussion
Vector-borne diseases are a growing concern worldwide, especially against a back-
drop of rapid environmental changes, such as rapid urbanization, expanding networks
for the mobility of goods and people, as well as changing climatic patterns. These factors
favor expansion of MBVs toward higher latitudes in Europe, as conditions become more
suitable for the mosquito vectors to colonize new regions. Furthermore, growing temper-
atures support virus transmission by supporting vector population reproduction and
Vector-borne diseases are a growing concern worldwide, especially against a backdrop
of rapid environmental changes, such as rapid urbanization, expanding networks for the
mobility of goods and people, as well as changing climatic patterns. These factors favor
expansion of MBVs toward higher latitudes in Europe, as conditions become more suitable
for the mosquito vectors to colonize new regions. Furthermore, growing temperatures
support virus transmission by supporting vector population reproduction and shortening
extrinsic incubation periods. This is especially the case of WNV, which, in the last two
decades, has established endemo-epidemic transmission in new regions of Europe [15,34]. Concomitantly, recurring autochthonous outbreaks of DENV in southern France, northern
Italy and Croatia [35–37] highlighted the high introduction risk associated with MBVs
transmitted by Aedes mosquitoes. Viruses 2023, 15, 186 10 of 14 10 of 14 Most mosquito taxa were found by our study in the natural sampling sites from
Ias,i and Tulcea, where ecological characteristics seem to support higher vector diversity. Aedes vexans were found positive for WNV for the first time in Romania. Studies of vector
competence have shown the ability of this species to transmit WNV [38,39]. However, its
mammophilic character suggests that Aedes vexans could play only a minor role in WNV
transmission, unlike the ornitophilic/generalist Culex pipiens s.l., which is the main WNV
vector and was repeatedly found infected in natural and anthropic areas from the east and
southeast of the country [40–42]. Previous investigations of WNV epidemic activity have
shown that the bioforms of Culex pipiens are thriving in urban settings where reservoir
host populations reach high densities and particular urbanization features enable high
mosquito densities near human habitation [43,44]. Hence, the detection of WNV in Culex
modestus and Culex pipiens s.l. from the urban site 2 from Ias,i warrants the implementation
of surveillance in urban and peri-urban settings. g
The prominent role of Culex pipiens s.l. as a WNV vector in the studied areas is also
indicated by the overall MIR. 4. Discussion Our results reflect this scenario, as we found WNV-positive mosquito pools in both
natural and urban sites sharing key ecological features (water bodies, high density of birds
and wooded areas). Of the nine endemic MBVs known in Europe, WNV, USUV and SINV are transmitted
by Culex mosquitoes [54]. The widespread presence of SINV in Europe was assessed mainly
by serological methods [21], and for some countries, the data are outdated. Isolation of
SINV has been performed to date only from mosquitoes collected in Sweden, Norway,
Finland, Germany, and Russia [55]. Recent serosurveys conducted in Romania on migratory
birds [56] did not confirm the SINV activity reported by Draganescu et al., in the 1970s [24]. Viruses 2023, 15, 186 11 of 14 11 of 14 Despite the SINV RNA detection, we could not isolate SINV, nor amplify it by PCR, thus
making it unclear whether we detected only SINV genome fragments. This issue represents
the main limitation of the study, and further studies are needed to assess the status of SINV
in Romania. Despite the SINV RNA detection, we could not isolate SINV, nor amplify it by PCR, thus
making it unclear whether we detected only SINV genome fragments. This issue represents
the main limitation of the study, and further studies are needed to assess the status of SINV
in Romania. The incidence of West Nile virus infection has increased in Romania and other Eu-
ropean countries in the last decade, as well as the geographic range of the virus. The
integrated surveillance approach used by several European countries is much needed in
a country such as Romania where WNV is firmly established. Annual counts of human
and equid infection in Romania in 2010–2018 were among the highest in Europe, surpassed
only by those of Italy, Serbia and Greece, countries which have integrated surveillance
programs [57]. The current study provides insights into the viral diversity and circulation
of MBVs in eastern/southeastern Romania. The CSEC clade of WNV was found to have a
wider distribution in Romania than previously known. Despite the first molecular detection
of SINV in the country, our results do not conclusively show its establishment in Romania. Such preliminary data, however, seem to indicate that a wider array of MBVs than the
ones historically reported could be present in Romania and should promote continuous
sampling efforts, surveillance and molecular characterization of MBVs in the country. 4. Discussion Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/v15010186/s1, Table S1: Virus species targeted in high-throughput
microfluidic real-time PCR and number of PCR designs per virus species, Table S2: Comparison of the
targets, the primers/probe sets used for WNV and SINV detection by high-throughput microfluidic
real-time PCR amplification and validation of the results by real-time PCRs screening for WNV
and SINV, Table S3: Mosquito species, collection site and Ct obtained in WNV microfluidic and
confirmatory real-time PCRs. Author Contributions: Conceptualization, L.A.C., G.S., S.M. and S.L. (Sylvie Lecollinet); method-
ology, L.A.C., G.S., S.M. and S.L. (Sylvie Lecollinet); software, A.T.; validation, G.S., S.M. and
S.L. (Sylvie Lecollinet); formal analysis, A.T. and L.A.C.; investigation, L.A.C., I.C.C., I.A.R., D.P.,
D.C.A., L.E.O., G.G. and S.L. (Steeve Lowenski); resources, L.A.C., L.E.O., D.C.A., S.M., G.S. and
S.L. (Sylvie Lecollinet); data curation, A.T. and L.A.C.; writing—original draft preparation, L.A.C.;
writing—review and editing, S.L. (Sylvie Lecollinet), A.T., S.M. and S.Z.; visualization, L.A.C., G.S.,
A.T. and S.L. (Sylvie Lecollinet); supervision, G.S., S.M. and S.L. (Sylvie Lecollinet); project administra-
tion, G.S. and S.L. (Sylvie Lecollinet); funding acquisition, G.S., S.Z., S.M. and S.L. (Sylvie Lecollinet). All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The sequences are submitted in the GenBank database under accession
numbers OQ134778 and OQ134779. Data Availability Statement: The sequences are submitted in the GenBank database under accession
numbers OQ134778 and OQ134779. Data Availability Statement: The sequences are submitted in the GenBank database under accession
numbers OQ134778 and OQ134779. Acknowledgments: We would like to thank Andreea Paula Cozma for help on the field and assistance
with sample processing, and S, tefan Răileanu for facilitating mosquito collection in Tulcea. Acknowledgments: We would like to thank Andreea Paula Cozma for help on the field and assistance
with sample processing, and S, tefan Răileanu for facilitating mosquito collection in Tulcea. 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. References 2017, 22, 28–36. [CrossRef] 9. Ciota, A.T. West Nile virus and its vectors. Curr. Opin. Insect Sci. 2017, 22, 28–36. [CrossRef] 10. Bondre, V.P.; Jadi, R.S.; Mishra, A.C.; Yergolkar, P.N.; Arankalle, V.A. West Nile virus isolates from India: Evidence for a distinct
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2021, 26, 2001095. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. References Epidemiol. 2019, 9, 1698903. [CrossRef] [PubMed] 55. Ayhan, N.; Hachid, A.; Thirion, L.; Benallal, K.E.; Pezzi, L.; Khardine, F.A.; Benbetka, C.; Benbetka, S.; Harrat, Z.; Charrel, R. Detection and Isolation of Sindbis Virus from Field Collected Mosquitoes in Timimoun, Algeria. Viruses 2022, 14, 894. [CrossRef]
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author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
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in-academy.uz/index.php/yo madaniyatida), “as false as fox”, “as sly as a fox”, “as cunning as a fox” (ingliz tilida). Shuningdek, o‘zbek tilining o‘ziga xos turg‘un o‘xshatishlari sifatida “qo‘ydek yuvosh”,
“arvohday ozg‘in”, “echkidek qaysar”,“musichaday beozor” kabilarni misol qilib ko‘rsatish
mumkin. “Musichaday beozor “o‘xshatish etalonida milliy-madaniy konnotatsiya mavjud,
unda “beozorlik” belgisining o‘zbek xalqiga xos ta’kidi yaqqol o‘z ifodasini topgan. Bora-bora
musichadek beozor dadam chindan ham oyimdan qo‘rqishiga ishondim. (O‘.Hoshimov. Ikki
eshik orasi). Ingliz lingvomadaniyatida esa “beozorlik” timsoli sifatida “kabutar” qabul qilinganini kuzatish
mumkin: As harmless as a dove (Kabutarday beozor). She spread about her beauty for a snare,
harmless as doves. (Christina Rosetti) Yoki koreys tilida “sigirday beozor” birikmasini uchratish mumkin. O‘zbek tilidagi ”qo‘yday
yuvosh“etaloni boshqa tillarda “yuvoshlik“ emas ,balki qo‘rqoqlik, qaramlik, bo‘ysinuvchanlik,
mustaqil fikrga ega emaslik belgisini ifodalash uchun ishlatiladi. Bu ham o‘ziga xos milliy-
madaniy qarash mahsulidir. Hamma narsaning oqibatini o‘ylayverib, qo‘yday yuvosh ,
chumchuqday qo‘rqoq bo‘p qopman-, Sadir nozir uning ko‘zlariga qattiq tikildi. (N.Norqobilov) People,like sheep, tend to follow a leader- occasionally in the right direction. (Alexander
Chase) Ingliz tilida sog‘lomlik, baquvvatlikni ifodalash uchun “as healthy as a goat” (takaday
sog‘lom) etaloni qo‘llaniladi, o‘zbek tilida esa “otday baquvvat” deb ishlatiladi. Yes, yes. That’s
it. You look healthy as a goat. (E.Xeminguey) Xo‘sh. Xo‘sh. Buni qarang-a. Otday baquvvat
ekansiz. Semiz insonlar salbiy munosabat ma’nosi bildirilganda ingliz tilida cho‘chqaga
qiyoslanadi: He is as fat as a pig. O‘zbek tilida bu o‘rinda “bo‘rdoqiga boqilgan qo‘ydek”
o‘xshatishi ishlatiladi. Yana yemishdan gapirasan-a!- deya o‘g‘liga zardasi qaynab tikildi Qo‘zi
qassob.-Bo‘rdoqiga boqilayotgan qo‘ydek yaltillab ketganingni qara (A.Obidjon. Oltin yurakli
avtobola) Qattiq bezovta, chorasiz insonlar uchun ingliz tilida “like a bat out of hell”
(do‘zaxdan chiqqan ko‘rshapalakday), o‘zbek tilida esa “oyog‘i kuygan tovuqday”
o‘xshatishlari qo‘llaniladi. Go‘zallikni ingliz lingvomadaniyatida as beautiful as a rainbow (kamalakday), as beautiful as a
picture (suratday), as beautiful as a spring time (bahorday) o‘xshatishlari bilan beriladi, lekin
o‘zbek tilida bu ma’noni ifodalash uchun “oyday”, “suqsurday” o‘xshatishlari mavjud bo‘lib,
suqsur o‘rdakning bir turi bo‘lgan qush nomidir. Ushbu o‘xshatish faqat o‘zbek tiliga xos
bo‘lib, unda milliy-madaniy qarashni ko‘rish mumkin. Mazkur o‘xshatish etalonlari milliy
dunyoqarash, dunyodagi hodisalarni milliy tasavvurlarga ko‘ra taqqoslash, qiyoslash
natijasida shakllangan. Demak, turli tillarda o‘xshatish etalonlarining tanlanishida ham farqli
milliy-etnik idrok hal qiluvchi rol o‘ynaydi.Aytish mumkinki, etalonlar dunyoning obrazli
qiyoslanishidir. Tilda etalonlar ko‘pincha turg‘un qiyoslamalar ko‘rinishida mavjud bo‘ladi,
shunga qaramay, insonning dunyoni taqqoslashdagi har qanday tasavvuri ham etalon bo‘lishi
mumkin. Xullas, o‘xshatish etalonlari predmetlar, obyektlar va hodisalarning xususiyatini, sifatini
qiyoslaydigan mohiyatdir. YOSH OLIMLAR
ILMIY-AMALIY KONFERENSIYASI
in-academy.uz/index.php/yo
ERTAKLARDA QO’LLANILGAN O’XSHATISHLAR
Jo’rayeva Nilufar Zokir qizi Annotatsiya: O‘xshatish etalonlari predmetlar, obyektlar va hodisalarning xususiyatini,
sifatini qiyoslaydigan mohiyatdir. Etalonlar allegorik birliklarda ham namoyon bo‘la oladi. Chunki allegoriya, o‘xshatish, metafora kabi stilistik troplar turli jihatlardan ham o‘xshash,
ham farqli jihatlarini namoyon qila oladi. Kalit so’zlar : ertaklar, o’xshatishlar, lingvokulturologiya, so’z. Kalit so zlar : ertaklar, o xshatishlar, lingvokulturologiya, so z. O‘xshatish turli xalqlarning madaniyatini yaqqol namoyon qilib beruvchi stilistik vositalardan
biri deya olamiz. Ularda gapiruvchining kechinmalari, tasavvurlari, shuningdek, butun
xalqning milliy va madaniy an’analari muhrlangan bo‘ladi. O‘xshatishlar o‘ziga xos obrazli
tafakkur mahsuli sifatida yuzaga keladi. O‘xshatish ham badiiy tasvir vositasi sifatida, ham
stilistik trop sifatida o‘rganiladi. Shuning uchun ular nutqda hamisha badiiy-estetik qiymatga
ega bo‘ladi, nutqning emotsional-ekspressivligi, ifodaliligi, ta'sirchanligini ta'minlashga xizmat
qiladi. O‘xshatish nutqning mazmundorligi va unumdorligini oshiradi va gapiruvchi yoki
yozuvchining estetik mahoratini ham ko‘rsata oladi. Professor Nizomiddin Mahmudov o‘zbek tilidagi o‘xshatishlarni to‘rt unsurga ajratib, ularni
o‘xshatiladigan narsa yoki subyekt, o‘xshatish etaloni, o‘xshatish asosi va o‘xshatishning
shakliy ko‘rsatkichi deb nomlaydi. «Tog‘lardagi qip-qizil lola Bo‘lib guyo yoqut piyola. Buloqlardan uzatadi suv El ko‘zidan qochadi uyqu» (H.Olimjon). Bunda o‘xshatish subyekti –
lola, o‘xshatish etaloni – piyola, o‘xshatish asosi – yoqut, o‘xshatish vositasi – go‘yo. O‘xshatishda chog‘ishtiriladigan narsa ham asosan uning to‘g‘ri ma’nosida qoladi. O‘rganish
jarayonida o‘xshatishlarning ikki turini uchratishimiz mumkin. Ular: 1) individual-muallif o‘xshatishlari yoki erkin o‘xshatishlar; 2) umumxalq yoki turg‘un (doimiy) o‘xshatishlar. Tildagi o‘xshatishlarni tadqiq etgan tilshunoslarning aksariyati turg‘un o‘xshatishlarning
idiomalarga yaqin turishini yoki idioma maqomida bo‘lishini, ular ko‘p asrlar mobaynida
kishilar nutqida qo‘llanish natijasi sifatida turg‘unlashib, so‘zlovchilar ongida muayyan
modellar shaklida mustahkamlanib qolishini, o‘xshatish etalonining, ya’ni o‘xshatish asosidagi
obrazning muayyan belgi-predmet bilan muntazam va qat’iy bog‘liq bo‘lishini ta’kidlaydi. Turg‘un o‘xshatishlar tarkibida o‘xshatish obrazi, ya’ni etaloni alohida ahamiyat kasb etadi. Bu
unsur o‘xshatishning markazini tashkil qiladi va boshqa unsurlar (o‘xshatish subyekti, asosi,
ko‘rsatkichi) ayni shu etalon atrofida birlashadi. V.Maslovaning fikricha, o‘xshatish etalonlari an’anaga kirgan obrazlar sifatida xalqning
dunyoni o‘ziga xos idrok etishning ifodachisi bo‘lganligi bilan til, madaniyat va mentalitet
munosabatlarini belgilash jihatidan juda muhimdir. Chunki bu o‘rinda ma’lum predmet bir
predmetga o‘xshatilsa, xuddi shu predmet boshqa o‘rinda ikkinchi narsaga o‘xshatilishi
mumkin va bu elementlar turli millatlarda turlicha ifodalanadi. O‘zbek tilining izohli lug‘atiga ko‘ra, turli lingvomadaniyatlarda muayyan bir narsaning,
masalan, hayvonning turg‘un o‘xshatish etaloni sifatida qo‘llanishini kuzatish mumkin. Masalan, tulki ayyorlik, aldoqchilik, yolg‘onchilik ramzi, etaloni sifatida o‘zbek, ingliz va
boshqa ko‘plab tillarda faol ishlatiladi, masalan: “tulkiday ayyor”, “хитрый как лиса” (rus 59 in-academy.uz/index.php/yo Etalonlar allegorik birliklarda ham namoyon bo‘la oladi. Chunki
allegoriya, o‘xshatish, metafora kabi stilistik troplar turli jihatlardan ham o‘xshash, ham farqli
jihatlarini namoyon qila oladi. O‘xshatish etalonlarini lingvomadaniy jihatdan o‘rganish va
tahlil qilish jarayonida ramzning belgilik jihati qiziqtiradi. Masalan, turli madaniyatlarda,
shuningdek g‘arbiy mamlakatlarda kabutar – tinchlik ramzi hisoblanadi, nasroniylikda 60 Muqaddas ruh ramzi, yarim oy – Islom dini ramzi, Xoch yoki unga o‘xshash belgi – nasroniylik
ramzi sanaladi. Daraxtlar, gullar, o‘simliklar ham ramz sifatida qo‘llaniladi. Jumladan,
xrizantema Xitoyda o‘lim, qabriston tushunchalarini anglatadi. Yoki qirqquloq yaponlar uchun
kirib kelayotgan yangi yilda omad tilash, ruslar uchun esa o‘lim, qabriston belgisi hisoblanadi. Bu kabi ramzlardan turli millatlar turli xil holatlarda ishlatishlari mumkin. Yuqorida berilgan
misollar buning tasdig‘i bo‘la oladi. Xususan, ranglardan ham ramz sifatida keng foydalaniladi. Aksariyat xalqlarda oq – yaxshilik, qora esa – yovuzlikni anglatsa, Afrikaliklar uchun har
ikkala rang ham neytral mazmun kasb etadi. Ya’ni ular uchun bu ranglar shunchaki, rang tus
hisoblanadi va asosiy mazmun kasb etmaydi. O‘xshatish tasvir obyektini boshqa narsa-
hodisaga o‘xshatish orqali yorqin bo‘rttirib tasvirlashga asoslangan badiiy tasvir vositasi
bo‘lib, bunda o‘xshatilayotgan narsa-hodisalar uchun umumiy belgi-xususiyatlarga tayaniladi. “As hungry as a bear” iborasiga e’tibor beradigan bo‘lsak, bu birikma ingliz tilida “ayiqday
och” ma’nosini beradi va o‘zbek tilidagi “och bo‘riday” o‘xshatish etaloniga teng keladi. “Och”
so‘zining bo‘ri bilan birga ishlatilishi o‘zbek lingvkulturologiyasiga xos bo‘lib, o‘zbek xalq
ertaklaridagi och bo‘ri tinglovchiga namoyon bo‘lishi mumkin. Jumladan, mif, ertak, doston va
badiiy asar qahramonlari ishtirok etgan muayyan o‘xshatishlar mavjud bolib, ular orqali
xalqning o‘ziga xos milliy madaniyatini, nutq uslublarini, va tasvir vositalarini chuqur anglab
yetish mumkin. Masalan, o‘zbek tilida “baquvvat, pahlavon, gavdali, juda ham kuchli, bahodir”
kabi sinonim so‘zlar ma’nosida ishlatiladigan “Alpomishdek” o‘xshatish etaloni, ingliz tilidagi
“jasur va botir” ma’nosida qo‘llanuvchi “as brave as Robin Hood” o‘xshatish birligi bilan
sinonimlik xosil qiladi. 1. O‘z o‘rnida ta’kidlash joizki, o‘xshatish etaloni, juda ko‘plab tillarda sinonimlikni hosil
qila
oladi. Yuqorida
ta’kidlab
o‘tilgan
birikmalar
bunga
misol
bo‘la
oladi. Lingvomadaniyatshunoslikning predmeti – madaniyatda ramziy, obrazli, metaforik ma’no
kasb etgan va natijalari inson ongida umumlashtirilib mif, afsona, folklor va diniy
diskurslarda, poetik va prozaik badiiy matnlarda, frazeologizmlarda, metaforalarda va
ramzlarda aks etadigan til birliklari hisoblanadi. Bunda muayyan lingvokulturologik birlik bir
paytning o‘zida bir qancha semiotik tizimlarga tegishli bo‘lishi mumkin: ma’lum bir odat
frazeologizmga, maqolga, matalga aylanishi mumkin . References:
1. .Афзалов М. Ўзбек халқ эртаклари. -Тошкент. Фан. Алимов С. Формирование и
развитие жанра литературной сказки в узбекской литературе. –автореф..дисс…
канд.фил.наук. Тошкент. Адабиёт назарияси. 2 томлик. Тошкент, Фан. 1978-1979. 2. Аристотель. Поэтика (Нафис санъатлар ҳақида). Русчадан Маҳмудов М. таржимаси. — Тошкент, “Янги аср авлоди”, 2004. 3. Бекназаров Қ. Ўзбек халқ маиший эртаклари (ўрганилиши, таснифи, поэтикаси). –автореф.дисс. канд.фил.наук.-Тошкент. 1993 61
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Sourcing the iron in the naturally fertilised bloom around the Kerguelen Plateau: particulate trace metal dynamics
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To cite this version: P. van Der Merwe, A. R. Bowie, F. Quéroué, L. Armand, S. Blain, et al.. Sourcing the iron in the
naturally fertilised bloom around the Kerguelen Plateau: particulate trace metal dynamics. Biogeo-
sciences, 2015, 12 (3), pp.739-755. 10.5194/bg-12-739-2015. hal-01207931 Sourcing the iron in the naturally fertilised bloom
around the Kerguelen Plateau: particulate trace metal
dynamics P. van Der Merwe, A. R. Bowie, F. Quéroué, L. Armand, S. Blain, Fanny
Chever, D. Davies, F. Dehairs, Frédéric Planchon, Géraldine Sarthou, et al. Distributed under a Creative Commons Attribution 4.0 International License Correspondence to: P. van der Merwe (pvander@utas.edu.au) Correspondence to: P. van der Merwe (pvander@utas.edu.au) Received: 26 August 2014 – Published in Biogeosciences Discuss.: 18 September 2014
Revised: 15 December 2014 – Accepted: 8 January 2015 – Published: 6 February 2015 Received: 26 August 2014 – Published in Biogeosciences Discuss.: 18 September 2014
Revised: 15 December 2014 – Accepted: 8 January 2015 – Published: 6 February 2015 Fe levels were only elevated by a factor of ∼2. Over the
Kerguelen Plateau, ratios of pMn / pAl and pFe / pAl resem-
ble basalt, likely originating from glacial/fluvial inputs into
shallow coastal waters. In downstream, offshore deep-waters,
higher pFe / pAl, and pMn / pAl ratios were observed, sug-
gesting loss of lithogenic material accompanied by retention
of pFe and pMn. Biological uptake of dissolved Fe and Mn
and conversion into the biogenic particulate fraction or ag-
gregation of particulate metals onto bioaggregates also in-
creased these ratios further in surface waters as the bloom
developed within the recirculation structure. While resus-
pension of shelf sediments is likely to be one of the impor-
tant mechanisms of Fe fertilisation over the plateau, fluvial
and glacial sources appear to be important to areas down-
stream of the island. Vertical profiles within an offshore re-
circulation feature associated with the Polar Front show pFe Abstract. The KEOPS2 project aims to elucidate the role of
natural Fe fertilisation on biogeochemical cycles and ecosys-
tem functioning, including quantifying the sources and pro-
cesses by which iron is delivered in the vicinity of the Ker-
guelen Archipelago, Southern Ocean. The KEOPS2 pro-
cess study used an upstream high-nutrient, low-chlorophyll
(HNLC), deep water (2500 m), reference station to compare
with a shallow (500 m), strongly fertilised plateau station and
continued the observations to a downstream, bathymetrically
trapped recirculation of the Polar Front where eddies com-
monly form and persist for hundreds of kilometres into the
Southern Ocean. Over the Kerguelen Plateau, mean partic-
ulate (1–53 µm) Fe and Al concentrations (pFe = 13.4 nM,
pAl = 25.2 nM) were more than 20-fold higher than at an off-
shore (lower-productivity) reference station (pFe = 0.53 nM,
pAl = 0.83 nM). In comparison, over the plateau dissolved HAL Id: hal-01207931
https://hal.sorbonne-universite.fr/hal-01207931v1
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destinée au dépôt et à la diffusion de documents
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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 Biogeosciences, 12, 739–755, 2015
www.biogeosciences.net/12/739/2015/
doi:10.5194/bg-12-739-2015
© Author(s) 2015. CC Attribution 3.0 License. Sourcing the iron in the naturally fertilised bloom around the
Kerguelen Plateau: particulate trace metal dynamics P. van der Merwe1, A. R. Bowie1,2, F. Quéroué1,2,3, L. Armand4, S. Blain5,6, F. Chever3,7, D. Davies1, F. Dehairs8,
F. Planchon3, G. Sarthou9, A. T. Townsend10, and T. W. Trull1,11 P. van der Merwe1, A. R. Bowie1,2, F. Quéroué1,2,3, L. Armand4, S. Blain5,6, F. Chever3,7, D. Davies1, F. Dehairs8,
F. Planchon3, G. Sarthou9, A. T. Townsend10, and T. W. Trull1,11 1Antarctic Climate and Ecosystems CRC, University of Tasmania, TAS 7004, Australia
2Institute for Marine and Antarctic Studies, University of Tasmania, Battery Point, TAS 7004, Australia
3Laboratoire des Sciences de l’Environnement Marin (LEMAR), Université de Bretagne Occidentale, CNRS,IRD,
UMR6539, IUEM, Technopole Brest Iroise, Place Nicolas Copernic, 29280 Plouzané, France
4Department of Biological Sciences and Climate Futures, Macquarie University, North Ryde, NSW 2109, Australia
5Sorbonne Universités, UPMC Univ Paris 06, UMR7621, Laboratoire d’Océanographie Microbienne, Observatoire 4Department of Biological Sciences and Climate Futures, Macquarie University, North Ryde, NSW 2109, Australia
5Sorbonne Universités, UPMC Univ Paris 06, UMR7621, Laboratoire d’Océanographie Microbienne, Observatoire
Océanologique, 66650 Banyuls/mer, France 4Department of Biological Sciences and Climate Futures, Macquarie University, North Ryde, NSW 2109, Australia
5Sorbonne Universités, UPMC Univ Paris 06, UMR7621, Laboratoire d’Océanographie Microbienne, Observatoire
Océanologique, 66650 Banyuls/mer, France 6CNRS, UMR7621, Laboratoire d’Océanographie Microbienne, Observatoire Océanologique, 66650 Banyuls/mer, France
7IFREMER/Centre de Brest, Département REM/EEP/Laboratoire Environnement Profond, 6CNRS, UMR7621, Laboratoire d’Océanographie Microbienne, Observatoire Océanologique, 66650 Banyuls/mer, France
7IFREMER/Centre de Brest, Département REM/EEP/Laboratoire Environnement Profond,
CS 10070, 29280 Plouzané, France 8Vrije Universiteit Brussel, Analytical, Environmental and Geo-Chemistry & Earth System Sciences
research group, Brussels, Belgium g
p
g
9CNRS, Université de Brest, IRD, Ifremer, UMR6539 LEMAR, IUEM ; Technopôle Brest Iroise,
Place Nicolas Copernic 29280 Plouzané France 10Central Science Laboratory, University of Tasmania, Sandy Bay, TAS 7005, Australia
11 11Commonwealth Scientific and Industrial Research Organisation, Oceans and Climate Flagship,
GPO Box 1538, Hobart, Tasmania, Australia 1
Introduction Small scale fertilisation experiments have now clearly estab-
lished that Southern Ocean primary production is limited by
the availability of the micronutrient iron (Fe) (Boyd et al.,
2007; de Baar, 2005). This limitation on the biological pump
means that the Southern Ocean does not realise its full po-
tential in transferring atmospheric CO2 into the ocean inte-
rior; a result illustrated in Antarctic continental ice records
over geological timescales and supported by modelling stud-
ies (Barnola et al., 1987; Bopp et al., 2003; Martin, 1990;
Watson et al., 2000). Less well understood is the overall sys-
tem response to the addition of Fe as efficiency estimates
(defined here as the amount of carbon exported relative to
Fe added above baseline conditions) can vary by an order of
magnitude (Blain et al., 2007; Pollard et al., 2009; Savoye
et al., 2008). Both the original and subsequent KEOPS mis-
sions aimed to resolve not only the efficiency estimate, but
also the response of the ecosystem and the overall effect on
biogeochemical cycles due to natural Fe fertilisation in the
vicinity of the Kerguelen Plateau. The KEOPS natural fertil-
isation experiment is complementary to artificial Fe enrich-
ment experiments due to the fact that its scale is much larger
and its time frame longer than that which is currently fea-
sible in artificial fertilisation experiments. Furthermore, due
to the sustained release of Fe into the fertilised region, as
opposed to a sudden pulse artificial experiment, the techni-
cal challenges of monitoring carbon export are reduced. Fur-
thermore, there is growing evidence that sustained Fe fertili-
sation favours large, highly silicified, slow growing diatoms
that are efficient at exporting carbon into the ocean interior
(Quéguiner, 2013). When the results of process studies such
as KEOPS are extrapolated over the whole Southern Ocean, The first KEOPS process study was conducted in 2005 and
specifically focused on processes affecting the demise of the
spring bloom over the Kerguelen Plateau (Blain et al., 2007). Blain et al. (2007) and Chever et al. (2010) demonstrated that
dFe fertilisation from the plateau increased primary produc-
tion in the area. From the data gathered it was proposed that
resuspension of plateau-derived sediments and entrainment
into the mixed layer during increased wind mixing that deep-
ened the mixed layer, was the primary source of particulate
and subsequently, dissolved Fe to the downstream blooms. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 740 and pMn levels that were 6-fold and 3.5-fold lower, respec-
tively, than over the plateau in surface waters, though still
3.6-fold and 1.7-fold higher respectively than the reference
station. Within the recirculation feature, strong depletions of
pFe and pMn were observed in the remnant winter water
(temperature-minimum) layer near 175 m, with higher values
above and below this depth. The correspondence between the
pFe minima and the winter water temperature minima im-
plies a seasonal cycle is involved in the supply of pFe into
the fertilised region. This observed association is indicative
of reduced supply in winter, which is counterintuitive if sedi-
ment resuspension and entrainment within the mixed layer is
the primary fertilising mechanism to the downstream recircu-
lation structure. Therefore, we hypothesise that lateral trans-
port of pFe from shallow coastal waters is strong in spring,
associated with snow melt and increased runoff due to rain-
fall, drawdown through summer and reduced supply in win-
ter when snowfall and freezing conditions predominate in the
Kerguelen region. a small change in the efficiency estimate could result in dif-
ferent conclusions as to the efficacy, for instance, of artificial
Fe fertilisation as a means of mitigating rising atmospheric
concentrations of anthropogenic CO2. p g
2
Dissolved
Fe
(dFe < 0.2 µm)
includes
colloidal
and
nanoparticulate Fe, which may only be partially bioavail-
able, as well as soluble Fe (sFe < 0.02 µm) which is highly
bioavailable (de Baar and de Jong, 2001). As a result, the
larger particulate fraction (> 0.2 µm) is often less studied due
to the perception that it has low bioavailability. However, the
particulate fraction can yield important information for sev-
eral reasons; firstly the dissolved fraction is constantly in a
state of change with uptake, particle scavenging and rem-
ineralisation occurring simultaneously and at varying rates
depending on many factors including complexation with or-
ganic ligands (Johnson et al., 1997) and the biological com-
munity present (Sunda, 2001). Thus, interpretation of dFe
data is difficult without a rarely obtained perspective on the
time varying aspects of the dFe distribution. Secondly, as
a fraction of the total Fe, the major sources of Fe into fer-
tilised regions (e.g. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources weathering products delivered by fluvial
and glacial processes, resuspension of sediments and pore-
waters, atmospheric and extra-terrestrial dust) are small par-
ticles (> 0.2 µm), with the concentration being more stable
over weeks to months, due to its abundance and relatively
slow biological uptake. The particulate fraction is primarily
lost from surface waters through sinking, either directly or
via adhesion to bioaggregates (Frew et al., 2006). However,
there is a constant transfer of dissolved Fe to particulate Fe,
either via biological uptake or precipitation and, particulate
Fe to dissolved Fe, via dissolution and biologically medi-
ated processes (Moffett, 2001). Thus, the particulate fraction
that is small enough to avoid sinking out of the water col-
umn rapidly (0.2–5 µm) can be considered as a significant
source of dissolved Fe, with the rate of supply into surround-
ing waters dependent on the dissolution and leaching rate. Furthermore, there is growing evidence that particles in this
size fraction are readily produced by mechanical erosion of
bedrock due to glacial processes at high latitudes and that
this large source may be partially bioavailable (Hawkings et
al., 2014; Poulton and Raiswell, 2005; Raiswell et al., 2008a,
b, 2006). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources www.biogeosciences.net/12/739/2015/ 1
Introduction Resolution of the Fe budget (accounting for all sources and
sinks of Fe in the system), from observations made during
the first mission, found that the vertical supply of dissolved Biogeosciences, 12, 739–755, 2015 www.biogeosciences.net/12/739/2015/ P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 741 SeaWiFS files courtesy of CLS (with support from CNES)
Reference
station
(HNLC)
Plateau
station
North Polar
Front station
Recirculation
structure within
a stationary
meander
Figure 1. SeaWiFS surface chlorophyll on 11 November 2011, ap-
proximately half way through the KEOPS2 sampling program. Ker-
guelen and Heard Island are visible in grey. Stations that were sam-
pled for suspended particles are indicated with black circles. Dis-
tinct regimes of interest for the KEOPS2 program are indicated in
red. SeaWiFS files courtesy of CLS (with support from CNES)
Reference
station
(HNLC)
Plateau
station
North Polar
Front station
Recirculation
structure within
a stationary
meander depths throughout the water column. Depths were chosen af-
ter viewing conductivity, temperature and depth (CTD) data
to sample within oceanographic features of interest as well
as obtaining a representative full water column profile. The
ISPs were fitted with 142 mm quartz micro fibre (QMA)
(Sartorius) filters with 53 µm Petex pre-filters and 350 µm
polyester supports. QMA filters were pre-combusted to re-
move particulate organic carbon and then acid-washed with
Seastar Baseline™HCl and rinsed with copious amounts of
ultra-pure water according to the methods outlined in the
GEOTRACES sample handling protocols handbook (Cutter
et al., 2010). The pre-filters and supports were carefully acid
washed and rinsed with copious amounts of ultra-pure wa-
ter before use. Both the Petex pre-filter and QMA filter were
analysed for every pump giving two size fractions at each
sampling location. Therefore, all particles greater than 53 µm
were collected on the pre-filter and all particles within the
1–53 µm size range were collected on the underlying QMA
filter. Lithogenics sourced from bedrock or sediments in the
larger size range (> 53 µm) would have a high sinking ve-
locity (> 500 m day−1) according to Stokes’ law and as such
would be expected to make up a relatively small fraction of
the total particles in this size range. In comparison, the 1–
53 µm size class can potentially capture both small biogenic
and lithogenic particles. 1
Introduction This is due to the prediction that
small lithogenic particles (1–5 µm) have significantly slower
sinking rates (0.1–10 m day−1) than large lithogenic particles
according to Stokes’ law. SeaWiFS files courtesy of CLS (with support from CNES) Figure 1. SeaWiFS surface chlorophyll on 11 November 2011, ap-
proximately half way through the KEOPS2 sampling program. Ker-
guelen and Heard Island are visible in grey. Stations that were sam-
pled for suspended particles are indicated with black circles. Dis-
tinct regimes of interest for the KEOPS2 program are indicated in
red. Figure 1. SeaWiFS surface chlorophyll on 11 November 2011, ap-
proximately half way through the KEOPS2 sampling program. Ker-
guelen and Heard Island are visible in grey. Stations that were sam-
pled for suspended particles are indicated with black circles. Dis-
tinct regimes of interest for the KEOPS2 program are indicated in
red. Fe was not sufficient to supply phytoplankton demand. Blain
et al. (2007) closed the KEOPS Fe budget by assuming that
dissolution of a small fraction of the unconstrained partic-
ulate Fe pool must occur. The KEOPS2 mission aimed to
improve on the successes of the first process study by ac-
counting for the missing Fe in the budget, namely particu-
late Fe (pFe). Thus, we aim to test the KEOPS1 hypothe-
sis that unconstrained particulate Fe is the missing Fe of the
KEOPS Fe budget by documenting the particulate metal en-
richment around the Kerguelen Plateau. Our goal is to de-
termine the sources of Fe enrichment within areas of inter-
est (i.e. reference, plateau and the recirculation structure; see
Fig. 1). Trace metal analysis of suspended particles, underly-
ing sediment and settling particulate material will elucidate
the source to sink progression of the particulate Fe pool. Fol-
lowing on from this work, and together with dissolved Fe
measurements (Quéroué et al., 2015), a focused Fe budget
will be constructed (Bowie et al., 2014). The ISPs were programmed to pump for up to four hours,
allowing up to 2000 L of seawater to be filtered. After re-
trieval, the filters were bagged and processed within an ISO
class 5, containerised clean room. Replicate 14 mm punches
were taken using an acid-washed polycarbonate punch and
stored frozen at −18 ◦C until analysis at the home labora-
tory. The 14 mm punches were then used for particulate metal
analysis, particulate organic carbon and particulate organic
nitrogen analysis. 2.2
Sediment traps (Technicap PPS3) For a full description of the sediment trap data dur-
ing KEOPS2 see Laurenceau et al. (2014) and Bowie et
al. (2014). Two Technicap PPS3 free-floating sediment traps
were deployed below the mixed layer at a depth of 200 m. The two sediment traps were deployed twice, giving a to-
tal of four deployments. The traps were prepared with acid-
cleaned sampling cups containing low-trace-metal brine so-
lution (salinity ∼60). The trap was programmed to sample
for 1.5–5.5 days, whilst the 12 individual sampling contain-
ers were open for an equal portion of the total deployment. Upon retrieval, the sampling containers were removed from
the carousel, sealed and processed within an ISO class 5, con-
tainerised clean room. The samples were filtered onto acid-
washed, 2 µm polycarbonate membrane filters via a 350 µm
pre-filter using a Sartorius™PTFE filtration unit. The 350 µm 2.1
In situ pumps (ISPs) All sample handling, processing and preparation was per-
formed in accordance with general GEOTRACES protocols
(http://www.geotraces.org/) and specific methodologies out-
lined in Bowie et al. (2010). Briefly, suspended particles
were collected using up to 11 in situ pumps (ISPs) (McLane
WTS and Challenger) suspended simultaneously at varying 2.4.1
ISP filters for particulate metals During the HF digest, a mixture of strong acids (250 µL
HNO3, 250 µL HF and 500 µL HCl) were used as per Bowie
et al. (Bowie et al., 2010). After 12 h at 95 ◦C the digest
PFA vials were uncapped and evaporated to dryness under
HEPA filtered air at 60 ◦C for 4 h. The digest was then resus-
pended in 10 % HNO3 with 10 ppb indium as internal stan-
dard. A 100 × dilution factor (v : v) was considered sufficient
to place the ∼20 mg sediment samples within the calibration
range of the SF-ICP-MS. All digestions and evaporations were carried out within a di-
gestion hood (SCP Science), where air was HEPA filtered
during intake and subsequently extracted through a fume
hood. Filter blanks and sample filters were digested in 15 mL
acid cleaned, Teflon perfluoroalkoxy (PFA) screw cap vials
(Savillex™) using ultra-pure nitric acid (1 mL 16 M HNO3)
(Seastar Baseline™) heated to 120 ◦C for 12 h on a Teflon
coated hotplate (SCP Science DigiPREP™), following the
method outlined in Bowie et al. (2010). Blanks containing
only HNO3 were also analysed to determine the contribution
of the digestion acid without filter material. 2.4.2
Sediment analysis for particulate metals An Oktopus Multicorer (www.oktopus-mari-tech.de) was
used to collect 8 replicate, 610 × 95 mm sediment cores, si-
multaneously within a 1 m2 area at each station. The upper-
most 5mm of surface sediment was subsampled according to
Armand et al. (2008) representing an approximate sedimen-
tation period of < 1000 years. Digestions and analysis were performed as per the ISP filters
except that HF acid was used to digest these highly refrac-
tory samples. The HNO3 digest used for the ISP filters is
relatively weak but digests the biogenic fraction completely
as evidenced by the excellent recoveries on the BCR414 cer-
tified reference material (trace metals in phytoplankton) (Ta-
ble A1) but has limited recoveries of the lithogenic fraction
as evidenced by the low recoveries of pAl and pTi in MESS-
3 and PACS-2 sediment certified reference material (Bowie
et al., 2010). P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 742 somewhat lower for the lithogenic suspended particles. How-
ever, a HNO3 only digestion will recover effectively 100 %
of the trace elements of biogenic suspended particles (Ta-
ble A1). For further information see Bowie et al. (2010). pre-filter was selected to exclude large copepods and other
large plankton that would lead to unrealistic sample variabil-
ity. 3
Results and discussion Quartz micro-fibre (QMA) filters were chosen as they
could be acid cleaned to a trace-metal-clean level and the
filter material allowed high particle loading and low wash-
off upon pump retrieval. Furthermore, the filters were com-
patible for use with both Inductively Coupled Plasma Mass
Spectrometry (ICP-MS) and elemental (CHN) analysis due
to their ability to be combusted. It should be noted that a
compromise was made here by using QMA filters on the
ISPs. The compromise is that HF acid cannot be used with
QMA filters as it digests the filter material completely and
leads to unacceptably high analytical blanks. Therefore, we
used HNO3 for the digestions of the QMAs and for consis-
tency regarding the suspended particles we also digested the
pre-filter with the same acid. On the other hand, we used a
full HF acid digestion for the underlying sediment analysis
(Sect. 2.4.2). Therefore, recoveries of lithogenic trace ele-
ments will be close to 100 % for the sediment analysis, but 2.4.3
Particulate organic carbon (POC) and nitrogen
(PN) The digest solution was diluted with 9 mL of ultra-high
purity water and spiked to a final concentration of 10 ppb in-
dium as an internal standard. Samples were analysed by Sec-
tor Field ICP-MS (Finnigan Element II, Thermo Scientific)
(Cullen and Sherrell, 1999; Townsend, 2000). A full suite of
trace elements was measured including Fe, Al, Mn, Ba and P. The data were quality controlled by comparison with a cer-
tified reference material with a similar composition to the
material collected (BCR-414 trace metals in phytoplankton,
European Commission) (Table A1). All glassware in contact with POC samples was pre-
combusted prior to field work (450 ◦C for 12 h). Total ni-
trogen, carbon and hydrogen were determined at the Central
Science Laboratory, University of Tasmania, using a Thermo
Finnigan EA 1112 Series Flash Elemental Analyzer (esti-
mated precision ∼1 %). www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 3.1
Station types The sampling locations of KEOPS2 (Fig. 1) were designed to
capture the key regime types of the Kerguelen Archipelago
including the high-nutrient, low-chlorophyll (HNLC) refer-
ence waters (station R-2), the high-trace-metal plateau wa-
ters (station A3), the northern Polar Front (station F-L) and a
quasi-stationary, bathymetrically trapped recirculation struc-
ture (E-1, E-3 and E-5) to the east of Kerguelen Island
(Table 1). Stations E-1, E-3 and E-5 can be thought of as
a pseudo-Lagrangian time series. In addition, two stations
were sampled at the eastern and western extremes of the re-
circulation structure (E-4W and E-4E) which proved to con-
trast in absolute concentrations as well as elemental ratios of
particulate trace metals. www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources
743
Table 1. KEOPS2 sampling locations and station types. A3-1
A3-2
R-2
F-L
E-1
E-3
E-5
E-4E
E-4W
Station type
Kerguelen
Kerguelen
HNLC reference
Northern
Recirculation
Recirculation
Recirculation
Eastern recirculation
Western recirculation
Plateau 1st visit
Plateau 2nd visit
station
Polar Front
structure
structure
structure
structure
structure
Sampling date
20/10/2011
16/11/2011
25/10/2011
6/11/2011
29/10/2011
3/11/2011
18/11/2011
13/11/2011
11/11/2011
Latitude (S)
50◦37.7574′
50◦37.4306′
50◦21.52′
48◦31.394′
48◦29.5728′
48◦42.1334′
48◦24.698′
48◦42.9218′
48◦45.927′
Longitude (E)
72◦4.8193′
72◦3.3366′
66◦43.00′
74◦40.036′
72◦14.1467′
71◦58.0027′
71◦53.7894′
72◦33.7792′
71◦25.51′
Bottom depth (m)
505
505
2528
2690
2050
1910
1920
2200
1400
Time series
Yes
Yes
No
No
Yes
Yes
Yes
No
No
Particulate trace metals
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
POC PON
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Sediment samples
Yes
Yes
Yes
Yes
No
Yes
No
No
Yes
Sediment trap samples
No
Yes
No
No
Yes
Yes
Yes
No
No
Figure 2. Surface (10 m) temperature in spring–summer (a) and autumn–winter (b) as well as surface salinity in spring–summer (c) and
autumn–winter (d) within the study area from 1970 until 2013. The PF is identified as a solid black line. Kerguelen and Heard Island are
visible in dark grey and black respectively and the Leclaire Rise can be identified as the shallow bathymetry, north of the PF, near the western
boundary of the map. Data obtained from the World Ocean Database (http://www.nodc.noaa.gov). P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources
T bl 1
O S2
li
l
i
d
i 743 Figure 2. 3.1
Station types Surface (10 m) temperature in spring–summer (a) and autumn–winter (b) as well as surface salinity in spring–summer (c) and
autumn–winter (d) within the study area from 1970 until 2013. The PF is identified as a solid black line. Kerguelen and Heard Island are
visible in dark grey and black respectively and the Leclaire Rise can be identified as the shallow bathymetry, north of the PF, near the western
boundary of the map. Data obtained from the World Ocean Database (http://www.nodc.noaa.gov). P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources these southern and northern water masses. As a result of the
mixing, eddies commonly form in this region. Also within
this mixing zone, surface filaments, identified by elevated
Chlorophyll a, can be seen in SeaWiFS images diverging
from the PF and entering the eastern boundary of the recir-
culation structure (see the Supplement in Trull et al., 2014). ilar to the ratio found in phytoplankton such as the BCR-
414 certified reference material (freshwater phytoplankton)
(Fe : Al = 1.01) used in this study (Table A1). Furthermore,
the pFe : pAl ratio of sinking particles captured by the free
floating PPS3 sediment traps (marine snow) had similar ra-
tios of pFe : pAl of 1.02, 1.05, 0.91 and 0.70 for stations E-
1, E-3, E-5 and A3-2, respectively (Table 2). These obser-
vations highlight the major contribution of sinking biogenic
material to the authigenic sediments in the area around the
Kerguelen Plateau which was in contrast to the signature at
station R-2 due to its low productivity. The pFe in the sedi-
ments at all stations and primarily at station R-2 (as a fraction
of its total weight) were similar to Weddell Sea surface sedi-
ments (Angino and Andrews, 1968) which ranged from 0.9–
3.2 %. In comparison, station R-2 has a mean value of 0.1 %
Fe while station E-3, A3, F-L and E-4W had mean values of
0.3, 0.8, 1.5 and 2.5 % respectively. The low fraction of Fe
within the authigenic sediment at R-2 indicates limited pFe
supply at this station in comparison to either the Weddell Sea
or the Kerguelen Plateau presented here. (
pp
)
The Kerguelen Archipelago is isolated, being a relatively
small and localised source of Fe fertilisation surrounded by
the large and deep, HNLC, low Fe, Southern Ocean. There-
fore, when identifying an Fe source to the region, our focus
is on the plateau and the two islands, Kerguelen and Heard. Over geological timescales, all pFe distributed throughout
the water column within this region must be derived from all
forms of weathering of bed rock including fluvial and glacial
outflow as well as dust from the islands, hydrothermal and
extra-terrestrial input. 3.4
Plateau, reference and Polar Front stations The reference station (R-2) has a bottom depth of 2528 m and
is characterised by low surface Chl a concentrations (Las-
bleiz et al., 2014) and nutrient concentrations characteristic
of HNLC waters (Blain et al., 2014). Station F-L is approx-
imately 313 km northeast of Kerguelen Island with a bottom
depth of 2690 m and represents the northern PF. Station F-
L is downstream of Kerguelen Island, with the PF deliver-
ing waters that originated near station R-2. In this case, the
waters crossing Station F-L have interacted with both the
plateau and shallow coastal waters of Kerguelen Island. In
contrast, station A3 is located over the Kerguelen Plateau
and has a bottom depth of 527 m, making it the shallowest
station sampled for trace metal analysis of suspended parti-
cles and one of the most likely to be influenced by resuspen-
sion of shelf sediments (Fig. 1). The proximity of the sta-
tion to Heard and Kerguelen Island (roughly half way be-
tween the two) means that fluvial and glacial runoff may also
drive fertilisation at this site. However, the hydrography of
the area dictates that waters which previously interacted with
upstream Heard Island are a more likely source to A3 than
downstream Kerguelen Island (Park et al., 2008). P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources Over shorter time frames, shelf sedi-
ments in the region contain recycled Fe as the vast major-
ity of these sediments are a combination of siliceous ooze
(Armand et al., 2008) and glacio-marine sediments; the ex-
ported product of the highly productive overlying waters to-
gether with some lithogenics (sourced from bed-rock) that
were unutilised or non-bioavailable and fast-sinking. There-
fore, understanding the pathways of supply of this new Fe is
important to understanding the processes controlling the long
term productivity and therefore, carbon sequestration, in the
area. 3.2
Surface water flow around the Kerguelen Plateau the Leclaire Rise. These surface waters are generally colder
and saltier than to the north (Fig. 2). The AASW also flows
around Heard Island and a weaker surface current flows
north-west over the Kerguelen Plateau towards the north-east
of Kerguelen Island where it is bound to the north by the PF. This cold surface current can be seen during winter in Fig. 2. The northern water mass has a source of easterly flowing
Sub Antarctic Surface Waters (SASW). The portion of the
SASW that interacts with Kerguelen Island is termed Ker-
guelen Island source waters and is bound to the south by the
PF (Fig. 2). A broad and poorly defined mixing zone to the
east of Kerguelen Island has been identified at the junction of During KEOPS1, van Beek et al. (2008), Zhang et al. (2008)
and Chever et al. (2010) revealed that the water column
south-east of Kerguelen Island was modified by passing over
the Heard Island Plateau. Park et al. (2008) demonstrated
that the interaction of the water masses over the Kerguelen
Plateau could be divided into the southern and northern wa-
ter masses separated by the Polar Front (PF, Fig. 1). The
southern water mass has source waters being derived from
the Antarctic surface waters (AASW), south-west of Kergue-
len which is bound to the north by the shallow bathymetry of www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 744 www.biogeosciences.net/12/739/2015/ P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 745 Table 2. Mean elemental molar ratios of marine snow particles captured in free-floating sediment traps, particulate matter (1–53 µm) below
the mixed layer and authigenic sediments at each station. Note that station TEW-1 is a near-coastal station located within Hillsborough Bay,
Kerguelen Island. Station TEW-1 is not discussed in detail in the MS as no samples were collected for suspended particles; however, details
are included here to show the influence of close proximity to the island and fluvial/glacial sources. Table 2. Mean elemental molar ratios of marine snow particles captured in free-floating sediment traps, particulate matter (1–53 µm) below
the mixed layer and authigenic sediments at each station. Note that station TEW-1 is a near-coastal station located within Hillsborough Bay,
Kerguelen Island. Station TEW-1 is not discussed in detail in the MS as no samples were collected for suspended particles; however, details
are included here to show the influence of close proximity to the island and fluvial/glacial sources. Sediment trap @ 210 m
pFe : pAl
pMn : pAl
pMn : pFe
pBa : pAl
A3-2
0.70
0.008
0.011
0.025
E-1
1.02
0.009
0.009
0.162
E-3
1.05
0.010
0.010
0.285
E-5
0.91
0.008
0.009
0.318
Suspended particles mean (> MLD)
A3-1
0.53
0.007
0.013
0.027
A3-2
0.63
0.009
0.014
0.034
R-2
0.65
0.036
0.059
0.322
F-L
0.77
0.020
0.027
0.190
E-4E
0.86
0.037
0.045
0.383
E-4W
0.63
0.014
0.021
0.078
E-1
0.68
0.023
0.034
0.185
E-3
0.71
0.024
0.033
0.258
E-5
0.68
0.020
0.030
0.260
Sediment analysis
TEW-1
1.10
0.013
0.012
0.003
A3-1
0.87
0.011
0.013
0.026
R-2
0.73
0.063
0.086
0.892
F-L
0.82
0.016
0.019
0.040
E-4W
0.81
0.013
0.016
0.013
E-3
0.93
0.015
0.016
0.125
Kerguelen Archipelago
0.08–0.49
0.004–0.010
0.021–0.050
0.002–0.004
Basalt mean
(Gautier et al., 1990)
Upper continental crust
0.19
0.003
0.017
0.002
(Wedepohl, 1995) explain the observed difference, given the strong decrease of
nepheloid layers away from the seabed (Blain et al., 2007;
Jouandet et al., 2014). all size classes combined (i.e. > 1 µm). However, if we look
closer at the pFe distribution only within the surface mixed
layer (165 m) between A3-1 and A3-2, we observe a loss of
70 % of the integrated total pFe (> 1 µm) (Fig. 4). Concur-
rently, using an Underwater Vision Profiler to track particle
size distribution, Jouandet et al. (2014) noted a four-fold in-
crease in particle numerical abundance through the full water
column. 3.3
Underlying sediment analysis Analysis of sediments sourced from cores taken at each sta-
tion revealed a distinctly different sediment signature at sta-
tion R-2 compared with any other station (Table 2). The
reference station signature was approximately six times en-
riched in Mn relative to Al (Mn : Al 0.063) in comparison
to the plateau station (A3) (Mn : Al 0.011). The Mn : Al
sedimentary signature at A3 was almost identical to authi-
genic sediments previously reported from the Amundsen Sea
(Angino, 1966). We consider that the enriched Mn at R-2
could be due to either MnO2 enrichment in the surface sedi-
ments during redox cycling of early diagenesis (Planquette et
al., 2013), or supplied via a Mn enriched source such as hy-
drothermal venting near the Leclaire Rise. The extremely low
carbon content of the sediment at station R-2, as evidenced
by its near white colour, low diatom content (L. Armand,
pers. obs., 2012) and low carbon export flux (Laurenceau et
al., 2014; Planchon et al., 2014), suggests that MnO2 enrich-
ment in the surface sediments during redox cycling is more
likely at R-2. The pFe, pAl and pMn concentrations at the reference sta-
tion (R-2) only increase slightly towards the sea floor; how-
ever, enrichment in pFe, pAl and primarily pMn is evident
at 500 m likely due to proximity to the Leclaire Rise (Fig. 3)
(discussed in detail below). The northern PF station (F-L) ex-
hibits moderate concentrations of pFe, pAl and pMn through-
out the water column, somewhat higher than the reference
station, and much higher concentrations are observed in close
proximity to the sea floor. It should be noted that the deep-
est sample at R-2 was 148 m above the seafloor, while at F-
L it was only 90 m above the sea floor and this could well The authigenic sediment ratio of Fe : Al was also lower at
station R-2 (Fe : Al = 0.73) in comparison to any other sta-
tion (Fe : Al range 0.81–1.1). However, all of our observed
pFe : pAl molar ratios were higher than upper crustal mo-
lar ratios (Fe : Al 0.19) (Wedepohl, 1995) or Amundsen Sea
surface sediments (Fe : Al 0.26) (Angino, 1966). Interest-
ingly, our observed pFe : pAl sedimentary ratios were sim- www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources Their one dimensional particle dynamic model sup-
ported the hypothesis that the increase in biogenic particles,
due to blooming conditions, resulted in the rapid formation
of large particles due to coagulation and subsequent vertical
transport to the base of the mixed layer. This result is sup-
ported in the current data set, in that we see a large decrease
in small pFe particles within the mixed layer and a moderate
increase in large pFe particles at the base of the mixed layer
when comparing pre (A3-1) to post (A3-2) bloom conditions
(Fig. 4). Furthermore, we observed an increase in biogenic
pFe within the surface mixed layer between A3-1 and A3-2
(Sect. 3.7). Profiles of pFe and pAl in the 1–53 µm size range from sta-
tion R-2, A3 and F-L are shown in Fig. 3. The plateau station
(A3) was sampled twice during the study (A3-1 and A3-2),
separated by 20 days. Surface chlorophyll images revealed
that between visits to the site, a large bloom developed in
the vicinity and extended over the site, and was beginning to
fade again by the time of the second sampling (Trull et al.,
2014). Thus, station A3-1 can be thought of as pre-bloom and
A3-2 as post-bloom conditions. Particulate Fe, Al and Mn
generally increased towards the sea floor at station A3, with
the exception of a slight enrichment below the mixed layer
during the second visit (A3-2) to the station in the > 53 µm
size fraction (Fig. 4). To investigate the progression of pFe
through time, we integrated the pFe throughout the full wa-
ter column, and observed a decrease in the pFe stock from
9.1 to 4.5 mmol m−2 between the first and second visit to
station A3. This translates to a 51 % reduction in pFe for Biogeosciences, 12, 739–755, 2015 www.biogeosciences.net/12/739/2015/ www.biogeosciences.net/12/739/2015/ Profiles of particulate Fe (a), Al (b) and Mn (c) (1–53 µm) at the reference HNLC station (R-2), the northern PF station (F-L)
and pre- and post-bloom over the plateau station (A3-1 A3-2) highlighting the contrasting particulate trace metal supply to these locations Ocean Data View
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D Figure 3. Profiles of particulate Fe (a), Al (b) and Mn (c) (1–53 µm) at the reference HNLC station (R-2), the northern PF station (F-L)
and pre- and post-bloom over the plateau station (A3-1, A3-2), highlighting the contrasting particulate trace metal supply to these locations. Biogenic Fe (d) (as a percentage of the total Fe) in surface waters shows a clear progression that can be explained by the location of each
station within the study area whereby, biogenic Fe at R-2 > > F-L > A3-2 > A3-1. Ocean Data View
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A3-2
Figure 4. Particulate Fe at the plateau station (A3) by size class. The integrated full water column pFe (> 1 µm) reduced by 51 % between
A3-1 and A3-2 (9.1–4.5 mMol m−2 at A3-1 and A3-2 respectively). The integrated mixed layer pFe reduced by 70 % between A3-1 and
A3-2 (1.4–0.56 mMol m−2 at A3-1 and A3-2 respectively). The mixed layer shoaled between A3-1 and A3-2 as illustrated by the dashed
horizontal line. The calculation of integrated mixed layer pFe used a constant mixed layer depth of 165 m for both A3-1 and A3-2 to allow
comparison between these stations. Ocean Data View
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pFe (nM) (>1 µm) Figure 4. Particulate Fe at the plateau station (A3) by size class. The integrated full water column pFe (> 1 µm) reduced by 51 % between
A3-1 and A3-2 (9.1–4.5 mMol m−2 at A3-1 and A3-2 respectively). The integrated mixed layer pFe reduced by 70 % between A3-1 and
A3-2 (1.4–0.56 mMol m−2 at A3-1 and A3-2 respectively). www.biogeosciences.net/12/739/2015/ www.biogeosciences.net/12/739/2015/ P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 746 g
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R-2
A3-1
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A3-1
A3-2
FL
R2
A
B
C
D
Figure 3. Profiles of particulate Fe (a), Al (b) and Mn (c) (1–53 µm) at the reference HNLC station (R-2), the northern PF station (F-L)
and pre- and post-bloom over the plateau station (A3-1, A3-2), highlighting the contrasting particulate trace metal supply to these locations. Biogenic Fe (d) (as a percentage of the total Fe) in surface waters shows a clear progression that can be explained by the location of each
station within the study area whereby, biogenic Fe at R-2 > > F-L > A3-2 > A3-1. Ocean Data View
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Figure 3. www.biogeosciences.net/12/739/2015/ The mixed layer shoaled between A3-1 and A3-2 as illustrated by the dashed
horizontal line. The calculation of integrated mixed layer pFe used a constant mixed layer depth of 165 m for both A3-1 and A3-2 to allow
comparison between these stations. conditions of A3-2 may be due to increased small particle
scavenging resulting from sinking phyto-aggregates or alter-
natively, small-scale variation in the thickness of the neph-
eloid layer. Thus, it appears that physical aggregation within the mixed
layer of the particles onto biogenic phyto-aggregates dur-
ing the bloom development and export to the base of the
mixed layer, combined with significantly lower concentra-
tions above the seafloor resulted in the observed 51 % reduc-
tion in pFe (> 1 µm) between A3-1 and A3-2. The signifi-
cantly lower concentration at 440 m during the post-bloom 3.5
Elemental ratios at stations R-2, F-L and A3 as well as Ba : Al are strikingly unique. At station R-2, be-
low 500 m, we see Mn : Fe 2 × higher than A3, Mn : Al 4.5 ×
higher and Ba : Al ratios 10 × higher than at A3, making this
source signature relatively clear (Table 2). The unique ratios
below 500 m at R-2 may arise from a source of dissolved
or particulate Mn (uncoupled from pFe and pAl) from the
Leclaire Rise. Furthermore, the elemental ratios over station
A3 are generally much lower (Fig. 5) due to high pFe and pAl
supply over the Kerguelen Plateau that is relatively deficient
in pMn, such as would be found in glacial runoff that has
a signature which reflects fresh weathering of basaltic rocks
(Doucet et al., 2005). This source theory is supported by the
observation of high dissolved Mn (dMn) near the Leclaire
Rise (Quéroué et al., 2015) and uniquely high pMn : pFe in
sediments found below station R-2 (Table 2). Surface water
particulate trace metals also reveal distinct differences. The
ratios of pFe : pAl, pMn : pAl and pMn : pFe all increase from
the bottom of the mixed layer to the surface at stations R-2
and F-L (Fig. 5). This profile characteristic is in contrast to
stations A3-1. The observed modification of the elemental ra-
tios in the surface mixed layer at R-2 and F-L is most likely
due to biological uptake of dissolved trace elements and con-
version into the biogenic particulate fraction. As station A3 is located over the Kerguelen Plateau and
also is in close proximity to fluvial and glacial runoff from
Heard Island, we would expect the trace metal source signa-
ture of suspended particles to be unique here in comparison
to our reference (R-2) and PF (F-L) stations. The particles
over the Kerguelen Plateau were characterised by very high
pFe (0.94–30.4 nM) and pAl (1.5–58.6 nM) with concentra-
tions an order of magnitude higher than R-2 (< DL–1.35 nM
Fe and < DL–2.08 nM Al). The reference station was char-
acterised by low surface Chl a concentrations characteristic
of HNLC waters (Lasbleiz et al., 2014), however, it is rel-
atively close to the Leclaire Rise; a seamount with its shal-
lowest point 135 km west north-west of station R-2 rising up
to approximately 395 m. www.biogeosciences.net/12/739/2015/ www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources P. van der Merwe et al.: Kerguelen Plateau: particulate tra
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pBa:pAl
A3-1
A3-2
F-L
R-2
Depth (m)
Figure 5. Profiles of elemental ratios at the reference station (R-2),
northern PF (F-L) and pre- and post-bloom over the plateau station
(A3-1, A3-2). Note the increase in pMn and pBa relative to pAl at
station R-2 below 500 m. 747 P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources
747
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Figure 5. Profiles of elemental ratios at the reference station (R-2),
northern PF (F-L) and pre- and post-bloom over the plateau station
(A3-1, A3-2). Note the increase in pMn and pBa relative to pAl at
station R-2 below 500 m. Depth (m)
3000
2500
2000
1500
1000
500
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3000
2500
2000
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.08
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E-5
E-4W
E-4E
E-3
E-1
A3-2
A3-1
Station
mean Kerguelen basalt
plateau sediment
reference station sediment
Figure 6. Molar ratio of pMn : pAl of suspended particles versus
depth, separated by station type. Vertical lines represent the median
molar ratios within Kerguelen Island basalts (Gautier et al., 1990)
(black), authigenic Kerguelen Plateau sediments (red) and station
R-2 authigenic sediments (green). www.biogeosciences.net/12/739/2015/ Depth (m)
3000
2500
2000
1500
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500
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2500
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1500
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.10
.08
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1500
1000
500
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A3-1 A3-2
R-2 and F-L
E-1 E-3 E-5 E-4W E-4E
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F-L
E-5
E-4W
E-4E
E-3
E-1
A3-2
A3-1
Station
mean Kerguelen basalt
plateau sediment
reference station sediment
Figure 6. Molar ratio of pMn : pAl of suspended particles versus
depth, separated by station type. Vertical lines represent the median
molar ratios within Kerguelen Island basalts (Gautier et al., 1990)
(black), authigenic Kerguelen Plateau sediments (red) and station
R-2 authigenic sediments (green). Ocean Data Vie
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mean Kerguelen basalt
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pBa:pAl Depth (m) Figure 6. Molar ratio of pMn : pAl of suspended particles versus
depth, separated by station type. Vertical lines represent the median
molar ratios within Kerguelen Island basalts (Gautier et al., 1990)
(black), authigenic Kerguelen Plateau sediments (red) and station
R-2 authigenic sediments (green). Figure 5. Profiles of elemental ratios at the reference station (R-2),
northern PF (F-L) and pre- and post-bloom over the plateau station
(A3-1, A3-2). Note the increase in pMn and pBa relative to pAl at
station R-2 below 500 m. 3.5
Elemental ratios at stations R-2, F-L and A3 The Leclaire Rise extends to 70km
north-west of station R-2 where it reaches a depth of approx-
imately 550 m. It is important to recall in this context that
the PF divides the northeast flowing AASW from the east-
ward flowing SASW to the north (Park et al., 2008). Classical
theory suggests that this oceanographic feature should block
much of the enrichment from the Leclaire Rise to station R-
2. However, enrichment was evident in the vertical profiles
of pFe, pMn and pAl at station R-2 at 500 m depth (Fig. 3). Figure 5 illustrates the full water column elemental ratios
at the reference station (R-2) in comparison to the Kergue-
len Plateau stations (A3-1 and A3-2) and reveals that Mn : Fe Particulate Al and pFe were closely coupled across all sta-
tions (Spearman’s ρ R = 0.91 P < 0.01 n = 70). However,
pAl and pMn, although still strongly correlated (P = 0.80 www.biogeosciences.net/12/739/2015/ P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources It is thought that
this dFe is leached from nanoparticulate Fe (oxyhydr)oxides
in glacial rock flour over time (Raiswell, 2011; Raiswell et
al., 2010) following an exponential decay, so it is possible
that this source could be excluded from the < 0.2 µm dis-
solved fraction, but included in the 1–53 µm particulate frac-
tion presented here. This is especially true of nanoparticulate
Fe that is attached to the surface of larger sediment grains
as has been observed previously in glacial sediments (Shaw
et al., 2011). Given that the particulate fraction is generally
an order of magnitude higher in concentration than the dis-
solved fraction, this source may well be more significant in
stimulating phytoplankton blooms than previously estimated. Overall, station A3 appeared to be directly fertilised by resus-
pension of shelf sediments at depth, and entrainment of this
pFe-rich water occurred during events that deepen the mixed
layer periodically. However, lateral supply above the mixed
layer of small particles from shallow coastal sources around
Heard Island, including glacial melt waters, cannot be ruled
out. 3.6
What are the sources of particulate trace metals
over the plateau and downstream? The high particulate trace metal concentrations found at
440 m, at A3-1 and A3-2, near the sea floor, most likely
originated from resuspension of deep (∼500 m) shelf sedi-
ments. The cause of the variability between A3-1 and A3-2
at this depth remains unclear, but could reflect small scale
variability in the depth of the nepheloid layer or be the result
of temporal variability due to the action of tides and inter-
nal waves (McCave, 1986). The pMn : pAl, pMn : pFe and
pBa : pAl ratios for A3-1 and A3-2 are similar from the sea
floor to the approximate base of the surface mixed layer, with
values higher than the mean crustal ratios (Wedepohl, 1995),
but lower than either basalt (Gautier et al., 1990) or the un-
derlying sediment ratios (Table 2). Within the surface mixed
layer, A3-1 maintains similar ratios to the deep water col-
umn, while at A3-2 the ratios diverge towards the surface. The particulate trace metal signature within the mixed layer
at A3-2 increases in pMn and pBa relative to pAl, which
is most likely driven by biogenic conversion of dissolved
bioessential elements into biogenic particles (Sect. 3.7). P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 748 P < 0.01 n = 70), appeared more variable than pFe versus
pAl. Figure 6 illustrates this variability in the pMn in com-
parison to pAl as a function of its location within the study
area. The observed variability in pMn but not pFe relative to
pAl highlights the uncoupling between some of the sources
of pMn and pFe. Specifically, the observed variability points
to a uniquely high Mn : Fe source either in the authigenic
sediments of the Leclaire Rise and/or a hydrothermal source
(German et al., 1991), or a process whereby pAl is prefer-
entially stripped out with distance from the source. A study
by Shigemitsu et al. (2013) showed that the concentration of
pAl in suspended particulate material in the intermediate wa-
ter of the Sea of Okhotsk (western Pacific Ocean) decreased
with distance from the shelf source preferentially in relation
to pFe and pMn. They concluded that increasing pFe : pAl
and pMn : pAl ratios occurred with distance from the source
and suggested that the denser, lithogenic particles settled out
preferentially, stripping out pAl. Furthermore, they found
pFe became associated with organic matter more readily than
pAl and therefore, remained suspended in the water column
more readily than pAl. These processes could explain the ob-
served modification of the elemental ratios between stations
R-2, A3 and possibly F-L. The stations that were in close
proximity to the shelf source such as A3-1 and A3-2 were
indeed enriched in lithogenic pAl and as such pFe : pAl was
relatively low (Fig. 5). In contrast, F-L was at the greatest
distance from a sedimentary source and displayed the high-
est pFe : pAl ratio. be a major source of bioavailable Fe to the Southern Ocean
(Poulton and Raiswell, 2005; Raiswell et al., 2008a, b, 2006). Assuming no mixing and a dilute suspension, Stokes’ law
predicts that the small grain size of glacial flour allows it to
remain suspended within a 500 m water column for between
2 and 2500 days or within a 165m mixed layer for 1–831 days
depending on particle size. Certainly, mixing within the sur-
face mixed layer would increase this duration significantly,
meaning that glacial and fluvial input from both Heard and
Kerguelen Island could remain suspended in the mixed layer
for long enough to travel well past any of the stations in the
present study, excluding the reference station (R-2). P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources Further-
more, it has been shown that 2–3 % of the Fe within glacial
rock flour can be leached into the dissolved size fraction
(< 0.2 µm) with ultrapure water; a large proportion of which
should be bioavailable (Schroth et al., 2009). It is thought that
this dFe is leached from nanoparticulate Fe (oxyhydr)oxides
in glacial rock flour over time (Raiswell, 2011; Raiswell et
al., 2010) following an exponential decay, so it is possible
that this source could be excluded from the < 0.2 µm dis-
solved fraction, but included in the 1–53 µm particulate frac-
tion presented here. This is especially true of nanoparticulate
Fe that is attached to the surface of larger sediment grains
as has been observed previously in glacial sediments (Shaw
et al., 2011). Given that the particulate fraction is generally
an order of magnitude higher in concentration than the dis-
solved fraction, this source may well be more significant in
stimulating phytoplankton blooms than previously estimated. Overall, station A3 appeared to be directly fertilised by resus-
pension of shelf sediments at depth, and entrainment of this
pFe-rich water occurred during events that deepen the mixed
layer periodically. However, lateral supply above the mixed
layer of small particles from shallow coastal sources around
Heard Island, including glacial melt waters, cannot be ruled
out. be a major source of bioavailable Fe to the Southern Ocean
(Poulton and Raiswell, 2005; Raiswell et al., 2008a, b, 2006). Assuming no mixing and a dilute suspension, Stokes’ law
predicts that the small grain size of glacial flour allows it to
remain suspended within a 500 m water column for between
2 and 2500 days or within a 165m mixed layer for 1–831 days
depending on particle size. Certainly, mixing within the sur-
face mixed layer would increase this duration significantly,
meaning that glacial and fluvial input from both Heard and
Kerguelen Island could remain suspended in the mixed layer
for long enough to travel well past any of the stations in the
present study, excluding the reference station (R-2). Further-
more, it has been shown that 2–3 % of the Fe within glacial
rock flour can be leached into the dissolved size fraction
(< 0.2 µm) with ultrapure water; a large proportion of which
should be bioavailable (Schroth et al., 2009). www.biogeosciences.net/12/739/2015/ www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 www.biogeosciences.net/12/739/2015/ P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources Given that the pFe and pMn minima coincides with
the remnant-winter-water temperature minimum, the total
amount of particulate trace metals distributed throughout the
winter mixed layer must be lower than during summer. This
is counterintuitive if sediment resuspension is the primary
source of particulate trace metals into the recirculation fea-
ture. During winter we would expect increased wind mixing,
leading to more entrainment of pFe over the plateau and more
supply into the recirculation feature leading to a maximum at
the temperature minimum. Given that we observe the inverse
situation, supply into the recirculation structure must be low
during winter. Thus, we suggest that the lateral supply of flu-
vial and glacial derived particulate trace metals must be an
important source. This source would be expected to reduce
in winter when precipitation as snow and glacial freezing is
at a maximum and conversely, during spring, snow and ice
melt and rainfall increases runoff into the coastal areas and
induces a fertilisation event downstream of Heard and Ker-
guelen Islands. Kerguelen Island is a subantarctic island, and
its climatology is cold and wet, with the Port-aux-Français
weather station recording mean daily temperatures of 2.1 ◦C
in winter and 8.2 ◦C in summer and year round consistent
precipitation (730 mm annually) (Meteo France). It should be
noted that due to its sheltered location and sea-level altitude,
the Port-aux-Français location is relatively mild compared to
the west coast and interior of the island which is estimated to
receive three times the rainfall of the east coast, or 2124 mm
annually. Therefore, having a climate of high precipitation
and seasonal thawing, increased seasonal runoff can be ex-
pected in spring and summer from Kerguelen Island. Alternatively, the relative importance of sedimentary in-
put at each of the stations can be gauged by observing the
pMn : pAl molar ratio within suspended particles and com-
paring these to known molar ratios of pMn : pAl within Ker-
guelen Island basalts, A3 authigenic sediments and R-2 au-
thigenic sediments (Fig. 6). At station A3 we see that almost
all the suspended particles lie within the ratio of plateau sed-
iments and Kerguelen Island basalts with the remaining sus-
pended particles associated with the development of a bloom
in surface waters at A3-2, which is also where we see an in-
crease in biogenic Fe. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources Biogenic Fe at stations A3-1 and A3-
2 constitutes less than 1 and 5 % respectively of the total Fe. The low biogenic fraction at station A3-1 most likely results
from an excess of lithogenic Fe, Al and Mn to the water col-
umn from the shelf sediments as well as fluvial/glacial runoff
from nearby islands of the Kerguelen Archipelago, which
are excess to demand. A similar pattern was observed dur-
ing a study located in the Amundsen Sea (Planquette et al.,
2013) where the percentage of biogenic Fe (full water col-
umn mean) reduced with proximity to the trace metal source. 1990; Wedepohl, 1995). Thus, the factor of 1000 increase in
pP observed within suspended particles compared to these
rock sources indicates that this pP is likely produced in situ
within the mixed layer from dissolved PO−
4 rather than sup-
plied from rock weathering together with Fe. Furthermore,
within the upper 200 m of the water column, biogenic Fe cor-
relates significantly with both fluorescence (Spearman’s ρ
R = 0.518, P < 0.05, n = 30) and dissolved oxygen (Spear-
man’s ρ R = 0.507, P < 0.05, n = 30) confirming the au-
totrophic composition of the particles identified as high in
biogenic Fe. Figure 3 illustrates the contribution of biogenic
Fe in surface waters at stations R-2, A3 and F-L. Station R-
2 and F-L have biogenic Fe fractions that are higher near
the surface than at depth (Fig. 3). In contrast, at stations
A3-1 and A3-2, biogenic Fe and Mn only make up a rela-
tively small fraction of the total pFe throughout the water
column although at station A3-2 we see a slight increase in
biogenic pFe towards the surface, corresponding with the de-
velopment of a bloom. Biogenic Fe at stations A3-1 and A3-
2 constitutes less than 1 and 5 % respectively of the total Fe. The low biogenic fraction at station A3-1 most likely results
from an excess of lithogenic Fe, Al and Mn to the water col-
umn from the shelf sediments as well as fluvial/glacial runoff
from nearby islands of the Kerguelen Archipelago, which
are excess to demand. A similar pattern was observed dur-
ing a study located in the Amundsen Sea (Planquette et al.,
2013) where the percentage of biogenic Fe (full water col-
umn mean) reduced with proximity to the trace metal source. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources Mid-depth suspended particles at E4-
W (red dots) also lie between Kerguelen Island basalts and
plateau sediments, indicating a similar source to station A3. The reference station exhibits highly modified pMn : pAl mo-
lar ratios within the suspended particles and its underlying
sediment. This modification is most likely due to biogenic
incorporation of bioessential elements such as Mn into par-
ticles. The remaining stations are intermediate between A-3
and R-2. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 749 approximately 150–175 m (Fig. 7). Our detailed depth pro-
file indicates that the pFe and pMn minima coincide with
the remnant winter water temperature minimum (Fig. 8). In-
terestingly, Blain et al. (2014) also estimated a winter water
depth of approximately 150 m. They observed, at 150 m, that
nitrate and phosphate profiles within the recirculation fea-
ture, from multiple years and seasons, converged with strik-
ing consistency. Particulate Fe and pMn, concentrations in-
crease above and below the temperature minimum, however,
pAl only increases below 175 m. Particulate Al is stripped
out preferentially with settling lithogenics while pFe and
pMn are retained either through conversion to the biogenic
particulate fraction (uptake) or adsorbed onto organic parti-
cles. It should be noted here that the work of Raiswell (2011)
indicates that iceberg and glacially derived Fe nanoparticu-
late material is typically high in Fe and low in Al. Thus, sup-
ply of glacially derived nanoparticulate Fe from Kerguelen
Island, via the north-east of the recirculation structure could
also cause the observed high Fe, low Al surface enrichment
within the recirculation structure. 1990; Wedepohl, 1995). Thus, the factor of 1000 increase in
pP observed within suspended particles compared to these
rock sources indicates that this pP is likely produced in situ
within the mixed layer from dissolved PO−
4 rather than sup-
plied from rock weathering together with Fe. Furthermore,
within the upper 200 m of the water column, biogenic Fe cor-
relates significantly with both fluorescence (Spearman’s ρ
R = 0.518, P < 0.05, n = 30) and dissolved oxygen (Spear-
man’s ρ R = 0.507, P < 0.05, n = 30) confirming the au-
totrophic composition of the particles identified as high in
biogenic Fe. Figure 3 illustrates the contribution of biogenic
Fe in surface waters at stations R-2, A3 and F-L. Station R-
2 and F-L have biogenic Fe fractions that are higher near
the surface than at depth (Fig. 3). In contrast, at stations
A3-1 and A3-2, biogenic Fe and Mn only make up a rela-
tively small fraction of the total pFe throughout the water
column although at station A3-2 we see a slight increase in
biogenic pFe towards the surface, corresponding with the de-
velopment of a bloom. 3.7
Biogenic and sedimentary particulate trace metals If we assume that all particulate phosphorus (pP) is of bio-
genic origin, we can calculate the biogenic Fe fraction of
the total Fe concentration by normalising to pP and com-
paring with published elemental ratios of Southern Ocean
diatoms (Planquette et al., 2013). For the calculations we
used the upper limit of Fe : P (1.93 mmol mol−1) reported
by Twinning et al. (2004) for Southern Ocean diatom assem-
blages. Given that pP and POC are remineralised through-
out the water column and are generated within the surface
mixed layer, calculations of biogenic trace metals will only
be valid within the surface mixed layer, as the concentra-
tion of pP and POC decreases strongly with depth. It should
also be noted that Kerguelen Island basalts and upper conti-
nental crust can contribute particulate phosphorus concomi-
tantly with pFe to the particulate pool. However the Fe : P ra-
tio found within Kerguelen Island basalts and the continental
crust is 12.8 and 25.8 (mol : mol) respectively (Gautier et al., Glacial flour is the result of mechanical erosion of bed
rock by glaciers. Typical particle sizes are within the silt size
range but can overlap with clays (0.002–0.063 mm). Recent
research suggests that Fe sourced from glacial erosion can Biogeosciences, 12, 739–755, 2015 www.biogeosciences.net/12/739/2015/ www.biogeosciences.net/12/739/2015/ 3.8
Pseudo-Lagrangian, recirculation structure Station E-4W has trace metal concentrations, elemental ra-
tios and community size structure (Trull et al., 2014) simi-
lar to A3 and as such, will be excluded from the discussion
in this section. The remaining recirculation structure stations
exhibit profiles of pFe and pMn which show a minimum at The importance of glacial/fluvial sources in supplying dis-
solved Fe and Mn into coastal waters to the north-east of Ker-
guelen, north of the PF, has been shown previously by Buc-
ciarelli et al. (2001). The authors found a linear relationship
between dissolved Fe and lithogenic silica and suggested www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 750
P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources
Ocean Data Vie
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pFe (1-53 µm) (nM)
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pAl (1-53 µm) (nM)
Depth (m)
71˚E
72˚E
73˚E
49˚S
48.6˚S
48.2˚S
Ocean Data View
E-3
E-4E
E-1
E-5
E-4W
E-1
E-3
E-5
E-4E
E-4W
Figure 7. Profiles of particulate trace metals during the pseudo-Lagrangian recirculation-structure study. Station E-4W (gray triangle) exhibits
unique trace metal profiles in comparison to the remaining stations (see text for details). Note the distinct pFe and pMn minima at 150–175 m. Particulate Al exhibits a similar profile albeit without surface enrichment. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 750 Ocean Data Vie
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pFe (1-53 µm) (nM) Depth (m) pMn (1-53 µm) (nM) 71˚E
72˚E
73˚E
49˚S
48.6˚S
48.2˚S
Ocean Data View
E-3
E-4E
E-1
E-5
E-4W Figure 7. Profiles of particulate trace metals during the pseudo-Lagrangian recirculation-structure study. Station E-4W (gray triangle) exhibits
unique trace metal profiles in comparison to the remaining stations (see text for details). Note the distinct pFe and pMn minima at 150–175 m. Particulate Al exhibits a similar profile albeit without surface enrichment. 3.8
Pseudo-Lagrangian, recirculation structure Ocean Data Vie
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1.5
2
2.5
3
3.5
500
400
300
200
100
0
Ocean Data View
Potential Temperature (°C)
Depth (m)
Figure 8. Temperature profiles within the upper 500 m within the
recirculation structure are shown alongside particulate Fe (1–53 µm)
within the upper 500 m within the recirculation structure. Corre-
spondence between the temperature minimum depth of winter water
and pFe minimum is illustrated. Ocean Data View
1.5
2
2.5
3
3.5
500
400
300
200
100
0
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Potential Temperature (°C)
Depth (m) Ocean Data Vie
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pFe (1-53 µm) (nM)
E-1
E-3
E-5
E-4E This exponential decrease would be expected to apply to the
particulate fraction also; however, it appears that even with
an exponential decrease in pFe with distance from the coast,
particulate Fe enrichment, sourced from fluvial runoff, is ev-
ident on the southern side of the PF within the recirculation
feature. This exponential decrease would be expected to apply to the
particulate fraction also; however, it appears that even with
an exponential decrease in pFe with distance from the coast,
particulate Fe enrichment, sourced from fluvial runoff, is ev-
ident on the southern side of the PF within the recirculation
feature. The hypothesis of pFe supply from north of the PF into the
eastern side of the recirculation feature via the mixing zone
is supported by radium isotope data (Sanial et al., 2014) col-
lected during the KEOPS2 mission. Apparent radium ages
derived from the ratio of 224Ra / 223Ra (and using the ratio
observed within the Baie des Baleiniers as the starting time)
suggest that the age of water since fertilisation within the
recirculation feature was only 5–8 days. This indicates that
there is likely rapid transfer across the PF of fertilised wa-
ters which were sourced from nearby shallow coastal areas
such as the Baie des Baleiniers, Kerguelen Island. The au-
thors go on to highlight that the heterogeneous distribution
of 224Ra and 223Ra indicates that transfer across the Polar
Front is sporadic in nature. Figure 8. Temperature profiles within the upper 500 m within the
recirculation structure are shown alongside particulate Fe (1–53 µm)
within the upper 500 m within the recirculation structure. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources fresher and warmer water within the upper 110 m than either
the R-2 or A3 stations, suggesting that glacial/fluvial runoff
from Kerguelen Island may well be delivering this high pFe,
low pAl surface enrichment. Over the mesoscale, it appears that physical processes as-
sociated with settling of refractory lithogenic particles was
an important process in modifying the particulate elemental
ratios. However, on the individual profile scale, biological
processes seem important in modifying the elemental ratios
in surface waters through preferential uptake of bio-essential
elements, even from the particulate fraction. 3.8
Pseudo-Lagrangian, recirculation structure Corre-
spondence between the temperature minimum depth of winter water
and pFe minimum is illustrated. that this was indicative of weathering of silicate rich min-
erals that characterise the Kerguelen Islands with a concomi-
tant release of dissolved Fe and Mn. Indeed, in the present
study, using the lithogenic and biogenic silica data presented
in Closset et al. (2014), total particulate Fe correlated signif-
icantly with total lithogenic silica (R = 0.76, P < 0.01) but
not with biogenic silica. However, this significant correlation
was not limited to the coastal regions in the present study
and instead was observed for all stations and depths com-
bined. Bucciarelli et al. (2001) found an exponential decrease
in dissolved Fe with distance from the coast, further support-
ing their theory of a dominant coastal source in this region. The observation of pFe enrichment in surface waters of
the recirculation structure without proportional concentra-
tions of pAl may be due to biological uptake and conver-
sion from a bioavailable pool into the biogenic particulate
pool. Settling of refractory lithogenics that are high in Al
may also partially explain the observation. Alternatively or
in combination, a high pFe, low pAl source such as nanopar-
ticulate Fe characteristic of glacial/fluvial runoff (Hawkings
et al., 2014; Raiswell et al., 2008b, 2006) on Kerguelen
Island could explain this observation. Indeed, temperature
and salinity profiles within the recirculation structure reveal www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 751 4
Conclusions Repeat sampling over the plateau provided a perspective
on the persistence of the particulate Fe availability. Small
particles containing pFe were efficiently transported out of
the mixed layer during a bloom event over stations A3. This
resulted in a 70 % reduction in the integrated pFe stock
within the mixed layer as a result of physical aggregation of
small particles onto phyto-aggregates, presumably decreas-
ing particle buoyancy and increasing export out of the mixed
layer. This is likely to be an important aspect of the complex
interaction between iron supply and biological availability,
capable of mediating bloom duration and thus the efficiency
of carbon sequestration. This study has identified two distinct areas of Fe fertilisation
in the vicinity of Kerguelen Island. Firstly, the plateau itself
is a major source of resuspended shelf sediments to station
A3 especially below the mixed layer. Secondly, fluvial and
glacial runoff into coastal waters in combination with resus-
pension of shallow coastal sediments fertilises areas to the
north of the PF, east of Kerguelen Island, but also across the
PF and into the recirculation feature from the north-east. In-
dications of particle transport across the PF were observed at
station R-2 sourced from the Leclaire Rise to the north of the
PF. Satellite imagery also revealed filaments clearly diverg-
ing from the main jet of the PF and into the north east of the
recirculation structure. Within the recirculation structure, the
correspondence of the winter water temperature minimum
with the particulate trace metal minimum implies that a sea-
sonal cycle is involved in the supply of trace elements. This
is most likely driven by increased fluvial and glacial runoff in
summer, associated with rainfall and basal melt and reduced
supply in winter when snowfall and freezing conditions pre-
dominate. In this complex region, it appears that weathering
of the islands themselves are direct sources of new Fe and
help stimulate the seasonal bloom that is significant in terms
of the regional carbon cycle. Biogeosciences, 12, 739–755, 2015 www.biogeosciences.net/12/739/2015/ www.biogeosciences.net/12/739/2015/ Biogeosciences, 12, 739–755, 2015 www.biogeosciences.net/12/739/2015/ www.biogeosciences.net/12/739/2015/ P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources 752 Biogeosciences, 12, 739–755, 2015 P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources using magnetic sector inductively coupled plasma-mass spec-
trometry, Anal. Chim. Acta, 676, 15–27, 2010. Acknowledgements. This work was supported by the Antarctic
Climate and Ecosystems Cooperative Research Centre, University
of Tasmania, Australia. This work was also supported by the
French research program of INSU-CNRS LEFE−CYBER (Les
enveloppes fluides et l’environnement – Cycles biogéochimiques,
environnement et ressources), the French ANR (Agence Nationale
de la Recherche, SIMI-6 program, ANR-10-BLAN-0614), the
French CNES (Centre National d’Etudes Spatiales) and the French
Polar Institute IPEV (Institut Polaire Paul−Emile Victor). We
would like to thank the captain and the crew of the R/V Marion
Dufresne, Stephane Blain and Bernard Quéguiner as chief scientist
and project coordinator of the KEOPS2 cruises, respectively. L. Ar-
mand was supported by grant Australian Antarctic Division, AAS
grant #3214. Access to Sector Field ICP-MS instrumentation was
supported through ARC LIEF funding (LE0989539). F. Dehairs
was supported by Belgian Science Policy grant SD/CA/05A; Flan-
ders Research Foundation grant G071512N and Vrije Universiteit
Brussel, Strategic Research Plan. Bowie, A. R., van der Merwe, P., Quéroué, F., Trull, T., Fourquez,
M., Planchon, F., Sarthou, G., Chever, F., Townsend, A. T.,
Obernosterer, I., Sallée, J.-B., and Blain, S.: Iron budgets
for three distinct biogeochemical sites around the Kerguelen
archipelago (Southern Ocean) during the natural fertilisation ex-
periment KEOPS-2, Biogeosciences Discuss., 11, 17861–17923,
doi:10.5194/bgd-11-17861-2014, 2014. Boyd, P. W., Jickells, T., Law, C. S., Blain, S., Boyle, E. A., Bues-
seler, K. O., Coale, K. H., Cullen, J. J., de Baar, H. J. W., Follows,
M., Harvey, M., Lancelot, C., Levasseur, M., Owens, N. P. J., Pol-
lard, R., Rivkin, R. B., Sarmiento, J., Schoemann, V., Smetacek,
V., Takeda, S., Tsuda, A., Turner, S., Watson, A. J., and Baar,
H. J. W. De: Mesoscale iron enrichment experiments 1993-2005:
Synthesis and future directions, Science, 315, 612–617, 2007. Bucciarelli, E., Bowie, A. R., and Tréguer, P.: Iron and manganese
in the wake of the Kerguelen Islands (Southern Ocean), Mar. Chem., 73, 21–36, 2001. Edited by: I. Obernosterer Chever, F., Sarthou, G., Bucciarelli, E., Blain, S., and Bowie, A. R.:
An iron budget during the natural iron fertilisation experiment
KEOPS (Kerguelen Islands, Southern Ocean), Biogeosciences,
7, 455–468, doi:10.5194/bg-7-455-2010, 2010. P. van der Merwe et al.: Kerguelen Plateau: particulate trace metal sources Closset, I., Lasbleiz, M., Leblanc, K., Quéguiner, B., Cavagna, A.-
J., Elskens, M., Navez, J., and Cardinal, D.: Seasonal evolution
of net and regenerated silica production around a natural Fe-
fertilized area in the Southern Ocean estimated with Si isotopic
approaches, Biogeosciences, 11, 5827–5846, doi:10.5194/bg-11-
5827-2014, 2014. Appendix A: Certified reference material analysis Table A1. Percentage recoveries of BCR-414 certified reference material. Certified and single lab values taken from the final report of the
Commission of the European Communities, Community Bureau of Reference for BCR-414, EUR14558. mg kg−1
Rep 1
Rep 2
Rep 3
Mean
SD
RSD (%)
Certified
% recovery
Single lab analysis
% recovery
Ba
34
26
32
31
4.0
13.1
31
99
Al
2243
1349
1943
1845
454.6
24.6
1800
102
Mn
278
284
283
282
3.4
1.2
299
94
Fe
1874
1850
1878
1867
15.0
0.8
1850
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Evaluating the “return on patient engagement initiatives” in medicines research and development: A literature review
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Health expectations
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R E V I E W A R T I C L E R E V I E W A R T I C L E Abstract Lidewij Eva Vat, Athena Institute, Vrije
Universiteit Amsterdam, Amsterdam,
Netherlands. Background: Showing how engagement adds value for all stakeholders can be an ef-
fective motivator for broader implementation of patient engagement. However, it is
unclear what methods can best be used to evaluate patient engagement. This paper
is focused on ways to evaluate patient engagement at three decision‐making points
in the medicines research and development process: research priority setting, clinical
trial design and early dialogues with regulators and health technology assessment
bodies. Evaluating the “return on patient engagement initiatives” in
medicines research and development: A literature review Lidewij Eva Vat MSc, Researcher1
| Teresa Finlay PhD, Researcher2
| Tjerk Jan
Schuitmaker‐Warnaar PhD, Assistant Professor1
| Nick Fahy DPhil, Researcher2
|
Paul Robinson FRCP, European Lead Patient Innovation3 | Mathieu Boudes PhD, PARADIGM
Coordinator4 | Ana Diaz PhD, Project Officer5 | Elisa Ferrer PhD, Patient Engagement
Senior Manager6 | Virginie Hivert PharmD, PhD, Therapeutic Development Director6 |
Gabor Purman MD, MBA, Senior Account Director7 | Marie‐Laure Kürzinger MSc, Benefit
Risk Expert8
| Robert A. Kroes MSc, Project Manager9 | Claudia Hey PhD, Senior
Director10 | Jacqueline E.W. Broerse PhD, Professor1 1Athena Institute, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
2Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK
3MSD (Merck Sharp & Dohme), London, UK
4European Patients' Forum (EPF), Brussels, Belgium
5Alzheimer Europe, Luxembourg, Luxembourg
6EURORDIS – Rare Diseases Europe, Paris, France
7Nexgen Healthcare Communications, London, UK
8Sanofi, Chilly-Mazarin, France
9Lilly Nederland BV, Utrecht, The Netherlands
10Merck Healthcare KGaA, Darmstadt, Germany 1Athena Institute, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
2Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, UK
3MSD (Merck Sharp & Dohme), London, UK
4European Patients' Forum (EPF), Brussels, Belgium
5Alzheimer Europe, Luxembourg, Luxembourg
6EURORDIS – Rare Diseases Europe, Paris, France
7Nexgen Healthcare Communications, London, UK
8Sanofi, Chilly-Mazarin, France
9Lilly Nederland BV, Utrecht, The Netherlands
10Merck Healthcare KGaA, Darmstadt, Germany 10Merck Healthcare KGaA, Darmstadt, Germany Received: 14 March 2019 | Revised: 2 July 2019 | Accepted: 17 July 2019 Received: 14 March 2019 | Revised: 2 July 2019 | Accepted: 17 July 2019 Received: 14 March 2019 | Revised: 2 July 2019 | Accepted: 17 July 2019
DOI: 10.1111/hex.12951 | 1
wileyonlinelibrary.com/journal/hex This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2019 The Authors Health Expectations published by John Wiley & Sons Ltd Health Expectations. 2019;00:1–14. 1 | INTRODUCTION to support engagement.2,14,15 Evaluation could also define the
genuine value of patients’ contributions, contributing to valued
rather than tokenistic inclusion for appearances’ sake.16 There is
also some resistance; people are concerned about assessing impact
too simplistically. 1 | INTRODUCTION Some question whether it is fair to evaluate the
value of patient input in isolation, and not that of others such as
key scientific leaders,12 not least because it may be the synergy of
working in partnership that produces benefit.17 As mentioned by
Staniszewska, it is important to recognize that “any form of mea-
surement sits within a political or ideological context that cannot
be ignored.”13 Nonetheless, there is a desire to assess the impact of
patient engagement, to demonstrate better decision making, avoid-
ance of previous errors and a contribution to continuous efficiency
and quality improvement.15,16,18 There is increasing consensus among stakeholders that patient en-
gagement in research and development (R&D) of medicines provides
benefits for patients, researchers, industry, regulatory bodies, pay-
ers and policy makers.1-3 The case for patient engagement is often
presented in ethical and political terms referring to fairness, trans-
parency and accountability.4,5 Methodological arguments consider
the experiential knowledge of patients—acquired by their personal
experience of a condition—as valuable to improving the quality and
relevance of the research.6-8 The inclusion of patients in decision
making about the development of new innovative medicines is a sub-
stantial change, requiring time and (financial) commitments from re-
searchers, industry and patients.2,4 Despite efforts to promote and
support patient engagement in research, the prevalence of patient
engagement in medicines research and development remains low.9,10
Patient engagement has not been fully embedded in the health re-
search system, partly because it is not yet clear to all involved what
the added value is.11 To address this need, an increasing number of
studies aim to evaluate the impact of patient engagement, under-
scoring the growing interest in the “return on engagement,” or why it
makes sense for patients, society and industry.2,12 Despite this perceived importance of assessing the return on pa-
tient engagement, little is known about “what” to evaluate, and even
less about “how.”19-21 A number of researchers have tried to assess
how patient engagement makes a difference.3,5,8,12,22-27 Although
there is no standardized way to assess the impact of patient en-
gagement, very similar benefits, costs and challenges are reported in
literature reviews.4,17,19,20,28-32 The current assessment of patient en-
gagement is considered weak, partly because much of the evidence
is mainly anecdotal17 and because methods used have not captured
the complexity, context or mechanisms of change.17,33 Previous stud-
ies have identified a number of gaps in the literature and identified
challenges such as the delayed nature of impact, inconsistent termi-
nology, absence of accepted criteria for judging the success or qual-
ity of research, no agreed evaluation methods or framework and few
reliable measurement tools. K E Y W O R D S evaluation, framework, impact, literature review, medicines development, metrics, patient and
public involvement, patient engagement, patient participation, research Funding information This study was funded by PARADIGM. PARADIGM is a public‐private partnership
and is co‐led by the European Patients’
Forum and the European Federation of
Pharmaceutical Industries and Associations
(EFPIA). PARADIGM is receiving funding
from the Innovative Medicines Initiative
Joint Undertaking 2. This Joint Undertaking
receives support from the European Union's
Horizon 2020 research and innovation
programme and EFPIA. Objective: Our aim was to review the literature on monitoring and evaluation of pa-
tient engagement, with a focus on indicators and methods. Search strategy and inclusion criteria: We undertook a scoping literature review
using a systematic search, including academic and grey literature with a focus on | 1
wileyonlinelibrary.com/journal/hex Health Expectations. 2019;00:1–14. 2 | 2 VAT et al. VAT et al. evaluation approaches or outcomes associated with patient engagement. No date
limits were applied other than a cut‐off of publications after July 2018. Data extraction and synthesis: Data were extracted from 91 publications, coded and
thematically analysed. Main results: A total of 18 benefits and 5 costs of patient engagement were identi-
fied, mapped with 28 possible indicators for their evaluation. Several quantitative and
qualitative methods were found for the evaluation of benefits and costs of patient
engagement. Discussion and conclusions: Currently available indicators and methods are of some
use in measuring impact but are not sufficient to understand the pathway to impact,
nor whether interaction between researchers and patients leads to change. We sug-
gest that the impacts of patient engagement can best be determined not by applying
single indicators, but a coherent set of measures. 1 | INTRODUCTION The absence of a control group—identi-
cal research carried out without patient engagement—is problematic
too, particularly in an area of science where direct comparison to The perceived value of patient engagement practices can vary
for different stakeholder groups, and the metrics of interest will
therefore differ accordingly.13 For example, for researchers and
industry partners it might be about evidence that patient en-
gagement improves the quality and efficiency of research and the
uptake of findings, whilst for patients it might be more about in-
fluencing the R&D agenda to develop medicines for unmet needs. Some argue that evidence is needed to justify the ‘business case’
for engagement. This could also help to establish a financial model | 3 | 3 3 VAT et al. TA B LE 1 Definitions
Concept
Description
Patient
engagement
The effective and active collaboration of patients, patient advocates, patient representatives and/or carers in the processes
and decisions within the medicines lifecycle, along with all other relevant stakeholders when appropriate1
Patient partner
A patient, patient advocate, patient representative and/or carer who contributes to any level of patient engagement activi-
ties; this can also be substituted for other terms such as patient contributor82
Research
participant
A person who participates in human subject research, also called a subject, study participant or volunteer of an experiment
or trial
Society
Includes all members of the public and patients who use health‐care services
Research priority
setting
Any process aimed at constructing priorities or agendas for health research and medicines development, to raise awareness
and change the way research funding is allocated
Design of clinical
trials
Any process aimed at the development or design of clinical trials for medicines development at any stage of that process. One example is changes made to inclusion and exclusion criteria for trial participants
Early dialogues
with regula-
tors and Health
Technology
Assessment
(HTA) bodies
Any process in which medical technology developers communicate with regulatory bodies and/or HTA bodies prior to
health technology assessment. 1 | INTRODUCTION Early dialogue can happen only with regulators (eg scientific advice), jointly with regulators
and HTA bodies (to discuss data requirements to support decision making on marketing authorization and reimbursement
simultaneously) or only with HTA bodies (eg EUnetHTA multi‐HTA dialogues)
Benefit
An advantage of engagement for research and development and stakeholders involved
Costs and
challenges
The expenditure and/or effort of engagement for research and development and the stakeholders involved
Outcomes
Decisions made and things produced as a direct result of patient engagement practices. One example is changes made in the
design of a clinical trial resulting in a more relevant and appropriate research protocol. Outcomes may lead to impact on
research and development
Impacts
Broader effect of outcomes, both positive and negative, of patient engagement. Impact may be direct or indirect, intended
or unintended. For example, this may include study quality benefits such as improved recruitment and retention of study
participants
Value
The benefits of patient engagement (in relation to the direct and indirect costs) for individuals and organizations involved
Monitoring
The formative evaluation of patient engagement practices in order to strengthen them
Evaluation
The ‘systematic acquisition and assessment of information to provide useful feedback about …’ patient engagement
practices.83 Summative evaluation examines the effects of patient engagement practices on various measures including
outcomes, impact and cost‐benefit
Criteria
Dimensions or parameters used for evaluation. These need to be translated into measurable entities called ‘indicators’ and
indicators are measured with ‘metrics’
Indicator
Qualitative or quantitative measure that provides a means of expressing achievement of a goal or ascertaining the con-
sequences of a specific change. Quantitative indicators are reported as numbers, such as rates of change and ratios. Qualitative indicators are reported as words, in statements, paragraphs and reports84
Metrics
Observations based on standardized data sources or agreed techniques for gathering information. Metrics could consist of
an agreed set of quantitative and/or qualitative indicators to measure evaluation criteria, with a set of agreed methods/
tools to collect this information
Methods
Ways to collect information for monitoring and evaluating the outcomes and impact of patient engagement practices, for
example quantitative, qualitative or mixed methods
Tools
Instruments to collect information about patient engagement practices. For example, interview guides, questionnaires, log
sheets and observation forms are all tools TA B LE 1 Definitions An advantage of engagement for research and development and stakeholders involved an existing standard is routinely demanded.8,16,34,35 It is argued that
to build an evidence base, some level of consensus on measurable
impacts is needed, whilst others state that the outcomes of engage-
ment cannot easily be quantified.13,30,36 In sum, it remains unclear
what methods can be best used to evaluate patient engagement. engagement and assessing the quality.37-39 Evaluation studies focus
mainly on qualitative methods and only occasionally link to specific
outcomes.12,33,40,41 Therefore, this paper is focused on ways to eval-
uate patient engagement with both qualitative and explicitly quan-
titative methods. To address the need for means of determining the “return on en-
gagement,” the aim of this paper was to scope, review and summarize
the literature on monitoring and evaluation of patient engagement. Many publications present useful guidance for conducting patient This work is part of the PARADIGM project, a public‐private
partnership that is developing ways to ensure that patients are al-
ways meaningfully involved in the development of medicines. The
impact of patient engagement may differ at different points in the 2 | METHODS We undertook a scoping review of published academic and grey
literature as recommended by Arksey and O’Malley, also drawing
on Mays et al and Peters et al42-44 Scoping reviews are similar to
systematic reviews in that they follow a structured search process;
however, they are performed for different reasons.45 Our aim was
not to answer a precise question addressing the effectiveness of a
certain practice, as in a meta‐analysis, but to provide an overview
of the breadth of the available literature about evaluating patient
engagement. Grey literature was searched using the same terms; items rec-
ommended by consortium partners and external stakeholders
were added manually, and reference lists of items included were
searched for additional publications. All searches were conducted
between 1 May 2018 and 31 July 2018. The search was limited
to publications in English. We excluded articles that did not pro-
vide information on possible evaluation approaches or outcomes
associated with patient or public engagement. No date limits
were applied other than a cut‐off of publications after 31 July
2018. Following completion of the search, duplicated items were
removed. Whilst the review is concerned with patient engagement at the
three key decision‐making points, we used broader search limits to
ensure capture of related publications in other areas of health re-
search. One of the challenges was the variety of terminology. For ex-
ample, the words “measure,” “metric” and “indicator” are often used
interchangeably and their definitions may vary, if they are stated
at all. Furthermore, the terms used for “patient engagement” differ
globally. In this paper, we use the term patient engagement; in our
search, we included terms such as public involvement, patient par-
ticipation, community engagement and user involvement. In Table 1,
we provide definitions of terms developed by the authors and as we
used them in this review. 4 | VAT et al. TA B LE 2 Search terms
Patient engagement
(title only)
Research
(title only)
Outcomes
(title/abstract only)
Patient participation
[MeSH]
Comparative
effective-
ness research
[MeSH]
Outcome(s)
Impact
Patient engagement
Research
Measurement(s)
Public engagement
Clinical trial
Metrics
Client engagement
Study design
Framework(s)
Community engagement
Trial design
Assessment
Public participation
Research design
Criteria
Patient participation
Health
technology
assessment
Indicator(s)
Public involvement
Agenda setting
User involvement
Client involvement
Consumer involvement development of a medicine. Accordingly, PARADIGM focuses on
three decision‐making points during R&D at which point integration
of the patient perspective is considered likely to be valuable, specif-
ically as part of research priority setting, design of clinical trials and
at early dialogues with regulators and health technology assessment
bodies. Each of these represents a point at which engagement can
influence effective planning and implementation, and demonstrate
impact on the final product. 2.4 | Consultation and validation The preliminary results of the review were presented and discussed
during a PARADIGM meeting held in London, 18 July 2018. This
session provided valuable input on how best to present and catego-
rize the results. Participants in the meeting included representa-
tives from patient organizations, pharmaceutical companies and
academia with an interest and considerable expertise in patient
engagement. Based on the discussions during the meeting, it was
agreed to structure the results per key decision‐making point and
per stakeholder, including benefits and costs. These members of the
PARADIGM consortium were involved in writing this article; their
interpretation of results informed the discussion and conclusion. Furthermore, their contributions to the entire research process in-
formed the direction of research, the terminology and definitions
used in this article. | VAT et al. VAT et al. FI G U R E 1 Article selection PRISMA flow diagram
Records from databases aer
duplicates removed
(n = 1305)
Records from grey literature and hand
searching aer duplicates removed
(n = 47)
Records included for full text reading
(n = 168)
Records excluded aer full text reading:
•
No informa on on possible evalua on
approaches or outcomes
•
No methods for evalua ng outcomes
and impacts of pa ent engagement
prac ces in health research or health
technology assessment
(n = 77)
Iden fica on
Screening
Eligibility
Included
Items included for data extrac on
(n = 91)
Records included for Ti/Ab screening
(n = 1352)
Records excluded aer screening Ti/Ab:
•
Not wrien in English
•
Not about evalua on of pa ent
engagement
(n = 1184) Records from grey literature and hand
searching aer duplicates removed
(n = 47) Records from databases aer
duplicates removed
(n = 1305) Records excluded aer screening Ti/Ab:
•
Not wrien in English
•
Not about evalua on of pa ent
engagement
(n = 1184) Records included for Ti/Ab screening
(n = 1352) eening
Records included for Ti/Ab screening
(n = 1352) Records excluded aer full text reading:
•
No informa on on possible evalua on
approaches or outcomes
•
No methods for evalua ng outcomes
and impacts of pa ent engagement
prac ces in health research or health
technology assessment
(n = 77) Records included for full text reading
(n = 168)
Eligibility Records included for full text reading
(n = 168) FI G U R E 1 Article selection PRISMA flow diagram When available, context and process criteria were also included. Both researchers extracted data independently from 30% of the
scientific articles and then compared their findings to agree the
approach to data extraction. Thereafter, all the peer‐reviewed
published papers’ data were extracted by LV and the grey litera-
ture by TF and LV. 2.3 | Analysis Data were thematically analysed following Braun and Clarke's ap-
proach.49 To achieve the summary, we coded data using the review
question, aim and objective as included on the data extraction sheet. Codes used were benefits (B), costs and challenges (C), outcome or
impact on research (O or I) and types of benefits or costs and chal-
lenges. Codes were then clustered into themes, which were agreed
by TF and LV; themes were identified deductively and include bene-
fits, costs and challenges for each stakeholder group, benefits, costs
and challenges for research per decision‐making point, indicators,
methods or tools. LV and NG clustered the indicators, methods and
tools into qualitative and quantitative types. Benefits and costs were
mapped to suggested or applied indicators and tools or methods. The
decision‐making point focus of articles was interpreted by the re-
searchers if not defined in the article. Benefits and costs that could
not easily be linked to one particular decision‐making point were
analysed separately. LV and TF agreed on the data analysis strategy,
and sections of the analysis were cross‐checked by comparing inter-
pretations of results; inconsistencies were discussed and agreed. Records excluded aer full text reading:
•
No informa on on possible evalua on
approaches or outcomes
•
No methods for evalua ng outcomes
and impacts of pa ent engagement
prac ces in health research or health
technology assessment
(n = 77) 2.1 | Search methods Two researchers (TF, LV) independently screened all items’ title and
abstract. To ensure inter‐rater reliability, items were marked for in-
clusion or exclusion with each researcher's initials, discrepancies
were discussed and consensus reached. Both researchers read all
the selected items in full and followed up references for final inclu-
sion. At this stage, further exclusions were made of items that did
not include methods for evaluating outcomes and/or impact of pa-
tient engagement practices in health research or health technology
assessment—discrepancies were discussed, and consensus agreed
for final inclusion in the data extraction and analysis. Figure 1 dem-
onstrates the number of articles identified, screened, selected and
reviewed. Prior to the database search, we did a search to identify a tentative
sample set of relevant studies for a snowballing exercise. Using broad
key words, we searched Google Scholar for published articles and
Google for grey literature. We also searched the Patient‐Centered
Outcomes Research Institute (PCORI) database46 and the INVOLVE
evidence library.47 A snowballing exercise using references and cita-
tions from these articles provided a starting set of publications that
informed the protocol for the main review. This is recommended for
the clarification of concepts and search terms when interrogating
large, diverse fields of literature.48 Accordingly, with the assistance of a specialist librarian, we
searched CINAHL, Embase, Medline, PsychINFO and PubMed da-
tabases for peer‐reviewed published literature. The following key
words were used “patient engagement” combined with « AND»
“research” « AND» “outcomes,” including a variation of terms com-
bined with « OR». An overview of all search terms can be found in
Table 2. TF and LV developed a data extraction sheet to record relevant
information from each item, including the publication year and
focus, country of origin, methodology, patients involved as part-
ners, use of a framework or model, definitions included, outcome
and/or impact on research, benefits and costs per stakeholder
group, measurement or evaluation methods suggested or applied. | 5 3.1.1 | Benefits of patient engagement in research
priority setting Literature suggests that patient engagement in research priority
setting has several benefits. We identified four unique benefits
and nine possible indicators. We clustered the benefits into three
domains: usability benefits, societal benefits and funding benefits. Usability benefits refer to impact on the topic generation and pri-
oritization process, for example more relevant topics and priorities
based on patients’ needs3,4,15,17,20,22,23,29,30,50-55 and the relevance of
studies, for example more relevant research questions and medical
interventions or technologies.30 Societal benefits refer to the way
public and private resources are allocated, for example more appro-
priate resource allocation based on patients’ needs.30 Funding ben-
efits refer to new funding and funding opportunities, for example
success in gaining funding due to enhanced credibility of research
proposals.25,29-32,56-58 In the literature, quantitative methods are used to collect infor-
mation about the perceived importance of studies by patients, the
perceived influence of stakeholders in research priority setting,23,59
or to compare academic and lay scores assigned to research pro-
posal evaluation.60 For example, studies suggest rating the impor-
tance or influence of partners in developing the research topics.23,59
Qualitative methods are used to explore the relevance of research
topics and how patients’ experiential knowledge helped shape the re-
search question.30 The Patient‐Centered Outcome Institute (PCORI)
uses mixed methods (survey, focus groups, database review) to ex-
plore the perceptions incorporated into the topic selection process
and the kinds of research gaps documented as important to pa-
tients and other stakeholders that were not previously identified.61
Quantitative methods could also be used for comparison of academic
and lay scores assigned to research proposals.60 Qualitative methods
are suggested for exploring similarities and differences in research
priorities.15 For example, Brown et al invited patients with diabetes
to focus groups to identify research priorities. Results were analysed
using the constant comparative method and compared with current
expert‐led research priorities in diabetes.62 Additionally, documen-
tary analyses (eg review of minutes, grant applications, reports) may
be conducted to compare patient input and responsiveness to pa-
tients’ ideas.54,61,63 that patients were involved in the study as partners. Table 3 provides
an overview of the characteristics of included documents. In this section, we present the findings of our review, first con-
sidering the three decision‐making points, which were relevant
to our search. Not all reported benefits and costs could easily be
linked to one decision‐making point. 6 | VAT et al. VAT et al. 6 TA B LE 3 Overview of characteristics of included documents
Characteristic
Output
Year
Last 8 y (2010‐2018) (n = 69)
10 y (2000‐2010) (n = 22)
Focus
Clinical trial (n = 24)
Health research (n = 47)
Regulation and HTA (n = 8)
Other (n = 12)
Country of origin
Canada (n = 11)
United Kingdom (n = 40)
Canada and the United Kingdom (n = 1)
United States (n = 27)
Europe (n = 6)
Netherlands (n = 4)
Germany (n = 1)
Denmark (n = 1)
Setting
Academia (n = 38)
Health care (n = 19)
Industry (n = 3)
Mixed (n = 14)
Other (n = 17)
Methodology (academic
literature only)
Quantitative study (n = 6)
Qualitative study (n = 23)
Mixed method study (n = 15)
Literature review (n = 25)
Commentary/Editorial/Opinion/other
(n = 8)
Patients involved as
partners in the study
(academic literature
only)
Yes (n = 16)
Unspecified (n = 61) domain. Please refer to Appendices S3–S5 for more detailed indica-
tors, evaluation methods and tools. domain. Please refer to Appendices S3–S5 for more detailed indica-
tors, evaluation methods and tools. TA B LE 3 Overview of characteristics of included documents 3.1.1 | Benefits of patient engagement in research
priority setting These referred to overall ben-
efits and costs for stakeholders and general costs or challenges for
research and development. Therefore, we report them separately. Additionally, reported benefits and costs were omitted where they
related to other phases of the research process (such as interpreta-
tion of research findings or dissemination of results). 3 | RESULTS A total of 91 documents met the eligibility criteria (academic litera-
ture n = 77 and grey literature n = 14). Included documents were
published between 2000 and 2018 and focused mainly on the health
research field. We found limited documents in the field of regulation
and health technology assessment. Most documents were published
in the United Kingdom in an academic setting. We found largely
qualitative studies and literature reviews. Sixteen studies reported 6 | 3.1 | Benefits, costs and challenges for research and
development We identified ten unique benefits of patient engagement for the
design of clinical trials, including 13 possible indicators. We clus-
tered the benefits into three domains: ethical benefits, meth-
odological benefits and study quality benefits. Several studies
described ethical benefits such as a more appropriate, inclu-
sive and sensitive research design.8,17,29,30,52,55,58 Furthermore, A total of 18 benefits and five costs of patient engagement at three
R&D decision‐making points were identified. These were grouped
into 11 domains and mapped with 28 possible indicators for their
evaluation. Tables 4 and 5 provides an overview of indicators per | 7 | 7 VAT et al. accessible recruitment materials.4,20,24,25,29,31,40,55,56,67,68 Study qual-
ity benefits are also reported, for example improved trial recruit-
ment and retention.23,24,29,40,69 3.1 | Benefits, costs and challenges for research and
development |
TA B LE 4 Summary of benefits for research and development mapped with reported indicators for evaluation
Research priority setting
Usability benefits (1)
Examples of indicators related to usability benefits (total: 6)
More relevant research topics and priorities, based
on patients’ needs3,4,15,17,20,22,23,29,30,50-55
Rating of influence of patients and other stakeholders61
Rating of relevance or importance of studies23,59
Perceptions or degree of contentment/satisfaction with the topic generation and prioriti-
zation process96
Similarities and differences in research priorities between stakeholder groups15
Types of research gaps reported that were not previously identified61
Perceptions on how patients’ experiential knowledge helped shaped the research
question30
Research questions, hypothesis, interventions and
medical technologies become more relevant and
usable for patients24,30
Societal benefits (2)
Examples of indicators related to societal benefits (total: 3)
More appropriate resource allocation, based on
patients’ needs30
Comparison of academic and lay scores assigned to research proposals60
Perceptions of public influence on funding decisions60
Indicators of dynamics in the panel discussion61
Funding benefits (3)
Examples of indicators related to funding benefits (total: 1)
Improved fundability and credibility of research
proposals25,29-32,56-58
Number of studies that had success in gaining research funding12
Design of clinical trials
Ethical benefits (4)
Examples of indicators related to ethical benefits (total: 1)
More appropriate, inclusive and sensitive research
design8,17,29,30,52,55,58
Number of studies that had success in gaining ethics approval12
Methodological benefits (5)
Examples of indicators related to methodological benefits (total: 4)
More appropriate wording and tim-
ing of research instruments and
interventions17,20,22,24,25,27,29,31,55,56,64-68
Number of changes made to clinical trial communication as a result of study participant
feedback59
Increased readability and accessibility of research
materials4,20,24,25,29,31,40,55,56,67,68
Reading level of research documents/instruments70
Rating or perceptions of understanding of the consent form70
More relevant research outcomes/endpoints32,41,93
Number and type of patient‐reported outcomes61
Study quality benefits (6)
Examples of indicators related to study quality benefits (total: 7)
Improved recruitment and retention23,24,29,40,69
Recruitment rates40,69,70
Number of study participants who dropout for reasons other than adverse reactions59
Increased diversity of study participants66
Recruitment and retention rates among hard‐to‐reach population, level of diversity61
Improved trial experience/satisfaction by study
participants2,80
Rating or explore feelings of satisfaction among study participants15,70
Rating convenience of study visits and procedures by study participants59
More adherence to the research protocol93
Number of protocol amendments59
Faster study completion2,23
Number of studies completed within a particular timeframe3,61
Regulatory and HTA processes
Instrumental benefits (7)
Examples of indicators related to instrumental benefits (total: 1)
Higher accuracy in measuring needs and preferences
of patients71,72
Perceptions on how patient input was used and added value for assessment75,76
Better quality of assessment (in terms of relevance
and reliability to local context)71,72
Study uptake benefits (8)
Examples of indicators related to study uptake benefits (total: 2)
Uptake of evidence/approval by regulators and HTA
bodies2,73
Time to approval/response of regulators52
Changes in the proportion of drugs recommended for reimbursement36
Developmental benefits (9)
Examples of indicators related to developmental benefits
Knowledge and public awareness of products72
None reported
Democratic accountability and transparency72 3.1.3 | Benefits of patient engagement in regulatory
processes and health technology assessment (HTA) We identified five unique benefits of patient engagement in regula-
tory processes and HTA, including four possible indicators. The ben-
efits can be categorized into three dimensions: instrumental benefits,
study uptake benefits and developmental benefits. Instrumental
benefits are related to improving the relevance of assessment to
making better quality decisions, for example higher accuracy in
measuring needs and preferences of patients and better quality of
assessment and relevance of reports to the local context.71,72 Study
uptake benefits refer to the usefulness of assessments for decision
makers and the uptake of evidence by decision makers, for example
gaining regulatory approval.2,73 Developmental benefits include, for
example, increasing the public's understanding of HTA and openness
of decision processes.72 Literature suggests a few methods to evaluate the bene-
fits of patient engagement in regulatory processes and HTA. Quantitative methods are suggested to assess study uptake ben-
efits such as the time to response/approval of regulators and a
change in the proportion of drugs recommended for reimburse-
ment.3,74 Furthermore, quantitative methods could be used to as-
sess the perceived impact. For example, the European Medicines
Agency has used a survey to assess the perceived added value
of patient input in scientific advice processes and feedback.75
Qualitative methods can also be used to explore measures of
change or uptake of patients’ input. For example, Abelson et al76
assessed how patients’ input informed the HTA process through
document analysis, interviews and observations. Dipankui et al77
used semi‐structured interviews and document analysis (eg HTA
reports, minutes) to evaluate how patient engagement changed
the HTA report and its recommendations. The literature suggests several indicators and methods for
the evaluation of patient engagement in the design of clinical
trials. For example, Guarino et al measured participants’ under-
standing of the study consent form, using the Informed Consent
Questionnaire‐4 questionnaire. 3.1.3 | Benefits of patient engagement in regulatory
processes and health technology assessment (HTA) The reading levels of the con-
sent forms were assessed using Flesch‐Kincaid reading level
scores.70 Rating the impact of patient engagement on study vol-
unteer attitudes about aspects of the participation process (eg
ease of understanding the informed consent form; convenience
of study visits and procedures) is also suggested.59 Other stud-
ies suggest collecting data on the number of studies that gain
research ethics committee approval,12 the number of protocol
amendments59 and the number and type of patient‐reported
outcomes.61 Furthermore, several studies have assessed study
quality benefits, for example recruitment rates, using differ-
ent quantitative methods.40,69,70 Iliffe, McGrath and Mitchell40
compared recruitment levels before and after the involvement
of the public. Guarino et al70 also conducted a comparison; they
assessed the effect of two different consent documents on re-
cruitment levels using one consent form developed by a con-
sumer focus group compared with another developed by the
study investigators. Ennis and Wykes conducted a quantitative
analysis of successful recruitment by studies where patient en-
gagement was undertaken. A change in patient engagement over
time was assessed by correlating study entry order (studies were
ordered by the date identified) with the level of patient engage-
ment. Additionally, suggested indicators include recruitment and
retention rates among hard‐to‐reach populations,61 the number
of dropouts for reasons other than adverse reactions, the total
number of changes made to clinical trial communications as a re-
sult of patient feedback,59 and the number of studies completed
within a particular time frame.3,61 Validated questionnaires such
as the Client Satisfaction Questionnaire‐8 measure overall satis-
faction of study participants.70 Qualitative methods are mostly
suggested for gathering information about participants’ experi-
ences of taking part in a clinical trial.15 8 | VAT et al. VAT et al. 8 TA B LE 5 Summary of costs for research and development
mapped with reported indicators for evaluation Study uptake benefits (8) accessible recruitment materials.4,20,24,25,29,31,40,55,56,67,68 Study qual-
ity benefits are also reported, for example improved trial recruit-
ment and retention.23,24,29,40,69 accessible recruitment materials.4,20,24,25,29,31,40,55,56,67,68 Study qual-
ity benefits are also reported, for example improved trial recruit-
ment and retention.23,24,29,40,69 studies described methodological benefits such as more appro-
priate wording and timing of research instruments and interven-
tions,17,20,22,24,25,27,29,31,55,56,64-68 and improved consent forms and 8 |
TA B LE 5 Summary of costs for research and development
mapped with reported indicators for evaluation
Various decision‐making points
Non‐financial costs (10)
Examples of indicators related to
non‐financial costs (total: 2)
Biases in recruitment or
findings24,67
Perceived negative impacts of
patient engagement for research
and development24
Total hours spent on engagement24
Scientific and ethical conflict
in protocol design20
Power struggles20
Increased time20
Financial costs (11)
Examples of indicators related to
financial costs (total: 1)
Increased costs20
Total monetary costs of en-
gagement for research and
development24 3.2 | Costs and challenges of patient engagement in
research and development Limited studies have published costs and challenges. Of those
which have, most studies reported increased time and costs for
researchers and research institutions due to the practical aspects
of planning and managing patient engagement. For example, there
are increased time and financial costs from building relationships
with the relevant community, setting up user groups, organizing
and providing training and education for users and researchers,
and the additional time needed for users to read and comment
on documentation.20 Only two studies suggest that patient en-
gagement could potentially result in a more homogenous sam-
ple or biases in recruitment.24,67 For example, Blackburn et al24
reported that a more homogenous study sample may have been
recruited, since the young contributors encouraged their friends
to participate in a study on reproductive health in young people. Furthermore, Brett et al found that studies indicated that patient
engagement led to scientific and ethical conflict in protocol de-
sign. Also, patient engagement may lead to tokenistic engagement | 9 9 VAT et al. 3.2 | Costs and challenges of patient engagement in
research and development and can lead to power struggles between researchers and patient
partners 20 Furthermore stakeholders have raised concerns that
practices, including the use of blinded, randomized contro
clinical trials 59
TA B LE 6 Summary of benefits, costs and challenges per stakeholder group
Individuals and
organizations
Benefits
Costs and challenges
Patient partners
• Empowerment8,19,20,29-31,55,85,86
• Enhanced well‐being29,30,87
• Learning about research and gaining research and
transferable skills20,24,29,55,97
• Learning about own condition and treatment
options54,79
• Enjoyment and satisfaction22,29,55,87
• Supportive, meaningful relationships29,31,79
• Possible remuneration8,54
• Future prospects29,30,79,87
• Confusion due to lack of clarity about roles and
procedures67,79
• Disappointment and frustration due to mismatched
expectations20,27
• Stress due to lack of knowledge and confidence and
burden of responsibility24,79
• Overburdened5,29,55,79
• Investment of time and possibly own
resources4,24,67,79,88
• Possible reduction of welfare payments24
Society
• Hope and trust in research/ers29,67,79
• Funding and prioritization of research relevant to the
community3
• Potentially more, relevant drugs recommended for
reimbursement74
• Increased awareness of and advocacy for condition
and associated research67,79
• Uncover or create conflict and power struggles in the
community79
• More time and resources67,79,85
• Difficulty representing vulnerable/hard‐to‐reach
groups67,79
Research participants
• Accessible information on all aspects of disease and
treatment24,55
• More positive experience of research
participation2,55,80
Researchers
• Learning about patients’ view of condition and
patient engagement's effects on research5,8,24,26,28-30
• Enhanced knowledge and skills8,28,55,79
• Fresh perspective on what research can
achieve19,20,22,85
• Enjoyment and satisfaction29
• Career benefits29,67
• Methodological concerns and costs20,32,59
• Stress due to new ways of working with patients
and advocacy groups and associated power
struggles4,20,32,55,67,79
• More resource‐intensive research
process19,55,67,79,86,87,89,90
Research institutes
• Increased research impact24
• Enhanced reputation24
• Diversion of research funds to patient engagement
(opportunity cost in terms of funded researcher time
etc)24
• IT and other support infrastructures 24
Research funders
• More relevant funding decisions24
• Increased transparency and accountability55,67
• Possible challenge to balance scientific integrity and
relevant research55
Industry
• More cost‐effective R&D2,85,91-93
• More regulatory success2
• Enhanced reputation2,31
• Better patient concordance with treatment93,94
• Enhanced knowledge94
• More resource‐intensive R&D20
Regulators and health
technology assessment
bodies
• Better understanding of real‐life context of
products71
• More efficient, relevant regulatory decisions95
• Increased transparency and accountability73,74
• Mutual respect between regulators and consumers73
• Increased uncertainty in policy‐making due to varied
views67
Others (decision mak-
ers and health‐care
providers)
• More useful evidence for clinical and health policy
decision making30
• Uncertainty about how to take the study recommen-
dations forward due to complexities of conflicting
clinical and health system goals between clinicians,
researchers, and users55 reported benefits, costs and challenges for stakeholders involved in
patient engagement initiatives. In this section, we reflect on the in-
dicators and methods found in this review and consider the review's
methodological strengths and limitations. al includes questions about costs for researchers such as total costs
associated with recruiting patients involved, the total costs associ-
ated with training patients involved, the total costs associated with
supporting patients, financial payment/rewards, total costs of ex-
penses reimbursed to all patients for their involvement and other
costs (including parking permits, room booking, audio‐visual, equip-
ment). A separate questionnaire developed for patients includes
questions about the hours spent on engagement, the costs they
incurred (eg travel, child care, food and drinks, accommodation) and
any costs related to arrangement and planning (for instance changed
shifts at work or arranged care for a relative).24 Log sheets are also
used to gather insights into time and costs.27 Open questions are
used to gather insights into (non‐financial) negative impacts.24 3.3 | Benefits, costs and challenges for stakeholders We agree with others that there is a need for new
evaluation methods and tools that focus on observable impact on the
research process and benefits for those involved.4,21,79,80 Some argue
for broadly applicable, quantitative methods whilst others contend
that more subjective, qualitative methods are necessary to capture
the nuances of outcomes and impacts of patient engagement.13,17,59,76
Universally applicable evaluation criteria that capture all aspects of
engagement are supported for reasons of consistency, reliability and
comparison across different projects.16 To build an evidence base,
conceptual and practical guidance and some level of consensus on
measurable impacts are needed. This has also been suggested by
other authors.13,30 However, whilst a standardized approach may
be appealing to health research and development communities, it is
problematic in the complex and contextually dependent arenas of
patient engagement.81 It might inhibit capacity‐building in projects
and makes changes difficult; arguably, this undermines the original This review also looked at methods for evaluation. We identified
several quantitative methods to measure the benefits of patient en-
gagement; these mostly assess the benefits on study quality, study
uptake and self‐reported benefits. Qualitative methods are mostly
suggested for gathering information about experiences, attitudes
and perceptions. We agree with others that there is a need for new
evaluation methods and tools that focus on observable impact on the
research process and benefits for those involved.4,21,79,80 Some argue
for broadly applicable, quantitative methods whilst others contend
that more subjective, qualitative methods are necessary to capture
the nuances of outcomes and impacts of patient engagement.13,17,59,76 Universally applicable evaluation criteria that capture all aspects of
engagement are supported for reasons of consistency, reliability and
comparison across different projects.16 To build an evidence base,
conceptual and practical guidance and some level of consensus on
measurable impacts are needed. This has also been suggested by
other authors.13,30 However, whilst a standardized approach may
be appealing to health research and development communities, it is
problematic in the complex and contextually dependent arenas of
patient engagement.81 It might inhibit capacity‐building in projects
and makes changes difficult; arguably, this undermines the original 4.1 A total of 18 benefits and five costs of patient engagement at the
three decision‐making points were identified in this review. These
were grouped into 11 domains and mapped with 28 possible indica-
tors for their evaluation. Little is known about the validity and per-
formance of these indicators as most were suggested rather than
applied, or used in single studies. Those studies mostly considered
a single indicator (eg recruitment rate) for trying to answer a single
question (eg Does patient engagement in research lead to better
recruitment?). Measuring this may be feasible but may not be use-
ful in predicting impact for other studies, as the factors influencing
impact may differ. This has been noted by other authors.36 We argue
that currently available indicators are of some use in measuring ben-
efits, but are not sufficient to understand the pathway to impact, or
whether the interaction between researchers and patients involved
could lead to change in the external environment (eg research cul-
ture, structure and practice). We argue that the impacts of patient
engagement can best be determined not by applying a single indica-
tor, but a coherent set of measures. Given the importance of con-
text and the complexity of evaluating patient engagement that this
review illustrates, we are developing a monitoring and evaluation
framework that considers various indicators for patient engagement
practices in medicines research and development. This framework is
informed by other frameworks and being tested in practice. We will
publish our findings of working with a more coherent evaluation ap-
proach in medicines research and development shortly. TA B LE 6 Summary of benefits, costs and challenges per stakeholder group TA B LE 6 Summary of benefits, costs and challenges per stakeholder group Costs and challenges
• Confusion due to lack of clarity about roles and
procedures67,79
• Disappointment and frustration due to mismatched
expectations20,27
• Stress due to lack of knowledge and confidence and a
burden of responsibility24,79
• Overburdened5,29,55,79
• Investment of time and possibly own
resources4,24,67,79,88
• Possible reduction of welfare payments24
• Uncover or create conflict and power struggles in the
community79
• More time and resources67,79,85
• Difficulty representing vulnerable/hard‐to‐reach
groups67,79 Costs and challenges • Methodological concerns and costs20,32,59
• Stress due to new ways of working with patients
and advocacy groups and associated power
struggles4,20,32,55,67,79
• More resource‐intensive research
process19,55,67,79,86,87,89,90
• Diversion of research funds to patient engagement
(opportunity cost in terms of funded researcher time,
etc)24
• IT and other support infrastructures 24
• Possible challenge to balance scientific integrity and
relevant research55
• More resource‐intensive R&D20 • Increased uncertainty in policy‐making due to varied
views67 • Increased uncertainty in policy‐making due to varied
views67 and can lead to power struggles between researchers and patient
partners.20 Furthermore, stakeholders have raised concerns that
engaged patients may want to see their clinical trials succeed,
and as a result, these patients may bias the study findings.59 It
was also reported that a number of clinical research professionals
fear that patient centricity is pushing them to discard traditional practices, including the use of blinded, randomized controlled
clinical trials.59 Methods to assess costs include qualitative methods to gather
insights into the perceived effort of engagement as well as a quan-
titative method to gather insights into financial costs. For example,
the costs and consequences framework developed by Blackburn et VAT et al. 10 VAT et al. 3.3 | Benefits, costs and challenges for stakeholders Studies that assessed patient engagement for individuals and or-
ganizations mostly highlighted benefits, costs and challenges for
patients engaged, with comparatively less published on the benefits
and costs for other groups. Based on our review, suggested dimen-
sions to measure the benefits, costs and challenges for the individu-
als and organizations involved relate to personal development, skills
and knowledge, emotions and meaningful relationships, financial,
performance and strategic value, transparency and awareness, trust
and mutual respect. A summary of reported benefits and costs for
stakeholders can be found in Table 6. Please refer to Appendices S1
and S2 for more detailed information on benefits, costs and chal-
lenges for patients and other stakeholders. Multiple tools have been developed to assess the benefits and
costs for stakeholders. The Evaluation Toolkit is a resource designed
for practitioners of the health sector, produced after the completion
of a rigorous systematic review of patient and public engagement
evaluation tools.78 Boivin et al reviewed the tools and concluded that
most tools were designed to collect information from patients and
the public; very few instruments measure the perspectives of other
stakeholder groups. The authors of the review reported that the out-
comes of patient engagement were least often evaluated (55.6% of
the tools), in contrast to the engagement process and context. The
most common focus of tools that measure outcomes was on per-
ceived, self‐reported impacts. Methods are qualitative (eg interviews,
focus groups) and quantitative for perceived self‐reported benefits
(eg surveys using Likert scales). Self‐administered questionnaires and
surveys were the most common types of tools identified.21 p
p
y
This review also looked at methods for evaluation. We identified
several quantitative methods to measure the benefits of patient en-
gagement; these mostly assess the benefits on study quality, study
uptake and self‐reported benefits. Qualitative methods are mostly
suggested for gathering information about experiences, attitudes
and perceptions. rationale for patient engagement. The tension between obtaining
comparable data on patient engagement by using metrics (standard-
ized or agreed techniques for gathering information) and tailored
participatory evaluative approaches should not be overlooked. By
implication, it should be recognized that measures can be valued and
applied differently in different contexts; therefore, we recommend
discussing relevant and feasible indicators and methods per setting. of priority setting, clinical trial design and regulatory and HTA
processes, benefits need to be demonstrable to all stakehold-
ers. This literature review has mapped benefits, costs and chal-
lenges with indicators in current literature. Discrete tools and
methods for evaluation are less apparent, as is evidence of their
application. The approaches to evaluation we found are largely
qualitative, and our review suggests that there are few quan-
titative tools and no standardized approaches to assessing the
outcomes and impact. The reported costs, challenges and ben-
efits are largely congruent, with agreement that there is a need
for consensus‐based monitoring and evaluation frameworks that
include metrics. of priority setting, clinical trial design and regulatory and HTA
processes, benefits need to be demonstrable to all stakehold-
ers. This literature review has mapped benefits, costs and chal-
lenges with indicators in current literature. Discrete tools and
methods for evaluation are less apparent, as is evidence of their
application. The approaches to evaluation we found are largely
qualitative, and our review suggests that there are few quan-
titative tools and no standardized approaches to assessing the
outcomes and impact. The reported costs, challenges and ben-
efits are largely congruent, with agreement that there is a need
for consensus‐based monitoring and evaluation frameworks that
include metrics. ORCID Lidewij Eva Vat
https://orcid.org/0000-0003-0077-6602
Teresa Finlay
https://orcid.org/0000-0003-2538-8366
Tjerk Jan Schuitmaker‐Warnaar
https://orcid. org/0000-0001-5158-581X
Nick Fahy
https://orcid.org/0000-0003-4957-0189
Marie‐Laure Kürzinger
https://orcid.org/0000-0002-2606-776X
Jacqueline E.W. Broerse
https://orcid.org/0000-0002-8478-3422 4.2 To our knowledge, this is the first literature review that attempts
to capture the existing publications about the evaluation of patient
engagement practice as it relates to medicines development. It both
maps outcomes and impacts of patient engagement with suggested
measures for each decision‐making point in R&D. We suggest that the development of a coherent set of measures
warrants further investigation and that the benefits, costs and chal-
lenges of patient engagement for all stakeholders should be given
more consideration (rather than the current focus on benefits for
research). To this end, we will co‐develop and test an evaluation
framework with stakeholders using a reflexive monitoring approach
in real‐life cases of patient engagement in medicines research and
development. Very few publications refer to costs or negative impact of en-
gagement, compared with positive findings. This may be because
people tend not to report negative outcomes and impacts despite
their being just as important. There were very few studies that con-
sidered patient engagement in the HTA process, and only, three
publications were authored by (and for) the pharmaceutical indus-
try. Furthermore, of the papers included in our review, very few
reported that they had involved patients; therefore, the conclusions
derived from the studies may be based on the perspectives of re-
searchers. For this review, a meeting was held to discuss prelimi-
nary findings with a broad range of stakeholders in our project and
the co‐authors of this paper work for patient representative groups
and industry. We therefore feel that our findings may be considered
relevant to a broader audience than a predominantly academic one. ACKNOWLEDGEMENTS We want to thank Nicole Goedhart from the Athena Institute for
assisting with the analysis. We also would like to thank Carina
Pittens from the Athena Institute for reviewing the first drafts. We are grateful for the support of Nia Roberts, librarian at the
University of Oxford. Furthermore, we very much appreciate the
feedback from Joanna Crocker from the University of Oxford and
María José Vicente Edo from Instituto Aragonés de Ciencias de la
Salud. Our focus on the measurement of impact of patient engagement
in the development of medicines has resulted in several limitations
to our review. Because this is a scoping review rather than a sys-
tematic review, we may have missed relevant articles. Our search
focused on titles and abstracts of publications and three decision‐
making points, which means that some articles (eg related to other
time points) have been excluded. We specifically searched for out-
comes and impact of patient engagement in the R&D of medicines;
therefore, our paper does not include context or process indicators,
or the indicators per stakeholder group. Furthermore, we cannot
draw hard conclusions about the relationship between input, out-
comes and impact with respect to the benefits and costs for the
people and organizations involved in patient engagement. Finally,
we had to exclude articles not published in English. Whilst we are
aware that most publications on this topic are written in English
originating from the UK and North America, we acknowledge that
we may have missed relevant publications in other languages. 4 | DISCUSSION To address the need for means of determining the “return on engage-
ment,” the aim of this paper was to review the literature on monitor-
ing and evaluation of patient engagement. This review identified a
range of benefits, costs and challenges that patient engagement can
have on R&D and describes several indicators associated with their
monitoring and evaluation. In addition, we summarized the overall VAT et al. 11 11 DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from
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be integrated in health technology assessments: an exploratory 14 |
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1–14. https://doi.org/10.1111/hex.12951 87. Minogue V, Boness J, Brown A, Girdlestone J. The impact of ser-
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https://openalex.org/W1785714480
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https://hal.sorbonne-universite.fr/hal-01197099/file/Despras_2015_H-Rubies%2C_a_new.pdf
|
English
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H-Rubies, a new family of red emitting fluorescent pH sensors for living cells
|
Chemical science
| 2,015
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cc-by
| 10,062
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To cite this version: Guillaume Despras, Alsu I. Zamaleeva, Lucie Dardevet, Céline Tisseyre, Joao Gamelas Magalhaes, et
al.. H-Rubies, a new family of red emitting fluorescent pH sensors for living cells †. Chemical Science,
2015, 6, pp.5928-5937 10.1039/c5sc01113b. hal-01197099 H-Rubies, a new family of red emitting fluorescent pH
sensors for living cells † Guillaume Despras, Alsu I. Zamaleeva, Lucie Dardevet, Céline Tisseyre, Joao
Gamelas Magalhaes, Charlotte Garner, Michel de Waard, Sebastian
Amigorena, Anne Feltz, Jean-Maurice Mallet, et al. Distributed under a Creative Commons Attribution 4.0 International License H-Rubies, a new family of red emitting fluorescent
pH sensors for living cells†
Guillaume Despras,‡a Alsu I. Zamaleeva,‡bf Lucie Dardevet,cd C´eline Tisseyre,cd
Joao Gamelas Magalhaes,f Charlotte Garner,a Michel De Waard,cde
Sebastian Amigorena,f Anne Feltz,b Jean-Maurice Malleta and Mayeul Collot§*a Guillaume Despras,‡a Alsu I. Zamaleeva,‡bf Lucie Dardevet,cd C´eline Tisseyre,cd
Joao Gamelas Magalhaes,f Charlotte Garner,a Michel De Waard,cde
Sebastian Amigorena,f Anne Feltz,b Jean-Maurice Malleta and Mayeul Collot§*a Monitoring intracellular pH has drawn much attention due to its undeniably important function in cells. The
widespread development of fluorescent imaging techniques makes pH sensitive fluorescent dyes valuable
tools, especially red-emitting dyes which help to avoid the overcrowded green end of the spectral band. Herein, we present H-Rubies, a family of pH sensors based on a phenol moiety and a X-rhodamine
fluorophore that display a bright red fluorescence upon acidification with pKa values spanning from 4 to
9. Slight structural modifications led to dramatic changes in their physicochemical properties and a
relationship between their structures, their ability to form H-aggregates, and their apparent pKa was
established. While molecular form H-Rubies can be used to monitor mitochondrial acidification of
glioma cells, their functionalised forms were linked via click chemistry to dextrans or microbeads
containing a near infrared Cy5 (Alexa-647) in order to provide ratiometric systems that were used to
measure respectively the phagosomal and endosomal pH in macrophages (RAW 264.7 cells) using flow
cytometry. Received 27th March 2015
Accepted 13th July 2015
DOI: 10.1039/c5sc01113b
www.rsc.org/chemicalscience internalisation pathways. Fluorescence spectroscopy and uo-
rescence imaging techniques have many advantages including
high sensitivity, high spatial and temporal resolution and are
not destructive to the cells. As such, molecular uorescent pH
indicators have become indispensable tools for observing pH
changes in cells. Although many uorescent indicators have
already been developed (for an extensive review see: Han and
Burgess),12 several factors have to be taken into account in order
to achieve efficient use within cells, including solubility in
biological media, hydrophobicity, photostability and bright-
ness. Our recent efforts to develop functionalisable red emitting
uorescent sensors13–15 were driven by the increasing use of cells
transfected with green or yellow uorescent proteins (FPs), thus
allowing multicolour imaging. Whilst recent progresses have
been reported in the development of red emitting genetically
encoded pH sensors,16 there is still a demand for efficient
molecular red uorescent indicators. Among red-shied uo-
rescent pH sensors, cyanines suffer from a weak photo-stability. Benzoxanthene dyes like SNARF, act as ratiometric probes but
generally suffer from weak quantum yields. H-Rubies, a new family of red emitting fluorescent
pH sensors for living cells†
Guillaume Despras,‡a Alsu I. Zamaleeva,‡bf Lucie Dardevet,cd C´eline Tisseyre,cd
Joao Gamelas Magalhaes,f Charlotte Garner,a Michel De Waard,cde
Sebastian Amigorena,f Anne Feltz,b Jean-Maurice Malleta and Mayeul Collot§*a In contrast, red-
emitting BODIPYs have recently attracted special attention due
to their photostability and high brightness.17,18 Having said this
they tend to suffer from high lipophilicity making them ineffi-
cient for cellular experiments as their apparent pKa shis in
lipophilic environment.19 aLaboratory of Biomolecules (LBM), UPMC Universit´e Paris 06, Ecole Normale
Sup´erieure (ENS), CNRS, UMR 7203, Paris F-75005, France. E-mail: mayeul.collot@
unistra.fr
bEcole Normale Sup´erieure, Institut de Biologie de l'ENS (IBENS), INSERM U1024,
CNRS UMR 8197, Paris F-75005, France
cInserm U836, LabEx Ion Channels, Science and Therapeutics, Grenoble Institute of
Neuroscience, chemin fortun´e ferrini, bˆatiment Edmond Safra, 38042 Grenoble
Cedex 09, France
dUniversit´e Joseph Fourier, Grenoble, France
eSmartox Biotechnology, Saint Martin d’H`eres, France
fINSERM U932, Institute Curie, 75248, Paris, Cedex 05, France
† Electronic supplementary information (ESI) available: Details of the organic
synthesis,
characterisations
(HPLC,
NMR,
mass
spectra)
as
well
as
measurements
of
their
photophysical
properties
can
be
found
in
the
supplementary information. See DOI: 10.1039/c5sc01113b
‡ Contributed equally to this work.
§ Current address: Laboratoire de Biophotonique et Pharmacologie, UMR 7213
CNRS, Universit´e de Strasbourg, Facult´e de Pharmacie, 74, Route du Rhin,
67401, Illkirch, France. aLaboratory of Biomolecules (LBM), UPMC Universit´e Paris 06, Ecole Normale
Sup´erieure (ENS), CNRS, UMR 7203, Paris F-75005, France. E-mail: mayeul.collot@
unistra.fr HAL Id: hal-01197099
https://hal.sorbonne-universite.fr/hal-01197099v1
Submitted on 11 Sep 2015 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 bEcole Normale Sup´erieure, Institut de Biologie de l'ENS (IBENS), INSERM U1024,
CNRS UMR 8197, Paris F-75005, France Introduction Intracellular pH plays many different and important roles in
cellular activity and is especially involved in ion transport,1
apoptosis,2,3 multidrug resistance4 and muscle contraction.5,6
Moreover, abnormal intracellular pH values are also associated
with diseases such as Alzheimer's7 and cancer.8,9 Monitoring
variations of intracellular pH in living cells is therefore of the
utmost importance and is an essential parameter for studying
phagocytosis,10
endocytosis11
and
consequently
cellular bEcole Normale Sup´erieure, Institut de Biologie de l'ENS (IBENS), INSERM U1024,
CNRS UMR 8197, Paris F-75005, France † Electronic supplementary information (ESI) available: Details of the organic
synthesis,
characterisations
(HPLC,
NMR,
mass
spectra)
as
well
as
measurements
of
their
photophysical
properties
can
be
found
in
the
supplementary information. See DOI: 10.1039/c5sc01113b In his review, Han et al.12 pointed out that despite their
advantageous spectral properties, pH probes based on rhoda-
mine were not extensively developed.20 Rhodamine-based pH ‡ Contributed equally to this work. § Current address: Laboratoire de Biophotonique et Pharmacologie, UMR 7213
CNRS, Universit´e de Strasbourg, Facult´e de Pharmacie, 74, Route du Rhin,
67401, Illkirch, France. § Current address: Laboratoire de Biophotonique et Pharmacologie, UMR 7213
CNRS, Universit´e de Strasbourg, Facult´e de Pharmacie, 74, Route du Rhin,
67401, Illkirch, France. This journal is © The Royal Society of Chemistry 2015 Chem. Sci. Chem. Sci. View Article Online View Article Online Fig. 1
Structures of the synthesised X-rhodamines and the first set of
H-Rubies. Edge Article Chemical Science Edge Article probes mostly rely on the spirolactam ring opening principle,
generally yielding pH sensors with high dynamic ranges but low
pKa values as well as high hydrophobicity due to their neutrally
charged OFF state.21–26 A key criteria for a pH sensor is its pKa value, which should
be in the physiological range. While the cytosol and some other
cellular compartments, such as the nucleus and endoplasmic
reticulum maintain their pH at 7.2, endosomes, lysosomes
and secretory vesicles on the other hand are acidic environ-
ments with the pH ranging from 4.5 to 6.7. Conversely, alkaline
pH can be found in mitochondria (8),27 phagosomes of
dendritic cells28,29 and neutrophils.30 As such, a collection of pH
probes with a large range of pKa values is required in order to
extend the scope of their application and thus a straightforward
method of tuning their pKa values without tedious chemical
modications would be a necessity. Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1
Structures of the synthesised X-rhodamines and the first set of
H-Rubies. and hydroxynaphthaldehydes (for synthesis see ESI†). The
use
of
phenyl-imidazol
and
(N-alkylated)-phenyl-pipera-
zine33,34
as
alternative
pH-sensitive
indicators
was
also
explored giving rise to X-rhodamines: Imidazole, Pip-H and
Pip-Alkyne (Fig. 1). The spectral properties and pKa values obtained for this rst
set of synthesised uorophores are given in Table 1. Although
X-rhodamine Imidazole did not behave as a uorescent pH
sensor (no signicant variation of uorescence intensity upon
protonation), phenyl-piperazine and phenol-based X-rhoda-
mines displayed clear pH sensitivity. Despite its physiologically
relevant pKa value and the capacity to be functionalised, Pip-
Alkyne was not developed further as its dynamic range (3-fold)
was found to be much lower than those obtained with phenol-
based X-rhodamines. Results and discussion Absorption spectra of H-Rubies at different pH values provided
us with information regarding their aggregation states (Fig. 2)
since rhodamines can form different patterns of aggregations
including H- and J-aggregates.35,36 The observed broadening of
absorption spectra for HR-pOH clearly indicated the formation
of non-dened aggregates at high pH values. This could be
explained by the equilibrium between the zwitterionic pheno-
late form and the neutral, hydrophobic and planar quinone
form (Fig. 2C). With regard to its isomers HR-mOH and HR-
oOH, the neutral quinone forms cannot be formed due to the
impossible delocalization of the electrons in the HR-mOH form
and a twisted structure imposed by hindered sterical effect in
HR-oOH; therefore, their absorption spectra only display slight
changes upon deprotonation with no sign of aggregation
(Fig. 2A and B). Interestingly, absorption spectra of HR-Br at pH
values above its pKa exhibited a second band indicating
formation of soluble H-aggregates (Fig. 2D). Basic forms of HR-
Cl and HR-Br both form H-aggregates, the stabilisation of which
could be attributed to intermolecular halogen bonding37,38
between the electron-rich oxygen of the phenolate and the
halogen atom forming typical head to head H-aggregate dimers. It is also noted that H-Rubies that forms H-aggregates in their This journal is © The Royal Society of Chemistry 2015 Introduction Finally, the large majority of
uorescent pH sensors do not offer an orthogonal and specic
site
of
attachment
required
for
functionalisation. Many
common pH probes are too lipophilic and tend to compart-
mentalise in hydrophobic domains of the cell, however, to date
no
alternative
methods
of
functionalisation
have
been
proposed. In this report we present the development of a new
family of red-emitting pH probes based on X-rhodamines and a
phenol moiety as the pH reporter (see abstract scheme). In
addition to their pKa values ranging from 4 to 9, these ON–
OFF type pH sensors, based on the PET (photoinduced electron
transfer) quenching phenomena, exhibit high brightness in
their acidic phenol form and can display very high turn ON of
their uorescence thanks to the formation of dark soluble H-
aggregates in their OFF state. Among this new family, a number
of H-Rubies were designed to bear a linker allowing the func-
tionalisation of dextran and microbeads in order to measure the
pH in living cells. Chem. Sci. Synthesis of phenolic X-rhodamines h Non-uorescent. i pKa value was too high. j No pH sensitivity. Synthesis of phenolic X-rhodamines Downloaded on 31/08/2015 13:41:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Table 1
Spectral and physico-chemical properties of the first set of synthesised X-rhodamines (5 mM in MOPS 30 mM, 100 mM KCl)a
Compound
labsmax
(nm)
lemd
(nm)
Log 3 (labsmax)
(M1 cm1)
F
FON/FOFF
Dynamic
rangee
Dynamic
range @ pH 1 pKa
f
pKa
HR-pOH
ONb
576
587
4.78
0.55
9
34
30
8.97
OFFc
579f
598
4.53
0.06
0.05
HR-mOH
ONb
578
602
4.70
0.30
3
132
75
9.33
OFFc
577
603
4,63
0.11
0.13
HR-oOH
ONb
581
603
4.89
0.75
3
866
460
9.68
OFFc
579
603
4.69
0.27
0.17
HR-Me
ONb
574
597
4.71
0.09
High
955
833
8.75
OFFc
N/Ag
598
N/Ag
N/Ah
0.16
HR-OMe
ONb
577
598
4,72
0.22
8
302
N/Ai
N/Ai
OFFc
576
600
4.74
0.03
HR-Cl
ONb
579
601
4.68
0.47
High
753
512
6.17
OFFc
544
599
4.52
N/Ah
0.10
Hr-Br
ONb
581
601
4.39
0.55
High
384
291
6.51
OFFc
544
600
4.40
N/Ah
0.10
p-Nph
ONb
581
600
4.79
0.28
6
23
9.7
8.51
OFFc
579
602
4.34
0.05
0.17
o-Nph
ONb
584
604
4.43
0.77
8
337
166
8.73
OFFc
576
604
4.46
0.10
0.17
Pip-H
ONb
578
600
4.88
0.16
4
6
3.9
8.67
OFFc
579
601
4.84
0.04
0.07
Pip-Alkyne
ONb
577
600
4.98
0.34
2
3
1.9
4.95
OFFc
579
602
4.45
0.15
0.09
Imidazole
ONb
583
609
4.80
0.40
N/Aj
N/Aj
N/Aj
N/Aj
OFFc
584
608
N/Aj
N/Aj
a 3 ¼ molar extinction coefficient, l ¼ wavelength, F ¼ quantum yield. b Protonated form: pH 4. c Deprotonated form: pH 10. d Excitation at 535 nm. e [(Imax Imin)/Imin] with I ¼ uorescence intensity. f Fluorescence enhancement between 1 pH unit around the pKa value. g Broad absorbance. h Non-uorescent. i pKa value was too high. j No pH sensitivity. d X-rhodamines (5 mM in MOPS 30 mM, 100 mM KCl)a Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. a 3 ¼ molar extinction coefficient, l ¼ wavelength, F ¼ quantum yield. b Protonated form: pH 4. c Deprotonated form: pH 10. d Excitation at 535 nm. e [(Imax Imin)/Imin] with I ¼ uorescence intensity. f Fluorescence enhancement between 1 pH unit around the pKa value. g Broad absorbance. This journal is © The Royal Society of Chemistry 2015 Synthesis of phenolic X-rhodamines ON–OFF pH probes generally require a pH-sensitive moiety
whereby protonation/deprotonation modies the quantum
yield of a linked uorophore either through PET (Photoinduced
Electron Transfer) quenching phenomena or ICT (Internal
Charge Transfer). Among these pH-sensitive moieties, phenol is
the most popular as illustrated by the wide use of uorescein
and its derivatives: FITC, BCECF, etc. Moreover, the pKa of
phenols can be easily lowered and tuned to a biologically rele-
vant range by introducing proper electron-withdrawing groups
on the phenyl ring. Boens' group successfully converted an
ortho-chlorophenol to obtain a pH-sensitive BODIPY with a pKa
of 7.6.31 Only few examples of phenolic rhodamines are given
in the literature32 and none of those were exploited as pH
probes, leading us to investigate the potential efficiency of
these entities as uorescent pH sensors. The red-emitting
uorophore X-rhodamine was chosen for its highly desirable
spectral properties including a high molar absorption, high
quantum yield and good photo-stability as well as its reduced
hydrophobicity compared to BODIPY. Thus, an initial set of 9
X-rhodamines was synthesised from hydroxybenzaldehydes This journal is © The Royal Society of Chemistry 2015 Chem. Sci. Edge Article basic forms have signicantly enhanced acidity. For example,
HR-Br is 1000-times more acidic than HR-pOH. This difference
in acidity cannot be exclusively attributed to the electronic
withdrawing effect of the bromine atom. These results tend to
suggest that H-aggregation stabilises the basic forms of H-
Rubies and hence, enhances their acidity. In this line of
thought, since aggregation is concentration dependent, absor-
bance spectra and titration curves of HR-Br were measured at
different concentrations of dye ranging from 500 nM to 5 mM
(see Fig. S3 and S4†). The results showed that aggregation of
basic form occurred at concentration above 500 nM and
impacted the pKa which varied from 6.5 at 5 mM to 7.3 at 500
nM. Moreover at a concentration of 500 nM where no aggre-
gation of the basic form occurs the uorescence enhancement
was diminished as the OFF state was only due to the PET
quenching phenomenon. H-aggregates are virtually non-uo-
rescent and thus, for the H-Rubies that are able to undergo
this type of aggregation, this property can confer impressive
levels of uorescence enhancement (dynamic range) between
their basic and acidic forms making them promising pH
sensor for bio-imaging purposes. Synthesis of phenolic X-rhodamines By controlling the H-
aggregation of uorophores one can achieve highly sensitive
uorogenic systems with a pronounced turning ON of uo-
rescence
upon
de-aggregation. This
principle
has
been
successfully used for bioimaging of ligand–receptor interac-
tions39 and was also applied in monitoring intracellular pH
with uorogenic polymers.40
Synthesis of functionalised H-Rubies
Despite the rst phenol-based X-rhodamines yielding
results as uorescent pH probes, the importance of the
functionalise such uorescent sensors must be emphas
thus, the basic principle was pushed further in order to
Table 1
Spectral and physico-chemical properties of the first set of synthesised X-rhodamines (5 mM in MOPS 30 mM, 100 mM KCl)
Compound
labsmax
(nm)
lemd
(nm)
Log 3 (labsmax)
(M1 cm1)
F
FON/FOFF
Dynamic
rangee
Dynamic
range @ pH 1 pKa
f
HR-pOH
ONb
576
587
4.78
0.55
9
34
30
OFFc
579f
598
4.53
0.06
HR-mOH
ONb
578
602
4.70
0.30
3
132
75
OFFc
577
603
4,63
0.11
HR-oOH
ONb
581
603
4.89
0.75
3
866
460
OFFc
579
603
4.69
0.27
HR-Me
ONb
574
597
4.71
0.09
High
955
833
OFFc
N/Ag
598
N/Ag
N/Ah
HR-OMe
ONb
577
598
4,72
0.22
8
302
N/Ai
OFFc
576
600
4.74
0.03
HR-Cl
ONb
579
601
4.68
0.47
High
753
512
OFFc
544
599
4.52
N/Ah
Hr-Br
ONb
581
601
4.39
0.55
High
384
291
OFFc
544
600
4.40
N/Ah
p-Nph
ONb
581
600
4.79
0.28
6
23
9.7
OFFc
579
602
4.34
0.05
o-Nph
ONb
584
604
4.43
0.77
8
337
166
OFFc
576
604
4.46
0.10
Pip-H
ONb
578
600
4.88
0.16
4
6
3.9
OFFc
579
601
4.84
0.04
Pip-Alkyne
ONb
577
600
4.98
0.34
2
3
1.9
OFFc
579
602
4.45
0.15
Imidazole
ONb
583
609
4.80
0.40
N/Aj
N/Aj
N/Aj
OFFc
584
608
N/Aj
N/Aj
a 3 ¼ molar extinction coefficient, l ¼ wavelength, F ¼ quantum yield. b Protonated form: pH 4. c Deprotonated form: pH 10. d Excitation a
e [(Imax Imin)/Imin] with I ¼ uorescence intensity. f Fluorescence enhancement between 1 pH unit around the pKa value. g Broad ab
h Non-uorescent. i pKa value was too high. j No pH sensitivity. Fig. 2
Absorption spectra of (A) HR-oOH, (B) HR-mOH, (C)
and (D) HR-Br at 5 mM in MOPS 30 mM, 100 mM KCl at differe
their basic form (blue) and acidic form (red) structures. Open Access Article. Published on 14 July 2015. Structure/properties relationship of functionalisable H-
Rubies Once again, it was noticed that slight modications of the base
structure led to dramatic changes in the photophysical prop-
erties of H-Rubies such as epsilon, quantum yields and pKa
values which ranged from 4 to 8 (Table 2). The most notable
differences were observed between secondary and tertiary
amides whereby secondary amides appeared much more acidic
than tertiary amides. To emphasise this phenomena, we syn-
thesised HR-PN3 and its methylated tertiary amide equivalent
HR-MPN3 and showed that the latter was 60-times less acidic. Moreover, absorption spectra of these functionalisable H-
Rubies clearly showed different behaviours of their basic forms
in water. While deprotonated tertiary amide H-Rubies showed Fig. 3
Synthesis and structures of the functionalisable H-Rubies: (a)
NHS, DCC, THF; (b) amine, DIEA, DMSO; (c) 8-hydroxyjulolidine, PTSA,
propionic acid then p-chloranil, DCM/MeOH (1 : 1); (d) 8-hydrox-
yjulolidine, TfOH, DCM, then p-chloranil. functionalisable H-Rubies. For this purpose, a linker was added
at the ortho position of the phenol via an amide bond: a mild
electron-withdrawing group that would ensure the conservation
of a biologically relevant pKa. Synthesis of functionalised H-Rubies basic forms have signicantly enhanced acidity. For example,
HR-Br is 1000-times more acidic than HR-pOH. This difference
in acidity cannot be exclusively attributed to the electronic
withdrawing effect of the bromine atom. These results tend to
suggest that H-aggregation stabilises the basic forms of H-
Rubies and hence, enhances their acidity. In this line of
thought, since aggregation is concentration dependent, absor-
bance spectra and titration curves of HR-Br were measured at
different concentrations of dye ranging from 500 nM to 5 mM
(see Fig. S3 and S4†). The results showed that aggregation of
basic form occurred at concentration above 500 nM and
impacted the pKa which varied from 6.5 at 5 mM to 7.3 at 500
nM. Moreover at a concentration of 500 nM where no aggre-
gation of the basic form occurs the uorescence enhancement
was diminished as the OFF state was only due to the PET
quenching phenomenon. H-aggregates are virtually non-uo-
rescent and thus, for the H-Rubies that are able to undergo
this type of aggregation, this property can confer impressive
levels of uorescence enhancement (dynamic range) between
their basic and acidic forms making them promising pH
sensor for bio-imaging purposes. By controlling the H-
aggregation of uorophores one can achieve highly sensitive
uorogenic systems with a pronounced turning ON of uo-
rescence
upon
de-aggregation. This
principle
has
been
successfully used for bioimaging of ligand–receptor interac-
tions39 and was also applied in monitoring intracellular pH
with uorogenic polymers.40 Despite the rst phenol-based X-rhodamines yielding excellent
results as uorescent pH probes, the importance of the ability to
functionalise such uorescent sensors must be emphasised and
thus, the basic principle was pushed further in order to develop Fig. 2
Absorption spectra of (A) HR-oOH, (B) HR-mOH, (C) HR-pOH
and (D) HR-Br at 5 mM in MOPS 30 mM, 100 mM KCl at different pH and
their basic form (blue) and acidic form (red) structures. Fig. 2
Absorption spectra of (A) HR-oOH, (B) HR-mOH, (C) HR-pOH
and (D) HR-Br at 5 mM in MOPS 30 mM, 100 mM KCl at different pH and
their basic form (blue) and acidic form (red) structures. This journal is © The Royal Society of Chemistry 2015 Chem. Sci. Chem. Sci. Edge Article
View Article Online Chemical Science Fig. Synthesis of functionalised H-Rubies 3
Synthesis and structures of the functionalisable H-Rubies: (a)
NHS, DCC, THF; (b) amine, DIEA, DMSO; (c) 8-hydroxyjulolidine, PTSA,
propionic acid then p-chloranil, DCM/MeOH (1 : 1); (d) 8-hydrox-
yjulolidine, TfOH, DCM, then p-chloranil. secondary amines bearing one or two orthogonal functions. Once the desirable aldehydes were obtained, they were trans-
formed into their corresponding X-rhodamines with a custom
optimized methodology allowing us to obtain the H-Rubies with
yields of up to 93% (see ESI†). This new set of sensors can be
categorised as follows: alkynes and azides, allowing linkage via
bio-orthogonal Huisgen cycloaddition “click chemistry”; acids
that can be coupled with amines and bifunctional H-Rubies,
bearing two different functional groups (Fig. 3). Among the
bifunctional H-Rubies that were developed is an Fmoc-pro-
tected Lysine H-Ruby (HR-LysF), which can be incorporated into
a peptide sequence. Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. a Protonated form: pH 4. b Deprotonated form: pH 10. c Excitation at 535 nm. d [(Imax Imin)/Imin] with I ¼ uorescence intensity. e Fluorescence
enhancement between 1 pH unit around the pKa value. f Complex titration curve. Chem. Sci.
This journal is © Th Structure/properties relationship of functionalisable H-
Rubies 4
Difference of acidity and H-aggregation capability between secondary amide H-Rubies (dashed lines) and tertiary amide H-Rubies (plain
lines). Concentration of probes was 5 mM in MOPS 30 mM, 100 mM KCl, excitation wavelength at 535 nm. probes (Fig. 5B and C plain lines). Finally, we checked the pH
sensitivity of these H-Rubies upon cytosolic acidication. The
intracellular pH was decreased to pH 6 and ow cytometry
assays were performed (Fig. 5C dashed lines). The results
showed that albeit HR-N3 and HR-Cl displayed non-signicant
uorescence enhancement (blue and green lines), HR-Br
(orange line) displayed an impressive in cellulo response to this
acidication (DpH ¼ 1.4, uorescence enhancement: up to 13-
fold). HR-Me
also
showed
an
interesting
uorescence
enhancement (5-fold) but behaved in an inconsistent manner
when the intracellular pH was alkalinised (Fig. S7†). Despite
HR-Br cannot be used to determine absolute pH values in cellulo
because of the effect of its concentration on its pKa, its cell
permeability combined with its high uorescence enhancement
in the physiological pH range make it an attractive tool for
tracking mitochondrial acidication which is a important eld
in cell biology as it is involved in mitophagy that is associated to
various pathologies including neurodegenerative and cardio-
vascular diseases.41,42 slight hypsochromic shis of their maximum absorption
wavelengths (1–5 nm), absorption spectra of secondary amide
H-Rubies displayed a second absorption band (530 nm) indi-
cating typical formation of soluble H-aggregates. These differences can be explained by the formation of a H-
bond between the phenolate and the proton of the secondary
amide whereby the former is stabilised and hence, the pKa is
lowered (Fig. 4). Moreover, the six membered ring formed via H-
bonding leads to a constrained conformation which aids the
formation of planar, head-to-head H-aggregates. As seen in our
initial observations, the acidity of H-Rubies is enhanced by their
ability to form H-aggregates in their OFF state (Fig. 4). Imaging of acidic domains in living cells with molecular H-
Rubies The impressive physical and optical properties of H-Rubies lead
us to investigate their ability to image acidic domains in living
cells. Aer establishing their insensitivity towards biologically
signicant metals (Zn2+, Mg2+, Ca2+, Fe3+, Mn2+) and their
nontoxicity (see ESI Fig. S5† for LDH cytotoxicity assay), HR-N3,
HR-Me, HR-Cl and HR-Br were involved in cellular experiments
(Fig. 5A). These probes were specically chosen for their
respective pKa values: HR-Me served as a positive control since
its pKa is more than two units above maximum physiological
pH, HR-N3 was expected to be a negative control since it has a
pKa of 4, whereas HR-Cl and HR-Br were expected to sense
variations of pH within the cell (Fig. 5B). Firstly, Fischer's rat
glioblastoma F98 cells (primary tumor) were incubated with the
four dyes (1 mM) for only 20 min and visualised by confocal laser
scanning microscopy. The obtained images indicated that these
H-Rubies are cell-permeant and stain the mitochondria; this
was conrmed by colocalisation experiments with mitotracker-
green (see Fig. S6†). In a second time, ow cytometry assays with
these cells showed a clear correlation between the observed
uorescence intensity of the cells and the measured pKa of the Structure/properties relationship of functionalisable H-
Rubies For this new set of functionalis-
able H-Rubies, 5-formylsalicylic acid 1 was converted into its
activated ester 2 onto which were condensed primary and Table 2
Spectral and physicochemical properties of the functionalisable H-Rubies (5 mM in MOPS 30 mM, 100 mM KCl)
Compound
labsmax
(nm)
lemc
(nm)
Log 3 (labsmax)
(M1 cm1)
F
FON/FOFF
Dynamic
ranged
Dynamic
range @ pH 1 pKa
e
pKa
HR-A
ONa
574
600
4.26
0.09
6
6
4.2
5.33
OFFb
534
598
4.58
0.02
0.13
HR-PA
ONa
580
602
4.87
0.46
1150
31
22
6.89
OFFb
573
596
4.86
4 104
0.04
HR-PiA
ONa
580
601
4.55
0.80
35
98
29
7.64
OFFb
574
600
4.32
0.02
0.10
HR-PiAC
ONa
580
601
4.86
0.64
40
86
45
7.68
OFFb
575
602
4.69
0.02
0.09
HR-N3
ONa
580
600
4.43
0.25
28
88
74
3.96
OFFb
523
596
4.43
0.01
0.03
HR-PN3
ONa
578
603
4.72
0.41
21
21
19
6.20
OFFb
573
597
4.74
0.02
0.03
HR-MPN3
ONa
578
605
5.20
0.65
65
78
66
8.00
OFFb
573
603
5.20
0.01
0.03
HR-Ala
ONa
577
601
4.81
0.15
8
9
8.3
7.85
OFFb
573
597
5.03
0.019
0.03
HR-bAla
ONa
577
602
5.10
0.20
11
11
N/Af
N/Af
OFFb
572
598
5.30
0.018
HR-LysF
ONa
583
607
4.73
0.11
37
45
33
4.84
OFFb
580
600
4.66
0.003
0.10
HR-CysA
ONa
577
602
4.69
0.09
N/Af
N/Af
N/Af
N/Af
OFFb
572
597
5.08
0.014
HR-LysA
ONa
577
602
4.75
0.25
13
13
11
7.51
OFFb
572
596
4.96
0.02
0.04
a Protonated form: pH 4. b Deprotonated form: pH 10. c Excitation at 535 nm. d [(Imax Imin)/Imin] with I ¼ uorescence intensity. e Fluorescence
enhancement between 1 pH unit around the pKa value. f Complex titration curve. physicochemical properties of the functionalisable H-Rubies (5 mM in MOPS 30 mM, 100 mM KCl) This journal is © The Royal Society of Chemistry 2015 Chem. Sci. Edge Article Fig. 4
Difference of acidity and H-aggregation capability between secondary amide H-Rubies (dashed lines) and tertiary amide H-Rubies (plain
lines). Concentration of probes was 5 mM in MOPS 30 mM, 100 mM KCl, excitation wavelength at 535 nm. g Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. This journal is © The Royal Society of Chemistry 2015 Measurements of intraorganellar pH using functionalisable
H-Rubies This effect has already been
noticed in our previous work involving a molecular uorescent
Ca2+ sensor linked to a quantum dot43 and can be attributed to
the proximity of the dye to the hydrophobic environment of the
polystyrene bead that enhances the basal uorescence of the
sensor. Compared to the titration curve of the free dye, the
uorescence ratio of the two dyes obtained by ow cytometry
shows an almost linear dependence when the pH is varied from
5 to 9. As a result, although these beads have a decreased
sensitivity in the pH of physiological domain, it provides a
ratiometric system which displays a pH sensitivity over a wider
range when compared to a free HR-PiAC (Fig. 6D). inuences the physicochemical properties of HR-PiAC, namely
its dynamic range and its pKa. This effect has already been
noticed in our previous work involving a molecular uorescent
Ca2+ sensor linked to a quantum dot43 and can be attributed to
the proximity of the dye to the hydrophobic environment of the
polystyrene bead that enhances the basal uorescence of the
sensor. Compared to the titration curve of the free dye, the
uorescence ratio of the two dyes obtained by ow cytometry
shows an almost linear dependence when the pH is varied from
5 to 9. As a result, although these beads have a decreased
sensitivity in the pH of physiological domain, it provides a
ratiometric system which displays a pH sensitivity over a wider
range when compared to a free HR-PiAC (Fig. 6D). For phagosomal pH measurement by ow cytometry,
adherent RAW 264.7 macrophages were incubated for 30 min at
37 C with the OVA-modied beads and let rest for 3 more hours
for phagosomal maturation. Aerwards the cells were harvested
and incubated with streptavidin-FITC that only binds to the
non-internalised
beads
containing
OVA-biotin. Two
pop-
ulations of phagocytic and non-phagocytic cells can be differ-
entiated by the uorescence intensity of Alexa Fluor 647 of the
beads. A cell population with beads attached to the cell surface Fig. 7
Confocal microscopy images of fixed RAW 264.7 cells after
bead phagocytosis. DAPI nuclear staining, concanavalin-Alexa 488
membrane staining and pH beads are in blue, green and red respec-
tively. (A) illustrates the orthogonal views of the cell that allow differ-
entiation of the internalized beads (B) and the beads attached to the
cell surface (C). Scale bars are 10 mm. Measurements of intraorganellar pH using functionalisable
H-Rubies To demonstrate the different biological applications of H-
Rubies we developed two systems to measure sub-organellar pH
in living cells. Our rst approach aimed at the calibration of
phagosomal pH in living cells using ow cytometry. As pH
reporter, HR-PiAC was chosen for its useful properties in
cellular experiments such as its high brightness, its high
dynamic range (80-fold) and its pKa value of 7.68 (Fig. 6A and
B). 3 mm latex beads containing amino groups were combined
with the bifunctional linker azido-PEG4-NHS ester followed by
double functionalisation using click chemistry with HR-PiAC
and a pH-insensitive near infrared emitting dye, Alexa Fluor
647, to allow ratiometric measurements (Fig. 6C). It was noticed
that the covalent binding of two dyes on the surface of the beads This journal is © The Royal Society of Chemistry 2015 Chem. Sci. Chem. Sci. Chem. Sci. Fig. 6
Absorption (A) and emission spectra (B) of HR-PiAC (5 mM,
MOPS 30 mM, 100 mM KCl) at different pH (excitation wavelength ¼
535 nm), inset is the titration curve fitted with the Hill equation
providing a pKa of 7.68 0.09 and an enhancement of fluorescence of
80-fold. Scheme of bead-based pH sensor, containing pH-sensitive
dye HR-PiAC and pH-insensitive Alexa Fluor 647 (C); titration curve of
the pH beads measured by flow cytometry (D). Edge Article
View Article Online Edge Article
View Article Online View Article Online Fig. 5
(A) Structures and pKa values of the H-Rubies used for cellular
experiments. (B) pH titration curves (Hill equation fit) of HR-N3 (blue),
HR-Cl (green), HR-Br (red) and HR-Me (brown) at 5 mM in MOPS (30
mM) KCl (100 mM), cytosolic and cellular acidic pH ranges are
delimited with coloured areas. (C) Comparison of fluorescence
intensity of F98 cells incubated for 20 min with 1 mM of HR-N3 (blue),
HR-Cl (green), HR-Br (red) and HR-Me (brown) at pH 7.4 (plain lines)
and pH 6 (dashed lines) assessed by flow cytometry. Each curve is an
average of 3 independent measurements. The control corresponds to
the intrinsic fluorescence of cells (black line). Bottom: laser scanning
confocal microscopy images of F98 cells incubated for 20 min with 1
mM of (D) HR-N3, (E) HR-Cl, (F) HR-Br, (G) HR-Me. The H-Rubies were
visualized in red, the nuclei were stained with Hoechst (blue) and the
membrane with
Alexa 647-conjugated
concanavalin
A
(green). Pictures show the three merged channels. Scale bars are 10 mm. Chem. Sci. Measurements of intraorganellar pH using functionalisable
H-Rubies Chemical Science Edge Article Chemical Science Chemical Science Chemical Science Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6
Absorption (A) and emission spectra (B) of HR-PiAC (5 mM,
MOPS 30 mM, 100 mM KCl) at different pH (excitation wavelength ¼
535 nm), inset is the titration curve fitted with the Hill equation
providing a pKa of 7.68 0.09 and an enhancement of fluorescence of
80-fold. Scheme of bead-based pH sensor, containing pH-sensitive
dye HR-PiAC and pH-insensitive Alexa Fluor 647 (C); titration curve of
the pH beads measured by flow cytometry (D). Fig. 5
(A) Structures and pKa values of the H-Rubies used for cellular
experiments. (B) pH titration curves (Hill equation fit) of HR-N3 (blue),
HR-Cl (green), HR-Br (red) and HR-Me (brown) at 5 mM in MOPS (30
mM) KCl (100 mM), cytosolic and cellular acidic pH ranges are
delimited with coloured areas. (C) Comparison of fluorescence
intensity of F98 cells incubated for 20 min with 1 mM of HR-N3 (blue),
HR-Cl (green), HR-Br (red) and HR-Me (brown) at pH 7.4 (plain lines)
and pH 6 (dashed lines) assessed by flow cytometry. Each curve is an
average of 3 independent measurements. The control corresponds to
the intrinsic fluorescence of cells (black line). Bottom: laser scanning
confocal microscopy images of F98 cells incubated for 20 min with 1
mM of (D) HR-N3, (E) HR-Cl, (F) HR-Br, (G) HR-Me. The H-Rubies were
visualized in red, the nuclei were stained with Hoechst (blue) and the
membrane with
Alexa 647-conjugated
concanavalin
A
(green). Pictures show the three merged channels. Scale bars are 10 mm. technique in this case will identify all uorescent objects, i.e. cells with internal and/or external beads. We, therefore, per-
formed an additional modication of the beads by coating them
with a biotinylated ovalbumin (OVA), via passive absorption. The coated beads, rstly, have a reduced non-specic binding to
the cell surface and favour phagocytosis through the interaction
between the mannosylated structure present on the surface of
OVA44 and the mannose receptors of macrophages.45 Secondly,
accessible biotin groups of OVA can be tagged by FITC-
streptavidin. inuences the physicochemical properties of HR-PiAC, namely
its dynamic range and its pKa. Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 8
Phagosomal pH measurement in RAW 264.7 cells by flow cytometry. (A) Density plot of the cells after bead internalisation showing three
distinct populations: non-phagocytic cells, cells with internalised beads and cells with non-internalised beads on which a biotin – streptavidin–
FITC interaction takes place; (B) the cells with only one internalised bead were used for analysis of phagosomal pH. (C) Typical calibration curve of
H-Ruby/Alexa Fluor 647 ratio of the cells clamped at different pHs with the ionophore nigericin; (D) phagosomal pH (5.3 0.2, in red) in RAW
264.7 cells measured after 3 hours of phagosomes maturation NH4Cl addition shows alkalinisation of phagolysosomes (7.2 0.1, in blue). The
data represent the mean of 3 independent experiments SD. Fig. 9
(A) Synthesis of HR-PN3 dextran conjugate via CuAAC Click
Chemistry. (B) Emission spectra of HR-PN3 dextran conjugate (MOPS
30 mM, 100 mM KCl) at different pH (excitation wavelength ¼ 535 nm),
inset is the titration curve fitted to the Hill equation yielding a pKa of
6.77 0.02 and an enhancement of fluorescence of 18-fold and 13
fold between 1 pH unit around the pKa. (C–E) Confocal images of
RAW 264.7 cells after incubation with 1 mg mL1 HR-PN3 dextran
conjugate (C), co-stained by Lysotracker Green (D) and a merged
image of two channels (E). HR-PN3 dextran conjugate labels lyso-
somes (in red) that colocalises with Lysotracker, and endosomes that
are shown in insets and do not colocalise with Lysotracker. Scale bars
are 10 mm. (F) Typical calibration curve of H-Ruby/Alexa Fluor 647 ratio
of the cells clamped at different pHs with the ionophore nigericin. (G)
Endosomal pH (6.3 0.05, in red) in RAW 264.7 cells measured after 3
hours of incubation with HR-PN3 dextran. NH4Cl addition shows
alkalinisation of endosomal organelles (7.0 0.06, in blue). The data
represent the mean of 3 independent experiments SD. can be separated by the higher uorescence intensity of FITC at
extracellular pH (Fig. 8A). Further detailed analysis of the
phagocytic population shows the presence of subpopulations
according to the uorescence intensities of Alexa Fluor 647 and
H-Ruby: cells that have internalised one bead per cell exhibit a
lower uorescence intensity (Fig. 8B). Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For precise analysis of
phagosomal pH the cells were gated on one bead population
and a ratiometric calibration curve of phagosomal pH was
obtained changing the external pH over the 5 to 8 range using a
citric acid/HEPES buffer and imposing the same pH intracel-
lularly using nigericin, a K+/H+ ionophore (Fig. 8C). Finally, to
determine the phagosomal pH, the ratio of the mean uores-
cence intensities of HRuby vs. Alexa Fluor 647 obtained from the
intact cells was interpolated to the calibration curve yielding a
phagolysosomal pH of 5.3 0.2. This result is in a good
agreement with a previously published study where authors
showed a phagosomal pH value of 5.22 0.03 in RAW cells
using FITC-sRBC particles.46 Addition of the weak base NH4
+/
NH3 caused fast alkalinisation, and demonstrated the intra-
cellular localisation of the beads (Fig. 8D). Another application of these newly synthesized H-Rubies is
the calibration of endosomal pH by ow cytometry and to
achieve this we used a polysaccharide dextran that is known to
target the endolysosomal pathway.47 40 kDa dextran was con-
verted to an alkyne containing dextran and was subsequently
clicked to HR-PN3 (Fig. 9A). In comparison to its parent
molecular form HR-PN3, the uorescent pH-sensitive dextran
displayed a slightly shied apparent pKa value (respectively 6.20
0.03 and 6.77 0.02; Fig. 9B) with a virtually non-uorescent
OFF state (see ESI†). Functionalisation did not signicantly
affect the uorescence enhancement between pH 10 and 4
(respectively 21- and 19-fold) making this water-soluble polymer
a valuable tool for monitoring endosomal pH. To demonstrate
this, adherent RAW 264.7 cells were incubated for 30 min with
HR-PN3 dextran conjugate and let rest for 3 more hours. As
shown in Fig. 9C, vesicles of different shapes and sizes could be
observed inside the cells due to the movement and segregation
of the dextran molecules between the endocytic organelles.48 Fig. 9
(A) Synthesis of HR-PN3 dextran conjugate via CuAAC Click
Chemistry. (B) Emission spectra of HR-PN3 dextran conjugate (MOPS
30 mM, 100 mM KCl) at different pH (excitation wavelength ¼ 535 nm),
inset is the titration curve fitted to the Hill equation yielding a pKa of
6.77 0.02 and an enhancement of fluorescence of 18-fold and 13
fold between 1 pH unit around the pKa. This journal is © The Royal Society of Chemistry 2015 Measurements of intraorganellar pH using functionalisable
H-Rubies The dyes-modied beads were then added to a suspension of
RAW 264.7 macrophages and incubated for 30 min at 37 C to
complete the process of phagocytosis. Aer several washes the
cells were xed and visualized by confocal microscopy to assess
the efficiency of bead internalisation. Orthogonal view analysis
of confocal sections shows partial internalisation of the beads
by macrophages. As shown in Fig. 7, in addition to fully
internalised beads, some beads are attached to the outer cell
membrane. On average only 30% of beads were fully internal-
ised by the cells. This could interfere with ow cytometric
measurements of phagosomal pH because the laser-based Fig. 7
Confocal microscopy images of fixed RAW 264.7 cells after
bead phagocytosis. DAPI nuclear staining, concanavalin-Alexa 488
membrane staining and pH beads are in blue, green and red respec-
tively. (A) illustrates the orthogonal views of the cell that allow differ-
entiation of the internalized beads (B) and the beads attached to the
cell surface (C). Scale bars are 10 mm. This journal is © The Royal Society of Chemistry 2015 Edge Article Fig. 8
Phagosomal pH measurement in RAW 264.7 cells by flow cytometry. (A) Density plot of the cells after bead internalisation showing three
distinct populations: non-phagocytic cells, cells with internalised beads and cells with non-internalised beads on which a biotin – streptavidin–
FITC interaction takes place; (B) the cells with only one internalised bead were used for analysis of phagosomal pH. (C) Typical calibration curve of
H-Ruby/Alexa Fluor 647 ratio of the cells clamped at different pHs with the ionophore nigericin; (D) phagosomal pH (5.3 0.2, in red) in RAW
264.7 cells measured after 3 hours of phagosomes maturation NH4Cl addition shows alkalinisation of phagolysosomes (7.2 0.1, in blue). The
data represent the mean of 3 independent experiments SD. LDH-cytotoxicity assay CHO-K1 cells (20 000 per well) were seeded in 96 multiple well
plates 24 h before the experiment. Cells (40 000 per well) were
incubated with the uorescent compounds in 100 mL DMEM for
2 hours at 37 C. The incubation milieu was then replaced by
100 mL fresh DMEM containing 10% of cell-counting solution
(Dojindo Laboratories) and incubated for a further 2 h at 37 C. The absorbance measured at 450 nm had a direct relationship
to the number of viable cells. Experiments were conducted in
triplicate and repeated twice. Fluorescence activated cell sorting analyses – effect of the pHi
on the uorescence intensity of the H-Rubies For the dose-response curve, F98 cells were incubated with
various concentrations of the H-Rubies in DMEM/F-12 culture
medium without serum at 37 C for 2 h. The cells were then
washed with PBS to remove excess extracellular H-Ruby and
were then treated with 0.05% Trypsin–EDTA for 2 min at 37 C
to detach cells from the surface, and centrifuged at 200 g in
DMEM/F-12 culture medium before suspension in PBS. Flow
cytometry analyses were performed with live cells using an
Accuri C6 ow cytometer (BD Biosciences, Le Pont de Claix,
France). For acquisition a 488 nm wavelength laser was used for
H-Rubies excitation, the uorescence emissions were recorded
in a 584 20 nm spectral detection channel. Data were
obtained and analyzed using CFlow Sampler (BD Biosciences). Live cells were gated by forward/side scattering for a total of
10 000 events. Confocal microscopy Cell cultures were incubated with the pH probes (in DMEM/F-12
nutrient medium only) for 20 min, and then washed twice with
phosphate-buffered saline (PBS) alone. Aer 4 hours, the
nucleus were stained with 60 mg mL1 Hoechst 34580 for 15
min, the cell cultures were then washed with PBS and the
plasma membrane was stained with 30 mg mL1 Alexa 647-
conjugated concanavalin A and the mitochondria stained with
50 nM mitotracker green for 5 min (stainings were purchased at
Invitrogen, Cergy Pontoise, France). Cells were washed once
more. Live cells were then immediately analysed by confocal
laser scanning microscopy using a Zeiss LSM operating system. Alexa 647 (633 nm), Hoechst 34580 (405 nm), pH probes (561
nm) and mitotracker green (488 nm) were sequentially excited
and emission uorescence was collected. To assess the efficiency of bead internalisation, the cells,
aer completing the process of phagocytosis (as described in
“Organellar pH measurement by ow cytometry”), were plated
on coverslips (d ¼ 12 mm) coated with 10 mg mL1 bronectin
and incubated at 37 C in 5% CO2: the cell membrane was
stained with 25 mg mL1 Alexa Fluor 488 – conjugated conca-
navalin (Invitrogen, USA) at 4 C for 5 min and 4% para-
formaldehyde was added for 20 min to x the cells. The
coverslips with the cells were then mounted on microscope Cell culture Undifferentiated malignant glioma rat (F98) and Chinese
Hamster Ovary (CHO) cell lines (from ATCC) were maintained at
37 C in 5% CO2 in DMEM/F-12 nutrient medium (Invitrogen,
Cergy Pontoise, France) supplemented with 2% (v/v) heat-inac-
tivated fetal bovine serum (Invitrogen) and 100 mg mL1 strep-
tomycin and 100 units per mL penicillin (Invitrogen). RAW
264.7 macrophages cell line was maintained at 37 C in 5% CO2
in DMEM nutrient medium (Gibco, Invitrogen) supplemented
with 10% fetal calf serum (Eurobio, France). The pHi of F-98 cells was modied using the pseudo-null
calibration method describe by Chow et al.49 F98 cells were
incubated with 1 mM of H-Rubies in DMEM/F-12 culture
medium without serum at 37 C for 20 min. The cells were then
washed with PBS to remove excess extracellular probe and were
then treated with 0.05% Trypsin–EDTA for 2 min at 37 C to
detach the cells from the surface, and centrifuged at 200 g in
DMEM/F 12 culture medium before suspension in culture
medium. Just before the analysis the pseudo null solution were
added (ratio of culture medium: pseudo-null solution 1 : 1) and
the data were acquired immediately (within 30 s). Pseudo-null
solutions were made according to the method of Chow et al. Six
times concentrated standard solution was made by adding 1 M
ammonium hydroxide and 1 M acetic acid to the basal solution
(culture medium). This provided a set of pseudo-null solution
as shown in Table S1 (see ESI†). Flow cytometry analyses were
performed with live cells using an Accuri C6 ow cytometer (BD
Biosciences, Le Pont de Claix, France). Data were obtained and
analyzed using CFlow Sampler (BD Biosciences). Live cells were
gated by forward/side scattering for a total of 10 000 events. Chem. Sci. Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (C–E) Confocal images of
RAW 264.7 cells after incubation with 1 mg mL1 HR-PN3 dextran
conjugate (C), co-stained by Lysotracker Green (D) and a merged
image of two channels (E). HR-PN3 dextran conjugate labels lyso-
somes (in red) that colocalises with Lysotracker, and endosomes that
are shown in insets and do not colocalise with Lysotracker. Scale bars
are 10 mm. (F) Typical calibration curve of H-Ruby/Alexa Fluor 647 ratio
of the cells clamped at different pHs with the ionophore nigericin. (G)
Endosomal pH (6.3 0.05, in red) in RAW 264.7 cells measured after 3
hours of incubation with HR-PN3 dextran. NH4Cl addition shows
alkalinisation of endosomal organelles (7.0 0.06, in blue). The data
represent the mean of 3 independent experiments SD. This journal is © The Royal Society of Chemistry 2015 Chem. Sci. Chem. Sci. Edge Article
View Article Online View Article Online Chemical Science Edge Article Addition of Lysotracker Green led to its partial colocalisation
with HR-PN3 dextran conjugate (Fig. 9C–E) suggesting that HR-
PN3 dextran accumulates both in the more acidic lysosomes and
in the mildly acidic vesicles of the endocytic pathway as well. This observation is consistent with the ow cytometry experi-
ments where, applying the same strategy as described above,
the pH value of 6.3 0.04 was obtained (Fig. 9F–G). slides
using
a
mounting
medium
DAPI-Fluoromount
G
(Southern Biotech, USA) and leat room temperature over-
night. Imaging was performed on a spinning disk microscope. The pH-sensitive beads were excited with 561 nm laser line and
detected with ET630/75 nm lter, DAPI was excited with 405 nm
laser line and detected with ET460/50 nm band-pass lter and
Concanavalin A-Alexa Fluor 488 was excited with 488 nm laser
line and detected with D505/40 nm band-pass lter. Images
were collected using microscope soware Soware: MetaMorph
7.8.2.0. The subsequent analysis of the images was conducted
on an open source image processing program ImageJ. Acknowledgements G. D. and A. Z. benetted of post-doctoral fellowships from the
Pierre Gilles de Gennes foundation. C.T. was supported by the
R´egion Rhˆone Alpes by an emergence fellowship. We would like
to thank Jean-Marie Carpier (Institute Curie, U932) for his help
with imaging, Dr. Sandrine Sagan and Dr Fabienne Burlina
(Laboratoire des biomolecules, UMR CNRS 7203) for the toxicity
assays. We also acknowledge PICT-IBiSA (Institute Curie),
member of the France-BioImaging national research infra-
structure. This work was supported by the Fondation Pierre-
Gilles de Gennes (to S. A., A. F. and J. M. M.), the European
Research Council (2013-AdG No. 340046), la Ligue Nationale
contre le Cancer (EL 2014.LNCC/SA) and the French Agence
National de la Recherche (ANR P3N, nanoFRET2 grant to A. F.,
M. D. W. and J. M. M., and ANR-11-LABX-0015 to M. D. W.). This
work is the subject of a European patent EP 13 199 575.5 – 1451
“uorescent red emitting functionalizable pH probes”. The calibration of phagosomal and endosomal pH was per-
formed using the K+/H+ ionophore nigericin that, together with
a high concentration of potassium in the buffer, equalises the
intracellular and extracellular pH.50 The cells containing
internalised pH beads or dextran were resuspended in buffers
containing 143 mM KCl, 1.17 mM MgCl2, 1.3 mM CaCl2, 5 mM
glucose and 10 mM nigericin with dened pH buffers ranging
from pH 5.0 to pH 8.0 with 0.5 increments. 20 mM citric acid
was used for a buffer range of pH 5.0–6.5 and 20 mM (4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) – for
buffers in the pH range 7.0–8.0. 0.05 mM DAPI was added to
exclude the dead cells. Aer incubation for 5 min at RT the
samples were analyzed by a ow cytometer (BD LSR Fortessa,
BD Biosciences). For acquisition, a 532 nm wavelength laser was
used for H-Rubies excitation, and its emission uorescence was
recorded in a 610 20 nm spectral detection channel. For Alexa
Fluor 647 a 640 nm wavelength laser was used for excitation and
a 670 30 nm spectral detection channel to record its emission
uorescence. Each time 10 000 events were acquired. For
analysis, a population of live cells was gated by forward/side
scatter, and within this population only the cells which have
phagocyted one pH bead were selected for precise
pH
determination. Notes and references 1 S. Grinstein and S. Dixon, Physiol. Rev., 1989, 69, 417. 1 S. Grinstein and S. Dixon, Physiol. Rev., 1989, 69, 417. 2 R. A. Gottlieb, H. A. Giesing, J. Y. Zhu, R. L. Engler and
B. M. Babior, Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 5965. 3 R. A. Gottlieb, J. Nordberg, E. Skowronski and B. M. Babior,
Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 654. 4 S. Simon, D. Roy and M. Schindler, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 1128. 5 G. Lamb and D. Stephenson, J. Physiol., 1994, 478, 331. 6 H. Westerblad and D. Allen, J. Physiol., 1993, 466, 611. 7 A. A. Golabek,
E. Kida,
M. Walus,
W. Kaczmarski,
M. Michalewski and K. E. Wisniewski, Mol. Genet. Metab.,
2000, 70, 203. Organellar pH measurement by ow cytometry Measurement of phagosomal or endosomal pH was performed
using macrophages cell line RAW 264. The cells were plated on a
Petri dish and at 80% conuence pH beads in a 4 : 1 ratio or 0.7
mg mL1 40 kDa HR-PN3 dextran conjugate and 0.3 mg mL1 40
kDa Alexa Fluor 647 dextran conjugate were added and incu-
bated 30 min at 37 C to complete internalisation. Aerwards
the non-internalised beads/dextran were washed out, fresh
medium was added and the cells were let rest at 37 C, 5% CO2
for 3 more hours. For FACS analysis the cells were harvested and
resuspended in CO2-independent medium (GIBCO, Invitrogen). For NH4
+/NH3 tests, 20 mM NH4Cl was added to the extracel-
lular medium and let to incubate for 3 min. This journal is © The Royal Society of Chemistry 2015 Functionalisation of the beads by H-Ruby Polybead® Amino Microspheres 3.00 mm (Polysciences) were
used. First the beads were washed with dH2O. In order to
convert the NH2 groups on the beads surface to N3 groups for
further click chemistry reactions, 100 nmol of the bifunctional
linker N3–PEG4–NHS was added to 100 mL of the beads This journal is © The Royal Society of Chemistry 2015 Chemical Science
View Article Online Edge Article containing 20 nmol of NH2 groups and incubated at room
temperature for 2 h. The alkyne-modied H-Ruby HR-PiAC and
alkyne-containing Alexa Fluor 647 (Molecular Probes®) were
then mixed with the bead solution in a ratio 1 nmol of NH2
group: 10 nmol of H-Ruby: 2.5 nmol of Alexa Fluor 647. 5 mL of
25 mM CuSO4$5H2O and 5 mL of 25 mM sodium ascorbate were
added to initiate a click-reaction. The mixture was incubated
overnight at room temperature. The dyes modied beads were
successively washed with 0.1 M EDTA, 10% ethanol and PBS to
remove non-reacted dyes. cross the plasma membrane to compartmentalise in mito-
chondria. Among them, HR-Br displays a strong uorescence
enhancement upon acidication of mitochondria in live cells. Functionalisable H-Rubies were linked to a dextran polymer or
to microbeads that were also decorated with a pH-insensitive
dye leading to ratiometric pH probe systems. These systems
were successfully used to measure phagosomal and endocytic
pHs in live cells. The authors believe that this new family of
uorescent indicators, with their large choice of pKa values and
spectral properties specically adapted to cellular imaging,
represents a valuable toolkit for imaging pH variations in living
cells. Moreover, their diverse functionalisable side arms allows
them to be attached to particles, polymers and biomolecules
such as peptides or antibodies for accurate pH readout of
subcellular micro-domains. Open Access Article. Published on 14 July 2015. Downloaded on 31/08/2015 13:41:59.
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Corrigendum to “Measurement of electrons from beauty hadron decays in pp collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt><mml:mo>=</mml:mo><mml:mn>7</mml:mn><mml:mtext> TeV</mml:mtext></mml:math>” [Phys. Lett. B 721 (1–3) (2013) 13–23] and “Beauty production in pp collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si2.gif" overflow="scroll"><mml:msqrt><mml:mi>s</mml:mi…
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Physics letters. B
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Table 1 Effect of the corrected treatment of the D-meson pT distribution on the d0 cut
efficiency for electrons from charm-hadron decays (ϵd0 ) and the resulting yield of
signal electrons (dNsignal/dpT). Effect of the corrected treatment of the D-meson pT distribution on the d0 cut
efficiency for electrons from charm-hadron decays (ϵd0 ) and the resulting yield of
signal electrons (dNsignal/dpT). 7 TeV pp collisions
pT interval (GeV/c)
1–2
2–3
3–8
ϵupdated
d0
/ϵprevious
d0
0.56–0.60
0.60–0.70
0.70–0.85
(dNsignal/dpT)updated/(dNsignal/dpT)previous
1.6–1.4
1.3–1.2
< 1.1
2.76 TeV pp collisions
pT interval (GeV/c)
1–2
2–3
3–8
ϵupdated
d0
/ϵprevious
d0
0.74–0.77
0.77-0.85
0.85–0.94
(dNsignal/dpT)updated/(dNsignal/dpT)previous
1.4–1.3
1.2–1.1
< 1.1 For weakly decaying hadrons with sufficiently high transverse
momentum (pT), the impact parameter distribution of the daugh-
ter particle at a given pT depends very weakly on the transverse
momentum of the mother hadrons. However, at low momentum
the impact parameter distribution of the decay particles depends
on the momentum distribution of the mother hadrons. Due to the
harder pT spectra of charm hadrons in the Monte Carlo simulation
[1,2] compared to the measured ones [3,4], the d0 cut efficiency of
decay electrons was biased towards larger values. Since the back-
ground was subtracted from the raw inclusive electron yield after
applying the d0 cut, the charm-hadron decay background was over-
estimated. The new value of the d0 cut efficiency (ϵupdated
d0
) of electrons
from charm-hadron decays is significantly smaller than that previ-
ously evaluated (ϵprevious
d0
) as summarized in Table 1. 0
In Fig. 1, the raw electron yield, as well as the non-beauty
electron background yield, which is subtracted in the analysis, are
shown after the application of the track selection criteria. Com-
pared to Fig. 3 in [2], the yield of electrons from charm-hadron
decays is smaller by the factor ϵupdated
d0
/ϵprevious
d0
given in Table 1. We have now computed the d0
distribution of electrons
from charm-hadron decays using a Monte Carlo and weighting
each
electron
by
the
ratio
(dN/dpT)measured/(dN/dpT)MC. (dN/dpT)measured and (dN/dpT)MC are the production yields eval-
uated at the pT of the mother charm-hadron of the electron, as
obtained from data [3,4] and in the Monte Carlo simulations [1,2],
respectively. In such a way, the measured mother pT spectra are
propagated to the impact parameter cut efficiency calculation for
the daughter electrons. The corresponding yield of beauty-signal electrons (dNsignal/dpT)
increases as listed in Table 1. a r t i c l e
i n f o We have identified a bias in the measurement of electrons from
beauty-hadron decays in pp collisions at center-of-mass energies
√
s = 2.76 TeV [1] and √
s = 7 TeV [2]. The efficiency corrections
were evaluated using a Monte Carlo simulation, based on PYTHIA
as described in [1,2]. When calculating the impact parameter (d0)
cut efficiency for the charm-hadron decay electrons, we did not
consider the difference between the impact parameter distribu-
tions using the measured D-meson pT distribution and the one
from Monte Carlo. DOIs of original articles: http://dx.doi.org/10.1016/j.physletb.2013.01.069,
http://dx.doi.org/10.1016/j.physletb.2014.09.026. DOIs of original articles: http://dx.doi.org/10.1016/j.physletb.2013.01.069,
http://dx.doi.org/10.1016/j.physletb.2014.09.026.
http://dx.doi.org/10.1016/j.physletb.2016.10.004
0370-2693 Corrigendum Corrigendum to “Measurement of electrons from beauty hadron decays
in pp collisions at √
s = 7 TeV” [Phys. Lett. B 721 (1–3) (2013) 13–23]
and “Beauty production in pp collisions at √
s = 2.76 TeV measured
via semi-electronic decays” [Phys. Lett. B 738 (2014) 97–108]
ALICE C ll b
ti ALICE Collaboration http://dx.doi.org/10.1016/j.physletb.2016.10.004
0370-2693 Physics Letters B 763 (2016) 507–509
Contents lists available at ScienceDirect
Physics Letters B
www.elsevier.com/locate/physletb
Corrigendum
Corrigendum to “Measurement of electrons from beauty hadron decays
in pp collisions at √
s = 7 TeV” [Phys. Lett. B 721 (1–3) (2013) 13–23]
and “Beauty production in pp collisions at √
s = 2.76 TeV measured
via semi-electronic decays” [Phys. Lett. B 738 (2014) 97–108]
ALICE Collaboration Physics Letters B 763 (2016) 507–509
Contents lists available at ScienceDirect
Physics Letters B
www.elsevier.com/locate/physletb
Corrigendum
Corrigendum to “Measurement of electrons from beauty hadron decays
in pp collisions at √
s = 7 TeV” [Phys. Lett. B 721 (1–3) (2013) 13–23]
and “Beauty production in pp collisions at √
s = 2.76 TeV measured
via semi-electronic decays” [Phys. Lett. B 738 (2014) 97–108]
ALICE Collaboration Physics Letters B 763 (2016) 507–509 Table 1 For pp collisions at √
s = 2.76 TeV,
where a similar bias was present, the same procedure has been
applied and the correct distributions are shown in Fig. 2 (to be
compared with Fig. 2 in [1]). Numerical values of the implication
for the d0 cut efficiency are given in Table 1. The uncertainty on the d0 efficiency was evaluated by propa-
gating the statistical and systematic uncertainties of the charm-
hadron pT distributions in [3] to the measurements discussed in
this corrigendum. The uncertainty was added in quadrature as an
independent contribution to the total systematic uncertainty. ALICE Collaboration / Physics Letters B 763 (2016) 507–509 508 Fig. 1. This figure replaces Fig. 3 from [2]. Caption is the same as Fig. 3 from [2]. Fig. 3. This figure replaces Fig. 4 from [2]. Caption is the same as Fig. 4 from [2]. Fig. 1. This figure replaces Fig. 3 from [2]. Caption is the same as Fig. 3 from [2] Fig. 1. This figure replaces Fig. 3 from [2]. Caption is the same as Fig. 3 from [2]. Fig. 2. This figure replaces Fig. 2 from [1]. Caption is the same as Fig. 2 from [1]. Fig. 3. This figure replaces Fig. 4 from [2]. Caption is the same as Fig. 4 from [2]. Fig. 4. This figure replaces Fig. 5 from [2]. Caption is the same as Fig. 5 from [2]. Fig. 2. This figure replaces Fig. 2 from [1]. Caption is the same as Fig. 2 from [1]. Table 2 This figure replaces Fig. 6 from [1]. Caption is the same as Fig. 6 from [1]. Fig. 5. This figure replaces Fig. 4 from [1]. Caption is the same as Fig. 4 from [1]. Fig. 7. This figure replaces Fig. 6 from [1]. Caption is the same as Fig. 6 from [1]. J/ψ
mesons from beauty-hadron decays [5] is also updated. After subtracting the new cross section of the electrons from
beauty-hadron decays from the measured cross section of the
electrons from heavy-flavour hadron decays [6], the production
cross section of electrons from charm-hadron decays was con-
verted into a charm production cross section. The charm pro-
duction cross section per unit rapidity at mid-rapidity (dσc¯c/dy)
and the total cross sections (σc¯c) at √
s = 7 TeV are also updated
in Table 2. Since the corresponding quantity at √
s = 2.76 TeV
was not explicitly evaluated in [1], there is no correspond-
ing entry in Table 2. All measured cross sections for 7 TeV
(2.76 TeV) have an additional normalization uncertainty of 3.5%
(1.9%) [7]. Fig. 5. This figure replaces Fig. 4 from [1]. Caption is the same as Fig. 4 from [1]. Fig. 6. This figure replaces Fig. 5 from [1]. Caption is the same as Fig. 5 from [1]. In Figs. 3, 4, 5, 6 and 7, we have updated accordingly the ALICE
data points. The main conclusion of the original papers remains valid: the
data and predictions are consistent within the experimental and
theoretical uncertainties. [1] B. Abelev, et al., ALICE Collaboration, Phys. Lett. B 738 (2014) 97.
[2] B. Abelev, et al., ALICE Collaboration, Phys. Lett. B 721 (2013) 13.
[3] K. Aamodt, et al., ALICE Collaboration, J. High Energy Phys. 01 (2012) 128.
[4] B. Abelev, et al., ALICE Collaboration, J. High Energy Phys. 1207 (2012) 191,
arXiv:1205.4007 [hep-ex].
[5] B. Abelev, et al., ALICE Collaboration, J. High Energy Phys. 11 (2012) 1.
[6] B. Abelev, et al., ALICE Collaboration, Phys. Rev. D 86 (2012) 112007, arXiv:
1205.5423 [hep-ex].
[7] B. Abelev, et al., ALICE Collaboration, Eur. Phys. J. C 73 (2013) 1. Table 2 Summary of the updated cross sections. Cross sections at 7 TeV pp collisions
Visible σb→e
9.03 ± 0.50 (stat) +2.72
−2.73 (sys) ± 0.32 (norm) μb
dσb¯b/dy
57.7 ± 3.2 (stat) +17.4
−17.4 (sys) +1.4
−2.3 (extr) ± 2.0 (norm) μb
σb¯b
383 ± 21 (stat) +116
−116 (sys) +10
−11 (extr) ± 13 (norm) ± 13 (br) μb
Weighted σb¯b
322 ± 45 (stat) +58
−62 (sys) +8
−9 (extr) μb
dσc¯c/dy
1.1 ± 0.2 (stat) +0.6
−0.7 (sys) +0.2
−0.1 (extr) mb
σc¯c
9.7 ± 1.7 (stat) +5.2
−5.6 (sys) +3.4
−0.5 (extr) ± 0.4 (br) mb
Cross sections at 2.76 TeV pp collisions
Visible σb→e
4.33 ± 0.38 (stat) +1.45
−1.75 (sys) ± 0.08 (norm) μb
dσb¯b/dy
29.1 ± 2.6 (stat) +9.8
−11.7 (sys) +0.6
−0.8 (extr) ± 0.6 (norm) μb
σb¯b
162 ± 14 (stat) +55
−65 (sys) +4
−4 (extr) ± 3 (norm) ± 6 (br) μb The relative systematic uncertainties on the charm-hadron de-
cay background increase by 3% (2%) at pT < 1.5 GeV/c for 7 TeV
(2.76 TeV) pp collisions. The change of the systematic uncertainties
at higher pT region is instead negligible. However, the amount of
background decreases and as a consequence the total uncertainty
on the beauty production measurement decreases. Fig. 4. This figure replaces Fig. 5 from [2]. Caption is the same as Fig. 5 from [2]. Fig. 4. This figure replaces Fig. 5 from [2]. Caption is the same as Fig. 5 from [2]. decays (visible σb→e), the beauty production cross section per unit
rapidity at mid-rapidity (dσb¯b/dy) and the total cross section (σb¯b)
are summarized in Table 2. For 7 TeV pp collisions, the weighted
average of this with the result of a previous measurement of decays (visible σb→e), the beauty production cross section per unit
rapidity at mid-rapidity (dσb¯b/dy) and the total cross section (σb¯b)
are summarized in Table 2. For 7 TeV pp collisions, the weighted
average of this with the result of a previous measurement of The production cross sections were also corrected correspond-
ingly. The integrated cross section of electrons from beauty hadron ALICE Collaboration / Physics Letters B 763 (2016) 507–509 509 Fig. 7. This figure replaces Fig. 6 from [1]. Caption is the same as Fig. 6 from [1]. Fig. 5. This figure replaces Fig. 4 from [1]. Caption is the same as Fig. 4 from [1]. Fig. 7. References [1] B. Abelev, et al., ALICE Collaboration, Phys. Lett. B 738 (2014) 97. [2] B. Abelev, et al., ALICE Collaboration, Phys. Lett. B 721 (2013) 13. [3] K. Aamodt, et al., ALICE Collaboration, J. High Energy Phys. 01 (2012) 128. [4] B. Abelev, et al., ALICE Collaboration, J. High Energy Phys. 1207 (2012) 191,
arXiv:1205.4007 [hep-ex]. [5] B. Abelev, et al., ALICE Collaboration, J. High Energy Phys. 11 (2012) 1. [6] B. Abelev, et al., ALICE Collaboration, Phys. Rev. D 86 (2012) 112007, arXiv:
1205.5423 [hep-ex]. [7] B. Abelev, et al., ALICE Collaboration, Eur. Phys. J. C 73 (2013) 1. Fig. 6. This figure replaces Fig. 5 from [1]. Caption is the same as Fig. 5 from [1].
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Quadruple Hydrogen Bond-Containing A-AB-A Triblock Copolymers: Probing the Influence of Hydrogen Bonding in the Central Block
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Keywords: quadruple hydrogen bonding; acrylic thermoplastic elastomer; supramolecular polymer;
microphase separation Publisher’s Note: MDPI stays neutral
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iations. molecules molecules molecules Article
Quadruple Hydrogen Bond-Containing A-AB-A Triblock
Copolymers: Probing the Influence of Hydrogen Bonding
in the Central Block Boer Liu 1, Xi Chen 2
, Glenn A. Spiering 2, Robert B. Moore 2 and Timothy E. Long 1,* 1
Biodesign Center for Sustainable Macromolecular Materials and Manufacturing, School of Molecular
Sciences, Arizona State University, Tempe, AZ 85281, USA; bliu117@asu.edu
2
Department of Chemistry, Macromolecules Innovation Institute (MII), Virginia Tech,
Blacksburg, VA 24061, USA; xichen4@vt.edu (X.C.); gaspiering@vt.edu (G.A.S.); rbmoore3@vt.edu (R.B.M.)
*
Correspondence: Timothy.E.Long@asu.edu Abstract: This work reveals the influence of pendant hydrogen bonding strength and distribution
on self-assembly and the resulting thermomechanical properties of A-AB-A triblock copolymers. Reversible addition-fragmentation chain transfer polymerization afforded a library of A-AB-A acrylic
triblock copolymers, wherein the A unit contained cytosine acrylate (CyA) or post-functionalized
ureido cytosine acrylate (UCyA) and the B unit consisted of n-butyl acrylate (nBA). Differential
scanning calorimetry revealed two glass transition temperatures, suggesting microphase-separation
in the A-AB-A triblock copolymers. Thermomechanical and morphological analysis revealed the
effects of hydrogen bonding distribution and strength on the self-assembly and microphase-separated
morphology. Dynamic mechanical analysis showed multiple tan delta (δ) transitions that correlated
to chain relaxation and hydrogen bonding dissociation, further confirming the microphase-separated
structure. In addition, UCyA triblock copolymers possessed an extended modulus plateau versus
temperature compared to the CyA analogs due to the stronger association of quadruple hydrogen
bonding. CyA triblock copolymers exhibited a cylindrical microphase-separated morphology ac-
cording to small-angle X-ray scattering. In contrast, UCyA triblock copolymers lacked long-range
ordering due to hydrogen bonding induced phase mixing. The incorporation of UCyA into the soft
central block resulted in improved tensile strength, extensibility, and toughness compared to the AB
random copolymer and A-B-A triblock copolymer comparisons. This study provides insight into
the structure-property relationships of A-AB-A supramolecular triblock copolymers that result from
tunable association strengths.
Citation: Liu, B.; Chen, X.; Spiering,
G.A.; Moore, R.B.; Long, T.E. Quadruple Hydrogen
Bond-Containing A-AB-A Triblock
Copolymers: Probing the Influence of
Hydrogen Bonding in the Central
Block. Molecules 2021, 26, 4705. https://doi.org/10.3390/
molecules26154705
Academic Editor: Feihe Huang
Received: 13 July 2021
Accepted: 30 July 2021
Published: 3 August 2021
Citation: Liu, B.; Chen, X.; Spiering,
G.A.; Moore, R.B.; Long, T.E.
Quadruple Hydrogen
Bond-Containing A-AB-A Triblock
Copolymers: Probing the Influence of
Hydrogen Bonding in the Central
Block. Molecules 2021, 26, 4705.
https://doi.org/10.3390/
molecules26154705
Academic Editor: Feihe Huang
Received: 13 July 2021
Accepted: 30 July 2021
Published: 3 August 2021 1
Biodesign Center for Sustainable Macromolecular Materials and Manufacturing, School of Molecular
Sciences, Arizona State University, Tempe, AZ 85281, USA; bliu117@asu.edu
2
Department of Chemistry, Macromolecules Innovation Institute (MII), Virginia Tech,
Blacksburg, VA 24061, USA; xichen4@vt.edu (X.C.); gaspiering@vt.edu (G.A.S.); rbmoore3@vt.edu (R.B.M.)
*
Correspondence: Timothy.E.Long@asu.edu 1. Introduction The UCy self-association constant was 2.5 × 105 M−1, significantly higher
than cytosine (Cy) (ca. 40 M−1) in chloroform (CHCl3) [19,20]. To further probe the ef-
fects of hydrogen bonding strength on the bulk morphology and mechanical properties,
Long et al. reported UCy and Cy functionalized A-B-A triblock polymers and observed
a 30 ◦C extended service window for the UCy-containing polymer compared to its Cy
counterpart due to higher association strength of the UCy unit [21]. The increased upper
service temperature afforded ureido cytosine acrylate (UCyA) copolymers as a novel high
performance TPE. p
Traditionally, A-B-A triblock copolymers represent the most common architecture for
TPEs. Wang et al. designed A-B-A triblock copolymer using polybenzofulvene (PBF) as
the hard A block and polyisoprene as the soft B block. The work extended the polymer
service temperature to 145 ◦C owing to an exceptional high Tg of the PBF block, which
is 40 ◦C higher than PS [7]. A-B-A triblock copolymer containing nucleobase on the pen-
dant group demonstrated a significant reinforcement of thermomechanical properties and
morphological orderings compared to the random copolymer analog [10,18]. With the
development of advanced polymerization techniques, recent TPE research has investigated
complex chain architectures, such as A-B-C triblock, A-BC-A triblock, pentablock, and
star-shaped copolymers [6,13,22–24]. A-B-C triblock copolymers, which bear different
external blocks, i.e., A and C, self-assembled into complex three-phase morphologies due
to the presence of three different blocks. The morphology of ABC triblock copolymer is
dependent on the chain length [22,25] and compatibility of A and C blocks [26]. Other
researchers developed A-BC-A triblock copolymers, where they incorporated the hydro-
gen bonding containing-C unit into the central block to form weak crosslinks in the soft
domain [27–29]. These crosslinks dissociated and associated dynamically during elonga-
tion, which effectively dissipated energy and prevented local stress concentration. As a
result, the toughness increased over 100 times compared to the polymer in the absence
of hydrogen bonding in the soft phase [27]. Yoshie et al. synthesized A-AB-A triblock
copolymers using ring-opening metathesis polymerization (ROMP), where the A unit was
ureidopyrimidone (UPy)-functionalized norbornene that formed self-complementary QHB,
and B unit was the flexible poly(dodecanyl norbornene) [30]. The designed TPE exhibited
tensile properties that were 36× toughness, 16× tensile strength, and 2.5× elongation of
the A-B-A counterpart [30]. Their following work synthesized A-AB-A triblock copolymers
with a varying number of A units (the hydrogen bonding unit) in the soft block. 1. Introduction In contrast to chemically crosslinked thermoset rubbers, thermoplastic elastomers
(TPEs) consist of thermoreversible physical crosslinks, which allow for recycling and
reprocessing through melt-extrusion and injection-molding [1,2]. Poly(styrene-b-butadiene-
b-styrene) (SBS) triblock copolymers as well as isoprene analogs and hydrogenated versions
collectively serve as prominent TPEs in the automotive industry and common consumer
products such as asphalt additives. This impact originates from similar mechanical prop-
erties to chemically crosslinked rubber counterparts as well as excellent thermal, oil, and
abrasion resistance [3,4]. The glass transition temperatures (Tg) of the poly(1,3-butadiene)
and polystyrene blocks are −90 ◦C and 100 ◦C, respectively. Thus, the SBS TPE exhibits a
wide working-temperature window between the two Tgs. However, the triblock copolymer
flows below 100 ◦C due to the partially miscible styrene and butadiene blocks, limiting
the upper service temperature [4,5]. Alternatively, supramolecular strategies including 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/). https://www.mdpi.com/journal/molecules Molecules 2021, 26, 4705. https://doi.org/10.3390/molecules26154705 Molecules 2021, 26, 4705 2 of 17 H-bonding, electrostatic interactions, and pi-pi stacking offer routes to extend the upper
service temperature due to the introduction of additional physical crosslinks into the hard
domain [2,6,7]. Moreover, these specific noncovalent interactions, which impart enhanced
non-covalent bond strengths compared to only van der Waals forces, improved the stiffness
of polymers [8,9]. p
y
Nucleobase-containing polymers bearing specific hydrogen bonding recognition are
a crucial design feature for tailoring supramolecular TPE properties [10]. In addition to
the incorporation of thermo- and solvent-reversible crosslinks [11–14], the presence of
nucleobase sites in TPEs afford novel properties such as self-healing and shape mem-
ory [15–17]. The physical properties of a nucleobase-functionalized polymer strongly
depend on the association strength of nucleobases. Long et al. discovered that the blend of
adenine-functionalized and thymine-functionalized A-B-A triblock copolymers exhibited
an extended modulus plateau compared to the adenine or thymine triblock copolymer
alone [18]. The improved thermomechanical properties resulted from the stronger associa-
tion of the adenine-thymine pair compared to the significantly weaker self-associations. Structural modification and molecular design further allowed tunability of the associa-
tion strength. Hailie et al. modified cytosine through the reaction of the primary amine
with an isocyanate to afford the ureido cytosine (UCy) with quadruple hydrogen bonding
(QHB) [19]. 1. Introduction The
authors observed a systematic increase of storage modulus with increasing A unit in the
central block [31]. Despite the advantages of dynamic bonds in TPEs, the effects of physical
crosslinking strength of the A unit on mechanical and morphological properties remain Molecules 2021, 26, 4705 3 of 17 unexplored. Moreover, understanding the relationship between block interaction and
physical crosslinking strength is crucial for predicting the 3D microstructure and tailoring
the thermomechanical performances of TPEs. unexplored. Moreover, understanding the relationship between block interaction and
physical crosslinking strength is crucial for predicting the 3D microstructure and tailoring
the thermomechanical performances of TPEs. p
Herein, we utilized Cy and UCy functional groups to design a library of A-AB-A
triblock copolymers, where the A unit contained Cy or UCy and the B unit consisted of a
flexible poly(n-butyl acrylate) (PnBA). This study detailed the synthesis and characteriza-
tion of supramolecular triblock copolymers and explored the manipulation of hydrogen
bonding strength and distribution in these copolymers for tunable physical properties. Reversible addition-fragmentation chain transfer (RAFT) copolymerization of CyA and
nBA prepared a macro chain-transfer agent (macro-CTA) with a statistical distribution of
monomers. Subsequently, chain extension using CyA afforded A-AB-A triblock copoly-
mers containing two CyA end blocks. Finally, post-functionalization of the CyA triblock
copolymer precursors yielded corresponding UCyA triblock copolymers with an identical
distribution of the hydrogen bonding units, allowing for a direct comparison of solid-state
properties. Systematic characterization of thermal, thermomechanical, and morphological
properties revealed the synergy of mechanical properties and self-assembled microstruc-
tures. In addition, tensile testing assessed the effects of architecture on the mechanical
properties of UCyA copolymers. The A-AB-A triblock copolymer exhibited improved
overall tensile properties compared to the AB and A-B-A counterparts. 2.1. Synthesis of CyA and UCyA Triblock Copolymer 2.1. Synthesis of CyA and UCyA Triblock Copolymer In the design of triblock copolymers, varying CyA/UCyA content in the central
block and maintaining the chain length of each block enabled investigation of the effects of
hydrogen bonding concentration and strength on mechanical and morphological properties. RAFT polymerization of nBA and CyA monomers yielded triblock copolymers with tunable
chain lengths (Scheme 1 and Table 1). To generate polymers with a defined A-AB-A
triblock architecture, a difunctional CTA, difunctional 2-(dodecylthiocarbonothioylthio)-
2-methylpropionic acid (di-DDMAT) was selected due to its excellent compatibility with
acrylic-based monomers [18,21]. A series of macro-CTAs, i.e., copolymers of nBA and CyA,
were prepared. A solvent mixture of dimethyl sulfoxide (DMSO) and CHCl3 dissolved
the reactants, and the resulting mixture maintained a homogeneous solution throughout
the reaction. Dialyzing in a CH3OH/dichloromethane (CH2Cl2) mixture for two days and
subsequently drying under a reduced pressure at room temperature effectively removed
unreacted monomers and high-boiling solvents. Proton nuclear magnetic resonance (1H
NMR) spectroscopy was the primary tool for determining molecular weight since the
challenging solubility of the nucleobase functionalized copolymers limited utilization of
size exclusion chromatography. The resonance at 7.4 ppm in Figure S1A corresponded to
the chemical shift of a proton on the cytosine site and was utilized to normalize the proton
integrations. The resonance at 0.8 ppm was associated with the methyl protons (3H) on
nBA. The integral ratio between acrylic protons (3H) at 5.8–6.3 ppm and the sum of CyA
and nBA protons yielded 97% conversion of the copolymerization. In addition, utilizing
the integrals of CyA and nBA protons elucidated the fraction of CyA in the purified
copolymer (4 mol %) (Figure S1B). Thus, monomer conversion of the copolymerization
was calculated as described in the ESI. Finally, the degree of polymerization (DP) of each
segment was calculated from the monomer feed and conversion (Table S1). The targeted
DP was utilized for nomenclature and the general expression of poly(CyA50-b-(nBA480-co-
CyA20)-b-CyA50) and poly(UCyA50-b-(nBA480-co-UCyA20)-b-UCyA50) was abbreviated as
C50-b-(B480-co-C20)-b-C50 and U50-b-(B480-co-U20)-b-U50, respectively. Molecules 2021, 26, 4705 4 of 17 Scheme 1. Synthesis of poly(CyA-b-(nBA-co-CyA)-b-CyA) and poly(UCyA-b-(nBA-co-UCyA)-b-UCyA) triblock copolymers
using RAFT polymerization. Scheme 1. Synthesis of poly(CyA-b-(nBA-co-CyA)-b-CyA) and poly(UCyA-b-(nBA-co-UCyA)-b-UCyA) triblock copolymers
using RAFT polymerization. 5 of 17 Molecules 2021, 26, 4705 Table 1. Compositions, thermal properties, and morphological properties of CyA and UCyA copolymers with varying
amounts of hydrogen bonding contents. Sample Name
CyA/UCyA in
Copolymer (mol %)
DP a
Td, 15 wt. 2.1. Synthesis of CyA and UCyA Triblock Copolymer %b
(◦C)
Tg1 c
(◦C)
Tg2 c
(◦C)
d d
(nm)
db d
(nm)
C50-b-(B480-co-C20)-b-C50
20
50–488/20–50
326
−37
85
41
N/A
C50-b-(B450-co-C50)-b-C50
25
51–453/50–51
317
−23
86
38
4.2
C50-b-(B420-co-C80)-b-C50
30
51–418/78–51
312
6
83
32
4.2
U50-b-(B480-co-U20)-b-U50
20
50–488/20–50
280
−35
N/A
41
N/A
U50-b-(B450-co-U50)-b-U50
25
51–453/50–51
265
−24
N/A
31
4.5
U50-b-(B420-co-U80)-b-U50
30
51–418/78–51
262
−9
N/A
N/A
4.4
a 1H NMR spectroscopy. b TGA, 10 ◦C min−1,23–600 ◦C, N2. c DSC, 10 ◦C min−1, −60–50 ◦C, N2. d SAXS, λ = 0.154 nm (Cu, Kα), and 23 ◦C. Interestingly, the CyA monomer displayed a different rate of conversion than nBA
during copolymerization, as evidenced from a significantly lower copolymer composition Interestingly, the CyA monomer displayed a different rate of conversion than nBA
during copolymerization, as evidenced from a significantly lower copolymer composition
(FCyA) than the starting monomer feed ratio (f CyA) (Table S1), resulting in a compositional
drift during copolymerization. In order to understand the origin of this phenomenon,
reactivity ratios of nBA and CyA (rCyA and rnBA, respectively) were determined for the nBA
and CyA copolymerization. The monomer conversion was analyzed at low conversion
(<10%) to avoid concentration effects on reactivity ratios [32]. Since the copolymers ex-
hibited a limited solubility in DMSO/CHCl3 solvent mixture when CyA content is higher
than 60 mol %, the CyA monomer feed content was set to f CyA < 0.6. Figure 1A showed
that FCyA at all investigated f CyA were located below the F = f line, i.e., an equal monomer
reactivity ratio towards the propagating chain (r1 = r2 = 1). The lower CyA copolymer
composition than the initial monomer feed ratio suggested a slower reactivity of CyA
compared to nBA. To provide more in-depth insights on the origin of the compositional
drift and relative reactivities of the two monomers, both Fineman-Ross (F-R) and Kelen-
Tüdös (K-T) approaches were used to estimate rCyA and rnBA, according to the linear fitting
of Equation (3) and (4), respectively. Both methods demonstrated excellent agreement in
predicting the reactivity ratios of the CyA and nBA monomers, where the F-R method
yielded rnBA = 1.03 and rCyA = 0.51 (Figure S4) and K-T yielded rnBA = 1.12 and rCyA = 0.64
(Figure 1A). 2.1. Synthesis of CyA and UCyA Triblock Copolymer rnBA (r2) ~ 1 indicated similar reactivity of nBA and CyA to the propagating
nBA radical chain ends, while rCyA (r1) < 1 suggested that nBA has a preference towards
adding to the CyA terminated propagating radical chain ends. The lower self-propagation
compared to cross-propagation of CyA monomers was presumably attributed to its bulkier
cytosine pendant unit, which inhibited the addition of another CyA monomer to a CyA-
terminated radical [33]. Although the calculated reactivity ratios demonstrated that nBA
propagates favorably at low conversions and thereby generating nBA-enriched polymer
chains, it is of great interest to probe if the polymer displays a composition gradient along
the polymer chain due to concentration effects at higher monomer conversions. Monitoring
the FCyA with respect to conversion at equal initial monomer feed ratio (50:50) (Figure S5)
revealed a relatively constant CyA composition from low (<10%) to high (>90%) conversion,
which demonstrated a consistent polymer composition along the chain. Molecules 2021, 26, 4705 6 of 17 Figure 1. (A) Dependence of the instantaneous copolymer composition FCyA on the initial comonomer feed composition
f CyA. The solid line represents a random copolymerization, where r1 = r2 = 1. (B) Calculation of reactivity ratios for the
copolymerization of CyA and nBA using Kelen-Tüdös method. Figure 1. (A) Dependence of the instantaneous copolymer composition FCyA on the initial comonomer feed composition
f CyA. The solid line represents a random copolymerization, where r1 = r2 = 1. (B) Calculation of reactivity ratios for the
copolymerization of CyA and nBA using Kelen-Tüdös method. In the second step, the chain extension of the macro-CTA with CyA monomer yielded
the CyA-containing triblock copolymer. An optimized ratio of CTA and initiator allowed
the reaction to achieve >60% conversion in 24 h. 1H NMR spectroscopy of the reaction
mixture yielded 65% monomer conversion, corresponding to an average of 49 CyA units for
each external block, assuming an equal propagation rate on both chain ends. Dialysis of the
reaction mixture in a CH3OH/CH2Cl2 mixture afforded a purified CyA-containing triblock
copolymer. 1H NMR spectroscopic analysis of the purified product revealed 20 mol % of
CyA incorporation in the triblock copolymer (Figure S2), which corresponds to an average
of 50 CyA units on each external block (Table 1). This calculated value was closely aligned
with the value calculated from monomer conversion. 2.1. Synthesis of CyA and UCyA Triblock Copolymer Post-functionalization of CyA copolymer precursors using hexyl isocyanate afforded
the corresponding UCyA triblock copolymers with an identical composition of hydrogen
bonding units. An excess of isocyanate (1.5 eq) was introduced to the reaction to achieve
quantitative conversion. A N,N-dimethylformamide (DMF)/DMSO mixture enabled the
dissolution of the UCyA triblock copolymers to allow the reaction to proceed in a homo-
geneous fashion. 1H NMR spectroscopy indicated that the signal at 7.0–7.5 ppm, which
originates from the amine hydrogen in the cytosine units, disappeared. In addition, two
peaks located at 8.5–10.5 ppm corresponded to the urea protons, indicating the quantita-
tive conversion of CyA- to UCyA-containing polymers (Figure S3). The resultant UCyA
copolymers were light-yellow solids that were stiffer than their CyA analogs, presumably
due to the stronger QHB for UCyA. 2.2. Thermal Analysis Figure 2A reveals a variation in the thermal weight loss profiles of the triblock copoly-
mers under N2 with increasing hydrogen bonding content. Derivatives of the weight loss
profile in Figure 2B indicate a two-step weight loss mechanism in the CyA copolymers. The first step of the weight loss occurred at ~240 ◦C and was primarily attributed to the
degradation of the butanediol diacrylate spacer. A second step occurred at ~300 ◦C, which
involved the degradation of the acrylic backbone [13]. Thus, increasing concentrations
of the hydrogen bonding sites in the central block resulted in a decreased Td, 15 wt. %, as
presented in Table 1. For the UCyA copolymers, an additional weight loss step occurred
at ~160 ◦C and involved the degradation of the urea bond at lower temperatures [34]. In
addition, the Td, 15 wt. % of CyA triblock copolymers were ~50 ◦C higher than the UCyA
triblock copolymers (Table 1), demonstrating a potential higher thermal stability for CyA
polymers under N2. However, it is worth noting that CyA copolymers crosslink at 130 ◦C as Molecules 2021, 26, 4705 7 of 17 evidenced by isothermal rheological experiments under air atmosphere, which is possibly
due to the reaction of amine with acrylate esters at elevated temperatures [34]. evidenced by isothermal rheological experiments under air atmosphere, which is possib
due to the reaction of amine with acrylate esters at elevated temperatures [34]. Figure 2. (A) TGA thermogram curves and (B) the corresponding weight derivative curves of CyA and UCyA triblock
copolymers with varied amounts of hydrogen bonding unit. Figure 2. (A) TGA thermogram curves and (B) the corresponding weight derivative curves of CyA and UCyA triblock
copolymers with varied amounts of hydrogen bonding unit. Differential scanning calorimetry (DSC) evaluated the glass transition temperatures of
CyA and UCyA triblock copolymers and their corresponding random copolymer analogs
with varied hydrogen bonding contents (Figure 3). The CyA random copolymers (dot-
ted lines, Figure 3A) displayed a single transition step, with the Tg of the copolymer
increasing with increasing CyA incorporation, which was consistent with our previous
observations [34]. A single Tg confirmed the statistical sequence distribution of the ran-
dom copolymers (Figure 3A). In addition, the glass transition step broadened as the CyA
content increased, where hydrogen bonding formed between the randomly associated
CyA units presumably resisted molecular mobility and expanded the timescale of molec-
ular segmental motion within the polymer structure. 2.3. Thermomechanical Analysis All CyA and UCyA copolymers afforded freestanding and optically transparent films
through solvent-casting and annealing, indicating the absence of macrophase separation. Copolymers bearing a higher content of UCy showed poor solubility in polar solvents such
as DMF or DMSO, but demonstrated good solubility in the mixture of DMF and DMSO. Therefore, the cosolvent system was used for solvent casting of all CyA and UCyA copoly-
mers. Both DMSO and DMF are polar solvents that assist solvation of the polymers by
dissociating strong hydrogen bonding interactions. In addition, utilizing high-boiling point
solvents such as DMSO and DMF decreased solvent evaporation during annealing and thus
facilitated polymer self-assembly [18]. Dynamic mechanical analysis (DMA) in Figure 4A,B
evaluated the viscoelastic properties of the annealed CyA and UCyA copolymer films. A
higher storage modulus was obtained with increased CyA/UCyA incorporation, indicating
that hydrogen bonding improved the rigidity of the polymers. Additionally, both copoly-
mer systems displayed multiple transitions verifying microphase-separated structures
revealed using DSC. The first tan δ peak at lower temperature was related to the Tg of the
soft central block. The value of Tg agreed well with the corresponding random copolymer
precursors (Figure S6). In addition, the tan δ peak shifted to a higher temperature as
CyA/UCyA content increased in the central block. The trend corroborated the observation
in DSC (Table 1 and Figure 3), where increasing the central CyA/UCyA content in the
central block restricted segmental motion of the polymer backbone due to the hydrogen
bonding pendant group. Further elevation of temperature resulted in the second tan δ peak for C50-b-(B480-
co-C20)-b-C50 and C50-b-(B450-co-C50)-b-C50 due to the dissociation of Cy (Figure 4A). In-
creasing Cy incorporation elongated the terminal flow to higher temperatures, suggesting
the formation of more robust networks due to the increased hydrogen bonding. Interest-
ingly, C50-b-(B420-co-C80)-b-C50 displayed a single tan δ peak before terminal flow. Further
investigation of its random precursor elucidated that the glass transition of the central
block occurred at a similar temperature to Cy dissociation (Figure S10). In contrast, UCyA
triblock copolymers exhibited multiple transitions on DMA (Figure 4B), which was dif-
ferent from CyA copolymers. Two tan δ transitions followed after the central block glass
transition, which correspond to the dissociation of UCy in the soft and hard domains,
respectively. The soft domains consist of UCyA units that were sparsely distributed in
the PnBA matrix forming lightly packed crosslinks. 2.2. Thermal Analysis In comparison, the CyA triblock
copolymers displayed two well-resolved transitions within the testing range, suggesting
a phase-separated morphology. The transition at lower temperatures agreed well with
the corresponding random copolymer precursors and thus corresponded to the central
random block. An additional higher temperature transition step at ~85 ◦C was associated
with the external blocks. It is noteworthy that the Tg of the CyA external block was slightly
lower compared with the Tg of a poly(CyA) homopolymer (~93 ◦C) [21], indicative of
phase mixing that originated from interactions among the CyA units between the central
and external blocks. Figure 3B depicts the thermal transition profiles of UCyA copolymers. Similar to CyA copolymers, the value of Tg agreed well with their random UCyA copoly-
mer counterparts. However, DSC thermograms only revealed the central block transition
within our testing regime. Lower thermal stability of the UCyA copolymers under N2
limited the upper testing temperature to 150 ◦C (degradation at ~160 ◦C). In fact, the slight
increase of the heat flow near 140 ◦C was attributed to the onset of the thermal transition
of the external blocks (Figure 3B) [21]. 8 of 17 Molecules 2021, 26, 4705 Figure 3. DSC thermograms of (A) CyA and (B) UCyA triblock copolymers with varied amounts of hydrogen bonding unit. 2 3 Thermomechanical Analysis Figure 3. DSC thermograms of (A) CyA and (B) UCyA triblock copolymers with varied amounts of hydrogen bonding unit. 2 3 Th
h
i
l A
l
i ermograms of (A) CyA and (B) UCyA triblock copolymers with varied amounts of hydrogen bonding unit. Figure 3. DSC thermograms of (A) CyA and (B) UCyA triblock copolymers with varied amounts of hydrog 2.3. Thermomechanical Analysis On the contrary, the hard domains Molecules 2021, 26, 4705 9 of 17 9 of 17 were composed of mostly UCyA that enabled the formation of densely packed crosslinks,
thus dissociated at higher temperatures. Similarly, Long et al. observed an increasing
terminal flow temperature when hydrogen bonding unit increased in the copolymer [21]. Additionally, all UCyA copolymers flowed at ~150 ◦C, suggesting minor effects of the
central block on the terminal flow temperature. The onset glass transition temperature of
the UCyA external block occurred at 140 ◦C, which was similar to the flow temperature
observed in DMA results as observed in Figure 3B. The strong QHB in the UCyA enabled
the retention of 3D networks until the temperature reached the external block Tg, regardless
of the hydrogen bonding concentration. Furthermore, UCyA copolymers increased the
terminal flow temperature up to 100 ◦C compared to the CyA triblock copolymers. Such
enhancement correlated to the highly oriented QHB for UCyA, which imparted higher
association strength than the dimeric hydrogen bonding of Cy unit [34]. Figure 4
Dynamic mechanical temperature ramps of storage modulus and tan δ of the solution casted (A) CyA and Figure 4. Dynamic mechanical temperature ramps of storage modulus and tan δ of the solution casted (A) CyA and
(B) UCyA triblock copolymer films. Figure 4. Dynamic mechanical temperature ramps of storage modulus and tan δ of the solution casted (A) CyA and
(B) UCyA triblock copolymer films. This section elucidates the influence of polymer architecture on mechanical proper-
ties using UCyA copolymer. The CyA A-AB-A triblock copolymers in this manuscript
displayed a low modulus that presented challenges to prepare films using compression-
molding, whereas the UCyA A-AB-A triblock copolymers formed mechanically robust
films. Additionally, the UCyA triblock copolymer is more desirable as a TPE compared
to the CyA triblock copolymer, as evidenced in Figure 4B. Specifically, the storage mod-
ulus of U50-b-(B450-co-U50)-b-U50 remained at the same magnitude from 10–30 ◦C, while
DMA revealed a steep decrease in storage modulus for C50-b-(B450-co-C50)-b-C50 over this Molecules 2021, 26, 4705 10 of 17 10 of 17 temperature range. Polymer samples with three different architectures: A-B-A triblock
copolymer, A-AB-A, and AB copolymers were compared using tensile testing. These
three samples have the same chain length (DP ~600) and the same UCy concentration
(25 mol %) in each polymer chain. 2.3. Thermomechanical Analysis First, as shown in Figure S9, U75-b-B450-b-U75 suffered
from cracking during film processing, where a concentrated UCy group in the outer block
afforded highly oriented physical networks that result in a brittle mechanical property. As
a result, U75-b-B450-b-U75 was too brittle to perform mechanical tests and tensile property
was not obtained. In contrast, A-AB-A triblock copolymer containing additional dynamic
crosslinks in the soft central block formed free-standing films that were suitable for tensile
testing. Furthermore, the A-AB-A triblock copolymer demonstrated improved mechanical
properties compared to the AB random copolymer counterpart, summarized in Table 2. The toughness of U50-b-(B450-co-U50)-b-U50 was twice of B450-co-U150, with a simultaneous
improvement of the elongation at break and ultimate stress that achieved 1.5 times and
1.3 times of the random polymer counterpart, respectively. The Young’s modulus of U50-b-
(B450-co-U50)-b-U50 was lower than B450-co-U150. Comparably, DMA results confirmed the
lower storage modulus of U50-b-(B450-co-U50)-b-U50 than B450-co-U150 (Figure S7) at room
temperature, due to a lower concentration of UCy units in the central block U50-b-(B450-co-
U50)-b-U50 that results in a lower Tg (~−3 ◦C) compared to that for B450-co-U150 (Tg ~47
◦C) (Figure S7). Table 2. Summary of tensile testing derived from the stress-strain curve (Figure S8). Sample
Young’s Modulus (MPa)
Strain at Break (%)
Stress at Break (MPa)
Toughness (MJ/m−3)
B450-co-U150
31 ± 2
89 ± 10
7.0 ± 0.5
4.3 ± 0.6
U50-b-(B450-co-U50)-b-U50
23 ± 4
131 ± 10
9.1 ± 0.3
8.6 ± 0.7
U75-b-B450-b-U75
N/A Table 2. Summary of tensile testing derived from the stress-strain curve (Figure S8). 2.4. Morphological Characterization Small angle X-ray scattering (SAXS) profiles in Figure 5 elucidated bulk morphologies
and chain ordering of the CyA and UCyA triblock copolymers. CyA copolymers displayed
a series of scattering maxima between q = 0.1 nm−1 and q = 1 nm−1, indicating a microphase-
separated morphology that was driven by the ordering of hard and soft blocks. The periodic
scattering maxima appeared at positions q, (2q), √7q, (3q), suggesting a hexagonally
packed cylindrical morphology. The concentration of CyA in the central block showed an
insignificant effect on the self-assembled morphology as all three CyA triblock copolymers
displayed the hexagonally packed cylindrical morphology. The nearly identical chain
length resulted in a similar volume fraction of the external blocks for all CyA copolymers,
which provided an explanation for the compositional independent morphology [35,36]. In
contrast, UCyA copolymers exhibited significantly less ordered morphologies with only
first-order q maxima observed for the U50-b-(B480-co-U20)-b-U50 and U50-b-(B450-co-U50)-
b-U50 and a loss of ordering for the U50-b-(B420-co-U80)-b-U50. Higher UCyA content in
the central block of the copolymer increased the interaction of the UCy groups between
in the central and external blocks, which disrupted the ordering for U50-b-(B420-co-U80)-b-
U50 [36]. Both CyA and UCyA copolymers exhibited a decrease of domain spacing with
increasing hydrogen bonds in the central block (Table 1), which also indicated a more
mixed microstructure at higher hydrogen bonding compositions. 11 of 17 Molecules 2021, 26, 4705 Figure 5. SAXS of solution casted (A) CyA and (B) UCyA triblock copolymer films with varied amounts of hydrogen
bonding unit. Curves are shifted vertically for clarity. Figure 5. SAXS of solution casted (A) CyA and (B) UCyA triblock copolymer films with varied amounts of hydrogen
bonding unit. Curves are shifted vertically for clarity. At larger scattering vectors q > 1 nm−1, a broad scattering peak centered at qb ~ 1.5 nm−1
remains relatively consistent for all CyA triblock copolymers. This peak was associated with
the phase separated Cy domains in the central random block, as evidenced by the SAXS
analysis of the B450-co-C150 random copolymer (Figure 5A). Additionally, the scattering peak
became sharper as CyA in the central block increased, which was attributed to enhanced
phase separation with increasing Cy incorporation. Figure 6 proposed a schematic rep-
resentation of the CyA copolymer morphology according to the SAXS result. The large
green dots represent the hard domains that are mainly composed of the external blocks. 3.2. Synthesis of Poly(nBA-co-CyA) Difunctional Macro-CTA (Scheme 1) Poly(nBA-co-CyA) was prepared using RAFT polymerization. Difunctional CTA,
1,6-bis(DDMAT)-hexane diamide (diDDMAT-NH2), and CyA was synthesized according
to our previous literature [34]. In a typical random copolymer synthesis, a 100-mL, single-
necked, Schlenk flask was charged with diDDMAT-NH2 (25.2 mg, 0.031 mmol, 1 eq), nBA
(2 g, 11.89 mmol, 501 eq), CyA (0.22 g, 0.71 mmol, 23 eq), AIBN (0.5 mg, 0.0031 mmol,
CTA/I = 10), and DMF (6.74 g, 25 wt. % solution). The solution was stirred at 70 ◦C for 24 h
after subjecting to four freeze–pump–thaw cycles and refilling with argon. The copolymer
was isolated from dialyzing in a CH3OH/CH2Cl2 mixture for two days and subsequent
drying under vacuum at 60 ◦C overnight to afford a light yellow tacky solid. 1H NMR
spectroscopy in CD3Cl/DMSO-d6 revealed the degree of polymerization (DP) of each unit:
20 for CyA and 488 for nBA (Table S1, Figure S1). 2.4. Morphological Characterization The characteristic dimension d described the distance between the hard domains and
decreased with increasing CyA content (Table 1). The small orange dots represent CyA
domains in the soft phase distributed in the nBA matrix with a characteristic dimension of
db = 4.2 nm that was invariant to CyA concentration (Table 1). Atomic Force Microscope
(AFM) (Figure S11) revealed a biphasic microstructure of C50-b-(B450-co-C50)-b-C50 where
the light and the dark regions were attributed to the hard and soft phase, respectively. In contrast, the characteristic scattering peak of UCyA copolymers is less prominent at
q > 1 nm−1 compared to the CyA copolymer, which confirmed the more mixed-phase
morphology of UCyA copolymer. The stronger QHB association in the UCyA copolymer
promoted the interactions between central and external blocks compared to the dimeric
hydrogen bonding interactions in the CyA triblock copolymers. In fact, increased phase
mixing in the UCyA triblock copolymer influenced the storage modulus above the second
tan δ transition where the hard domains underwent QHB dissociation. The hard domain
contained not only the external blocks but also the central blocks that were incorporated
due to phase mixing. Increasing UCyA in the central block from C50-b-(B480-co-C20)-b-C50
to C50-b-(B420-co-C80)-b-C50 increased the physical crosslinks in the hard domain and thus
enhanced the storage modulus from 1.9 to 22 MPa (Figure 4B). 12 of 17 Molecules 2021, 26, 4705 db
Figure 6. Schematic representation of the CyA triblock copolymer morphology based on SAXS. 3 M t
i l
d M th d Figure 6. Schematic representation of the CyA triblock copolymer morphology based on SAXS. 3.1. Materials n-butyl acrylate (nBA, 99+%) was purchased from Sigma-Aldrich and passed through
basic alumina columns before use. α,α’-Azobis-(isobutyronitrile) (AIBN, Fluka, 99%) was
recrystallized twice from methanol. 1,4-butanediol diacrylate (Aldrich, 90%), cytosine (TCI,
>98%), hexyl isocyanate (Aldrich, 97%), 2-(dodecylthiocarbonothioylthio)-2- methylpropi-
onic acid (DDMAT, Sigma-Aldrich, 98%), N,N’-dicyclohexylcarbodiimide (Sigma-Aldrich,
99%), 4-(dimethylamino) pyridine (Sigma-Aldrich, ≥99%), potassium carbonate (anhy-
drous, Aldrich, 99%), and sodium sulfate (anhydrous, Spectrum, 99%) was used without
further purification. Dimethyl sulfoxide (DMSO, HPLC grade), N,N-dimethylformamide
(DMF, HPLC grade), dichloromethane (ACS grade), hexane (HPLC grade), and methanol
(ACS grade) were purchased from Spectrum and used as received. 3.3. Reactivity Ratio Determination Fineman-Ross and Kelen-Tüdös presented methods to calculate the reactivity ratio
values (rnBA and rCyA), and thus, a variety of different monomer feed ratios (f CyA) were
explored. Limiting the copolymerization at a low conversion (<10%) allowed to deter-
mine the instantaneous copolymer composition (FCyA) [32]. Copolymerization equation Molecules 2021, 26, 4705 13 of 17 13 of 17 (Equation (1)) correlated the monomer feed (f CyA and f nBA) and copolymer composition
(FCyA and FnBA) to the reactivity ratios (rCyA and rnBA) (Equation (1)) correlated the monomer feed (f CyA and f nBA) and copolymer composition
(FCyA and FnBA) to the reactivity ratios (rCyA and rnBA) FCyA =
rCyA f 2
CyA + fCyA fnBA
rCyA f 2
CyA + 2fCyA fnBA + rnBA f 2
nBA
(1) (1) Fineman-Ross method rearranged Equation (1) to afford linearization expression of
reactivity ratios (Equation (2)). f1(2F1 −1)
(1 −f1)F1
= f 2
1 (1 −F1)
(1 −f1)2F1
r1 −r2
(2) (2) where G and H are where G and H are G = f1(2F1 −1)
(1 −f1)F1
H = f 2
1 (1 −F1)
(1 −f1)2F1 G = f1(2F1 −1)
(1 −f1)F1
H = f 2
1 (1 −F1)
(1 −f1)2F1 Therefore, Equation (2) can also be converted to Therefore, Equation (2) can also be converted to Therefore, Equation (2) can also be converted to Therefore, Equation (2) can also be converted to G = Hr1 −r2
(3) (3) where G and H were readily determined by monomer feed and copolymer composi-
tion at different monomer conversions calculated from 1H NMR spectroscopy. Based on
Equation (3), a linear relationship was generated through plotting G against H, where −r2
and r1 corresponding to the intercept and slope of the linear relationship, respectively. However, Fineman-Ross method resulted in a local concentration of data points. To solve
this problem, Kelen-Tüdös method distributed data points evenly through adding an
arbitrary correction factor α to modify Equation (3) to Equation (4). 3.3. Reactivity Ratio Determination η =
j
r1 +
r2
α
k
ξ −
r2
α
(4) (4) where η, ξ, and α are where η, ξ, and α are η =
G
α + H
ξ =
H
α + H
α =
p
HminHmax η =
G
α + H
ξ =
H
α + H
α =
p
HminHmax α =
p
HminHmax α =
p
HminHmax Therefore, a new linear relationship was applied with −rnBA/α and rCyA correspond-
ing to the intercept and slope of the curve, respectively. 3.7. Instrumentation Proton nuclear magnetic resonance (1H NMR) spectroscopy was collected on an
Agilent U4-DD2 400 Hz spectrometer at 23 ◦C in a mixture of CDCl3 and DMSO-d6
(1:1, v/v). Thermogravimetric analysis (TGA) of cytosine and UCy-containing triblock
copolymers was conducted on a TA Instruments TGA Q500 ramping from 30 ◦C to 550 ◦C
under N2 purge at a heating rate of 10 ◦C/min. Differential scanning calorimetry (DSC)
was performed on a TA Instruments DSC Q2000 using a heat/cool/heat cycle between
−60 ◦C and 150 ◦C under N2 purge at a heating/cooling rate of 10 ◦C/min. Dynamic
mechanical analysis (DMA) was conducted on a TA Instruments Q800 Dynamic Mechanical
Analyzer in tension mode at a frequency of 1 Hz, an oscillatory amplitude of 5 µm, and a
static force of 0.01 N. The annealed film was subjected to a temperature ramp of 3 ◦C/min,
starting from −80 to 180 ◦C. An Instron 5500R universal testing instrument measured the
uniaxial tensile properties of the compression-molded sample at a rate of 5 mm/min. The
compression-molded film was cut using ASTMD-638-V cutter to generate tensile specimens. Tensile analysis data represented an average of five specimens with calculated standard
deviations. Toughness was determined by calculating the area under the stress-strain curve. g
y
g
Small angle X-ray scattering (SAXS) experiments were performed using a Rigaku
S-Max 3000 3 pinhole SAXS system, equipped with a rotating anode emitting X-ray with a
wavelength of 0.154 nm (Cu Kα). The sample-to-detector distance was 1600 mm, and the
q-range was calibrated using a silver behenate standard. Two-dimensional SAXS patterns
were obtained using a 2D multiwire, proportional counting, gas-filled detector, with an
exposure time of 2 h. The SAXS data were corrected for sample thickness, transmission,
and background, and were put on an absolute scale by correction using a glassy carbon
standard from the Advanced Photon Source (APS). All the SAXS data were analyzed using
the SAXSGUI software package to obtain radially integrated SAXS intensity versus the
scattering vector q, where q = (4π/λ) sin (θ), θ is one half of the scattering angle and λ is the
X-ray wavelength. The scattering profiles were vertically shifted to facilitate a comparison
of peak positions. Domain spacing was calculated according to the equation d = 2π/q. Tapping-mode AFM imaging was performed using a Veeco MultiMode scanning probe
microscope, at a set-point ratio of 0.3 with a magnification of 1 × 1 µm. 3.6. Polymer Film Preparation and Annealing Conditions Triblock copolymer films for DMA, SAXS, and AFM were prepared by dissolving
polymer samples in a mixture of DMSO and DMF at 6 wt. % concentration, and casting into
poly(tetrafluoroethylene) molds to minimize deformation during solvent annealing and
film removal. The molds were covered with a glass Petri dish and maintained at 50 ◦C for
three days on a hot plate to allow slow evaporation of the solvents. The copolymer films
were further dried in the vacuum at room temperature overnight and were then annealed
at 100 ◦C for 12 h. The annealed films were stored in a desiccator prior to characterization. 3.5. Synthesis of Poly(UCyA-b-(nBA-co-UCyA)-b-UCyA) Triblock Copolymers (Scheme 1) 3.5. Synthesis of Poly(UCyA-b-(nBA-co-UCyA)-b-UCyA) Triblock Copolymers (Scheme 1) 3.5. Synthesis of Poly(UCyA-b-(nBA-co-UCyA)-b-UCyA) Triblock Copolymers (Scheme 1) Poly(UCyA-b-(nBA-co-UCyA)-b-UCyA) was prepared using post-functionalization
of poly(CyA-b-(nBA-co-CyA)-b-CyA). In a representative triblock copolymer synthesis,
1 g (1 eq) of CyA block copolymer was dissolved in an anhydrous DMF/DMSO mixture
(10 mL) in a sealed round-bottomed flask, followed by adding hexyl isocyanate (0.26 mL,
1.5 eq) dropwise using a syringe and then stirred at 20 ◦C for 20 min. The reaction flask
was then reacted at 80 ◦C until a quantitative conversion of CyA to UCyA (indicated by 1H
NMR spectroscopy) (Figure S3). The reaction was quenched by adding methanol into the
solution, and the copolymer was isolated through dialysis in a CH3OH/CH2Cl2 mixture
for two days and was then dried under reduced pressure at 60 ◦C for 18 h to afford a
slightly yellow solid. 3.4. Chain Extension of Poly(nBA-co-CyA) with CyA (Scheme 1) A typical synthesis of poly(CyA-b-(nBA-co-CyA)-b-CyA) was conducted as follows:
CyA (1.12 g, 3.64 mmol, 150 eq), AIBN (0.80 mg, 0.0048 mmol, CTA/I = 5), poly(nBA-co-
CyA) macro-CTA (1.63 g, 0.024 mmol, 1 eq), and DMF (15.62 g, 15 wt. % solution) were
charged into a single-necked Schlenk flask. The solution was stirred at 70 ◦C for 24 h after
subjecting to four freeze-pump-thaw cycles and refilling with argon. 1H NMR spectroscopy
in DMSO-d6 determined a monomer conversion of 67%. The copolymer was isolated from
dialyzing in a CH3OH/CH2Cl2 mixture for three days, subsequent drying under vacuum
at 60 ◦C for overnight afforded a light-yellow solid. 1H NMR spectroscopy of the purified
polymer in CD3OD/DMSO-d6 revealed an average of 50 CyA units in each external block
assuming an equal propagation rate on both chain ends (Figure S2, Table 1). 14 of 17 Molecules 2021, 26, 4705 4. Conclusions Synthesis of CyA and UCyA-containing A-AB-A triblock copolymers provided a
strategy to design high-performance TPEs. Manipulation of hydrogen bonding strength
and distribution enabled tunable mechanical properties and bulk morphology. Triblock
copolymers containing weakly associated Cy units formed hexagonally packed cylindrical
morphologies, whereas the UCy units with highly oriented and stronger QHB association
promoted phase mixing, resulting in less-ordered microstructures in the UCyA copoly-
mers. Surprisingly, such phase mixing did not impair the mechanical properties of UCyA
copolymers. On the contrary, the UCyA copolymer with strong QHB interactions retained
the three-dimensional structure of the copolymer film until 150 ◦C. Additionally, the topo-
logical design endowed A-AB-A UCyA copolymer with exceptional tensile properties
compared to their random and A-B-A triblock copolymer counterparts. Balancing these de-
sign parameters in concert provides a roadmap for tuning the thermomechanical properties
and morphologies of supramolecular TPEs. Supplementary Materials: The following are available online, Figure S1: 1H NMR spectroscopy
of poly(CyA-co-nBA) macro-CTA. (A) The crude product in DMSO-d6 from reaction solution for
conversion calculation and (B) purified polymer in DMSO-d6 + CDCl3 for determining molecular
weight, Figure S2: 1H NMR spectroscopy of purified C50-b-(B480-co-C20)-b-C50 in DMSO-d6 + CDCl3,
Figure S3: 1H NMR spectroscopy of the purified C50-b-(B480-co-C20)-b-C50 and U50-b-(B480-co-U20)-b-
U50 in DMSO-d6 + CDCl3, calculation of monomer conversion, Figure S4: Determination of reactivity
ratios for the copolymerization of CyA and nBA using the Fineman−Ross method, Figure S5: Fraction
of CyA segment in the polymer as a function of overall conversion. Figure S6: Dynamic mechanical
temperature ramps of storage modulus and tan δ of the solution cast C50-b-(B450-co-C50)-b-C50 and
B450-co-C50, Figure S7: Dynamic mechanical temperature ramps of storage modulus and tan δ of
the solution cast U50-b-(B450-co-U50)-b-U50 and B450-co-C150, Table S1: Compositions and thermal
properties of CyA and UCyA copolymer macro-CTA with varying amounts of hydrogen bonding
content, Figure S8: Stress-strain curves of B450-co-U150 and U50-b-(B450-co-U50)-b-U50, Figure S9:
The cracked film of U75-b-B450-b-U75 during thermal annealing, Figure S10: Dynamic mechanical
temperature ramps of storage modulus and tan δ of the solution cast C50-b-(B420-co-C80)-b-C50 and
B420-co-C80, Figure S11: AFM phase image for the solution casted C50-b-(B450-co-C50)-b-C50 film. Author Contributions: B.L., X.C., and T.E.L. developed the concept and designed the experiments. B.L. and X.C. performed synthesis. B.L. and G.A.S. conducted material characterization. G.A.S. and
R.B.M. contributed SAXS measurements and fruitful comments. B.L. and T.E.L. wrote the paper with
major contributions from X.C. for critical revision and figure design. 3.7. Instrumentation Phase images were
mapped using Veeco’s Nanosensor silicon tips with a spring constant of 42 N m−1. Molecules 2021, 26, 4705 15 of 17 15 of 17 References 1. Pekkanen, A.M.; Zawaski, C.; Stevenson, A.T., Jr.; Dickerman, R.; Whittington, A.R.; Williams, C.B.; Long, T.E. Poly (ether ester)
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ing Supramolecular Elastomers for Melt Extrusion Additive Manufacturing. ACS Appl. Mater. Interfaces 2020, 12, 32006–32016. [CrossRef] [
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through contributions of all authors. All authors have read and agreed to the published version of
the manuscript. Funding: This material is based in-part upon work supported by the National Science Foundation
under grant no. DMR-1809291. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in insert article or
Supplementary Materials. Acknowledgments: The authors acknowledge Jae Sang Lee, Matthew D. Green, and Diana Convey
for the expertise and instrumentation for AFM in Eyring Materials Center at Arizona State University. The authors thank the insightful discussion with Kailong Jin and Tyler B. White. The authors
thank the support from Department of Chemistry and Macromolecules Innovation Institute (MII) at
Virginia Tech, and School of Molecular Sciences and Biodesign Center for Sustainable Macromolecular
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https://openalex.org/W2735385051
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https://napier-repository.worktribe.com/preview/962317/DielePestana%20_et_al_2017.pdf
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English
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The influence of crab burrows on sediment salinity in a Rhizophora-dominated mangrove forest in North Brazil during the dry season
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Hydrobiologia
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cc-by
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The influence of crab burrows on sediment salinity
1
during the dry season in a Rhizophora- dominated mangrove forest
2
in North Brazil
3
Accepted Manuscript published in Hydrobiologia
4
Cite this article as:
5
Pestana, D.F., Pülmanns, N., Nordhaus, I. et al. Hydrobiologia (2017).
6
doi:10.1007/s10750-017-3282-4
7
8
Debora F. Pestana1*, Nathalie Pülmanns1, Inga Nordhaus1, Karen
9
Diele2,3, Martin Zimmer1,4,5
10
1 Leibniz-Centre for Tropical Marine Research
11
Fahrenheitstr. 6
12
28359 Bremen
13
Germany
14
2 School for Applied Sciences, Edinburgh Napier University
15
Sighthill Campus
16
Edinburgh EH11 4 BN
17
UK
18 The influence of crab burrows on sediment salinity
1
during the dry season in a Rhizophora- dominated mangrove forest
2
in North Brazil
3
Accepted Manuscript published in Hydrobiologia
4
Cite this article as:
5
Pestana, D.F., Pülmanns, N., Nordhaus, I. et al. Hydrobiologia (2017). 6
doi:10.1007/s10750-017-3282-4
7
8
Debora F. Pestana1*, Nathalie Pülmanns1, Inga Nordhaus1, Karen
9
Diele2,3, Martin Zimmer1,4,5
10
1 Leibniz-Centre for Tropical Marine Research
11
Fahrenheitstr. 6
12
28359 Bremen
13
Germany
14
2 School for Applied Sciences, Edinburgh Napier University
15
Sighthill Campus
16
Edinburgh EH11 4 BN
17
UK
18 3 St Abbs Marine Station 19 St Abbs Marine Station
19
St Abbs
20
Berwickshire
21
TD14 5QF
22
UK
23
4 University of Bremen, Faculty 02: Biology/Chemistry
24
Germany
25
5 IUCN-SSC Mangrove Specialist Group
26
27
*: corresponding author:
28
deborafpestana@gmail.com
29
30
31
32
33
34
35 35 Abstract
36 Many ecological processes are influenced by salinity. Burrowing
37
crabs, abundant fauna of mangrove forests around the world, can facilitate
38
sediment water fluxes, which may decrease the salinity in mangrove
39
sediments. We investigated whether and how crab burrow density and
40
secondary fine root biomass interact to drive sediment salinity during the
41
dry season in a northern Brazilian mangrove forest. Areas with high density
42
of Rhizophora mangle prop roots and areas free of such roots were
43
compared. We found no correlation between burrow density and sediment
44
salinity in areas with dense prop and fine roots, while crab density
45
correlated negatively with sediment salinity in areas without prop roots,
46
where fine root density was low. Hence, the strength of sediment
47
desalination effects of crabs appears to be context-dependent, and high root
48
density of a salt-excluding mangrove species (R. mangle) seems to
49
counteract the crabs’ effects. Our results complement those of a former
50
study conducted in the same area during the rainy season, highlighting that
51
the findings are independent from seasonality and should be considered
52
when evaluating the overall ecological effects of crabs in mangrove
53
ecosystems
54 55 56 Key words: Rhizophora mangle, mangrove, Ucides cordatus, sediment
57
salinity
58
Introduction
59
Mangrove forests are species-rich, highly productive systems, and
60
they provide numerous ecosystem services. These forests can considerably
61
affect the biogeochemical cycles of coastal regions (Alongi et al., 1989;
62
Alongi, 2008; Rivera-Monroy et al., 1995; Sakho et al., 2015;
63
Schwendenmann et al., 2006), serve as feeding grounds and nursery sites
64
for oceanic and coastal nekton, and provide habitat for a range of
65
terrestrial, intertidal and marine fauna and flora (Ellison, 2008; Faunce and
66
Serafy, 2006). 67 Mangrove forests are species-rich, highly productive systems, and
60
they provide numerous ecosystem services. These forests can considerably
61
affect the biogeochemical cycles of coastal regions (Alongi et al., 1989;
62
Alongi, 2008; Rivera-Monroy et al., 1995; Sakho et al., 2015;
63
Schwendenmann et al., 2006), serve as feeding grounds and nursery sites
64
for oceanic and coastal nekton, and provide habitat for a range of
65
terrestrial, intertidal and marine fauna and flora (Ellison, 2008; Faunce and
66
Serafy, 2006). Abstract
36 67 Adaptations of mangrove trees to the intertidal environment include
68
various mechanisms of handling high salt concentrations in the sediment
69
pore water. For example, some species (e.g., Avicennia germinans (L.) L.,
70
Acanthaceae, common in Brazil) excrete salt by aboveground tissues, while
71
others exclude salt upon water-uptake through roots. The latter results in
72
salt accumulation in the sediment near the roots, potentially producing
73
deleterious effects on mangrove trees (Passioura et al., 1992). The Red
74
mangrove, Rhizophora mangle L., Rhizophoraceae, the dominant species in
75
North Brazilian mangrove forests, is one example for a salt-excluding
76
species (Parida and Jha, 2010; Passioura et al., 1992). 77 Crabs are one of the most abundant faunal groups in mangrove
78
forests in terms of number and biomass (Legat et al., 2006; Smith III et al.,
79
1991; Kristensen, 2008; Lee, 2008). Burrowing crabs can play an important
80
functional role as they can enhance the exchange of nutrients, salt, oxygen,
81
and pollutants between surface water and sediment. 82 Crabs are one of the most abundant faunal groups in mangrove
78
forests in terms of number and biomass (Legat et al., 2006; Smith III et al.,
79
1991; Kristensen, 2008; Lee, 2008). Burrowing crabs can play an important
80
functional role as they can enhance the exchange of nutrients, salt, oxygen,
81
and pollutants between surface water and sediment. 82 78 Two morphologically and functionally different groups of crabs are
83
common in North Brazil, fiddler crabs (Uca spp., Leach, 1814,
84
Ocypodidae) and Ucides cordatus (Linnaeus, 1763, Ucididae). Fiddler crab
85
burrows are usually shallow, typically with a maximum depth of
86
approximately 20 cm, and with a single opening (Lim, 2006). By contrast,
87
burrows of U. cordatus reach as deep as 2 m into the sediment and can
88
exhibit up to 2 burrow openings, while one opening is the most common
89
type (Wunderlich et al., 2008). The tidal flushing effect of U-shaped and
90
multiple-looped crab burrows is a well-studied topic (Heron and Ridd,
91
2008; 2003; Wolanski and Gardiner, 1981; Stieglitz et al., 2000; Xin et al.,
92
2009; Lim, 2006). Abstract
36 Crab burrows can facilitate the cycling of nutrients, CO2
93
and oxygen, and are therefore considered an important pathway for the
94
export of solutes between the mangrove sediment and the creek and for
95
land-ocean organic and inorganic solutes exchange (Xin et al., 2009;
96
Stieglitz et al., 2000 and 2013; Hollins et al. 2009; Pülmanns et al., 2014). 97
The desalinating influence of animal burrows has also been investigated
98 (Pissoura et al., 1992; Smith III et al., 1991; Stieglitz et al., 2000; 2013). 99
However, the first authors that have addressed specifically the relation
100
between burrows with one opening from larger crabs, like Ucides cordatus,
101
and salt contents in rooted sediments were Pülmanns et al. (2015). In a
102
laboratory microcosm study, salinity in mangrove sediment with artificial
103
crab burrows with one opening was significantly lower than in sediment
104
without burrows. However, no evidence for a desalination effect in rooted
105
areas was found for natural Ucides burrows in a field study conducted by
106
the same authors during the pronounced North Brazilian rainy season. 107
Pülmanns et al. (2015) suggested that any such effect of the burrows may
108
have been masked by leaching of salt through precipitation. They predicted
109
that the burrows’ influence on sediment salinity would be revealed during
110
the dry season. Sediment salinity drives many processes in mangrove
111
sediments (e.g. Kida et al., 2017) and it is important to understand how it is
112
influenced by burrowing crabs, particularly in light of the harvesting
113
pressures that many crab species experience. Ucides cordatus for example,
114
our study species, is heavily fished in many areas (e.g. Nascimento et al.,
115
2017), and mass mortalities caused by fungal pathogens (Boeger et al.,
116
2007; 2005) have caused population crashes, possibly affecting ecosystem
117
processes (Schmidt et al., 2013). 118 Here we investigate whether the density of both Ucides and fiddler
119
crab burrows and/or the density of secondary fine roots affect the salinity
120
of the upper sediment (up to 50 cm depth) of Rhizophora mangle stands
121
during the dry season. We hypothesize that sediment salinity is lower in
122
areas with higher crab burrow density due to the tidal flushing of the
123
burrows. 124 119 Location
126 The study was carried out in an intertidal mangrove forest located at
127
the Ajuruteua Peninsula, close to the channel Furo Grande (46°38’W;
128
0°50’S), at the Caeté Estuary, about 30 km northwest of the city of
129
Bragança in Pará, Brazil (Fig. 1). Situated within the Amazonian Coastal
130
Zone (ZCA), this mangrove forest is part of the largest and best-preserved
131
continuous mangrove belt on earth (Nascimento Jr. et al., 2013). These
132
extensive mangrove forests can extend up to 40 km inland. The Furo
133
Grande channel has a length of approximately 12 km with many smaller
134
tributaries and connections with the Atlantic Ocean (Acheampong, 2001). 135
The Ajuruteua Peninsula has a characteristic well-developed forest made
136
up by the Red mangrove (Rhizophora mangle,), the Black mangrove
137
(Avicennia germinans,) and the White mangrove (Laguncularia racemosa
138
(L.) C. F. Gaertrn, Combretaceae). At the Furo Grande channel, the
139 The study was carried out in an intertidal mangrove forest located at
127
the Ajuruteua Peninsula, close to the channel Furo Grande (46°38’W;
128
0°50’S), at the Caeté Estuary, about 30 km northwest of the city of
129
Bragança in Pará, Brazil (Fig. 1). Situated within the Amazonian Coastal
130
Zone (ZCA), this mangrove forest is part of the largest and best-preserved
131
continuous mangrove belt on earth (Nascimento Jr. et al., 2013). These
132
extensive mangrove forests can extend up to 40 km inland. The Furo
133
Grande channel has a length of approximately 12 km with many smaller
134
tributaries and connections with the Atlantic Ocean (Acheampong, 2001). 135
The Ajuruteua Peninsula has a characteristic well-developed forest made
136
up by the Red mangrove (Rhizophora mangle,), the Black mangrove
137
(Avicennia germinans,) and the White mangrove (Laguncularia racemosa
138
(L.) C. F. Gaertrn, Combretaceae). At the Furo Grande channel, the
139 mangrove forest consists of a mix of Rhizophora and Avicennia, with a
140
mean density of 520 trees per ha (Reise, 2003). 141 The tides at the study area are semidiurnal with a range between 3
142
and 5 m. The forest at the study site is located in the high intertidal zone
143
which is not flooded during neap high tides (Pülmanns et al., 2015). The
144
region has two very distinct seasons. Location
126 The dry season lasts for about 3 to 5
145
months, generally from August/September until November/December, and
146
the rainy season lasts from January until June/July (INMET, 2015). 147
Throughout the year, air temperatures vary between 24ºC and 34°C
148
(Mehlig, 2006; Menezes et al., 2003). No significant precipitation was
149
recorded during the experiment; the only rain event during the study
150
occurred on October 5 2014 (5mm; Source: INMET, 2015). 151 Burrowing crabs
152 Ucides cordatus is a semi-terrestrial crab that lives only in
153
mangrove forests and occurs throughout the western Atlantic Ocean, from
154
Florida (USA) to Santa Catarina State (Brazil) (Pinheiro and Hattori,
155
2006). The crabs have a life span of more than 10 years (Ostrensky et al.,
156
1995; Diele, 2000; Pinheiro et al., 2005), are slow-growing (Diele and
157
Koch, 2010) and can reach sizes of up to 9 cm carapace width in the Caeté
158
Estuary (Diele et al., 2005). They construct up to 2 m deep burrows and
159
feed mostly upon leaf litter (Goes et al., 2010; Nordhaus et al., 2006;
160 Nordhaus et al., 2009). Ucides cordatus preferably lives among the roots of
161
Rhizophora mangle, as this tree species provides shelter and food through
162
litter fall (Diele et al., 2005; Piou et al., 2009). Their density can be
163
extremely heterogeneous, with very few crab holes in dry habitats,
164
especially among pneumatophores of Avicennia germinans (Schories et al.,
165
2003), and higher densities in humid habitats and underneath the aerial
166
roots of Rhizophora mangle, with an average of 1.7 crab burrows m−2
167
(Diele et al., 2005). 168 Species of the genus Uca, commonly known as fiddler crabs, are
169
widespread throughout the Western Atlantic and abundant in mangroves
170
from Southern Florida (USA) to Santa Catarina (Brazil) (Crane, 1975). 171
Fiddler crabs feed on organic matter that they sieve out from the sediment
172
(Moura et al., 1998; Twilley et al., 1995). Inside the mangrove forests of
173
the Caeté Estuary, two species are abundant, Uca rapax (S. I. Smith, 1870)
174
and Uca vocator (J. F. W. Herbst, 1804) (Diele et al., 2010), with average
175
densities of 19 and 18 crabs burrows/m2 (Koch et al., 2005). Male Uca
176
rapax can reach a carapace width of 26 mm, while the females can grow up
177
to 24 mm (Castigllioni and Negreiros-Fransozo, 2006). Uca vocator varies
178
in size from 13 mm (females) to 17 mm carapace width (males) (Crane,
179
1975). 180 Two field sampling campaigns were conducted throughout the dry
182
season of 2014 during slack low tide. The first campaign was held on 15th
183
and 16th of October, and the second on 16th and 17th of November, both
184
during waning moon phase. Burrowing crabs
152 At seven sites (up to 200 m apart from each
185
other), three replicate sediment cores of 50 cm length and 5 cm diameter
186
were collected with a peat sampler (Eijkelkamp), from two areas with
187
different root densities: areas with high density of aerial prop roots
188
(“rooted” areas, samples were collected within an area of approximatively
189
5 m of diameter) and areas without prop roots (“gap” areas, again
190
approximatively 5 m in diameter)
191 182 On both sampling occasions, a 1.0 m x 1.0 m quadrat was placed
192
three times in both “rooted” and “gap” areas at each of the seven sites. The
193
number of crab burrows assigned to either fiddler crabs or U. cordatus was
194
estimated within each quadrat. This differentiation was made by the size of
195
the burrow opening. In R. mangle dominated forest stands of the Caeté
196
Estuary, the average and minimum size of U. cordatus burrows is 5.08 cm
197
(SD = 1.39) and 1.45 cm, respectively (Korting, 2012). In our study area,
198
most crab burrows were either large, i.e. with a diameter of 5 cm or above,
199
or small, i.e. 1 cm or below. The former could clearly be assigned to U. 200
cordatus, whereas all small burrows, often with characteristic chimneys,
201
were assigned to the much smaller fiddler crabs that are abundant in the
202 forest, according to visual observations. In contrast, intermediate and
203
smaller Ucides crabs are often more aggregated at the margins of the forest
204
and near creeks (Diele et al., 2005; Schmidt et al., 2013). The few
205
intermediate sized burrows present (approximatively 5%) (Korting, 2012),
206
that could belong to Ucides or fiddler crabs, were not considered in this
207
study. 208 From inside each quadrat, one core was sampled for analysis of
209
sediment salinity and fine root biomass. From the core, samples were taken
210
at depths of 1, 5, 10, 20, 30, 40, and 50 cm. For salinity, at each of these
211
depths, 1 cm segments of the sediment core were collected and stored in
212
sealed plastic tubes until further analysis. Burrowing crabs
152 For the analyses of the first
213
sediment layer (1 cm), the segment from 1 to 2 cm was collected; for the
214
second layer (5 cm), the segment from 5 to 6cm, for the third (10 cm), the
215
segment from 10 to 11 cm. For the determination of fine root biomass, 4
216
cm segments of the sediment were collected from the core and stored in
217
plastic bags until processing. All samples were stored in a styrofoam box
218
on ice in the field and then transferred into a refrigerator in the laboratory
219
where they were kept at 4°C until processing. 220 Laboratory Analyses
221 Salinity and water content
222 In the laboratory, sediment samples were homogenized and then
223
divided into two parts, to measure salinity and water content. For salinity, 2
224
g of the sediment were mixed with 10 mL of distilled water and shaken for
225
24 hours using a mechanical shaker (MA 136, Marconi). After 24 hours,
226
salinity was measured with a WTW TetraCon 325 connected to a WTW
227
portable meter (Multi 340i). The water content was determined through
228
mass loss upon oven-drying at 104° C to constant mass. 229 223 Fine Root Biomass
230 In this study, only the secondary thin roots were considered as fine
231
root biomass. Due to their small size, no separation between living and
232
dead material was made. Samples were washed with tap water using a sieve
233
with 0.5 mm mesh size and stored at 4° C until further processing. Fine
234
roots (including live and dead ones) were oven-dried at 104°C to constant
235
mass and weighed. Herein, we report root biomass as grams of dried roots
236
per unit soil volume (g/cm3). 237 Statistical data analyses
238 The analyses were performed in R (R Core Team 2012, version
239
2.15.2). The protocol for data exploration from Zuur et al. (2009; 2010)
240
was followed to check for outliers and collinearity between explanatory
241
variables. Then a linear mixed effect model (LME) (Pinheiro and Bates,
242
2000; Zuur et al., 2009) was used to analyze differences in sediment
243 salinity among area types (gap and rooted area) and sediment depths and all
244
their interaction terms. The random part of the LME model allowed for
245
heterogeneity among individual sediment cores and different sampling
246
sites. A variance function was applied to account for variance heterogeneity
247
between sediment depth levels (Pinheiro and Bates, 2000; Zuur et al.,
248
2009). For this the package “nlme” (Pinheiro et al., 2012) was used. 249
Differences in burrow density among area types, sediment salinity and
250
amount of fine root biomass were tested with a linear model of covariance
251
(ANCOVA). For this analysis, the density of aerial prop roots (“rooted”
252
and “gap” areas) was used as a fixed factor, the salinity as a dependent
253
variable and the fine root biomass and crab density as co-variables. 254 salinity among area types (gap and rooted area) and sediment depths and all
244
their interaction terms. The random part of the LME model allowed for
245
heterogeneity among individual sediment cores and different sampling
246
sites. A variance function was applied to account for variance heterogeneity
247
between sediment depth levels (Pinheiro and Bates, 2000; Zuur et al.,
248
2009). For this the package “nlme” (Pinheiro et al., 2012) was used. 249
Differences in burrow density among area types, sediment salinity and
250
amount of fine root biomass were tested with a linear model of covariance
251
(ANCOVA). For this analysis, the density of aerial prop roots (“rooted”
252
and “gap” areas) was used as a fixed factor, the salinity as a dependent
253
variable and the fine root biomass and crab density as co-variables. 254 Results
255 Fine root biomass changed significantly with depth in areas with
256
high density of aerial prop roots (“rooted”) (p < 0.001, R2 = 0.036), No
257
such significant change was observed within “gap” areas (p = 0.583, R2 = -
258
0.002, Fig. 2). Average fine root biomass was significantly higher in the
259
“rooted” areas (0.274 g/cm3 ± 0.012 SE) than in “gaps” (0.163 g/cm3 ±
260
0.023 SE) (p < 0.001, L Ratio = 36.4, df = 1). The difference of fine root
261
biomass between the “rooted” and “gap” areas at the surface (depth ≤ 5 cm)
262
and the deeper layers (≥ 40 cm) remained constant, around 0.1 g/cm3, but
263 was more pronounced at intermediate depths, between 10 and 30 cm depth,
264
being around 0.4 g/cm3 at 20 cm depth (Fig. 2). 265 was more pronounced at intermediate depths, between 10 and 30 cm depth,
264
being around 0.4 g/cm3 at 20 cm depth (Fig. 2). 265 was more pronounced at intermediate depths, between 10 and 30 cm depth,
264
being around 0.4 g/cm3 at 20 cm depth (Fig. 2). 265 Sediment salinity was higher in “rooted” areas than in “gap” areas (p
266
< 0.001, L. Ratio = 23.6, df = 1), with the highest values at the surface,
267
regardless of the aerial root density (at 1 cm: “rooted” 38 ± 1.23 and “gaps”
268
36 ± 0.52 SE; Fig. 3). In the “gap” areas, salinity dropped drastically from
269
1 to 5 cm depth (from 36 ± 3 to 31 ± 1 SE), then decreased gradually until
270
50 cm depth, where it reached 25 ± 1 SE. In the “rooted” areas the salinity
271
varied likewise, dropping from 38 ± 8 SE at the surface to 35 ± 1 SE at 5
272
cm depth, but remained relatively constant between 5 and 30 cm depth. 273
Below 30 cm, salinity dropped gradually until reaching 31 ±1 SE at 50 cm
274
depth in the “rooted" areas,, being significantly higher than at the same
275
depth in root-free gaps (31 ± 1 in contrast to 25 ± 1 SE, p = 0.002, R2 =
276
0.101, df = 1; Fig. 3)
277 Overall, salinity as a function of depth followed a similar pattern in
278
both areas, despite the higher overall values in the “rooted” areas. Results
255 Sediment
279
salinity and fine root biomass were positively correlated, both when
280
samples from the two areas were pooled together (p <0.05; Fig. 4A) and in
281
the "rooted" area (p = 0.026; Fig. 4B). In contrast, in gap areas without
282
aerial roots, the correlation was not significant (p = 0.398; Fig. 4C). When
283
comparing fine root biomass and Ucides burrow density, no significant
284 relation was found when pooling the two treatments together (p = 0.804;
285
Fig. 5A) and at “gap” areas (p = 0.236; Fig. 5C). However, Ucides burrow
286
density and fine root biomass were positively correlated in "rooted" areas
287
(p = 0.028; Fig. 5B). No correlation was observed for fiddler crabs (all
288
samples: p = 0.665, “rooted” areas: p = 0.797, “gaps”: p = 0.352; Fig. 6). 289
Sediment salinity did not show a relation with crab burrow density, when
290
samples from the two areas were pooled together (U. cordatus: p = 0.465;
291
Fig. 7A, Fiddler: p = 0.750; Fig. 8A), and there was also no relation
292
between crab density and sediment salinity in "rooted" areas (U. cordatus:
293
p = 0.331; Fig. 7B, Fiddler: p = 0.673; Fig. 8B). However, in gaps without
294
aerial roots, sediment salinity decreased with both increasing Ucides
295
burrow density (p = 0.026; Fig. 7C) and fiddler crab burrow density (p =
296
0.052; Fig. 8C). 297 The area (“rooted” versus “gap”) had a significant influence on
298
salinity (p = 4.6 e-10) and on the burrow density of U. cordatus (p = 0.004),
299
but did not have a significant effect on fiddler crab burrow density (p =
300
0.194). Burrow densities of both Ucides and fiddler crabs were higher in
301
the “rooted” areas than in the “gaps”. In the “rooted” areas the average
302
density was 7.4 m-2 (± 0.4 SE) for Ucides cordatus and 7.6 m-2 (± 0.5 SE)
303
for fiddler crabs. In the “gaps” the average density for U. cordatus was 5.5
304
m-2 (± 0.7 SE) and 6.6 m-2 (± 0.5 SE) for fiddlers. 305 Discussion
306 Salinity influences many processes in mangrove sediments. For
307
example, Kida et al. (2017) recently demonstrated that high salinity
308
flocculates and thereby accumulates humic substances, which could be one
309
of the mechanisms underlying carbon belowground accumulation in these
310
wetlands. Our understanding of the effects of the abundant crab burrows in
311
mangrove forests on sediment salinity (and depending processes) is sparse. 312
Crab burrows extend the contact surface of these sediments. In the case of
313
U. cordatus, this increase in contact surface amounts to 43% to 128%
314
(Korting, 2012), while it is only approximately 1% per each fiddler crab
315
burrow (Kristensen, 2008). Any increase in contact surface is likely to
316
enhance tidal flushing (Katz, 1980; Heron and Ridd, 2003; 2008). By
317
reworking and bioturbating the sediment (Kristensen, 2008), burrowing
318
crabs can play an important direct role in carbon storage in mangrove
319
sediments (Iribarne et al., 1997) and their burrowing also changes the
320
vertical and horizontal transfer of soil nutrients (Wang et al., 2010), a
321
further important ecological function of these crabs in mangrove
322
ecosystems. 323
h
ili
d
d
d d
i
h
d In our North-Brazilian study, conducted during the dry season,
324
sediment salinity was higher in areas with a higher R. mangle prop root
325
density (despite higher Ucides and fiddler crab burrow densities) than in
326 the gap areas. This refutes our hypothesis of lower sediment salinity at
327
areas with higher density of crab burrows during this time of year. The
328
result corroborates the findings of Pülmanns et al. (2015) in the rainy
329
season, when sediment salinity in “rooted” areas was also higher than in
330
“gap” areas (27 and 31 in gap areas and rooted areas, respectively, at the
331
end of the rainy season) Findings by Smith III (1987), demonstrating a
332
salinity of 57.5 in areas with high amounts of aerial roots versus 55.2 in
333
gaps, are also in concordance with the present results. A microcosm
334
experiment by Pülmanns et al. (2016), showed lower salinity in treatments
335
with (41) than without (47) artificial burrows after 6 months (both
336
treatments started with sediment salinity of 37.5 at the first centimeter),
337
showcasing that crab burrows can have a desalinating effect. 338 the gap areas. Discussion
306 This refutes our hypothesis of lower sediment salinity at
327
areas with higher density of crab burrows during this time of year. The
328
result corroborates the findings of Pülmanns et al. (2015) in the rainy
329
season, when sediment salinity in “rooted” areas was also higher than in
330
“gap” areas (27 and 31 in gap areas and rooted areas, respectively, at the
331
end of the rainy season) Findings by Smith III (1987), demonstrating a
332
salinity of 57.5 in areas with high amounts of aerial roots versus 55.2 in
333
gaps, are also in concordance with the present results. A microcosm
334
experiment by Pülmanns et al. (2016), showed lower salinity in treatments
335
with (41) than without (47) artificial burrows after 6 months (both
336
treatments started with sediment salinity of 37.5 at the first centimeter),
337
showcasing that crab burrows can have a desalinating effect. 338 In our study, secondary fine root biomass was highest at a sediment
339
depth of 20 cm in areas with aerial roots, roughly coinciding with the
340
average depth of fiddler crab burrows (Lim, 2006). Fiddler crab densities
341
were higher than U. cordatus in both “rooted” and “gap” areas, whereas
342
previous studies state that fiddler crabs preferentially colonize areas with a
343
less dense canopy, since they feed on microphytobenthos (Miller, 1961;
344
Bouillon et al., 2002). By contrast, Ucides cordatus preferably settles in
345
areas with high density of R. mangle aerial roots, probably due to the
346 shelter and burrow structure stability that these roots provide (Piou et al.,
347
2009). 348 According to Heron and Ridd (2008), a multiple-loop crab burrow
349
can decrease sediment salinity by up to 5 units within one week. In the
350
present study, sediment salinity decreased with increasing density of
351
Ucides burrows in gap areas with low density of fine roots. In “rooted”
352
areas, no such effect of crab burrows on salinity was found, indicating that
353
any potential existing crab effect was overruled (masked) by the salt-
354
accumulating effects of the activity of the fine roots. Overall, our results
355
indicate that the magnitude of the desalinating effects of the crab burrows
356
seems to be context dependent, driven by the density of Rhizophora fine
357
roots. 358 We conclude that neither Uca spp. Discussion
306 nor Ucides cordatus are the key
359
drivers for sediment salinity underneath mangrove trees in the studied
360
mangrove forest. The areas where these crabs do have a clear desalinating
361
effect, the gaps, are much smaller in area coverage than the rooted areas in
362
the Rhizophora dominated mangrove forest in Northern Brazil, part of the
363
largest continuous mangrove ecosystem of the world. These results need to
364
be considered when evaluating the overall ecological effect(s) of crabs in
365
mangrove ecosystems. 366 We conclude that neither Uca spp. nor Ucides cordatus are the key
359
drivers for sediment salinity underneath mangrove trees in the studied
360
mangrove forest. The areas where these crabs do have a clear desalinating
361
effect, the gaps, are much smaller in area coverage than the rooted areas in
362
the Rhizophora dominated mangrove forest in Northern Brazil, part of the
363
largest continuous mangrove ecosystem of the world. These results need to
364
be considered when evaluating the overall ecological effect(s) of crabs in
365
mangrove ecosystems. 366 367 Acknowledgements
368 The authors would like to thank the University of Pará (UFPA), and
369
Dr. Moirah Menezes and Dr. Ulf Mehlig for the support during the field
370
work. Acknowledgments also to Dr. Thiago Branquinho de Queiroz from
371
the Universidade Federal do ABC (São Paulo) for all support provided. 372
Karen Diele received funding from the MASTS pooling initiative (The
373
Marine Alliance for Science and Technology for Scotland), and its support
374
is gratefully acknowledged. MASTS is funded by the Scottish Funding
375
Council (grant reference HR09011) and contributing institutions. 376 377
Figure 1: Location of the study area in North Brazil and the detail of
378
the Ajuruteua's Peninsula. Source: Pülmanns. 2014. 379 377 Figure 1: Location of the study area in North Brazil and the detail o Figure 1: Location of the study area in North Brazil and the detail of
378
h Aj
' P
i
l
S
P l
2014 Figure 1: Location of the study area in North Brazil and the detail of
378
the Ajuruteua's Peninsula. Source: Pülmanns. 2014. 379 Figure 1: Location of the study area in North Brazil and the detail of
378
the Ajuruteua's Peninsula. Source: Pülmanns. 2014. 379 the Ajuruteua's Peninsula. Source: Pülmanns. 2014. 379 the Ajuruteua's Peninsula. Source: Pülmanns. 2014. 379 380
Figure 2: Relationship between fine root biomass (g) and the sediment
381
depth. Data represent means and standard error (L. Ratio = 36.4, df = 1, p <
382
0.001). 383 380 Figure 2: Relationship between fine root biomass (g) and the sediment
381
depth. Data represent means and standard error (L. Ratio = 36.4, df = 1, p <
382
0.001). 383 Figure 2: Relationship between fine root biomass (g) and the sediment Figure 2: Relationship between fine root biomass (g) and the sediment
381 Figure 2: Relationship between fine root biomass (g) and the sediment
381
depth. Data represent means and standard error (L. Ratio = 36.4, df = 1, p <
382
0.001). 383 384 385 386
Figure 3: Relationship between sediment salinity and sediment depth. 387
Data represent means and standard error. (L. Ratio = 23.6, df = 1, p <
388
0.001). 389 Figure 3: Relationship between sediment salinity and sediment depth Figure 3: Relationship between sediment salinity and sediment depth. 387
Data represent means and standard error. (L. Ratio = 23.6, df = 1, p <
388
0.001). indicates the number of measurements.
421 Acknowledgements
368 389 Figure 3: Relationship between sediment salinity and sediment depth. 387
Data represent means and standard error. (L. Ratio = 23.6, df = 1, p <
388
0.001). 389 393 394
395 394 395 Figure 4: Relationship between fine root biomass (g) and sediment salinity. 396
A) all samples (p = 0.004, R2 = 0.011), B) densely rooted areas (p = 0.0266,
397
R2= 0.094), C) root-free gaps (p = 0.398, R2 = -0.006). The red line
398
indicates the trend line, when significant. “n” indicates the number of
399
measurements. 400 404 405
406 405 406 Figure 5: Relationship between fine root biomass (g) and Ucides cordatus
407
density. A) all samples (p = 0.804, R2 = -0.011), B) densely rooted areas (p
408
= 0.028, R2= 0.092), C) root-free gaps (p = 0.236, R2 = 0.010). The red line
409
indicates the trend line, when significant. “n” indicates the number of
410
measurements. 411 Figure 5: Relationship between fine root biomass (g) and Ucides cordatus
407
density. A) all samples (p = 0.804, R2 = -0.011), B) densely rooted areas (p
408
= 0.028, R2= 0.092), C) root-free gaps (p = 0.236, R2 = 0.010). The red line
409
indicates the trend line, when significant. “n” indicates the number of
410
measurements. 411 415 416
417 416 417 Figure 6: Relationship between fine root biomass (g) and Fiddler crab
418
density. A) all samples (p = 0.665, R2 = -0.009), B) densely rooted areas (p
419
= 0.797, R2= -0.023), C) root-free gaps (p = 0.352, R2 = -0.002). “n”
420 indicates the number of measurements. 421 424 425
426 425 426 426 Figure 7: Relationship between sediment salinity and Ucides cordatus
427
density. A) all samples (p = 0.465, R2 = -0.005), B) densely rooted areas (p
428
= 0.331, R2= 0.008), C) root-free gaps (p = 0.026, R2 = 0.0957. The red line
429
indicates the trend line, when significant. “n” indicates the number of
430
measurements. 431 433 434
435
Figure 8: Relationship between fine root biomass (g) and Fiddler crab
436
density. A) all samples (p = 0.750, R2 = -0.010), B) densely rooted areas (p
437
= 0.673, R2= -0.020), C) root-free gaps (p = 0.052, R2 = 0.068). The red
438
line indicates the trend line, when significant. “n” indicates the number of
439
measurements. Acknowledgements
368 440 434 435 435 435 Figure 8: Relationship between fine root biomass (g) and Fiddler crab
436
density. A) all samples (p = 0.750, R2 = -0.010), B) densely rooted areas (p
437
= 0.673, R2= -0.020), C) root-free gaps (p = 0.052, R2 = 0.068). The red
438
line indicates the trend line, when significant. “n” indicates the number of
439
measurements. 440 Figure 8: Relationship between fine root biomass (g) and Fiddler crab
436
density. A) all samples (p = 0.750, R2 = -0.010), B) densely rooted areas (p
437
= 0.673, R2= -0.020), C) root-free gaps (p = 0.052, R2 = 0.068). The red
438
line indicates the trend line, when significant. “n” indicates the number of
439
measurements. 440 Figure 8: Relationship between fine root biomass (g) and Fiddler crab
436
density. A) all samples (p = 0.750, R2 = -0.010), B) densely rooted areas (p
437
= 0.673, R2= -0.020), C) root-free gaps (p = 0.052, R2 = 0.068). The red
438
line indicates the trend line, when significant. “n” indicates the number of
439
measurements. 440 References
441
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442
biomass in intertidal soft sediments of North-East Brazil. Master
443
Thesis of the University of Bremen, Germany, 69 p. 444
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Alongi, D. M., 2014. Carbon cycling and storage in mangrove forests. 448
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Ecology Progress Series 56, pp. 133-144. 453
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Castilho, G. G., 2007. Histopathology of the mangrove land crab
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biomass in intertidal soft sediments of North-East Brazil. Master
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and Shelf Science 76, pp. 1-13. 447 Alongi, D. M., 2014. Carbon cycling and storage in mangrove forests. 448 Annual Review of Marine Science 6, pp. 195 - 219. 449 Alongi, D. M., Boto, K. G. & Tirendi, F., 1989. Effect of exported
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mangrove litter on bacterial productivity and dissolved organic
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carbon fluxes in adjacent tropical nearshore sediments. Marine
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Ecology Progress Series 56, pp. 133-144. 453 Boerger, W. A., Pie, M. R., Ostresky, A. & Patella, L., 2005. Lethargic
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crab disease: multidisciplinary evidence supports a mycotic etiology. 455
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Castilho, G. G., 2007. Histopathology of the mangrove land crab
458
Ucides cordatus (Ocypodidae) affected by lethargic crab disease. 459
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Diele, K. 2000. Life history and population structure of the exploited
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Experimental Biology and Ecology 395, 171-180. 476
Diele, K., Koch, V. & Saint-Paul, U., 2005. Population structure, catch
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Indications for overfishing? Aquatic Living Resources 18, 169–178. 480 Bouillon, S., Koedam, N., Raman, A.V. References
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https://openalex.org/W2032365913
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https://translational-medicine.biomedcentral.com/track/pdf/10.1186/1479-5876-10-S2-A64
|
English
| null |
Metformin improves IKCa-mediated endothelial dilative dysfunction of arteriole in diabetic rats
|
Journal of translational medicine
| 2,012
|
cc-by
| 562
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Results Increased HbA1c level and reduceded endothelium-
dependent dilative response mediated by IKCa in mesen-
tery arterioles were observed in diabetic rats, and metfor-
min treatment (300 mg/kg/day by gavage) restored the
adverse condition. The vasodilatation mediated by IKCa
was also impaired in 200 μg/mL AGE-BSA-incubated
mesentery arterioles. AGE-BSA at 200 μg/mL concentra-
tion and H2O2 (100 μmol/L) significantly decreased the
mRNA and protein expression of IKCa. AGE-BSA also
increased the production of MDA and inhibited Cu-Zn
SOD activity in HUVECs. Metformin of 10-6 mol/L and
10-7 mol/L reversed those effects. Activation of intermediate conductance Ca2+-activated K+
channel (IKCa) in endothelial cells has been shown to con-
tribute to vasodilation, especially in small vessels. The aim
of this study is to observe the effect of metformin on
endothelial dilative dysfunction in diabetic rats and investi-
gate whether the alteration of IKCa are involved in the
underlying mechanism. Methods Diabetic rat model was induced by a single intraperitoneal
injection of 30 mg/kg STZ after high fat and glucose diet
for 8 weeks. Animals whose blood glucose > 11.1 mmol/L
were included in diabetic and metformin group. Age-
matched animals fed with standard chow and injected
with citric acid buffer were served as control. Four weeks
after STZ injection, rats in three groups were fed with nor-
mal diet for additional 8 weeks. After that, fasting blood
was drawn and third-order mesenteric arteries were sepa-
rated. Hemoglobin A1c (HbA1c) was measured with an
automatic analyzer. The changes of Ach- and NS309
(opener of IKCa and small conductance Ca2+-activated K+
channel, SKCa) -induced vasodilatation mediated by IKCa
in mesentery arterioles of each group and mesentery arter-
ioles of normal rats incubated with 200 μg/mL AGE-BSA
(200 μg/mL BSA as control) for 3 hours were measured by
multi-myograph system. The effect of metformin on AGE-
BSA (200 μg/mL) and H2O2 (100 μmol/L) induced
changes of IKCa mRNA and protein expression in cultured
human umbilical vein endothelial cells (HUVECs) were
detected by RT-PCR and Western blot. The level of mal-
ondialdehyde (MDA) and the activity of Cu-Zn superoxide
dismutase (Cu-Zn SOD) in cellular supernatant were
determined by colorimetric method. Conclusion Metformin significantly improves endothelium dilative
dysfunction mediated by IKCa in diabetic rats, which is
likely related to the inversion of AGEs-induced oxidation
and downregulation of IKCa expression in endothelial cells. Zhao et al. Journal of Translational Medicine 2012, 10(Suppl 2):A64
http://www.translational-medicine.com/content/10/S1/A64 Zhao et al. Journal of Translational Medicine 2012, 10(Suppl 2):A64
http://www.translational-medicine.com/content/10/S1/A64 Zhao et al. Journal of Translational Medicine 2012, 10(Suppl 2):A64
http://www.translational-medicine.com/content/10/S1/A64 Open Access © 2012 Zhao 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: dengxl@mail.xjtu.edu.cn
Department of Physiology and Pathophysiology, School of Medicine, Xi’an
Jiaotong University, 76 West Yanta Road, Xi’an, 710061, Shanxi, China Acknowledgments g
This work was supported by the National Nature Science Foundation of
China (grant number 81070129) and Nature Science Foundation of Shaanxi
province (No. 2011JQ4021). Published: 17 October 2012 Published: 17 October 2012 © 2012 Zhao 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.
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Glutamine Metabolism in Both the Oxidative and Reductive Directions Is Triggered in Shrimp Immune Cells (Hemocytes) at the WSSV Genome Replication Stage to Benefit Virus Replication
|
Frontiers in immunology
| 2,019
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Glutamine Metabolism in Both the
Oxidative and Reductive Directions Is
Triggered in Shrimp Immune Cells
(Hemocytes) at the WSSV Genome
Replication Stage to Benefit Virus
Replication 1 Department of Biotechnology and Bioindustry Sciences, College of Biosciences and Biotechnology, National Cheng Kung
University, Tainan, Taiwan, 2 Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan,
3 International Center for the Scientific Development of Shrimp Aquaculture, National Cheng Kung University, Tainan, Taiwan ORIGINAL RESEARCH
published: 04 September 2019
doi: 10.3389/fimmu.2019.02102 White spot syndrome virus (WSSV) is the causative agent of a shrimp disease that
has caused huge global economic losses. Although its pathogenesis remains poorly
understood, it has been reported that in the shrimp immune cells (hemocytes) targeted
by WSSV, the virus triggers both the Warburg effect and glutamine metabolism
at the WSSV genome replication stage (12 h post infection). Glutamine metabolism
follows two pathways: an oxidative pathway mediated by α-KGDH (α-ketoglutarate
dehydrogenase) and an alternative reductive pathway mediated by IDH1 and IDH2
(isocitrate dehydrogenase 1 and 2). Here we used isotopically labeled glutamine
([U-13C]glutamine and [1-13C]glutamine) as metabolic tracers to show that, at the
replication stage, both the oxidative and reductive glutamine metabolic pathways
were activated. We further show that the mRNA expression levels of α-KGDH and
IDH1 were increased in WSSV-infected shrimps and that silencing of α-KGDH,
IDH1, and IDH2 with their respective dsRNAs led to a decrease in WSSV gene
expression and WSSV replication. Taken together, our findings provide new evidence for
WSSV-induced metabolic reprogramming in hemocytes and demonstrate its importance
in virus replication. Edited by:
Shoichiro Kurata,
Tohoku University, Japan
Reviewed by:
Anchalee-Tassanakajon,
Chulalongkorn University, Thailand
Chia-Ying Chu,
National Taiwan University, Taiwan
*Correspondence:
Han-Ching Wang
wanghc@mail.ncku.edu.tw
†These authors have contributed
equally to this work Edited by:
Shoichiro Kurata,
Tohoku University, Japan Reviewed by:
Anchalee-Tassanakajon,
Chulalongkorn University, Thailand
Chia-Ying Chu,
National Taiwan University, Taiwan
*Correspondence:
Han-Ching Wang
wanghc@mail.ncku.edu.tw †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Comparative Immunology,
a section of the journal
Frontiers in Immunology Keywords: white spot syndrome virus, IDHs, oxidative glutaminolysis, reductive carboxylation, hemocytes Received: 25 June 2019
Accepted: 20 August 2019
Published: 04 September 2019 Keywords: white spot syndrome virus, IDHs, oxidative glutaminolysis, reductive carboxylation, hemocytes Experimental Animals and WSSV Inoculum Cloning of Full-Length cDNA of LvGLS1,
LvGLS2, LvIDH1, LvIDH2, and Lvα-KGDH
By using next generation sequencing, an in-house L. vannamei
stomach transcriptomic database was established (data not
shown) and this was used to search for the target genes. Two
contigs,
PVHP259804.2
and
PVHP193998,
were
found to show high homology with Rat GLS (Accession
number:
M65150.1),
and
PVHP193998
also
matched
P. vannamei GLS (Accession number: XP_027218904.1) with
98% identity. These contig sequences were used to design
primer sets to amplify the two GLS isoforms (Table 1)
and these two genes were named LvGLS1(PVHP259804.2)
and
LvGLS2
(PVHP193998). Three
other
contigs,
PVHP107410.1,
PVHP176973.1,
and
PVHP203127.2,
respectively showed high homology with Penaeus vannamei
IDH1
(Accession
number:
XP_027219531.1),
Penaeus
vannamei
IDH2
(Accession
number:
XP_027239404.1),
and
Penaeus
vannamei
α-KGDH
(Accession
number:
XP_027220285.1). These contig sequences were also used
to design primer sets to amplify the corresponding genes
(Table 1). We have recently shown that glutamine anaplerosis also
occurs in WSSV-infected shrimp: we found that WSSV increases
the expression of GDH, an enzyme which converts glutamate
into α-KG (8), and also that direct in vivo replenishment of α-
KG rescued WSSV replication after the down-regulation of GDH
by dsRNA-mediated gene silencing. These results all suggest that,
in conjunction with the WSSV-induced Warburg effect, WSSV-
infected cells may activate glutamine metabolism to fuel the
TCA cycle. However, it was not known whether WSSV triggered
glutamine metabolism in both the oxidative and reductive
directions. In the present study, we therefore look more closely at
the glutamine metabolism induced by WSSV. To do this, we use
LC-ESI-MS and isotopically labeled glutamine (uniformly-13C
[U-13C] glutamine and [1-13C] glutamine) as metabolic tracers. We also provide additional evidence of the importance of the
reductive carboxylation glutaminolysis for virus replication. Experimental Animals and WSSV Inoculum Experimental Animals and WSSV Inoculum
The shrimp (Litopenaeus vannamei) of around 3 g body weight
used in the study were obtained from the International Center
for the Scientific Development of Shrimp Aquaculture, National
Cheng Kung University (NCKU), and the Department of
Aquaculture, National Pingtung University of Science and
Technology (NPUST). Before the experiments, shrimp were
cultured for 1∼3 days in sterilized seawater (30 ppt at
26∼27◦C). The WSSV (Taiwan isolate, GenBank accession no. AF440570) stock (3.3 × 104 WSSV copies/µl) was prepared from
hemolymph of WSSV-infected moribund SPF (specific pathogen
free) shrimp as described previously (6, 9). The viral inoculum
was prepared from the stock for intramuscular injection by
dilution (10−4) with 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 2 mM KH2PO4). The WSSV challenge dosage (100
µl/3 g shrimp) resulted in an ∼50% cumulative mortality at
3 days post WSSV challenge. Shrimp in the control group
were treated with PBS (100 µl/3 g shrimp). At 12 and 24 h
post WSSV challenge, hemocyte samples and stomach tissue
were collected and used for the glutamine-metabolism-related
enzyme activity assays. The hemocyte samples were also used
to measure the expression of host genes and viral genes, for the
stable-isotope metabolic tracing experiments, and to measure the
copy number of the WSSV genomic DNA as described in Su
et al. (9). When aerobic glycolysis is activated, most of the carbon
atoms from the glucose that has been taken up become
diverted into lactate production instead of being routed into
the mitochondrial TCA (tricarboxylic acid) cycle (2, 14). Thus,
in order to allow the TCA cycle to continue to produce
energy and biosynthetic products during aerobic glycolysis,
glutamine, an amino acid that is abundant in the circulation
system, is used as an alternative carbon source in a process
known as glutamine anaplerosis (11, 15, 16). In normal cells,
after glutamine is converted (via glutamate) into α-KG, it
is subsequently metabolized to succinate by α-ketoglutarate
dehydrogenase (α-KGDH) through oxidative glutaminolysis. However, cells undergoing the Warburg effect are also able
to convert α-KG into isocitrate either by cytoplasmic IDH1
or by mitochondrial IDH2 through the reductive glutamine
metabolic pathway (17, 18). Although mammalian studies have
shown that glutamine metabolism can have both immune-related
benefits as well as pathogenesis-related effects (3–5), the pathway
primarily seems to favor virus replication in shrimp infected by
WSSV (8). Citation: He S-T, Lee D-Y, Tung C-Y, Li C-Y and
Wang H-C (2019) Glutamine
Metabolism in Both the Oxidative and
Reductive Directions Is Triggered in
Shrimp Immune Cells (Hemocytes) at
the WSSV Genome Replication Stage
to Benefit Virus Replication. Front. Immunol. 10:2102. doi: 10.3389/fimmu.2019.02102 Although in many kinds of tumors and cancers, metabolic reprogramming has been found to be
crucial for the cell’s aberrant proliferation and potentially unlimited self-renewal (1, 2), immune
cells are also able to control pathogens by triggering particular metabolic pathways to aid immune
responses, such as increasing amino acid catabolism (3). However, there is also increasing evidence
that some vertebrate viruses, especially oncogenic viruses can utilize the reprogramming of host
metabolism to complete their replication cycle (4, 5). Quite recently, the white spot syndrome virus
(WSSV), a large dsDNA virus that is the causative agent of a devastating viral shrimp disease, September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org He et al. WSSV-Induced Glutamine Metabolism MATERIALS AND METHODS became the first invertebrate virus that was shown to induce host
metabolic reprogramming in shrimp immune cells (hemocytes). These changes include the Warburg effect (aerobic glycolysis),
amino acid catabolism (i.e., glutaminolysis [glutamate-driven
anaplerosis]), lipid metabolism, activation of the pentose
phosphate pathway, nucleotide biosynthesis, and amino acid
biosynthesis (6–9). The effects are most noticeable at the genome
replication stage (12 h post infection), and like the metabolic
reprogramming that is seen in cancer cells and cells infected by
some vertebrate viruses (10–13), the WSSV-induced metabolic
changes benefit the virus by meeting the both its energy
requirements and its biosynthetic needs. Frontiers in Immunology | www.frontiersin.org Measurement of Host Genes and the WSSV
Major Structural Gene VP28 by Real-Time
PCR Abbreviations:
WSSV,
white
spot
syndrome
virus;
LC-ESI-MS,
Liquid
chromatography-electrospray ionization mass spectrometry; PBS, phosphate-
buffered saline; GLS, glutaminase; GDH: glutamine dehydrogenase; ASAT,
aspartate
aminotransferase;
IDH,
isocitrate
dehydrogenase;
α-KGDH,
α-ketoglutarate dehydrogenase. Gln, glutamine; Glu, glutamate; α-KG, α-
ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Oac, oxaloacetate; Cit,
citrate; Ict, isocitrate; Lac, lactate; Asp, aspartate. After shrimp tissues were collected at the 12 and 24 hpi time
points, total RNA was extracted and subjected to cDNA synthesis
by using Superscriptase II Reverse Transcriptase (Invitrogen) and
Anchor-dTv primer (Table 1). The cDNA samples were used September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 2 WSSV-Induced Glutamine Metabolism He et al. He et al. TABLE 1 | Primer sets used in the present paper. Gene
Primer
Primer sequence (5′-3′)a
Usag
EF1α
EF1α-F
5′-ATGGTTGTCAACTTTGCCC-3′
Cloni
EF1α-R
5′-TTGACCTCCTTGATCACACC-3′
Cloni
EF1α-qF
5′-ACGTGTCCGTGAAGGATCTGAA-3′
Real-
EF1α-qR
5′-TCCTTGGCAGGGTCGTTCTT-3′
Real-
LUCIFERASE
Luc-F
5′-CTGAATACAAATCACAGAATC-3′
Cloni
Luc-R
5′-GTAAGACCTTTCGGTACTTCG-3′
Cloni
T7-Luc-F
5′-TAATACGACTCACTATAGGGAGACTGAATACAAATCACAGAATC-3′
dsRN
T7-Luc-R
5′-TAATACGACTCACTATAGGGAGAGTAAGACCTTTCGGTACTTCG-3′
dsRN
GDH
GDH-qF
5′-TGAGGAGAAGCGCAACAAGA-3′
Real-
GDH-qR
5′-TGGCAGGGCTCCATGATC-3′
Real-
GLS1
GLS1-qF
5′-CATTGGCGACACTGACAT-3′
Real-
GLS1-qR
5′-CTGCAGAAGGCCATTGACTA-3′
Real-
GLS2
GLS2-F
5′-GACCGCAAGAACCTCCTCAA-3′
Cloni
GLS2-R
5′-GTGATGACAGAAGCCACGGA-3′
Cloni
T7-GLS2- F
5′-TAATACGACTCACTATAGGGAGAGACCGCAAGAACCTCCTCAA-3′
dsRN
T7-GLS2- R
5′-TAATACGACTCACTATAGGGAGAGTGATGACAGAAGCCACGGA-3′
dsRN
GLS2-qF
5′-AACTACATGGGGATGGAG-3′
Real-
GLS2-qR
5′-GATTGAAATCCAGGCAAAGCTC-3′
Real-
IDH1
IDH1-F
5′-GAGGATTTGCTCATGCTTC-3′
Cloni
IDH1-R
5′-TCAGGCAGTGATCTTCTTCTGC-3′
Cloni
T7-IDH1-F
5′-TAATACGACTCACTATAGGGAGAGAGGATTTGCTCATGCTTC-3′
dsRN
T7-IDH1-R
5′-TAATACGACTCACTATAGGGAGATCAGGCAGTGATCTTCTTCTGC-3′
dsRN
IDH1-qF
5′-GGCATGATGACCTCGGTACTG-3′
Real-
IDH1-qR
5′-GGCAGCCTCAGACTCCAGAGT-3′
Real-
IDH2
IDH2-F
5′-GCAAGAACTACGATGGTG-3′
Cloni
IDH2-R
5′-ATGCAGCCAGCCAGATCCT-3′
Cloni
T7-IDH2-F
5′-TAATACGACTCACTATAGGGAGAGCAAGAACTACGATGGTG-3′
dsRN
T7-IDH2-R
5′-TAATACGACTCACTATAGGGAGAATGCAGCCAGCCAGATCCT-3′
dsRN
IDH2-qF
5′-CCAACCCTGTTGCTTCCATT-3′
Real-
IDH2-qR
5′-AAGCTTGGCACGATGTTCAAG-3′
Real-
α-KGDH
KGDH-F
5′-ATGGGTTTGAGGCATTCTTG-3′
Cloni
KGDH-R
5′-CCTGAGAAAGCAGCATCTCC-3′
Cloni
T7-KGDH-F
5′-TAATACGACTCACTATAGGGAGAATGGGTTTGAGGCATTCTTG-3′
dsRN
T7-KGDH-R
5′-TAATACGACTCACTATAGGGAGACCTGAGAAAGCAGCATCTCC-3′
dsRN
KGDH-qF
5′-TCCAGCCTCGCATTTCCA-3′
Real-
KGDH-qR
5′-GACGGCCAGCATATGAAA-3′
Real-
VP28
vp28-real-F
5′-AGTTGGCACCTTTGTGTGTGGTA-3′
Real-
vp28-real-R
5′-TTTCCACCGGCGGTAGCT-3′
Real-
aThe added T7 promoter sequence is underlined. to quantify the mRNA expression of the target genes by using
l
h
(
)
gene VP28 and EF1α. The specific primer sets for ea
l
d
bl
l
l
d aThe added T7 promoter sequence is underlined. to quantify the mRNA expression of the target genes by using
real-time PCR with KAPA SYBR1 FAST Master Mix (KAPA)
and the Bio-Rad detection system. The gene expression levels
measured in this study were for the two GLS isoforms (GLS1
and GLS2), IDH1, IDH2, α-KGDH, the WSSV major structural to quantify the mRNA expression of the target genes by using
real-time PCR with KAPA SYBR1 FAST Master Mix (KAPA)
and the Bio-Rad detection system. Determination of the Enzyme Activity of
IDH in Shrimp Stomachs and Hemocytes
During WSSV Infection Hemocytes and stomachs from shrimp were collected at 12
and 24 h after WSSV or PBS injection (6 shrimp/pool and
4 pools/group), and the GLS activity was measured with a
commercial Glutaminase Microplate Assay Kit (MyBiosource). The hemocyte and stomach samples were homogenized with 100
and 300 µl ice cold assay buffer, respectively. After centrifugation
at 4◦C at 13,000 g for 10 min, the cell debris was removed and the
protein concentration in the supernatant was determined. The
hemocyte lysates (3∼52 µg) and stomach lysates (6 µg) were
mixed with 200 µl substrate, and the reaction were incubated
at 37◦C for 1 h. The reactions were stopped by adding 300 µl
Stop solution, incubating for 10 min and then centrifuging at
4◦C at 8,000 g for 5 min. One hundred and thirty microliter of
each supernatant was collected and mixed with a 70 µl reaction
mixture containing 50 µl reaction buffer and 20 µl dye reagent. The controls were prepared as per the samples except that
the protein lysates were replaced by distilled water. Standards
and blanks were prepared according to the manufacturer’s
instructions. The absorbance of the final mixtures was measured
at 450 nm. The GLS activity was calculated by the following
equation: GLS activity (U/mg) =
4×T×CStandard× (ODSample−ODControl)
(ODStandard−ODBlank)
CProtein
(note: CStandard: reference standard [1 mg/ ml]; CProtein: protein
concentration; ODblank: buffer only; T: reaction time in hours). Statistical analysis was performed as described above. g
Hemocytes and stomachs from shrimp were collected at 12
and 24 h after WSSV or PBS injection (6 shrimp/pool and 4
pools/group) to measure the activity of IDH. The hemocyte and
stomach samples were homogenized with 100 and 200 µl ice
cold IDH assay buffer, respectively, from a commercial Isocitrate
Dehydrogenase Activity Assay Kit (Sigma). The cell debris was
removed by centrifugation at 4◦C at 13,000 g for 10 min. After
using a Bio-Rad protein assay to measure protein concentrations,
samples with the appropriate amounts of protein (hemocyte
lysate: 15 ∼30 µg; stomach lysate: 10 µg) were collected and
adjusted to a final volume of 50 µl using IDH assay buffer. The
lysates were then mixed with 50 µl reaction mixture containing
38 µl IDH Assay Buffer, 8 µl Developer 2 µl IDH Substrate, and
2 µl NADP+. The final mixtures were incubated at 37◦C and
protected from light. Measurement of the WSSV Genome Copy
Number Using the IQ RealTM WSSV
Quantitative System mixtures were then incubated at 37◦C and protected from light. The NADH standards supplied with the kit were prepared in the
same way as the shrimp tissue samples. α-KGDH activity was
measured at A450 every 3–5 min until the value of the most active
sample was greater than the value for the NADH standard with
the highest concentration (12.5 nmole). The NADH standard
curve at this time point was then used to convert the difference
in absorbance at the initial (Tinitial) and final (Tfinal) time points
to the amount of NADH (B). The activity was calculated by
the following equation: enzyme activity (mU/mg) = B/([Tfinal –
Tinitial] × [total amount of protein in the reaction]). Statistical
analysis was performed as described above. Shrimp hemocytes were collected from each group at the
12 and 24 hpi time points and subjected to genomic DNA
extraction using a DTAB/CTAB DNA extraction kit (GeneReach
Biotechnology Corp.). The viral genome copy numbers were
then quantified by the real-time PCR-based IQ Real
TM WSSV
quantitative system (GeneReach Biotechnology Corp.). Statistical
analysis was performed as described above. Measurement of Host Genes and the WSSV
Major Structural Gene VP28 by Real-Time
PCR The gene expression levels
measured in this study were for the two GLS isoforms (GLS1
and GLS2), IDH1, IDH2, α-KGDH, the WSSV major structural gene VP28 and EF1α. The specific primer sets for each target
gene are listed in Table 1. Data values were normalized to EF1α
cDNA (internal control) and calculated by the 2−1CT method. Statistically significant differences between groups were analyzed
by Student’s t-test as described in Tseng et al. (19). September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 3 WSSV-Induced Glutamine Metabolism He et al. He et al. Determination of the Enzyme Activity of
IDH in Shrimp Stomachs and Hemocytes
During WSSV Infection The NADH standards supplied with the kit
were prepared in the same way as the tissue samples. IDH activity
was measured at A450 every 3–5 min until the value of the most
active sample was greater than the value of the standard with the
highest concentration (10 nmole). The difference in absorbance
at the initial (Tinitial) and final (Tfinal) time points was converted
to the NADPH amount (B) using the NADH standard curve at
the final time point. The enzyme activity was calculated by the
following equation: IDH activity (mU/mg) = B/([Tfinal – Tinitial]
× [total amount of protein in the reaction]). Statistical analysis
was performed as described above. In vivo Gene Silencing of LvIDH1, LvIDH2,
Lvα-KGDH Mediated by dsRNA
Interference Using Stable Isotope-Labeled Glutamine
Tracer and Liquid Chromatography
Electrospray Ionization Mass Spectrometry
(LC-ESI-MS) to Monitor Metabolites in the
Hemocytes of WSSV-Infected Shrimp
Stable isotope-labeled glutamine can be used together with LC-
ESI-MS to identify and quantify metabolites in the oxidative and
reductive glutamine metabolic pathways that have incorporated
the labeled glutamine carbon atoms. In this study, we used both
[U-13C]glutamine (M5 gln) and [1-13C]glutamine (M1 gln) to
trace the metabolites of interest as shown in Figure 1. As no
shrimp cell line or alternative cell line is presently available, we
established an in vivo, 13C-labeled shrimp hemocyte metabolic
analysis platform ab initio. Based on preliminary tests (data not
shown), our experimental protocol was to treat WSSV-challenged
shrimp with stable isotope-labeled glutamine by haemocoel
injection at 12 and 24 hpi after challenge. At 10 and 30 min
after injection of the M5 or M1 gln, pooled hemocyte samples
(4 pools; 3 shrimp in each pool) were collected and analyzed as
described below. Using Stable Isotope-Labeled Glutamine
Tracer and Liquid Chromatography
Electrospray Ionization Mass Spectrometry
(LC-ESI-MS) to Monitor Metabolites in the
Hemocytes of WSSV-Infected Shrimp y
p
Stable isotope-labeled glutamine can be used together with LC-
ESI-MS to identify and quantify metabolites in the oxidative and
reductive glutamine metabolic pathways that have incorporated
the labeled glutamine carbon atoms. In this study, we used both
[U-13C]glutamine (M5 gln) and [1-13C]glutamine (M1 gln) to
trace the metabolites of interest as shown in Figure 1. As no
shrimp cell line or alternative cell line is presently available, we
established an in vivo, 13C-labeled shrimp hemocyte metabolic
analysis platform ab initio. Based on preliminary tests (data not
shown), our experimental protocol was to treat WSSV-challenged
shrimp with stable isotope-labeled glutamine by haemocoel
injection at 12 and 24 hpi after challenge. At 10 and 30 min
after injection of the M5 or M1 gln, pooled hemocyte samples
(4 pools; 3 shrimp in each pool) were collected and analyzed as
described below. g
Data were acquired by HyStar and micrOTOF control
software (Bruker Daltonics) and processed by DataAnalysis
and TargetAnalysis software (Bruker Daltonics) to generate the
signals corresponding to the integrated areas for each extracted
ion chromatogram. To monitor the change in the quantity of the 13C labeled
metabolites, fold changes in the WSSV group were calculated
relative to the corresponding PBS group (WSSV/PBS group). All the signal counts were normalized by the sample’s weight
(mg). In vivo Gene Silencing of LvIDH1, LvIDH2,
Lvα-KGDH Mediated by dsRNA
Interference Statistically significant differences between WSSV and PBS
groups were analyzed by Student’s t-test as described above. Stable isotope tracer solutions were prepared by dissolving [U-
13C]glutamine (M5 gln; Cambridge Isotope Laboratories, Inc)
or [1-13C]glutamine (M1 gln; Cambridge Isotope Laboratories,
Inc) in PBS. At 12 or 24 h after the experimental shrimp had
been injected with WSSV or PBS, one of the stable isotope
tracer solutions ([U-13C]glutamine: 400 µg /g shrimp; [1-
13C]glutamine: 800 µg /g shrimp) was injected into the shrimp’s
abdominal hemal sinus. At 10 and 30 min after treatment with the
tracer, 4 pooled hemocyte samples (3 shrimp in each pool) were
collected from each group using a cold anticoagulant (1x PBS,
10 mM EDTA, pH8.0). After immediately centrifuging at 1,000x
g for 10 min by using a swinging bucket, the pellet of shrimp
hemocytes from each sample was collected, washed with ice-cold
1x PBS and the hemocytes were lysed with sterilized ddH2O on In vitro Synthesis of LvGLS2, LvIDH1,
LvIDH2, and Lvα-KGDH dsRNAs g
Hemocytes and stomachs from shrimp were collected at 12
and 24 h after WSSV or PBS injection (6 shrimp/pool and 4
pools/group) to measure the activity of α-KGDH. The hemocyte
and stomach samples were homogenized with 100 and 200 µl
ice cold KGDH assay buffer, respectively, from a commercial
α-Ketoglutarate Dehydrogenase Activity Colorimetric Assay Kit
(Sigma). The cell debris was removed by centrifugation at 4◦C
at 13,000 g for 10 min. After using a Bio-Rad protein assay to
measure protein concentrations, samples with the appropriate
amounts of protein (hemocyte lysate: 3.6 ∼50 µg; stomach lysate:
10 ∼20 µg) were collected and adjusted to a final volume of 50 µl
using the KGDH assay buffer. The lysates were then mixed with
50 µl reaction mixture containing 46 µl KGDH Assay Buffer,
2 µl KGDH Developer, and 2 µl KGDH Substrate. The final dsRNAs were prepared as described in our previous study (9,
20). In short, the partial sequences (300∼600 bp) of each host
gene and the non-specific Luciferase control were amplified by
PCR with the following respective primer sets: GLS2-F/GLS2-
R, IDH1-F/IDH1-R, IDH2-F/IDH2-R, α-KGDH-F/α-KGDH-R,
and Luc-F/Luc-R (Table 1). After obtaining the corresponding
PCR amplicons, T7 promoter sequence was added to the 5
′ and
3
′ ends of each amplicon by a second PCR with the primer sets:
T7-GLS2-F/GLS2-R, GLS2-F/ T7-GLS2-R, T7-IDH1-F/IDH1-R,
IDH1-F/T7-IDH1-R, T7-IDH2-F/IDH2-R, IDH2-F/T7-IDH2-
R, T7-α-KGDH-F/α-KGDH-R, α-KGDH-F/T7-α-KGDH-R, T7-
Luc-F/Luc-R, and Luc-F/T7-Luc-R (Table 1). The ssRNAs were
then synthesized by the T7 RiboMAX Express large-scale RNA September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org 4 He et al. WSSV-Induced Glutamine Metabolism production system (Promega) and the corresponding ssRNA
pairs were mixed together to form the dsRNAs. After the dsRNAs
were purified by phenol/chloroform extraction, quantified by UV
spectrophotometer and checked by agarose gel electrophoresis,
the final dsRNA products were stored at −80◦C before use. ice. Next, 100% MeOH was added to the cell lysate at a ratio
of 1:3 to quench the metabolic reactions, and the samples were
kept at −80◦C for 10 min. The samples were then centrifuged
at 4◦C at 18,000 g for 10 min, and 75% MeOH was added to
the cell lysate for secondary extraction. The supernatants, which
now contained the metabolites, were then collected, lyophilized,
and stored at −80◦C before being analyzed by LC-ESI-MS as
described previously (9). Frontiers in Immunology | www.frontiersin.org In vivo Gene Silencing of LvIDH1, LvIDH2,
Lvα-KGDH Mediated by dsRNA
Interference p
y
Briefly, for LC-ESI-MS analysis the samples were dissolved
in 35 µl of ddH2O and 5 µl of reaction buffer (0.3 M aniline
[Sigma-Aldrich, USA] in 60 mM HCl), and 5 µl of N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride
(EDC; Sigma-Aldrich, USA) was added. After incubating the
mixture at 25◦C for 2 h, 5 µl of 10% ammonium hydroxide was
added to stop the reaction. The derivatives were analyzed on
an LC-ESI-MS system comprising an ultra-performance liquid
chromatography (UPLC) system (Ultimate 3000 RSLC, Dionex)
and a quadrupole time-of-flight (QTOF) mass spectrometer
with an electrospray ionization (ESI) source (maXis HUR-
QToF
system,
Bruker
Daltonics). Reversed-phase
liquid
chromatography (RPLC) on a BEH C18 column (2.1 × 100 mm,
Waters) was used. The elution started from 99% mobile phase
A (0.1% formic acid in ddH2O) and 1% mobile phase B (0.1%
formic acid in ACN), held at 1% B for 0.5 min, raised to 60% B
in 6 min, further raised to 90% B in 0.5 min, held at 90% B for
1.5 min, and then lowered to 1% B in 0.5 min. The column was
then equilibrated by pumping 1% B for 4 min. The flow rate was
set at 0.3 ml/min with an injection volume of 10 µl. LC-ESI-MS
chromatograms were acquired under a capillary voltage of either
4,500 or 3,500 V in negative ion mode, a dry temperature of
190◦C, a dry gas flow maintained at 8 l/min, nebulizer gas at 1.4
bar, and an acquisition range of m/z 100–1,000. Shrimp (∼3 g body weight) were injected with the LvIDH1,
LvIDH2, Lvα-KGDH dsRNAs at a concentration of 1 µg /g
shrimp. Shrimp treated with Luc dsRNA were used as the control. At 72 h (3 days) post the dsRNA injection, shrimp hemocyte
samples were collected from each group and real-time PCR was
used to confirm that the LvIDH1, LvIDH2, and Lvα-KGDH genes
had been specifically silenced by the respective dsRNA. At the
same time, the remaining shrimp in each group were challenged
by injection with WSSV inoculum or PBS. At 24 h post injection,
shrimp hemocyte samples were collected (3 shrimp in each
sample, 4 samples for each group) and used to measure the
expression of host genes, viral genes and the WSSV genome copy
number. Statistical analysis was performed as described above. Effect of the Inhibitors Salirasib (S35),
Torin1, and LY294002 on the mRNA
Expression of Key Host Genes Involved in
Glutamine Metabolism During WSSV
Infection Following the protocol described in previous studies (8, 9,
19), shrimp were intramuscularly injected with 100 µl of the
inhibitors Salirasib, Torin1, and LY294002 or their vehicles 2 h
before being challenged with WSSV. Samples were then collected
at 12 and 24 h post WSSV injection. To evaluate the involvement
of shrimp Ras, shrimp were treated with Salirasib (dissolved in
99% EtOH and diluted with PBS, pH 8.0; 35 µg/g shrimp) or with September 2019 | Volume 10 | Article 2102 5 He et al. WSSV-Induced Glutamine Metabolism He et al. FIGURE 1 | Schematic showing how the metabolites derived from (A) [U-13C]glutamine can be traced through the oxidative and reductive glutamine pathways and
(B) [1-13C]glutamine can be traced through the reductive glutamine pathway. In the Gln-Glu-a-KG pathway, white circles represent 12Carbon, while solid black circles
represent 13Carbon. Blue circles represent 13C in the oxidative glutamine metabolism pathway of the TCA cycle and the citrate shuttle, while red circles represent 13C
in the reductive carboxylation pathways in either mitochondria or cytoplasm. The green arrow indicates the shift toward lipid metabolism. For [U-13C]glutamine in (A),
abbreviations are as follows: for the Gln-Glu-α-KG pathway: Gln, M5 glutamine; Glu, M5 glutamate; α-KG, M5 α-ketoglutarate. For oxidative glutamine metabolism:
Suc, M4 succinate; Fum, M4 fumarate; Mal, M4 malate; Oac, M4 oxaloacetate; Cit, M4 citrate; Lac, M3 lactate. For reductive carboxylation: Ict, M5 isocitrate; Cit, M5
citrate; Oac, M3 oxaloacetate; Asp, M3 aspartate; Fum, M3 fumarate; Mal, M3 malate. Similar abbreviations apply for [1-13C]glutamine (M1 glutamine) in (B). FIGURE 1 | Schematic showing how the metabolites derived from (A) [U-13C]glutamine can be traced through the oxidative and reductive glutamine pathways and
(B) [1-13C]glutamine can be traced through the reductive glutamine pathway. In the Gln-Glu-a-KG pathway, white circles represent 12Carbon, while solid black circles
represent 13Carbon. Blue circles represent 13C in the oxidative glutamine metabolism pathway of the TCA cycle and the citrate shuttle, while red circles represent 13C
in the reductive carboxylation pathways in either mitochondria or cytoplasm. The green arrow indicates the shift toward lipid metabolism. For [U-13C]glutamine in (A),
abbreviations are as follows: for the Gln-Glu-α-KG pathway: Gln, M5 glutamine; Glu, M5 glutamate; α-KG, M5 α-ketoglutarate. For oxidative glutamine metabolism:
Suc, M4 succinate; Fum, M4 fumarate; Mal, M4 malate; Oac, M4 oxaloacetate; Cit, M4 citrate; Lac, M3 lactate. Effect of the Inhibitors Salirasib (S35),
Torin1, and LY294002 on the mRNA
Expression of Key Host Genes Involved in
Glutamine Metabolism During WSSV
Infection For reductive carboxylation: Ict, M5 isocitrate; Cit, M5
citrate; Oac, M3 oxaloacetate; Asp, M3 aspartate; Fum, M3 fumarate; Mal, M3 malate. Similar abbreviations apply for [1-13C]glutamine (M1 glutamine) in (B). target genes, all samples were analyzed by real-time PCR as
described above. vehicle only (0.3% ethanol). Samples from Salirasib-pretreated
shrimp were collected into 4 pooled hemocyte samples, with
each sample being taken from 3 shrimp (19). To investigate
the involvement of the PI3K-mTORC1 pathway, shrimp were
treated with LY294002 (dissolved in 10% DMSO and diluted
with PBS; 0.625 µg/g shrimp) or with vehicle only (0.01%
DMSO). Hemocyte samples from LY294002-pretreated shrimp
were collected individually for each group (8). To observe the
involvement of mTORC1/mTORC2, shrimp were treated with
Torin 1 (dissolved in DMSO and diluted with PEG solvent;
20 µg/g shrimp) or with vehicle only (0.25% PEG, 0.25% Tween
20, and 0.15 M NaCl) five or six pleopod samples were collected
from each group, with each sample being taken from 10
shrimp (9). To determine the mRNA expression levels of the RESULTS In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Showed
That Both Glutaminolysis and Reductive
Carboxylation Were Activated at the WSSV
Genome Replication Stage (12 hpi)
To investigate the effect of WSSV in WSSV-infected hemocytes,
shrimp were challenged with WSSV, and then injected with [U-
13C]glutamine (M5 gln) to allow tracing of this stable carbon In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Showed
That Both Glutaminolysis and Reductive
Carboxylation Were Activated at the WSSV
Genome Replication Stage (12 hpi) In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Showed
That Both Glutaminolysis and Reductive
Carboxylation Were Activated at the WSSV
Genome Replication Stage (12 hpi) p
g
(
p )
To investigate the effect of WSSV in WSSV-infected hemocytes,
shrimp were challenged with WSSV, and then injected with [U-
13C]glutamine (M5 gln) to allow tracing of this stable carbon September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org 6 WSSV-Induced Glutamine Metabolism He et al. isotope through the glutamine metabolic pathways as shown
in Figure 1A. reductive carboxylation (Figure 1B). Compared to the PBS-
treated shrimp, there was generally an increase of M1 metabolites
in the glutamine-glutamate-αKG (Gln-Glu-α-KG) pathway in
shrimp hemocytes at the WSSV genome replication stage (12
hpi; Figures 4A,B), and there was also a general increase in
the amounts of the subsequent metabolites in the reductive
carboxylation pathway (Figures 4A,B). As before, these results
suggest that reductive carboxylation is triggered at the WSSV
genome replication stage. However, once again, there was
no clear pattern of increase in these metabolites at the late
stage of WSSV replication (Figures 4C,D). Figures 4E,F provide
graphical summaries of these results. Raw metabolomic data is
given in Table S2. Compared to the PBS treated shrimp, at 12 hpi, there
was an 8-fold increase in the amount of M5 glutamine
in shrimp hemocytes at 10 min after treatment with [U-
13C]glutamine (Figure 2A). At the same point, there was also
a significant increase in the final downstream product of
oxidative glutaminolysis, M3 lactate (Figure 2A). These data
suggest that, at 12 hpi, WSSV infection triggers glutamine
uptake into shrimp hemocytes and that at least some of this
glutamine is converted into lactate via oxidative glutaminolysis. Figure 2A also shows a significant increase in M5 isocitrate
and M5 citrate which suggests that the glutamine-dependent
reductive carboxylation pathway is also active in WSSV-infected
shrimp hemocytes at 12 hpi. RESULTS At 30 min after [U-13C]glutamine
treatment, similarity elevated amounts of the oxidative and
reductive pathway metabolites were still detected (Figure 2B),
but the relatively low levels of glutamine and downstream
products such as M4 citrate and M3 oxaloacetate suggest that
most of the labeled glutamine input might already have flushed
through these pathways. Figure 2C summarizes the changes
in these 13C glutamine metabolites in both the oxidative and
reductive directions at 12 hpi and 10 min after [U-13C]glutamine
treatment. The raw metabolomic data for [U-13C]glutamine and
its metabolites are given in Table S1. In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Suggests
That Glutaminolysis and Reductive
Carboxylation are No Longer Activated at
the Late Stage of the WSSV Replication
Cycle (24 hpi) In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Suggests
That Glutaminolysis and Reductive
Carboxylation are No Longer Activated at
the Late Stage of the WSSV Replication
Cycle (24 hpi) For GLS, which functions as the initial enzyme in glutamine
metabolism by converting glutamine to glutamate, we found
that although there was no significant change in the expression
of the two GLS isoforms, GLS1 and GLS2, the enzyme
activity was significantly decreased at both stages of the WSSV
replication cycle (12 and 24 hpi) in both hemocytes and
stomach (Figure 6A). For IDH1 and IDH2, which are the
key enzymes in reductive glutamine metabolism, significant
upregulation of IDH1 mRNA expression was observed at 12
and 24 hpi, while there was no change in IDH2. There was
also a significant increase in the enzyme activity of IDH
in shrimp hemocytes at 12 hpi, and a significant decrease
at 24 hpi; meanwhile in the stomach there was no change
(Figure 6B). For α-KGDH, which is a key enzyme in oxidative
glutamine metabolism, the mRNA levels and enzyme activity
were significantly increased in hemocytes at both 12 and 24
hpi (Figure 6C). α-KGDH enzyme activity was also significantly
increased in shrimp stomach (Figure 6C). Taken together, at
least at 12 hpi, WSSV seems to trigger both IDH1-mediated
reductive glutamine metabolism as well as α-KGDH-mediated
glutamine metabolism. At 24 hpi, although [U-13C]glutamine still seems to be taken up
by the WSSV-infected hemocytes, the elevated accumulation of
M5 α-KG and the reduced levels of the glutamine-derived TCA
intermediates (i.e., the M4 forms of succinate, fumarate, malate,
oxaloacetate, and the M3 forms of lactate) suggest that oxidative
metabolism of [U-13C]glutamine was not triggered at this stage
(Figures 3A,B). Similarly, there was no evidence of reductive
carboxylation, i.e., no obvious increase in the M5 forms of
isocitrate and citrate or the M3 forms of oxaloacetate, aspartate,
fumarate, and malate (Figures 3A,B). Figure 3C provides a
graphical summary of the above results. In vivo Tracking of [1-13C]
Glutamine-Derived Metabolites
Reconfirmed That Reductive Carboxylation
Was Activated at the WSSV Genome
Replication Stage (12 hpi) WSSV Activates Glutamine
Metabolism-Related Genes in L. vannamei
at the Genome Replication Stage Previous reports have shown that at the WSSV genome
replication stage (12 hpi), WSSV activates GDH and ASAT to
induce glutamine metabolism, which in turn fuels the TCA
cycle via α-KG (8). Here, to further investigate the enzymes
involved in WSSV-induced glutamine metabolism, we next
isolated and characterized 5 other important genes involved
in this pathway, LvGLS1, LvGLS2, LvIDH1, LvIDH2, and Lvα-
KGDH (Figure 5). The full-length or partial cDNAs of these 5
genes were identified from our in-house transcriptomic database,
and after amplification by PCR with the corresponding primer set
(Table 1), the amplicons were sequenced and confirmed to match
their respective genes (data not shown). Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication The above isotope-labeled tracing experiments were repeated
using [1-13C]glutamine instead of [U-13C]glutamine. With [1-
13C]glutamine, the first isotope-labeled carbon is lost during
oxidative conversion from α-KG to succinate, while this
same labeled carbon is kept in the metabolites produced by To further investigate the importance of GLS2, IDH1, IDH2, and
α-KGDH in WSSV replication, we performed dsRNA-mediated September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 7 He et al. WSSV-Induced Glutamine Metabolism ncreased 13C-labeled metabolites in oxidative glutaminolysis and reductive carboxylation at the WSSV replication stage (12 hpi). At 12 h after
V or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of
mples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
e PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the
control group (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Summary of changes in 13C glutamine metabolites in the oxidative and reductive
and 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows:
crease), yellow (no significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as Figure 1. of these genes with the corresponding dsRNAs. GLS1 dsRNA could not be synthesized because
his gene has not yet been completely determined,
and we failed to find any primer set that could successfully
produce any PCR amplicons.) As Figure 7A shows, at 72 h-
post dsRNA treatment, although the gene expression of GLS2 URE 2 | WSSV increased 13C-labeled metabolites in oxidative glutaminolysis and reductive carboxylation at the WSSV replication stage (12 hpi). At 12 h after
enge with WSSV or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of
ed hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the FIGURE 2 | WSSV increased 13C-labeled metabolites in oxidative glutaminolysis and reductive carboxylation at the WSSV replication stage (12 hpi). Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication At 12 h after
challenge with WSSV or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of
pooled hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
13C metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the
corresponding PBS control group (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Summary of changes in 13C glutamine metabolites in the oxidative and reductive
directions at 12 hpi and 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows:
green (significant decrease), yellow (no significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as Figure 1. FIGURE 2 | WSSV increased 13C-labeled metabolites in oxidative glutaminolysis and reductive carboxylation at the WSSV replication stage (12 hpi). At 12 h after
challenge with WSSV or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of
pooled hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
13C metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the
corresponding PBS control group (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Summary of changes in 13C glutamine metabolites in the oxidative and reductive
directions at 12 hpi and 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows:
green (significant decrease), yellow (no significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as Figure 1. in vivo silencing of these genes with the corresponding dsRNAs. (Unfortunately, GLS1 dsRNA could not be synthesized because
the sequence of this gene has not yet been completely determined, and we failed to find any primer set that could successfully
produce any PCR amplicons.) As Figure 7A shows, at 72 h-
post dsRNA treatment, although the gene expression of GLS2 in vivo silencing of these genes with the corresponding dsRNAs. Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication (Unfortunately, GLS1 dsRNA could not be synthesized because
the sequence of this gene has not yet been completely determined, September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 8 He et al. He et al. WSSV-Induced Glutamine Metabolism suppressed oxidative glutaminolysis and reductive carboxylation at the late stage of WSSV replication (24 hpi). At 24 h after challenge with WSSV
ere injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of pooled hemocyte samples
MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding 13C metabolite in the PBS
presents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the corresponding PBS control group (*p
***p < 0.001). (C) Schematic representation of 13C metabolic expression of glutamine metabolism in oxidative and reductive directions at 24 hpi
3C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows: green (significant
o significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as for Figure 1. relative to PBS control, the gene expressions
and α-KGDH were all specifically decreased. point, the shrimp were then injected with
WSSV and hemocytes were collected 24 h later. We found that
although the mRNA expression of IDH1 and α-KGDH were
still significantly suppressed by the corresponding dsRNAs and FIGURE 3 | WSSV suppressed oxidative glutaminolysis and reductive carboxylation at the late stage of WSSV replication (24 hpi). At 24 h after challenge with WSSV
or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of pooled hemocyte samples
by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding 13C metabolite in the PBS
group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the corresponding PBS control group (*p
< 0.05, **p < 0.01, ***p < 0.001). (C) Schematic representation of 13C metabolic expression of glutamine metabolism in oxidative and reductive directions at 24 hpi
at 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows: green (significant
decrease), yellow (no significant difference), red (significant increase), and white (non-detectable). Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication Abbreviations are the same as for Figure 1. FIGURE 3 | WSSV suppressed oxidative glutaminolysis and reductive carboxylation at the late stage of WSSV replication (24 hpi). At 24 h after challenge with WSSV
or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of pooled hemocyte samples
by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding 13C metabolite in the PBS
group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the corresponding PBS control group (*p
< 0.05, **p < 0.01, ***p < 0.001). (C) Schematic representation of 13C metabolic expression of glutamine metabolism in oxidative and reductive directions at 24 hpi
at 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows: green (significant
decrease), yellow (no significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as for Figure 1. WSSV and hemocytes were collected 24 h later. We found that
although the mRNA expression of IDH1 and α-KGDH were
still significantly suppressed by the corresponding dsRNAs and was unchanged relative to PBS control, the gene expressions
of IDH1, IDH2, and α-KGDH were all specifically decreased. After this time point, the shrimp were then injected with September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 9 He et al. WSSV-Induced Glutamine Metabolism RE 4 | WSSV activates reductive carboxylation at the WSSV genome replication stage (12 hpi) and suppresses it at the late stage of WSSV replication (24 hpi). h after challenge with WSSV or PBS, shrimps were injected with [1-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. At 24 h after
enge with WSSV or PBS, shrimps were also injected with [1-13C]glutamine and hemocytes were collected (C) 10 min or (D) 30 min later. Metabolomic analysis of
d hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the
sponding PBS control group (*p < 0.05, **p < 0.01, ***p < 0.001). Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication WSSV-Induced Glutamine Metabolism He et al. FIGURE 5 | Simplified schematic of oxidative and reductive glutamine metabolic pathways. Glutamine metabolism in the oxidative direction (glutaminolysis) is shown
in blue, glutamine metabolism in the reductive direction (reductive carboxylation) is shown in red, and shared metabolic pathways are shown in black. Enzymes: GLS,
glutaminase; GDH, glutamine dehydrogenase; ASAT, aspartate aminotransferase; α-KG, α-ketoglutarate; IDH, isocitrate dehydrogenase; α-KGDH, α-ketoglutarate
dehydrogenase. Metabolites: abbreviations are the same as for Figure 1. FIGURE 5 | Simplified schematic of oxidative and reductive glutamine metabolic pathways. Glutamine metabolism in the oxidative direction (glutaminolysis) is shown
in blue, glutamine metabolism in the reductive direction (reductive carboxylation) is shown in red, and shared metabolic pathways are shown in black. Enzymes: GLS,
glutaminase; GDH, glutamine dehydrogenase; ASAT, aspartate aminotransferase; α-KG, α-ketoglutarate; IDH, isocitrate dehydrogenase; α-KGDH, α-ketoglutarate
dehydrogenase. Metabolites: abbreviations are the same as for Figure 1. the mRNA level of GDH, but also drives an increase in reductive
glutamine metabolism by increasing the mRNA levels of IDH2 at
the WSSV genome replication stage. that GLS2 was also suppressed at this time, the mRNA level of
IDH2 was not significantly different from the PBS or Luciferase
controls (Figure 7B). We further found that there was significant
suppression of WSSV VP28 mRNA expression and WSSV viral
copy numbers in the WSSV-injected groups pretreated with
IDH1, IDH2, and α-KGDH dsRNA, while in the GLS2 group,
VP28 mRNA was significantly suppressed even though the viral
copy number was not (Figures 7C,D). Taken together, these data
suggest that IDH1, IDH2, α-KGDH, and to a lesser extent, GLS
might all be important for WSSV replication. To investigate the role of the PI3K-Akt-mTOR pathway,
shrimps were pretreated with LY294002 to inhibit both PI3K and
mTORC1. In LY294002-treated shrimps, at both 12 and 24 hpi,
there was no significant difference in the mRNA levels of IDH1,
IDH2, or α-KGDH compared to the control group (Figure 8C). In shrimps treated with Torin1, which was used to inhibit both
of the mTOR complexes, there was no impact on any of the
three enzymes at 12 hpi, whereas, at 24 hpi, gene expression
of IDH1 decreased while IDH2 and α-KGDH mRNA increased
(Figure 8D). Taken together, these results suggest that at 12 hpi,
glutamine metabolism is regulated by Ras but not by the PI3K-
Akt-mTOR pathway. Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication (E,F) Schematic representation of 13C metabolic expression of glutamine metabolism in the
ctive direction at (E) 12 and (F) 24 hpi at 10 min after [1-13C]glutamine treatment. Changes in the WSSV groups relative to the corresponding PBS controls are
coded as follows: green (significant decrease), yellow (no significant difference), red (significant increase), gray (not measured), and white (non-detectable). eviations are the same as for Figure 1. FIGURE 4 | WSSV activates reductive carboxylation at the WSSV genome replication stage (12 hpi) and suppresses it at the late stage of WSSV replication (24 hpi). At 12 h after challenge with WSSV or PBS, shrimps were injected with [1-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. At 24 h after
challenge with WSSV or PBS, shrimps were also injected with [1-13C]glutamine and hemocytes were collected (C) 10 min or (D) 30 min later. Metabolomic analysis of
pooled hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding FIGURE 4 | WSSV activates reductive carboxylation at the WSSV genome replication stage (12 hpi) and suppresses it at the late stage of WSSV replication (24 hpi). At 12 h after challenge with WSSV or PBS, shrimps were injected with [1-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. At 24 h after
challenge with WSSV or PBS, shrimps were also injected with [1-13C]glutamine and hemocytes were collected (C) 10 min or (D) 30 min later. Metabolomic analysis of
pooled hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
13C metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the
corresponding PBS control group (*p < 0.05, **p < 0.01, ***p < 0.001). (E,F) Schematic representation of 13C metabolic expression of glutamine metabolism in the
reductive direction at (E) 12 and (F) 24 hpi at 10 min after [1-13C]glutamine treatment. Changes in the WSSV groups relative to the corresponding PBS controls are
color coded as follows: green (significant decrease), yellow (no significant difference), red (significant increase), gray (not measured), and white (non-detectable). Abbreviations are the same as for Figure 1. September 2019 | Volume 10 | Article 2102 10 Frontiers in Immunology | www.frontiersin.org He et al. Genome Replication Stage Previous reports have suggested that the PI3K-Akt-mTOR
pathway is involved in triggering both the WSSV-induced
Warburg effect at 12 hpi (9) and WSSV-induced lipogenesis at 24
hpi (7). Meanwhile, mTORC2 is a key regulator for upregulating
the expression of GDH mRNA independently of PI3K-mTORC1
at 12 hpi (8), while LvRas has been shown to play a positive role
in triggering WSSV-induced PI3K-Akt-mTOR activation (19). To investigate which of these pathways might regulate oxidative
and reductive glutamine metabolism during WSSV infection,
shrimp were treated with three specific inhibitors (Figure 8A)
and the mRNA expression of key genes involved in glutamine
metabolism was monitored. Frontiers in Immunology | www.frontiersin.org Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication Meanwhile, at 24 hpi, mTORC2 is pre-
dominantly responsible for the down-regulation of glutamine
metabolism, and it acts independently of Ras and the PI3K-
mTORC1 pathway. DISCUSSION We have shown here that, as in other cancer or virally infected
cells that are in a state of aerobic glycolysis (10, 22, 23), both
oxidative glutaminolysis and reductive carboxylation have been
triggered in WSSV-infected shrimp hemocytes (Figures 2–4). The present results are mostly consistent with previous findings
for WSSV-induced glutaminolysis. For example, the significantly
increased levels of M4 citrate in Figure 2A can be accounted for
by the upregulation of citrate synthase at the protein level, as
reported by Su et al. (9). Further, the patterns of changes in the
levels of various metabolites suggest that these pathways might be
more active at the genome replication stage (12 hpi) than in the
late stage (24 hpi). This conclusion is further supported by our In shrimps treated with Salirasib, a Ras inhibitor which
disrupts the spatiotemporal localization of active Ras (21), the
gene expression of GDH and IDH2 was significantly decreased
at 12 hpi (Figure 8B). At the same time, IDH1 mRNA was
significantly increased. At 24 hpi, inhibition of Ras caused no
change in the mRNA levels of these glutamine metabolism-
related genes. These data suggest that Ras not only upregulates September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 11 WSSV-Induced Glutamine Metabolism He et al. He et al. FIGURE 6 | WSSV induces IDH and α-KGDH expression at the WSSV replication stage (12 hpi) in shrimp hemocytes. (A) The mRNA levels of GLS1 and GLS2 and
the GLS enzyme activity in shrimp hemocytes and stomachs during WSSV infection. (B) The mRNA levels of IDH1 and IDH2 and the IDH enzyme activity in shrimp
hemocytes and stomachs during WSSV infection. (C) The mRNA level and the enzyme activity of α-KGDH in shrimp hemocytes and stomachs during WSSV infection. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the corresponding PBS control group (*p <
0.05, **p < 0.01). FIGURE 6 | WSSV induces IDH and α-KGDH expression at the WSSV replication stage (12 hpi) in shrimp hemocytes. (A) The mRNA levels of GLS1 and GLS2 and
the GLS enzyme activity in shrimp hemocytes and stomachs during WSSV infection. (B) The mRNA levels of IDH1 and IDH2 and the IDH enzyme activity in shrimp
hemocytes and stomachs during WSSV infection. September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org DISCUSSION (C) The mRNA level and the enzyme activity of α-KGDH in shrimp hemocytes and stomachs during WSSV infection. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the corresponding PBS control group (*p <
0.05, **p < 0.01). WSSV replication was shown by the significant reduction in
VP28 mRNA and viral genome copies when α-KGDH expression
was silenced by dsRNA (Figures 7C,D). We note too that since
enzymes such as IDH1 and α-KGDH appear to be necessary for
the virus to replicate, they might also be useful as biomarkers for
developing disease-resistant shrimp. dsRNA silencing results for several key enzymes. In particular,
we found that in L. vannamei hemocytes, IDH activity was
induced at 12 hpi, although only cytoplasmic IDH1 showed an
increase in mRNA expression, while the mitochondrial IDH2
was unchanged (Figure 6). Shrimp IDH1 silencing also led
to stronger suppression of the WSSV genome copy number
(Figure 7). From this we infer that cytoplasmic IDH1 is likely
to play a relatively more important role in triggering WSSV-
induced reductive carboxylation. We also note that IDH1 plays
a key role in NADPH production and acts as a potential regulator
of lipid metabolism in cancer cells and in virus-infected cells
with reductive metabolism (23, 24). At the same time (12 hpi),
there was also an increase in α-KGDH activity (Figure 6B),
suggesting that the TCA cycle was also upregulated in the
oxidative direction. Again, the importance of this enzyme to While oxidative glutamine metabolism provides an alternative
mechanism to produce ATP and, like glycolysis, results
in the accumulation of lactate as an end product, the
reductive carboxylation pathway is important for providing the
macromolecular precursors of lipid synthase (18, 23, 24). In a
previous study, we found that lipogenesis, which is essential for
the creation of the lipid-containing fraction of the WSSV viral
envelope, was induced at 24 hpi (7). Here, however, our results
suggest that reductive carboxylation might be more active at 12 September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 12 He et al. He et al. WSSV-Induced Glutamine Metabolism FIGURE 7 | IDH1, IDH2, α-KGDH, and to a lesser extent, GLS are involved in WSSV replication. DISCUSSION WSSV-Induced Glutamine Metabolism FIGURE 8 | Ras is a primary regulator of reductive glutamine metabolism at the WSSV genome replication stage. (A) Schematic representation of the Ras and
PI3K-Akt-mTOR pathways and the three inhibitors used in this experiment (black boxes). Shrimp were treated with the various inhibitors or vehicle only 2 h before
WSSV infection. (B) Real-time PCR analysis shows that the RAS inhibitor Salirasib had a significant effect on the expression of GDH, IDH1, and IDH2 in shrimp
hemocytes after WSSV infection. (C) Real-time PCR analysis shows that the PI3K-mTOR inhibitor LY29400 had no significant effect on the expression of IDH1, IDH2,
and α-KGDH in shrimp hemocytes after WSSV infection. (D) Real-time PCR analysis shows that the mTORC1/C2 inhibitor Torin 1 had a significant effect on the
expression of IDH1, IDH2, and α-KGDH in shrimp pleopods after WSSV infection. Bars represent the mean ± SD. Asterisks indicate statistically significant differences
in WSSV-injected shrimp between the inhibitor treated groups and the corresponding vehicle-only control (*p < 0.05, **p < 0.01). synthesis in WSSV-infected cells that are in a state of aerobic
We found in an earlier study that although WSSV stimulates FIGURE 8 | Ras is a primary regulator of reductive glutamine metabolism at the WSSV genome replication stage. (A) Schematic representation of the Ras and
PI3K-Akt-mTOR pathways and the three inhibitors used in this experiment (black boxes). Shrimp were treated with the various inhibitors or vehicle only 2 h before
WSSV infection. (B) Real-time PCR analysis shows that the RAS inhibitor Salirasib had a significant effect on the expression of GDH, IDH1, and IDH2 in shrimp
hemocytes after WSSV infection. (C) Real-time PCR analysis shows that the PI3K-mTOR inhibitor LY29400 had no significant effect on the expression of IDH1, IDH2,
and α-KGDH in shrimp hemocytes after WSSV infection. (D) Real-time PCR analysis shows that the mTORC1/C2 inhibitor Torin 1 had a significant effect on the
expression of IDH1, IDH2, and α-KGDH in shrimp pleopods after WSSV infection. Bars represent the mean ± SD. Asterisks indicate statistically significant differences
in WSSV-injected shrimp between the inhibitor treated groups and the corresponding vehicle-only control (*p < 0.05, **p < 0.01). FIGURE 8 | Ras is a primary regulator of reductive glutamine metabolism at the WSSV genome replication stage. (A) Schematic representation of the Ras and
PI3K-Akt-mTOR pathways and the three inhibitors used in this experiment (black boxes). DISCUSSION (A) Gene expression of GLS2, IDH1, IDH2, and α-KGDH in shrimp
hemocytes was analyzed by real-time PCR at 72 h-post injection of the corresponding dsRNA and before WSSV challenge. (B) Gene expression of the above genes
was measured again in dsRNA-treated shrimp at 24 h post WSSV injection. (C,D) The effect of gene silencing of GLS2, IDH1, IDH2, and α-KGDH on the expression
of the WSSV gene VP28 and WSSV genome copy numbers at 24 h post WSSV injection. Groups treated with PBS only or with non-specific luciferase (Luc) dsRNA
were used as control groups. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the indicated groups (*p < 0.05,
**p < 0.01, ***p < 0.001). hpi rather than 24 hpi (Figures 2 4)
hich
o ld impl
that
follo ing M nger et al (25)
ho sho ed that HCMV
as able FIGURE 7 | IDH1, IDH2, α-KGDH, and to a lesser extent, GLS are involved in WSSV replication. (A) Gene expression of GLS2, IDH1, IDH2, and α-KGDH in shrimp
hemocytes was analyzed by real-time PCR at 72 h-post injection of the corresponding dsRNA and before WSSV challenge. (B) Gene expression of the above genes
was measured again in dsRNA-treated shrimp at 24 h post WSSV injection. (C,D) The effect of gene silencing of GLS2, IDH1, IDH2, and α-KGDH on the expression
of the WSSV gene VP28 and WSSV genome copy numbers at 24 h post WSSV injection. Groups treated with PBS only or with non-specific luciferase (Luc) dsRNA
were used as control groups. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the indicated groups (*p < 0.05,
**p < 0.01, ***p < 0.001). following Munger et al. (25), who showed that HCMV was able
to divert glucose-derived carbon away from lactate and into
fatty acid synthesis, it is also possible that in the same way,
the glycolysis pathway might act as the carbon source for lipid hpi rather than 24 hpi (Figures 2–4), which would imply that
although lipogenesis might still occur at the late stage, the lipid
macromolecular precursors are in fact being produced at an
earlier time point than was previously proposed. Alternatively, September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 13 He et al. He et al. DISCUSSION Further, although our experiments here
demonstrate that labeled glutamine can be used as the carbon
source, as noted above, our previous study found that WSSV-
induced glutaminolysis was driven by glutamate rather than
glutamine (8). This is consistent with the observed down-
regulation of GLS activity (Figure 6A), because although this
enzyme would still provide some of the carbon input, it would
no longer be essential for driving this metabolic pathway. Figure 9
provides
a
comprehensive
schematic
that
summarizes the increased mRNA levels of all of the above
enzymes in shrimp hemocytes at 12 hpi when the Warburg
effect is activated. The same figure also shows the larger context,
including the PI3K-Akt-mTOR pathway [by which the Warburg
effect is triggered; (9)] and the means by which WSSV is able to
replenish the TCA cycle by triggering glutamine metabolism, i.e.,
the Gln-Glu-α-KG pathway, oxidative glutamine metabolism
and reductive carboxylation. the WSSV-infected hemocytes. The first step in this metabolic
pathway is the conversion of glutamine to glutamate by GLS,
and it is interesting to note that while WSSV upregulates the
activity of both GDH and ASAT (8), we found here that the
mRNA expression levels of shrimp GLS were unchanged and its
enzyme activity was actually reduced (Figure 6A). This is also in
contrast to other similar instances of metabolic reprogramming
during aerobic glycolysis, such as HCMV for example, where
glutaminolysis is associated with the increased activity of both
GLS and GDH (26). In the case of WSSV, we therefore
hypothesize that the increased uptake of glutamine might instead
be driven by the high demand created by the conversion of
glutamate to α-KG. Further, although our experiments here
demonstrate that labeled glutamine can be used as the carbon
source, as noted above, our previous study found that WSSV-
induced glutaminolysis was driven by glutamate rather than
glutamine (8). This is consistent with the observed down-
regulation of GLS activity (Figure 6A), because although this
enzyme would still provide some of the carbon input, it would
no longer be essential for driving this metabolic pathway. In our previous research, we found that WSSV induces
glutamine metabolism independently of the PI3K-Akt-mTORC1
pathway and instead uses mTORC2 to up-regulate GDH
mRNA (8). DISCUSSION Shrimp were treated with the various inhibitors or vehicle only 2 h before
WSSV infection. (B) Real-time PCR analysis shows that the RAS inhibitor Salirasib had a significant effect on the expression of GDH, IDH1, and IDH2 in shrimp
hemocytes after WSSV infection. (C) Real-time PCR analysis shows that the PI3K-mTOR inhibitor LY29400 had no significant effect on the expression of IDH1, IDH2,
and α-KGDH in shrimp hemocytes after WSSV infection. (D) Real-time PCR analysis shows that the mTORC1/C2 inhibitor Torin 1 had a significant effect on the
expression of IDH1, IDH2, and α-KGDH in shrimp pleopods after WSSV infection. Bars represent the mean ± SD. Asterisks indicate statistically significant differences
in WSSV-injected shrimp between the inhibitor treated groups and the corresponding vehicle-only control (*p < 0.05, **p < 0.01). synthesis in WSSV-infected cells that are in a state of aerobic
glycolysis. Unfortunately, in the present study, we were unable
to resolve this question because the Ac-CoA metabolite could
not be detected, but we are currently working on a new set
of experiments that use 13C-labeled glucose to trace Ac-CoA’s
carbon source. We found in an earlier study that although WSSV stimulates
glutamine metabolism to fuel the TCA cycle via α-KG (8),
the carbon source that was taken up by the WSSV-infected
hemocytes was glutamate rather than glutamine. In the present
study, our stable isotope results (Figures 2–4) clearly show
that glutamine is also being taken up and metabolized by September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 14 He et al. He et al. WSSV-Induced Glutamine Metabolism FIGURE 9 | Schematic representation of metabolism in WSSV-infected hemocytes showing the aerobic glycolysis, glutamine metabolism and lipogenesis pathways FIGURE 9 | Schematic representation of metabolism in WSSV-infected hemocytes showing the aerobic glycolysis, glutamine metabolism and lipogenesis pathways
at the WSSV genome replication stage (12 hpi). This figure was compiled from data obtained in the present study and previous studies (7–9, 19). Enzymes are shown
in boxes where red indicates an upregulated enzyme and yellow indicates the enzyme was unchanged. Oxidative glutamine metabolism and reductive carboxylation
are indicated by blue and red arrows, respectively. Green arrows represent aerobic glycolysis and the pentose phosphate pathway (PPP). WSSV-triggered lipogenesis,
which is triggered at the late stage, is shown in purple. Round boxes and gray arrows indicate enzyme activity and signaling pathways. Pyr, pyruvate; CS, citrate
synthase; FA, fatty acids. DISCUSSION All other abbreviations are the same as for Figure 1. FIGURE 9 | Schematic representation of metabolism in WSSV-infected hemocytes showing the aerobic glycolysis, glutamine metabolism and lipogenesis pathways
at the WSSV genome replication stage (12 hpi). This figure was compiled from data obtained in the present study and previous studies (7–9, 19). Enzymes are shown
in boxes where red indicates an upregulated enzyme and yellow indicates the enzyme was unchanged. Oxidative glutamine metabolism and reductive carboxylation
are indicated by blue and red arrows, respectively. Green arrows represent aerobic glycolysis and the pentose phosphate pathway (PPP). WSSV-triggered lipogenesis,
which is triggered at the late stage, is shown in purple. Round boxes and gray arrows indicate enzyme activity and signaling pathways. Pyr, pyruvate; CS, citrate
synthase; FA, fatty acids. All other abbreviations are the same as for Figure 1. FIGURE 9 | Schematic representation of metabolism in WSSV-infected hemocytes showing the aerobic glycolysis, glutamine metabolism and lipogenesis pathways
at the WSSV genome replication stage (12 hpi). This figure was compiled from data obtained in the present study and previous studies (7–9, 19). Enzymes are shown
in boxes where red indicates an upregulated enzyme and yellow indicates the enzyme was unchanged. Oxidative glutamine metabolism and reductive carboxylation
are indicated by blue and red arrows, respectively. Green arrows represent aerobic glycolysis and the pentose phosphate pathway (PPP). WSSV-triggered lipogenesis,
which is triggered at the late stage, is shown in purple. Round boxes and gray arrows indicate enzyme activity and signaling pathways. Pyr, pyruvate; CS, citrate
synthase; FA, fatty acids. All other abbreviations are the same as for Figure 1. the WSSV-infected hemocytes. The first step in this metabolic
pathway is the conversion of glutamine to glutamate by GLS,
and it is interesting to note that while WSSV upregulates the
activity of both GDH and ASAT (8), we found here that the
mRNA expression levels of shrimp GLS were unchanged and its
enzyme activity was actually reduced (Figure 6A). This is also in
contrast to other similar instances of metabolic reprogramming
during aerobic glycolysis, such as HCMV for example, where
glutaminolysis is associated with the increased activity of both
GLS and GDH (26). In the case of WSSV, we therefore
hypothesize that the increased uptake of glutamine might instead
be driven by the high demand created by the conversion of
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his helpful criticism of the manuscript. We thank Mr. Paul Barlow, National Cheng Kung University, for
his helpful criticism of the manuscript. FUNDING This study was supported financially by the Ministry of Science
and Technology (MOST 107-2313-B-006-006-MY3, MOST 107-
3017-F-006-001, MOST 108-2314-B-006-096-MY3, MOST 107-
2311-B-039-001, and CMU-107-N-16). DATA AVAILABILITY The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02102/full#supplementary-material All datasets generated for this study are included in the
manuscript/Supplementary Files. DISCUSSION Here we further showed that although Ras can
sometimes act as an upstream regulator of the PI3K-Akt-
mTOR and Raf-MEK-ERK pathways (27, 28), in WSSV-infected
hemocytes, it acts independently to significantly upregulate
the gene expression of GDH (Figure 8B). Taken together,
it thus appears that Ras and mTORC2 may both regulate
the gene expression level of GDH. As shown in Figure 9,
however, their exact relationship remains unclear. Although September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 15 He et al. WSSV-Induced Glutamine Metabolism AUTHOR CONTRIBUTIONS it is possible that Ras acts directly as a GDH regulator, in
Dictyostelium it promotes cell migration via the activation of
mTORC2 (29). mTORC2 has also been reported to encode a
Ras binding domain that may be correlated to Ras regulation
(30), and in cancer research, oncogenic Ras binds to mTORC2
and stimulates its activation to promote cell proliferation and
tumorigenesis (31). Further research will be needed to establish
whether or not the effect of Ras on GDH is mediated by
mTORC2 in a similar way. Lastly, there remaining the question
of which viral factors might be involved in this metabolic
reprogramming. In the case of dengue virus, which leads to
increased glycolysis in the host, Allonso et al. found that
viral NS1 protein interacts with glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and upregulates its activity (32). To
address the question of how WSSV might regulate glutamine
metabolism, we are now working with a yeast two-hybridization
platform that has so far been used to select several viral factor
candidates that can interact with enzymes related to this pathway. We hope to further explore these potential mechanisms in a
future study. S-TH, C-YT, and C-YL designed and performed in vivo animal
experiments and analyzed data. D-YL performed LC-ESI-MS-
based isotopic labeled metabolomic analysis. S-TH, D-YL,
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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
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Sci. (2011)
36:320–8. doi: 10.1016/j.tibs.2011.03.006 28. Robey RB, Hay N. Is Akt the “Warburg kinase”?-Akt-energy metabolism
interactions
and
oncogenesis. Semin
Cancer
Biol. (2009)
19:25–31. doi: 10.1016/j.semcancer.2008.11.010 September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 17
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Editorial - CITAR Journal, Volume 9, No. 3 – Special Issue: xCoAx 2017
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Journal of science and technology of the arts
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Mario Verdicchio
University of Bergamo
-----
mario.verdicchio@unibg.it
----- André Rangel
CITAR / Portuguese Catholic University
-----
a@3kta.net
----- André Rangel
CITAR / Portuguese Catholic University
-----
a@3kta.net
----- Luísa Ribas
Faculty of Fine Arts,
University of Lisbon
-----
l.ribas@belasartes.ulisboa.pt
----- Miguel Carvalhais
ID+ / Faculty of Fine Arts,
University of Porto
Portugal
-----
mcarvalhais@fba.up.pt
----- rely on algorithms to fulfil their desires, how humans
adapt to algorithms, how humans show algorithmic
characteristics, like breaks, flows, halts, especially
when they are making art. This special issue of the CITAR Journal of Science
and Technology of the Arts is dedicated to the 5th
International
Conference
on
Computation,
Communication, Aesthetics and X (xCoAx), which
took place in Lisbon, Portugal, from July 5 to 7, 2017. Nake and Grabowski wade in similar waters, but in
the opposite direction: by focusing on re-coding, they
propose
the
intellectual
exercise
of
reverse
engineering a visual artwork back to the original
algorithm that generated it. To prevent the risk of
reducing a human being to a data source while
reducing worldly processes such as creativity to
computational ones, the authors propose to insert re-
coding into an art education framework. xCoAx is a multimodal event, including an art
exhibition, a performance night, and a conference
where scholars from all over the world present and
discuss their ideas. We are very grateful to the CITAR
journal for having us as guest editors and for giving
us this space where some of the more stimulating
ideas that emerged from xCoAx could be further
elaborated. Alves da Veiga expands the discourse of generative
art from the realm of visual art to performance. He
proposes a model for the creation of performances
based on stochastic systems that generate numeric
values interpreted as components of a theatrical
experience. As in any computational system that is
meant to interact with the real world, such
interpretation is based on a mapping technique, and
the one proposed here addresses seven fundamental
variables: light, space, plane, form, motion, sound
and, last but not least, the human. The papers included in this issue are possibly among
the most ambitious we have ever had the pleasure to
read in our still short yet intense history with xCoAx. The ambition, in this case, is not to go onwards or
upwards, but inwards: towards the core of what
computation is and towards the core of what it means
to be human. Journal of Science and Technology of the Arts, Volume 9, No. 3 – Special Issue: xCoAx 2017 Journal of Science and Technology of the Arts, Volume 9, No. 3 – Special Issue: xCoAx 2017 Journal of Science and Technology of the Arts, Volume 9, No. 3 – Special Issue: xCoAx 2017 Editorial Mario Verdicchio
University of Bergamo
-----
mario.verdicchio@unibg.it
----- BIOGRAPHICAL INFORMATION Information technology is indeed ubiquitous, and
Papadimitraki brings our attention back to the outside,
with a focus on the environment surrounding us, in
terms of space and architecture. The hot topic on
everybody’s mouth nowadays is smart cities, but she
uses them only as a starting point of a much deeper
discourse on spatial ontology, to which the use of
code to create architectural objects adds a new
temporal dimension that deserves investigation. Luísa Ribas holds a PhD in Art & Design (2012), a
Master in Multimedia Art and a Degree in
Communication Design from the Faculty of Fine Arts,
University
of Porto. Her research addresses
interactive systems as aesthetic artifacts, their design
and experience, while focusing on sound-image
relations. She has contributed to publications and
events on digital art and design as a collaborator of
ID+ and CIEBA research centers. As a professor at
the Faculty of Fine-Arts, University of Lisbon, she
teaches Communication Design with a focus on print
and digital computational media, being currently the
scientific coordinator of the Master in Communication
Design and New Media. Coding can be an extremely useful instrument in
many endeavours, but it is different from the physical
tools we use to build or fix objects: it is, after all,
language. Temkin reminds us of the intrinsically
linguistic nature of code in the most playful way, by
giving us an overview of the most bizarre
programming languages out there, some of which are
designed in a way that it is impossible to write a
functioning program with them. Those who create
such languages do not consider themselves artists,
but their challenge to the very idea of coding is
nothing short of creative. André Rangel, 1971. Intermedia artist-cum-designer
that holds a PhD in Science and Technology of the
Arts, a Master in Digital Arts and a Degree in
Communication Design. Currently Guest Assistant
Professor at the Faculty of Fine Arts of the University
of Porto and Researcher at the Research Center for
Science and Technology of the Arts. 3kta.net founder
and director, xCoAx.org co-founder and co-organizer. Even without venturing into the extremes of non-
functioning code, complex systems always pose
several problems, glitches, hiccups, due to the great
number of interacting parts. Eustáquio invites us to a
radical
change
of
perspective,
by
adopting
interference as a paradigm to describe and design
interactive systems. Mario Verdicchio
University of Bergamo
-----
mario.verdicchio@unibg.it
----- Of course, if ultimate answers are still
(and possibly forever) out of our reach, the
comparative results we obtain from the search for
commonalities and differences between computers
and humans are interesting enough for us to continue
with the quest. Computing technology is indeed a way to model the
human, but Hernández-Ramírez takes the discourse
one step further, analysing what happens when
technology creeps into the human to modify it from Rutz goes back to analyse what an algorithm is with
a specific focus on the relation humans have with
algorithms in their creative endeavours: how humans CITARJ
1 itself is already pushing us to rethink who we are, how
we communicate, create, and modify the environment
we live in. the inside. If self-modification is an ancient practice,
he argues, new technologies enable us to tinker with
the human dimension in a new way, based not only
on the materiality of the body, but on the information
that describes us in all our aspects, bodily and
mental. Come join in the conversation. Come join in the conversation. BIOGRAPHICAL INFORMATION He analyses a number of works
in which interference in no longer seen as an
unwanted side effect, but it allows for the discovery of
new potential, and provides a new space for creative
expression and collaborative engagement. Mario Verdicchio was born in Milano, Italy in 1975. He
obtained a PhD in Information Engineering in 2004 at
Politecnico di Milano, where he worked in the Artificial
Intelligence and Robotics group. He co-founded
xCoAx in 2012 while he was a researcher at the
School of Engineering at the University of Bergamo,
Italy. His collaborations include the University of
Virginia, USA, the University of Porto, Portugal, and
the University of the West of Scotland, UK, where he
currently works as a lecturer at the School of Media,
Culture and Society. We are back at it again: we have never ceased
wondering what we are, and we will not leave any
path untapped in this quest. From this very general,
existential and epistemological perspective, art and
computation are just two different ways to pursue the
same goal. In trying to understand ourselves, we
have built computational artefacts that are meant to
work with us, for us, like us and, in some cases,
instead of us. Whether computational technology is a
new way of doing old things or a radically new realm
remains to be seen. What is sure is that the question Miguel Carvalhais is a designer and musician. He’s
an Assistant Professor at the Faculty of Fine Arts of
the University of Porto, a researcher at INESC TEC
and a fellow at V2_ Lab for the Unstable Media. He’s
the author of “Artificial Aesthetics: Creative Practices
in Computational Art and Design”. CITARJ
2 CITARJ
2 2
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The Draft Genome Sequence of Paenibacillus polymyxa Strain CCI-25 Encompasses High Potential for Secondary Metabolite Production
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Genome announcements
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The Draft Genome Sequence of Paenibacillus polymyxa Strain CCI-25
Encompasses High Potential for Secondary Metabolite Production The Draft Genome Sequence of Paenibacillus polymyxa Strain CCI-25
Encompasses High Potential for Secondary Metabolite Production jender Aleti, Livio Antonielli, Erika Corretto, Branislav Nikolic´, Angela Sessitsch, Günter Brader Bioresources Unit, Health and Environment Department, Austrian Institute of Technology (AIT), Tulln, Austria We report here the draft genome sequence of Paenibacillus polymyxa strain CCI-25, which displays strong antifungal and anti-
bacterial activities in vitro. The genome encompasses nonribosomal peptide synthetases predicted to encode a tridecaptin, poly-
myxin, fusaricidin, an iturin-like synthetase, a lantibiotic similar to paenicidin A, as well as a type 1 polyketide synthase. Received 24 March 2016 Accepted 29 March 2016 Published 19 May 2016 Received 24 March 2016 Accepted 29 March 2016 Published 19 May 2016 Citation Aleti G, Antonielli L, Corretto E, Nikolic´ B, Sessitsch A, Brader G. 2016. The draft genome sequence of Paenibacillus polymyxa strain CCI-25 encompasses high potential
for secondary metabolite production. Genome Announc 4(3):e00366-16. doi:10.1128/genomeA.00366-16. yright © 2016 Aleti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International licen
ess correspondence to Günter Brader, guenter.brader@ait.ac.at. synthetases with sequence similarities to published genes (1, 18–
22), and the prediction includes the encoding of a tridecaptin with
valine instead of isoleucine at the 13th position compared to
Paenibacillus terrae NRRL B-30644 and fusaricidin C, and a poly-
myxin with leucine instead of phenylalanine at the 6th position
compared to P. polymyxa M1. In addition, an iturin- and
paenilarvin-like compound with altered monomer composition
(D-Gly-D-Orn-D-Glu-D-nrp-L-nrp-L-Ile-L-Val) compared to the
published metabolites from Bacillus amyloliquefaciens FZB42
(58% identity) and Paenibacillus larvae DSM 25430 (40%
identity) (1, 23) has been predicted. CCI-25 contains a lantibiotic
gene similar to paenicidin A and a predicted polyketide synthase
with a different number of acyl carrier domains with 61% identity
to bacillaene synthase from B. amyloliquefaciens FZB42 and 87%
identity to P. polymyxa M-1 polyketide synthase (23). Given the
fact that about 370 kb (6.6% of the total genome) is dedicated to
secondary metabolite biosynthesis, CCI-25 has high potential to
be exploited for medical or agricultural applications. P
l P
lant-associated Paenibacillus polymyxa strains are well noted
for their production of a wide range of secondary metabolites
(1–3), predominantly lipopeptides and polyketides involved in
plant growth promotion and biocontrol of fungi (4–6). Here, we
highlight the secondary metabolite capacity of P. polymyxa strain
CCI-25 isolated from vermicompost. ACKNOWLEDGMENT This work was supported by the life science project LS11-014 of the Nie-
derösterreichische Forschungs-und Bildungsges.m.b.H (NFB). This work was supported by the life science project LS11-014 of the Nie-
derösterreichische Forschungs-und Bildungsges.m.b.H (NFB). FUNDING INFORMATION This work, including the efforts of Günter Brader and Branislav Nikolic´,
was funded by NFB under grant number LS11-04. This work, including the efforts of Günter Brader and Branislav Nikolic´,
was funded by NFB under grant number LS11-04. genomea.asm.org
1 The Draft Genome Sequence of Paenibacillus polymyxa Strain CCI-25
Encompasses High Potential for Secondary Metabolite Production Both colonies and lipopep-
tide and polyketide crude extracts (7, 8) exhibited strong antimi-
crobial activity against Escherichia coli and fungi, including Fusar-
ium oxysporum ACC01, Botrytis cinerea ofi501-E (Austrian
Institute of Technology [AIT] collection), and Rhizoctonia solani
CBS101769, on plate assays. To evaluate the molecular basis for secondary metabolite pro-
duction, genomic DNA was isolated by phenol-chloroform ex-
traction, and a library was prepared, according to the manufactur-
er’s protocol, using the Nextera XT kit (Illumina, San Diego, CA). Library sequencing was performed using an Illumina MiSeq plat-
form (MiSeq reagent kit version 3). Sequencing generated
2,213,773 paired-end reads with 124 53-fold coverage after
PhiX sequence removal by Bowtie2 (9). Adapter and quality trim-
ming were performed using Trimmomatic-0.32 (10). Overlap-
ping reads were merged with FLASH (11), and paired-end reads
were assembled by SPAdes 3.1.0 (12). Quality control of mapping
data was carried out by Qualimap 2.2 (13), and assembly quality
was estimated by QUAST 3.2 (14). Assembly resulted in 117 con-
tigs 1,000 bp, with an N50 size of 95,765 bp. The draft genome
size is 5.61 Mb, with a GC content of 44.95%. The identification
of 40 highly conserved single-copy marker genes in the assembly
by PhyloSift version 1.0.1 (15) indicated completeness of the ge-
nome and excluded contaminant sequences. Genomic BLAST
showed similarities to P. polymyxa CR1. The NCBI Prokaryotic
Genome Annotation Pipeline (PGAP) identified 5,146 genes,
4,953 coding sequences (CDSs), 15 complete 5S rRNAs, 30 partial
16S rRNAs, 37 partial 23S rRNAs (for a total of 15 putative rRNA
operons), 107 tRNAs, 4 noncoding RNAs (ncRNAs), and 241
pseudogenes. The rRNAs were further confirmed by RNAmmer
1.2 (16). Prediction of secondary metabolite-encoding sequences
was performed by antiSMASH (17). Nucleotide sequence accession number. The nucleotide se-
quences have been deposited at the DDBJ/EMBL/GenBank under
the accession no. LTYJ00000000. The version described in this
paper is the first version. May/June 2016
Volume 4
Issue 3
e00366-16 crossmark smark REFERENCES 1. Niu B, Rueckert C, Blom J, Wang Q, Borriss R. 2011. The genome of the
plant growth-promoting rhizobacterium Paenibacillus polymyxa M-1
contains nine sites dedicated to nonribosomal synthesis of lipopeptides
and polyketides. J Bacteriol 193:5862–5863. http://dx.doi.org/10.1128/
JB.05806-11. 1. Niu B, Rueckert C, Blom J, Wang Q, Borriss R. 2011. The genome of the
plant growth-promoting rhizobacterium Paenibacillus polymyxa M-1
contains nine sites dedicated to nonribosomal synthesis of lipopeptides
and polyketides. J Bacteriol 193:5862–5863. http://dx.doi.org/10.1128/
JB.05806-11. 2. Catch JR, Jones TSG, Wilkinson S. 1949. The chemistry of polymyxin A. Ann N Y Acad Sci 51:917–923. http://dx.doi.org/10.1111/j.1749
-6632.1949.tb27318.x. 2. Catch JR, Jones TSG, Wilkinson S. 1949. The chemistry of polymyxin A. Ann N Y Acad Sci 51:917–923. http://dx.doi.org/10.1111/j.1749
-6632.1949.tb27318.x. The CCI-25 draft genome encompasses nonribosomal peptide The CCI-25 draft genome encompasses nonribosomal peptide May/June 2016
Volume 4
Issue 3
e00366-16 Genome Announcements Aleti et al. next-generation sequencing alignment data. Bioinformatics 28:
2678–2679. http://dx.doi.org/10.1093/bioinformatics/bts503. next-generation sequencing alignment data. Bioinformatics 28:
2678–2679. http://dx.doi.org/10.1093/bioinformatics/bts503. 3. He Z, Kisla D, Zhang L, Yuan C, Green-Church KB, Yousef AE. 2007. Isolation and identification of a Paenibacillus polymyxa strain that copro-
duces a novel lantibiotic and polymyxin. Appl Environ Microbiol 73:
168–178. http://dx.doi.org/10.1128/AEM.02023-06. p
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14. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality
assessment tool for genome assemblies. Bioinformatics 29:1072–1075. http://dx.doi.org/10.1093/bioinformatics/btt086. p
g
4. Beatty PH, Jensen SE. 2002. Paenibacillus polymyxa produces fusaricidin-
type antifungal antibiotics active against Leptosphaeria maculans, the caus-
ative agent of blackleg disease of canola. Can J Microbiol 48:159–169. http://dx.doi.org/10.1139/w02-002. 15. Darling AE, Jospin G, Lowe E, Matsen FA IV, Bik HM, Eisen JA. 2014. PhyloSift: phylogenetic analysis of genomes and metagenomes. PeerJ 2:
http://dx.doi.org/10.7717/peerj.243. p
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5. Raza W, Yang X, Wu H, Wang Y, Xu Y, Shen Q. 2009. Isolation and
characterisation of fusaricidin-type compound-producing strain of
Paenibacillus polymyxa SQR-21 active against Fusarium oxysporum f. sp. nevium. Eur J Plant Pathol 125:471–483. http://dx.doi.org/10.1007/
s10658-009-9496-1. 16. Lagesen K, Hallin P, Rødland EA, Staerfeldt HH, Rognes T, Ussery
DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal
RNA genes. Nucleic Acids Res 35:3100–3108. http://dx.doi.org/10.1093/
nar/gkm160. 17. Blin K, Medema MH, Kazempour D, Fischbach MA, Breitling R,
Takano E, Weber T. 2013. antiSMASH 2.0 a versatile platform for ge-
nome mining of secondary metabolite producers. Nucleic Acids Res 41:
W204–W212 http://dx.doi.org/10.1093/nar/gkt449. 6. Dijksterhuis J, Sanders M, Gorris LG, Smid EJ. 1999. REFERENCES Antibiosis plays a
role in the context of direct interaction during antagonism of Paenibacillus
polymyxa towards Fusarium oxysporum. J Appl Microbiol 86:13–21. http://dx.doi.org/10.1046/j.1365-2672.1999.t01-1-00600.x. W204–W212 http://dx.doi.org/10.1093/nar/gkt449. 18. Choi SK, Park SY, Kim R, Kim SB, Lee CH, Kim JF, Park SH. 2009. Identification of a polymyxin synthetase gene cluster of Paenibacillus poly-
myxa and heterologous expression of the gene in Bacillus subtilis. J Bacte-
riol 191:3350–3358. http://dx.doi.org/10.1128/JB.01728-08. p
g
j
7. Aktuganov G, Jokela J, Kivelä H, Khalikova E, Melentjev A, Galimzi-
anova N, Kuzmina L, Kouvonen P, Himanen JP, Susi P, Korpela T. 2014. Isolation and identification of cyclic lipopeptides from Paenibacillus
ehimensis, strain IB-X-b. J Chromatogr B 973:9–16. http://dx.doi.org/
10.1016/j.jchromb.2014.09.042. 19. Choi SK, Park SY, Kim R, Lee CH, Kim JF, Park SH. 2008. Identification
and functional analysis of the fusaricidin biosynthetic gene of Paenibacil-
lus polymyxa E681. Biochem Biophys Res Commun 365:89–95. http://
dx.doi.org/10.1016/j.bbrc.2007.10.147. 8. Smyth TJP, Perfumo A, McClean S, Marchant R, Banat IM. 2010. Isolation and analysis of lipopeptides and high molecular weight biosur-
factants, p 3687–3704. In Timmis KN (ed), Handbook of hydrocarbon
and lipid microbiology. Springer, Heidelberg, Germany. 20. Lohans CT, Huang Z, van Belkum MJ, Giroud M, Sit CS, Steels EM,
Zheng J, Whittal RM, McMullen LM, Vederas JC. 2012. Structural
characterization of the highly cyclized lantibiotic paenicidin A via a partial
desulfurization/reduction strategy. J Am Chem Soc 134:19540–19543. http://dx.doi.org/10.1021/ja3089229. 9. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie
2. Nat Methods 9:357–359. http://dx.doi.org/10.1038/nmeth.1923. p
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10. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer
for Illumina sequence data. Bioinformatics 30:2114–2120. 21. Aleti G, Sessitsch A, Brader G. 2015. Genome mining: prediction of
lipopeptides and polyketides from Bacillus and related Firmicutes. Com-
put Struct Biotechnol J 13:192–203. 11. Mago T, Salzberg SL. 2011. FLASH: fast length adjustment of short reads
to improve genome assemblies. Bioinformatics 27:2957–2963. http://
dx.doi.org/10.1093/bioinformatics/btr507. 22. Lohans CT, Van Belkum MJ, Cochrane SA, Huang Z, Sit CS, McMullen
LM, Vederas JC. 2014. Biochemical, structural, and genetic characteriza-
tion of tridecaptin A1, an antagonist of Campylobacter jejuni. Chembi-
ochem 15:243–249. http://dx.doi.org/10.1002/cbic.201300595. 12. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov
AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV,
Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single
cell sequencing. J Comput Biol 19:455–477. http://dx.doi.org/10.1089/
cmb.2012.0021. 2
genomea.asm.org May/June 2016
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Issue 3
e00366-16 REFERENCES p
g
23. Sood S, Steinmetz H, Beims H, Mohr KI, Stadler M, Djukic M, von der
Ohe W, Steinert M, Daniel R, Müller R. 2014. Paenilarvins: iturin family
lipopeptides from the honey bee pathogen Paenibacillus larvae. Chembi-
ochem 15:1947–1955. http://dx.doi.org/10.1002/cbic.201402139. 13. García-Alcalde F, Okonechnikov K, Carbonell J, Cruz LM, M, Götz S,
Tarazona S, Dopazo J, Meyer TF, Conesa A. 2012. Qualimap: evaluating May/June 2016
Volume 4
Issue 3
e00366-16 Genome Announcements Genome Announcements 2
genomea.asm.org 2
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Immunoadjuvant Properties of the Rho Activating Factor CNF1 in Prophylactic and Curative Vaccination against Leishmania infantum
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PloS one
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RESEARCH ARTICLE Abstract Citation: Michel G, Ferrua B, Munro P, Boyer L,
Mathal N, Gillet D, et al. (2016) Immunoadjuvant
Properties of the Rho Activating Factor CNF1 in
Prophylactic and Curative Vaccination against
Leishmania infantum. PLoS ONE 11(6): e0156363. doi:10.1371/journal.pone.0156363 There is a need to develop new effective immunoadjuvants for prophylactic or therapeutic
vaccines against intracellular pathogens. The activation of Rho GTPases by bacterial cyto-
toxic necrotizing factor 1 (CNF1) elicits humoral protective responses against protein anti-
gens. Here, we set out to investigate whether CNF1 activity initiates humoral immunity
against co-administered parasite antigens and anti-microbial immune signaling. We report
that co-administration of wild-type (WT) CNF1 with Leishmania (L.) promastigote antigens
at the nasal mucosa triggered prophylactic and curative vaccine responses against this par-
asite. Vaccination of the mucosa with promastigote lysate antigens combined with WT
CNF1 conferred protection against high inoculum L. infantum infection, which reached 82%
in the spleen. Immune parameter analysis by antigen recall indicated robust T-helper (Th)1
polarization of immune memory cells, with high IL-2 and IFN-γ production combined with
decreased IL-4 production. Additionally, we explored the curative effect of WT CNF1 on pre-
viously infected animals. We observed that PL combined with WT CNF1, but not the inac-
tive C866S mutant CNF1 (mCNF1), induced a 58% decrease in the parasite burden in the
spleen. Editor: Michel R. Popoff, Institute Pasteur, FRANCE
Received: March 30, 2016
Accepted: May 12, 2016
Published: June 3, 2016 Copyright: © 2016 Michel 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. Funding: This work was supported by the Groupe
d’Action Contre la Leishmaniose (GACL), by
institutional funding from INSERM, grants from the
Fondation Infectiopôle Sud, the Agence Nationale de
la Recherche (ANR 11BSV3 004 01), the
"Investments for the Future" LABEX SIGNALIFE
ANR-11-LABX-0028-01 and the C3M animal facility. NM was supported by DGA and the joint ministerial
program of R&D against CBRNE threats. SIMOPRO
is a member of the laboratory of Excellence LERMIT Immunoadjuvant Properties of the Rho
Activating Factor CNF1 in Prophylactic and
Curative Vaccination against Leishmania
infantum Grégory Michel1,2,3*, Bernard Ferrua1,2, Patrick Munro1,2, Laurent Boyer1,2,
Nassim Mathal4, Daniel Gillet4, Pierre Marty1,2,3, Emmanuel Lemichez1,2* a11111 a1111 1 Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, Team “Microbial toxins in host pathogen
interactions”, Equipe labellisée ligue contre le cancer, Nice, France, 2 Université de Nice-Sophia Antipolis,
Faculté de Médecine, Nice, France, 3 Centre Hospitalier Universitaire de Nice, Laboratoire de Parasitologie-
Mycologie, Nice, France, 4 CEA, iBiTecS, SIMOPRO, Paris Saclay University, LabEx LERMIT, Gif sur
Yvette, France * gmichel@unice.fr (GM); lemichez@unice.fr (EL) * gmichel@unice.fr (GM); lemichez@unice.fr (EL) Immunoadjuvanticity of CNF1 Activity virulence factors as a danger signal that is translated into effective anti-bacterial immune
responses [5–7]. Here, we address whether a microbial effector targeting Rho GTPases can be
translated into an adjuvant for vaccination against Leishmania infantum. supported by the grant from the Agence Nationale de
la Recherche (ANR-10-LABEX-33). supported by the grant from the Agence Nationale de
la Recherche (ANR-10-LABEX-33). Competing Interests: The authors have declared
that no competing interests exist. Host Rho GTPases are essential elements in host-pathogen interactions. The cytotoxic nec-
rotizing factor-1 (CNF1) is an A-B toxin produced by uropathogenic strains of Escherichia coli. Wild-type (WT) CNF1 specifically deamidates the glutamine 61 in Rac1/Cdc42 (Q63 in RhoA)
into a glutamic acid [8–10]. WT CNF1, but not the catalytically inactive mutant C866S of
CNF1 (mCNF1), catalyzes the activation of small Rho GTPases [9,10]. Consequently, WT
CNF1 can be used to increase the flux of activated Rho GTPases in host cells and the down-
stream Rho GTPase signaling pathways [11]. Although the small GTPases of the Rho protein
family are frequent targets of post-translational modifications that are catalyzed by bacterial
toxins, they contribute to sensing bacterial virulence [6,12]. Genome-wide gene expression
analysis in cells treated with WT CNF1 has revealed the induction of a large panel of NF-κB-
driven pro-inflammatory cytokines and chemokines [2]. More recent studies have begun to
underscore the importance of the RIPK/NF- κB and ASC/Caspase-1 signaling axis in host anti-
bacterial responses modulated by WT CNF1 and the Rac GTPases [5–7,13]. It is important to
determine the spectrum of pathogens for which the CNF1 activity can be exploited to develop
vaccines. Leishmania infantum/chagasi is the causative agent of visceral leishmaniasis (VL), which is
endemic in numerous southern countries, notably in the Mediterranean basin [14,15]. VL is
fatal if left untreated and represents the second most challenging infectious disease worldwide
[15]. Hence, part of the human population is chronically affected by poorly understood health
consequences. Apart from humans, dogs are the main victims and reservoir. The current treat-
ments are based on antibiotherapy and have serious limitations, such as high costs and toxicity
[16]. For these reasons, and on the basis of the robust immunity to reinfection observed in
cured patients, several vaccine trials against VL have been performed [17]. Leishmania para-
sites harness phagocytic cells, notably monocytes, in order to survive and replicate. Clinical
studies of VL have suggested that decreased T-helper (Th)1 and increased Th2 responses are
the hallmarks of the disease [15]. Hence, treatments that actively increase Th1 immune
responses can promote the clearance of the parasites [18]. CNF1 activity and the downstream activation of Rac are sufficient to promote efficient host
immune responses against bacteria [7,13]. We have begun to divert this toxin’s Rho activating
property in the development of an immunoadjuvant for mucosal vaccination. We established
that CNF1 activity stimulates the systemic and mucosal production of IgG and IgA antibodies
against ovalbumin and tetanus toxoid [2,3]. Mice immunized against tetanus toxoid together
with WT CNF1 show specific and long-lasting protection against a challenge by 10-fold of the
LD50 of tetanus toxin [4]. It is now of interest to determine whether WT CNF1 can also stimu-
late Th-1 cellular immunity against an intracellular pathogen. Introduction The discovery of the molecular basis of innate immunity has boosted the development of vac-
cine adjuvants on the basis of their capacity to stimulate innate immune receptors [1]. The
family of bacterial effectors catalyzing the activation of Rho proteins has attracted growing
attention because of their capacity to stimulate the immune system [2–6]. It has now been
established in different model systems that cells perceive robust activation of Rho GTPases by 1 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 L. infantum parasites, antigens and CNF1 L. infantum MON-1 (MHOM/FR/94/LPN101) was isolated from a patient with Mediterranean
visceral Leishmania that was contracted in Nice, France. L. infantum promastigotes were rou-
tinely grown at 26°C in Schneider’s medium, as previously described [19]. L. infantum clones
encoding firefly luciferase were generated as previously described [20]. For the promastigote lysate (PL) preparation, stationary phase L. infantum promastigotes
were washed and suspended at 109/ml in distilled water [19]. The suspension was submitted to
5 freeze/thaw cycles to generate PL. Typically, 5 mg of Leishmania protein was obtained from
109 parasites. Recombinant wild-type CNF-1 (WT CNF1) and its catalytically inactive form
(CNF1-C866S; mCNF1) were produced and purified as previously reported [21]. Both recom-
binant proteins were passed through a polymixin B column (Affinity pack TM-detoxy gel TM,
Pierce), and the lack of endotoxin content was verified using a colorimetric LAL assay (LAL
QCL-1000, Cambrex). Each CNF1 preparation stock (2 mg/ml) was shown to contain less than
0.5 endotoxin units/ml. Endonasal immunization and challenge in BALB/c mice Groups of 7 mice were immunized 3 times at 2-weeks intervals with 15 μg of PL together with
1 μg WT CNF1 or 1 μg catalytically inactive CNF1 C866S (mCNF1). PL preparations were
delivered into the nasal mucosa with a micropipette in 10 μl volumes of Dulbecco’s phosphate-
buffered saline (PBS, from Gibco life technologies) (5 μl per nostril). Fourteen days after the
last boost, mice were challenged via the intraperitoneal route with 108 stationary phase L. infantum metacyclic parasites. One month later, the mice were sacrificed, and spleen section
were collected and analyzed for parasite content by ELISA sandwich technique [22]. Briefly,
parasite antigens in infected tissues were extracted with Nonidet-P40 detergent and were cap-
tured by anti-L. infantum human IgG that were insolubilized onto microtiter plate and were
subsequently revealed using anti-L. infantum F(ab)' fragments labelled with peroxidase. Mice and ethics statement The protocol was approved by the Committee on the Ethics of Animal Experiments of the
School of Medicine of the University of Nice, France (Permit Number: 2010–45). Groups of
BALB/c female mice were purchased from Charles River at 6 weeks of age (Le Genest St. Isle,
France). The mice were maintained and handled according to the regulations of the European
Union and the French Ministry of Agriculture as well as to the FELASA (the Federation of Lab-
oratory Animal Science Associations) recommendations. All efforts were made to minimize or
avoid suffering. 2 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 Immunoadjuvanticity of CNF1 Activity Analysis of vaccine-induced immune responses To assess total IgG titers, blood samples were recovered from the tail vein after vaccination
(one day before infection) and before mouse dissection (one month after infection). IgG anti-
body responses were assessed at a 1/100 dilution by ELISA using PL-coated plates, as reported
[19]. Vaccine-induced cellular immunity was measured post-vaccination using in vitro antigen
recall experiments on spleen homogenates as follows: the spleens from each individual mouse
(5 per group) were homogenized in sterile PBS, and erythrocytes were lysed at room tempera-
ture using 10 mM NaHCO3 containing 155 mM NH4Cl and 0.1 mM EDTA. Splenocytes were
then washed twice with PBS, counted and suspended at 5×106 cells/ml in DMEM containing 2
mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM
2-mercaptoethanol and 10% fetal calf serum. Cell suspensions were cultured for 48 h in the
presence or absence of 50 μg/ml of PL. Supernatants were harvested and assayed for IL-2, IL-4
and IFN-γ content by indirect sandwich ELISA (Pharmingen, Clinisciences). The threshold
sensitivities of the techniques were in the range of 20–30 pg/ml. Non-parametric Mann-Whitney tests were performed using GraphPad Prism version 5.0d for
Mac (GraphPad Software, San Diego California USA, www.graphpad.com). Statistical analysis Statistical analysis
Non-parametric Mann-Whitney tests were performed using GraphPad Prism version 5.0d for
Mac (GraphPad Software, San Diego California USA, www.graphpad.com). 3 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 Immunoadjuvanticity of CNF1 Activity CNF1 activity stimulates humoral IgG responses against L. infantum
antigens In this study, we first sought to determine the efficacy of WT CNF1 as a specific immunoadju-
vant for the induction of protective responses against an intracellular pathogen. Additionally, we
sought to evaluate the efficacy of this adjuvant for needle-free vaccination by topical delivery
through the nasal mucosa. The immuno-modulatory effects of WT CNF1 rely on its catalytic
activity, with mCNF1 catalytically inactive mutant having no effect on humoral responses [3,4]. Therefore, we directly compared the effect of WT CNF1, to that of mCNF1. As infectious model,
we choose mice infection with L. infantum. Mice were immunized 3 times at 2-week intervals
with promastigote lysate (PL), which was supplemented with either WT CNF1 (PL + WT CNF1)
or the catalytically inactive mutant CNF1-C866S (PL + mCNF1) as a control. At first, we moni-
tored the adjuvant effect of WT CNF1 by measuring the IgG antibody titers against PL in the
sera. Under these conditions, we observed a modest but reproducible 4-fold increase in the
serum IgG-titer of PL + WT CNF1 immunized mice (Fig 1). We concluded that CNF1 activity
enhances immune responses against L. infantum antigens. Fig 1. Antibody responses to L. infantum antigens post-vaccination. The anti-PL IgG antibody
responses were measured post-vaccination by ELISA (one day before infection). Mice were immunized
intranasally with 3x15 μg promastigote lysate (PL) plus either wild-type CNF1 (PL + WT CNF1) or catalytically
inactive CNF1 (PL + mCNF1). The controls represent infected but non-immunized animals. Serum samples
were tested at a 1/100 dilution and evaluated using HRP-labeled anti-mouse IgG. The interquartile ranges as
well as the 10–90% percentiles are presented for each group. ***: p<0.001. The results are representative of
2 independent experiments. n = 7. doi:10.1371/journal.pone.0156363.g001 Fig 1. Antibody responses to L. infantum antigens post-vaccination. The anti-PL IgG antibody
responses were measured post-vaccination by ELISA (one day before infection). Mice were immunized
intranasally with 3x15 μg promastigote lysate (PL) plus either wild-type CNF1 (PL + WT CNF1) or catalytically
inactive CNF1 (PL + mCNF1). The controls represent infected but non-immunized animals. Serum samples
were tested at a 1/100 dilution and evaluated using HRP-labeled anti-mouse IgG. The interquartile ranges as
well as the 10–90% percentiles are presented for each group. ***: p<0.001. The results are representative of
2 independent experiments. n = 7. Fig 1. Antibody responses to L. infantum antigens post-vaccination. CNF1 activity stimulates humoral IgG responses against L. infantum
antigens The anti-PL IgG antibody
responses were measured post-vaccination by ELISA (one day before infection). Mice were immunized
intranasally with 3x15 μg promastigote lysate (PL) plus either wild-type CNF1 (PL + WT CNF1) or catalytically
inactive CNF1 (PL + mCNF1). The controls represent infected but non-immunized animals. Serum samples
were tested at a 1/100 dilution and evaluated using HRP-labeled anti-mouse IgG. The interquartile ranges as
well as the 10–90% percentiles are presented for each group. ***: p<0.001. The results are representative of
2 independent experiments. n = 7. doi:10.1371/journal.pone.0156363.g001 4 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 Immunoadjuvanticity of CNF1 Activity Fig 2. Protective effects of nasal immunizations against L. infantum infection. BALB/c mice were
immunized with promastigote lysate plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive
CNF1 (PL + mCNF1). Fourteen days after the last boost, the mice were intraperitoneally challenged with 108
stationary phase L. infantum metacyclic parasites. The controls represent infected but non-immunized
animals. Spleen parasite burdens were quantified 1 month later by ELISA. The bars indicate the mean
parasite loads ± SEM. **: p<0.01. The results are representative of 2 independent experiments. n = 7. doi:10.1371/journal.pone.0156363.g002 Fig 2. Protective effects of nasal immunizations against L. infantum infection. BALB/c mice were
immunized with promastigote lysate plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive
CNF1 (PL + mCNF1). Fourteen days after the last boost, the mice were intraperitoneally challenged with 108
stationary phase L. infantum metacyclic parasites. The controls represent infected but non-immunized
animals. Spleen parasite burdens were quantified 1 month later by ELISA. The bars indicate the mean
parasite loads ± SEM. **: p<0.01. The results are representative of 2 independent experiments. n = 7. doi:10.1371/journal.pone.0156363.g002 doi:10.1371/journal.pone.0156363.g002 doi:10.1371/journal.pone.0156363.g002 We then established the extent of protection against L. infantum in animals under different
immunization conditions. Groups of 7 mice were immunized with PL supplemented with
either WT CNF1 (PL + WT CNF1) or mCNF1 (PL + mCNF1), prior to infection with high
loads of 108 infective metacyclic parasites. Mice were sacrificed one month later to analyze the
parasite content in the spleen (Fig 2). In the naïve group, we measured a typical parasite burden
ranging from 6-15x106 parasites/spleen; mean = 8.5x106 (Fig 2). The live parasite level dramati-
cally decreased by 23-fold in mice that were immunized with PL together with WT CNF1 com-
pared with controls. CNF1 activity stimulates humoral IgG responses against L. infantum
antigens To evaluate the effects of CNF1 activity, we also quantified the parasite
levels in a group of mice immunized with PL + mCNF1. The results revealed that the WT
CNF1 catalytic activity produced a marked 6-fold increase of protection compared with
mCNF1. Together, these experiments suggest that mice immunized against PL together with
active WT CNF1 develop a strong resistance to infection. In parallel, we assessed the IgG-titer against PL in infected mice (Fig 3). The results showed
a 3-fold increase in the IgG level in PL + WT CNF1 immunized mice compared with naïve and
PL + mCNF1 conditions (Fig 3). Our data are in good agreement with our findings that active
CNF1 together with promastigote lysate conferred a high resistance to infection in vaccinated
mice. 5 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 Immunoadjuvanticity of CNF1 Activity Fig 3. Antibody responses to L. infantum antigens post-infection. Anti-PL IgG antibody responses
measured by ELISA post-infection in vaccinated mice. Mice were immunized intranasally with 3x15 μg
promastigote lysate plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1). Serum samples were collected one month after infection and tested at a 1/100 dilution and were evaluated
using HRP-labeled anti mouse IgG. The interquartile ranges as well as the 10–90% percentiles are presented
for each group. ***: p<0.001. The results are representative of 2 independent experiments. n = 7. doi:10.1371/journal.pone.0156363.g003 Fig 3. Antibody responses to L. infantum antigens post-infection. Anti-PL IgG antibody responses
measured by ELISA post-infection in vaccinated mice. Mice were immunized intranasally with 3x15 μg
promastigote lysate plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1). Serum samples were collected one month after infection and tested at a 1/100 dilution and were evaluated
using HRP-labeled anti mouse IgG. The interquartile ranges as well as the 10–90% percentiles are presented
for each group. ***: p<0.001. The results are representative of 2 independent experiments. n = 7. doi:10.1371/journal.pone.0156363.g003 doi:10.1371/journal.pone.0156363.g003 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 WT CNF1 primes T-cell stimulatory responses against L. infantum WT CNF1 primes T-cell stimulatory responses against L. infantum Th1 cellular immune responses confer animals and humans with a capacity to control Leish-
mania multiplication and dissemination [23,24]. We investigated whether WT CNF1 might
stimulate T-helper Th1 responses. This was assessed in isolated splenocytes by means of anti-
gen recall. Fig 4 shows IL-2, IFN-γ and IL-4 production levels recorded after PL-antigen recall. No cytokine production was recorded after in vitro PL-antigen stimulation in naïve mice spleen
cells. In contrast, robust cytokine responses were recorded in mice immunized with PL. Inter-
estingly, these responses differed among the different immunization conditions with catalyti-
cally active or inactive CNF1. The highest IL-2 and IFN-γ memory responses to PL recall were
measured in mice immunized with PL + WT CNF1 compared with PL + mCNF1 (Fig 4A and
4B). Additionally, we measured a 2-fold decrease in IL-4 production in mice immunized with a
catalytic form of CNF1 (Fig 4C), which produced an IFN-γ/IL-4 ratio approximately 4-fold
higher in the PL + WT CNF1 vaccinated mouse group compared with the group immunized
with PL + mCNF1. This profile of immune cell responses against PL, which included an
increase in IL-2 and IFN-γ combined with a decrease in IL-4, indicates that CNF1 activity 6 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 Immunoadjuvanticity of CNF1 Activity Fig 4. In vitro antigen recall experiments. Spleen homogenates from mice immunized via the nasal route
with promastigote lysate (PL) plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL +
mCNF1) and infected with 108 stationary phase L. infantum metacyclic parasites were challenged with 50 μg/ml
PL for 48 hours. The supernatants were collected and assayed for IL-2 (A), IFN-γ (B) and IL-4 (C) by ELISA. The bars represent the mean cytokine production ± SEM. *: p<0.05, **: p<0,01, ***: p<0,001. n = 7. doi:10.1371/journal.pone.0156363.g004
e 0156363
June 3 2016
7 / 13 Fig 4. In vitro antigen recall experiments. Spleen homogenates from mice immunized via the nasal route
with promastigote lysate (PL) plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL +
mCNF1) and infected with 108 stationary phase L. infantum metacyclic parasites were challenged with 50 μg/ml
PL for 48 hours. The supernatants were collected and assayed for IL-2 (A), IFN-γ (B) and IL-4 (C) by ELISA. The bars represent the mean cytokine production ± SEM. Immunoadjuvanticity of CNF1 Activity stimulates pro-T-helper Th1 cellular responses. This result is in agreement with the capacity of
WT CNF1 to promote protection against L. infantum infection in mice. WT CNF1 shows curative activity against L. infantum The above data revealed a previously unknown property of WT CNF1 in stimulating a cytokine
response, thus demonstrating its strong capacity to stimulate pro T-helper Th1 immune
responses. This prompted us to assess whether the CNF1 activity might also be endowed with
adjuvant curative properties. Mice were first infected and later treated with or without PL in
the presence or absence of either WT CNF1 or the catalytically inactive mutant, mCNF1, as a
control. Therapeutic vaccination was repeated twice at one-week intervals, and the infection
was monitored at day 42 post-immunization. Fig 5 depicts the parasite burden in the spleen. The infected mice treated with WT CNF1 had a reduced parasite burden compared with the
mCNF1 and control mice. Second, the infected mice treated with PL alone had a significantly
reduced parasite burden. Third, the PL + WT CNF1 treatment produced a maximal parasite
clearance effect. Altogether, our results show a robust reduction in parasite burden triggered by
PL in combination with WT CNF1. Our previous observations indicated a higher IL-2 and
IFN-γ response after PL-antigen recall when mice were vaccinated with PL + WT CNF1 com-
pared with PL + mCNF1. We tested whether the curative properties of WT CNF1, PL and PL
+ WT CNF1 correlated with an increase in IL-2 and IFN-γ levels after PL-antigen recall. We
measured a significant increase in IL-2 and IFN-γ cytokine responses with the PL treatment Fig 5. CNF1 activity confers curative immunoadjuvant properties. BALB/c mice were first infected with
3x106 of stationary phase parasites. Fourteen days post-infection, mice were immunized via the nasal route
with promastigote lysate (PL), wild-type CNF1 (WT CNF1), catalytically inactive CNF1 (mCNF1), PL plus
either WT CNF1 (WT CNF1 + PL) or catalytically inactive CNF1 (mCNF1 + PL). The controls represent
infected but non-immunized animals. Twenty-one and twenty-eight days post infection, the mice were
immunized again. At day 42, the mice were sacrificed, and parasite numbers were determined by quantitative
PCR using mouse spleen DNA extracts. The bars represent the mean cytokine production ± SEM. *: p<0.05,
**: p<0,01, ***: p<0,001. n = 5. doi:10 1371/journal pone 0156363 g005 Fig 5. CNF1 activity confers curative immunoadjuvant properties. BALB/c mice were first infected with
3x106 of stationary phase parasites. doi:10.1371/journal.pone.0156363.g005 WT CNF1 primes T-cell stimulatory responses against L. infantum *: p<0.05, **: p<0,01, ***: p<0,001. n = 7. doi:10.1371/journal.pone.0156363.g004 Fig 4. In vitro antigen recall experiments. Spleen homogenates from mice immunized via the nasal route
with promastigote lysate (PL) plus either wild-type CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL +
mCNF1) and infected with 108 stationary phase L. infantum metacyclic parasites were challenged with 50 μg/ml
PL for 48 hours. The supernatants were collected and assayed for IL-2 (A), IFN-γ (B) and IL-4 (C) by ELISA. The bars represent the mean cytokine production ± SEM. *: p<0.05, **: p<0,01, ***: p<0,001. n = 7. 7 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 Discussion Here, we report the use of WT CNF1 as an immunoadjuvant in a prophylactic and curative
vaccination against L. infantum Infection. We linked this property of WT CNF1 to its enzy-
matic activity, and we provide evidence that CNF1 activity induces an immunostimulatory
cytokine profile that is biased toward a Th1 response. This study suggests that Rho GTPases
are targets of great value to stimulate cellular immunity against L. infantum intracellular
parasite. A limited number of vaccine trials against the visceral species, L. infantum/chagasi, have
been reported to date [15,17,25]. Second- and third-generation vaccine candidates are based
on the use of various Leishmania antigen preparations combined with different adjuvants [15]. Second- and third-generation vaccines using purified or recombinant L. infantum subfractions
represent a feasible option for mass vaccination campaigns; however, their efficacy generally
requires the co-administration of an adjuvant [15,17]. Several compounds with adjuvant prop-
erties, including cytokines, monophosphoryl lipid A, saponins, Cryptosporidium parvum, Pro-
pionibacterium acnes and Complete Freund Adjuvant have been described in vaccination trials
against L. infantum [26]. However, to our knowledge, the adjuvant effect of WT CNF1, a Rho
GTPase activating protein, during vaccination against Leishmania species has not yet been
reported. In this study, we found that catalytically active CNF1 exerted a protective effect
against L. infantum infection when mice were immunized in the nasal mucosa with a promasti-
gote lysate. Notably, WT CNF1 significantly increased the resistance to Leishmania infection
in animals despite the use of high doses of metacyclic parasites. Extending previous reports
showing that vaccination with Leishmania antigens confers some protection in animals [27],
here, we established that this protection was dramatically improved by using WT CNF1 reach-
ing 82% protection in the spleen and 94% in liver tissues (S1 Fig). WT CNF1 confers protection against L. infantum infection through molecular mechanisms
that remain to be fully elucidated; however, they involve the toxin catalytic activity toward Rho
GTPases. Here, we showed that treatment with WT CNF1 elicited specific cellular responses
characterized by increased secretion of IFN-γ and IL-2 cytokines and decreased secretion of IL-
4. WT CNF1 had no effect on the levels of IL-10 production after antigen recall, a down regula-
tor of Th1 responses (not shown). IFN-γ production has a major role in eliciting anti-parasite
macrophage responses, notably it induces production of H2O2 and induction of NO synthase,
which are required for intracellular parasite killing. Immunoadjuvanticity of CNF1 Activity but not with the WT CNF1 treatment alone, thus suggesting a role for WT CNF1 in stimulat-
ing the Th1 response initiated by PL (Fig 6A and 6B). The highest IFN-γ cytokine response
level was found when PL + WT CNF1 were combined, a result consistent with maximal para-
site clearance in this treatment condition (Figs 5 and 6B). Additionally, in these experiments,
we measured a decreased IL-4 production of approximately 2.1-fold (Fig 6C). The ratio of
IFN-γ/IL-4 was approximately 3.8-fold higher for the mice treated with PL + WT CNF1 com-
pared with the mice immunized with PL + mCNF1 (Fig 6). Both of our curative and prophylac-
tic vaccination settings suggest a similar mode of action for CNF1 activity, which exacerbates
the Th1 cellular responses induced by PL. PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 WT CNF1 shows curative activity against L. infantum Fourteen days post-infection, mice were immunized via the nasal route
with promastigote lysate (PL), wild-type CNF1 (WT CNF1), catalytically inactive CNF1 (mCNF1), PL plus
either WT CNF1 (WT CNF1 + PL) or catalytically inactive CNF1 (mCNF1 + PL). The controls represent
infected but non-immunized animals. Twenty-one and twenty-eight days post infection, the mice were
immunized again. At day 42, the mice were sacrificed, and parasite numbers were determined by quantitative
PCR using mouse spleen DNA extracts. The bars represent the mean cytokine production ± SEM. *: p<0.05,
**: p<0,01, ***: p<0,001. n = 5. 8 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 S1 Fig. Liver Protective effects of nasal immunizations against L. infantum infection. S1 Fig. Liver Protective effects of nasal immunizations against L. infantum infection. BALB/c mice were immunized with promastigote lysate plus either wild-type CNF1 (PL + WT
CNF1) or catalytically inactive CNF1 (PL + mCNF1). Fourteen days after the last boost, the mice
were intraperitoneally challenged with 108 stationary phase L. infantum metacyclic parasites. The
controls represent infected but non-immunized animals. Liver parasite burdens were quantified
1 month later by ELISA. The bars indicate the mean parasite loads ± SEM. : p<0.01. The
results are representative of 2 independent experiments. n = 7. (TIFF) Discussion At day 42, the mice were sacrificed, and spleen homogenates were challenged with 50 μg/ml PL for
48 hours. Supernatants were collected and assayed for IL-2 (A), IFN-γ (note, on graph it is INF) (B) and IL-4
(C) by ELISA. The bars represent the mean cytokine production ± SEM. *: p<0.05, *: p<0.05, **: p<0,01,
***: p<0,001. n = 5. doi:10.1371/journal.pone.0156363.g006 polarization toward the Th1 phenotype. The mechanistic behind this Th1 polarization trig-
gered by WT CNF1 requires further investigation. One possibility is that WT CNF1 directly
targets the T cell compartment. A second possibility is that WT CNF1 targets other immune
cells allowing them to produce signals triggering the polarization of T cells toward the Th1 phe-
notype. We also reveal a significant curative effect triggered by WT CNF1 during the treatment
of Leishmania infected animals. This curative effect is greatly enhanced in the presence of
Leishmania antigen. While the exact mechanism by which CNF1 activity confers protection
against Leishmania remains to be uncovered, interestingly we show here that this curative
effect of WT CNF1 is specifically promoted by the co-administered antigens. Collectively, our data provide the first indication that active WT CNF1 has vaccinal properties
in promoting prophylactic and curative protection against L. infantum intracellular parasites. Acknowledgments We are grateful to Dr Mery Tulic and Carmelo Luci for critical reading of the manuscript. This
work was supported by the Groupe d’Action Contre la Leishmaniose (GACL), by institutional
funding from INSERM, grants from the Fondation Infectiopôle Sud, the Agence Nationale de
la Recherche (ANR 11BSV3 004 01), the "Investments for the Future" LABEX SIGNALIFE
ANR-11-LABX-0028-01 and the C3M animal facility. NM was supported by DGA and the
joint ministerial program of R&D against CBRNE threats. SIMOPRO is a member of the labo-
ratory of Excellence LERMIT supported by the grant from the Agence Nationale de la Recher-
che (ANR-10-LABEX-33). We thank Veronique Corcelle and the C3M animal facility team
(Yannick Michelle, Sandrine Verger and Alexandre Ipekdjian) for their help and valuable assis-
tance with animal care. Author Contributions Conceived and designed the experiments: GM BF EL. Performed the experiments: GM BF P. Munro. Analyzed the data: GM BF LB EL. Contributed reagents/materials/analysis tools: GM
BF P. Munro LB EL. Wrote the paper: GM BF P. Munro DG NM LB P. Marty EL. 1.
Mbow ML, De Gregorio E, Valiante NM, Rappuoli R (2010) New adjuvants for human vaccines. Curr
Opin Immunol 22: 411–416. doi: 10.1016/j.coi.2010.04.004 PMID: 20466528 Discussion Additionally, IL-2 production and lympho-
proliferation contribute to conferring cellular immunoprotection. These protective immune
responses are balanced by the immunosuppressive responses triggered by the parasite. Immu-
nosuppressive cytokines, notably IL-4, are involved in the exacerbation of infection. Although
WT CNF1 had no effect on IL-10 production, we found that catalytically active CNF1 was able
to decrease IL-4 production. Thus, decreased IL-4 production combined with increased IFN-γ
and IL-2 production indicates that CNF1 activity enhances immune T-cell response 9 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
June 3, 2016 Immunoadjuvanticity of CNF1 Activity Fig 6. In vitro antigen recall for curative experiments. BALB/c mice were first infected with 3x106
stationary phase parasites. Fourteen days post-infection, the mice were immunized via the nasal route with
promastigote lysate (PL), wild-type CNF1 (WT CNF1), catalytically inactive CNF1 (mCNF1), or promastigote
lysate plus either WT CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1). Twenty-one and
28 days post infection, the mice were immunized again. The controls represent infected but non-immunized Fig 6. In vitro antigen recall for curative experiments. BALB/c mice were first infected with 3x106
stationary phase parasites. Fourteen days post-infection, the mice were immunized via the nasal route with
promastigote lysate (PL), wild-type CNF1 (WT CNF1), catalytically inactive CNF1 (mCNF1), or promastigote
lysate plus either WT CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1). Twenty-one and
28 days post infection, the mice were immunized again. The controls represent infected but non-immunized Fig 6. In vitro antigen recall for curative experiments. BALB/c mice were first infected with 3x106
stationary phase parasites. Fourteen days post-infection, the mice were immunized via the nasal route with
promastigote lysate (PL), wild-type CNF1 (WT CNF1), catalytically inactive CNF1 (mCNF1), or promastigote
lysate plus either WT CNF1 (PL + WT CNF1) or catalytically inactive CNF1 (PL + mCNF1). Twenty-one and
28 days post infection, the mice were immunized again. The controls represent infected but non-immunized 10 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156363
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Mutational synergy with CREBBP loss in lymphomagenesis identified through forward insertional mutagenesis in a new DLBCL mouse model
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bioRxiv preprint Abstract Loss-of-function mutations in the gene encoding the acetyltransferase CREBBP have been
reported in numerous cancers but are particularly frequent in lymphoid malignancies. However, the functional significance of CREBBP loss in transformation and disease
progression, most likely through cooperation with secondary genetic hits, has not yet been
fully unravelled. Similarly, the contribution of the initial cell population sustaining CREBBP loss
in the course of disease remains elusive. Here, we developed a new lymphoma mouse model
integrating Crebbp loss at various stages of B cell development with a transposon-based
insertional mutagenesis system. We demonstrated that Crebbp loss from the HSPC
compartment resulted in an aggressive DLBCL-like disease, recapitulating well-characterised
histological and molecular features of the human disease, as well as the recently described
enhanced CD24 expression. More importantly, we identified candidate genes functionally
equivalent to patient mutated genes. Those genes, mainly related to B cell development and
cellular signalling, may represent novel therapeutic targets. Overall, this new model provides
a powerful resource in which to conduct future mechanistic and therapeutic studies. Mutational synergy with CREBBP loss in lymphomagenesis identified through forward
insertional mutagenesis in a new DLBCL mouse model Nathalie Sakakini1,2, Roy Straver3,*, Dhoyazan M. A Azazi1,2,*, Sarah J. Horton1,2, Ryan Asby1,2,
Simon E. Richardson1,2,6, Pedro Madrigal1,2, Elizabeth J Soilleux4, Rachael Bashford-Rogers5,
Jeroen de Ridder3, Brian J. P Huntly1,2,6,$ 1. Department of Haematology, University of Cambridge, Cambridge, UK 2. Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
3. Centre for Molecular Medicine, University Medical Centre, Utrecht University, the
Netherlands
4. Department of Pathology, University of Cambridge, UK
5. Department of Biochemistry, University of Oxford, UK
6. Hematology Service, Cambridge University Hospitals, Cambridge, UK
* Co-second authors
$ corresponding author: bjph2@cam.ac.uk Prof Brian Huntly
Department of Haematology
Wellcome Trust—Medical Research Council Cambridge Stem Cell Institute
Cambridge, UK CB2 0AW Prof Brian Huntly
Department of Haematology
Wellcome Trust—Medical Research Council Cambridge Stem Cell Institute
Cambridge, UK CB2 0AW Prof Brian Huntly
Department of Haematology
Wellcome Trust—Medical Research Council Cambridge Stem Cell Institute
Cambridge, UK CB2 0AW Introduction The acetyltransferase CREBBP (Cyclic AMP Response Element Binding (CREB) binding protein,
also called CBP and KAT3A) is a pivotal transcriptional regulator. Its effects on transcription
are mediated at multiple levels. By acetylating histone proteins (including H3K18, K27, K56), . CC-BY 4.0 International license
made available under a
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made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
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bioRxiv preprint it favours chromatin accessibility at active promoters and enhancers and facilitates
transcription factor binding [1]. It also acetylates and modulates the activity of non-histone
proteins, such as the tumour suppressor p53, whose transcriptional activity is enhanced by
acetylation [2,3]. Finally, via its scaffolding properties it recruits members of the basal
transcriptional machinery (TBP, TFIIB, RNA polymerase II) and other proteins to gene
regulatory complexes to facilitate transcription [4]. it favours chromatin accessibility at active promoters and enhancers and facilitates
transcription factor binding [1]. It also acetylates and modulates the activity of non-histone
proteins, such as the tumour suppressor p53, whose transcriptional activity is enhanced by
acetylation [2,3]. Finally, via its scaffolding properties it recruits members of the basal
transcriptional machinery (TBP, TFIIB, RNA polymerase II) and other proteins to gene
regulatory complexes to facilitate transcription [4]. CREBBP has a broad interactome (with more than 300 interactants identified), thus it is
implicated in numerous biological processes. It is essential for embryonic development, as
Crebbp-null mouse embryos die between E9.5-10.5 from severe anaemia and neurological
defects [5]. CREBBP is also critical for the formation and maintenance of haematopoietic stem
cells (HSC) [6,7]. Germline CREBBP loss-of-function mutations have been reported in Rubinstein-Taybi
syndrome [8]. Mice Mice
C57Bl6 Mx1Crebbp-/- (CrebbpFl/Fl; Mx1Cre/wt) and C57Bl6 Cd19Crebbp-/- (CrebbpFl/Fl;
Cd19Cre/wt) have previously been described [3]. Both conditional Crebbp knockout strains
were independently bred with individual components of a forward mutagenesis system as
illustrated in Supp Fig 1A, B. This system utilizes the GrOnc transposon [15] along with a Piggy
Bac (PB) transposase under the transcriptional control of a modified Vk promoter (Vk*)
containing perfect substrate sequences for somatic hypermutation, leading to the restoration
of frame expression of the transposase within the germinal centre (GC) B-cell compartment
[16]. Cre-mediated recombination was induced by intraperitoneal injections of 5 doses of
poly(I) poly(C) (pIpC) (400 ug per dose; Sigma) administered every other day for 10 days to 6-
to 12-week-old mice, or occurred naturally upon Cd19 expression in the B cell lineage (Supp
Fig 1C). Deletion efficiency was determined by PCR of genomic DNA extracted from various
tissues (Supp Fig 1D). Somatic hypermutation and expression of the transposase was
triggered by Sheep Red Blood Cells (SRBC) stimulation. Three injections of 1x10^6 cells were
a administered fortnightly (Supp Fig 1C). Peripheral blood samples were taken from the lateral
saphenous vein and collected in EDTA-treated tubes. Peripheral blood cells were counted
using a Vet abc automated counter (Scil Vet abc Plus+, Horiba Medical). Mice were closely
monitored for signs of disease. Necropsy was performed to assess the presence of
abnormalities. All mice were housed in a pathogen-free animal facility. Mice were maintained
in a 12h-12h dark-light cycle, at a temperature of 21 degrees and 40% humidity. Experiments
were conducted under UK Home Office regulations (under the Animals (Scientific Procedures)
Act 1986, Amendment Regulations (2012)) and following ethical review by the University of
Cambridge Animal Welfare and Ethical Review Body. Flow cytometry analyses
Single-cell suspensions were obtained from various tissues. Cells were filtered through a
70um nylon cell strainer (EASYstrainer, Greiner) and treated with red blood cell lysis buffer
(Thermo Scientific) prior to staining. All staining were performed in DPBS (Invitrogen)
supplemented with 2% FBS (Sigma) for 20 min at 4°C. A list of the antibodies used for the flow
cytometry analyses is shown in Supplemental Table 1. Dead cells were excluded by gating on
7AAD negative cells (BD Pharmingen). Unstained, single stained and Fluorescence Minus One
(FMO) controls were used to determine the background, fix the compensation in each
channel and to determine the gates. Introduction CC-BY 4.0 International license
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bioRxiv preprint relevance of our new mouse models to investigate the molecular basis of this disease. Taken
together this work provides a rational framework to design future studies. Mice Flow cytometry experiments were acquired on a Fortessa
LSR (BD Bioscience). Data were analysed with FlowJo v10.8.2 software. Introduction This condition confers a predisposition to cancer, suggesting that CREBBP has
a tumour suppressor role. This has been confirmed by numerous studies describing
inactivating somatic mutations in a number of solid tumours and haematological malignancies
[9,10]. However, CREBBP mutations are particularly prevalent in lymphoid malignancies. They
occur in up to 40% of diffuse large B-cell lymphoma (DLBCL) [11] and up to 60% of follicular
lymphoma (FL) [12], the two most common lymphoma subtypes. These mutations are mainly
monoallelic and result in a partial loss of acetyltransferase activity [11]. Evidence from mouse
models [3,13,14] demonstrates that, although Crebbp loss of function constitutes an early
event in lymphomagenesis, it is not sufficient to drive full malignant transformation. This
suggests the acquisition of secondary mutations are required, and we have shown that this
is, at least in part, due to a defective DNA damage response related to the failure of p53
acetylation by Crebbp [3]. Despite global efforts to elucidate the molecular basis of DLBCL, the identity of the initial cell
that acquires CREBBP mutations and the dynamics with cooperating mutations driving
lymphomagenesis still remain to be fully address. To address these questions, we generated
a new DLBCL mouse model that combines Crebbp loss with a transposon-based insertional
mutagenesis (IM) system. Through the usage of haematopoietic stage-specific promoters
controlling a recombinase Cre, this model was constructed in two versions (referred as IM-
Mx1 or IM-Cd19) offering the flexibility to investigate Crebbp loss at different stages during
lymphoid development. Both models synergize with dynamic insertions of the GrOnc
transposon within the B-cell lineage, allowing evaluation of the effects of stochastic genomic
insertions on disease burden. With this advanced approach we have shown that early Crebbp
loss from the haematopoietic stem and progenitor (HSPC) compartment considerably
increased the penetrance of B-cell lymphomas and considerably accelerated disease
progression. Detailed analyses of murine tissues uncovered an aberrant B220low B cell
population expressing a germinal centre-like transcriptional program. In contrast, the loss of
Crebbp at a later stage during B cell ontogeny resulted a longer latency period prior to B-cell
lymphoma development. Sequencing analyses of the neoplastic B220low cells isolated from
our lymphoma models mapped GrOnc insertions to several key genes for B-cell development
including Pax5 and Ebf1, as well as in targetable signalling pathways. Comparisons with DLBCL
patient data revealed significant functional overlaps between both datasets, validating the . Transplantation assay Total splenocytes (1 x 10^6) isolated from individual malignant mice were injected
intravenously into lethally irradiated (5.5Gy + 5Gy) recipient mice that constitutively express
EGFP. Helper bone marrow cells (0.5 x 10^6) from the recipient strain were mixed with the
donor cells prior to the injection. EGFP+ cells were gated out to assess engraftment. Histopathology Histopathology
Tissues were fixed in a 10% neutral buffered formalin solution (CellPath Ltd.). They were
washed in PBS then transferred to 70% ethanol and embedded in paraffin blocks. Tissue
sections (4um) were stained with Hematoxylin and Eosin (Thermo Fisher Scientific) according
to the manufacturer’s protocol. Scoring of the slides was performed by an experienced
histopathologist blinded to mouse genotypes. . CC-BY 4.0 International license
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bioRxiv preprint BCR amplification and library preparation p f
y p
p
Total RNA was isolated from FACS-sorted B220low/CD19+ splenocytes from sick mice (B-cell
lymphoma only) using an RNeasy Micro kit (Qiagen). Control RNA was isolated from normal
B220high/CD19+ splenocytes from control healthy mice that had also received pIpC and SRBC
injections similarly to the experimental mice. Mice were age-matched, and gender balanced
between each cohort whenever possible. RNA quantity and quality were checked by Qubit
and Tape Station. Samples with a RIN score > 7 were used for library preparation. 200ng
mRNA was used for BCR-sequencing library preparation. Briefly, mRNA was first
retrotranscribed using a pool of primers targeting the IgH mouse locus. cDNA was then PCR
amplified with 1ul of VH forward primer set pools (group 1 or group 2, 10uM stock per primer)
and 1ul of a universal constant reverse primer in two independent reactions, using 25ul of
KAPA High Fidelity master mix (Roche). The following PCR program was used: 5 min at 95 °C;
five cycles of 5 s at 98 °C and 2 min at 72 °C; five cycles of 5 s at 98 °C, 10 s at 65 °C, and 2 min
at 72 °C; and 30 cycles of 20 s at 98 °C, 30 s at 60 °C, and 2 min at 72 °C; with a final extension
cycle of 7 min at 72 °C. Group 1 and group 2 PCR products from each sample were pooled and
purified using SPRI beads and eluted in 50ul water. Libraries were generated with a KAPA
Hyper Kit (Roche), incorporating adaptors and index primers provided by NEBNext® Multiplex
Oligos for Illumina® (96 Unique Dual Index Primer Pairs). Final libraries were quantified and
multiplexed for paired-end 300bp sequencing on an Illumina MiSeq platform. BCR-sequencing data processing and analysis BCR sequencing data processing and analysis
Forward and reverse reads generated from NGS sequencing were merged together if they
contained an identical overlapping region >7 bp, or otherwise discarded, using FLASHv1.2.11
[17]. Joined reads were filtered for base quality (median Phred score >32) using QUASR v.7.01
(http://sourceforge.net/projects/quasr) [18] and converted to fasta format. Universal
barcoded regions were identified in reads and oriented to read from V-primer to constant
region primer. The barcoded region within each primer was identified and checked for
conserved bases. Primers and constant regions were trimmed from each sequence, and
sequences were retained only if there was >80% per base sequence similarity between all
sequences obtained with the same barcode, otherwise they were discarded. The constant
region allele with the highest sequence similarity was identified by 10-mer matching to the
reference constant region genes from the IMGT database [19], and sequences were trimmed
to give only the region of the sequence corresponding to the variable (VDJ) regions. Isotype
usage information for each BCR was retained throughout the analysis hereafter. Sequences
without complete reading frames and non-immunoglobulin sequences were removed and
only reads with significant similarity to reference IgHV and J genes from the IMGT database
using BLAST (v2.10.0) [20] were retained. Immunoglobulin gene usages and sequence
annotation were performed in IMGT V-QUEST (v.3.4.9) [19], and class-switch recombination
analysis was performed as in Bashford-Rogers et al. [21]. Repertoire statistics were performed
in R using MANOVA for significance. . CC-BY 4.0 International license
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bioRxiv preprint The network generation algorithm and network properties were calculated as in Bashford-
Rogers et al. [21]: each vertex represents a unique sequence, where relative vertex size is
proportional to the number of identical reads. Edges join vertices that differ by single-
nucleotide nonindel differences and clusters are collections of related, connected vertices. BCR-sequencing data processing and analysis A
clone (cluster) refers to a group of clonally related B cells, each containing BCRs with identical
CDR3 regions and IgHV gene usage or differing by single point mutations. Each cluster should
originate from the same pre-B cell. RNA-sequencing library preparation
Total RNA was isolated from FACS-sorted B220low/CD19+ splenocytes from sick mice (B-cell
lymphoma only) using a RNeasy Micro kit (Qiagen). Control RNA was isolated from normal
B220high/CD19+ splenocytes from control healthy mice that also received pIpC and SRBC
injections similarly to the experimental mice. Mice were age-matched, and gender balanced
between each cohort whenever possible. RNA quantity and quality were checked by Qubit
and Tape Station. Samples with a RIN score > 7 were used for library preparation. RNA-
sequencing libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep
kit for Illumina® (NEB) following supplier’s recommendations. Poly(A) mRNA was first
separated using magnetic beads (NEB) and subsequently converted to cDNA. After adaptor
ligation, libraries were amplified and barcoded by PCR using NEBNext® Multiplex Oligos for
Illumina® (96 Unique Dual Index Primer Pairs). Libraries were quantified and multiplexed for
paired-end 50bp sequencing on the Illumina Novaseq platform. RNA-sequencing data processing and analysis
Paired-end RNA-sequencing sequences were first quality-assessed before any further
processing using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). RNA-sequencing libraries that were found to pass the minimum quality thresholds across all
quality control metrics were then subjected to adapter sequence trimming using TrimGalore
package (https://github.com/FelixKrueger/TrimGalore). This was followed by alignment to
the mouse genome (mm10) using STAR version=2.7.10a [22]. These flags (NH:i:1 and
MAPQ=255) were used to extract uniquely-mapping high-confidence reads from alignment
files, and used for transcriptome quantification with featureCounts version 2.0.1 [23]. Only
genes with a raw read count of >= 10 in at least two samples were considered for all
downstream analysis. Differential expression analysis was carried out on normalised gene-
level transcriptomic reads using the Bioconductor package DESeq2 [24]. The R package
‘clusterProfiler’ (version 4.4.4) was used to perform KEGG Pathway enrichment, Gene Set
Enrichment Analyses (GSEA) and Gene Ontology (GO) analyses on statistically-significant
differentially-expressed genes [25]. A false-discovery rate (FDR) or a multiple-testing adjusted
p-value of 0.05 was used across all tests to establish statistical significance. Differentially
expressed genes were filtered using an FDR < 5% and Log2 Fold Change >|1|. RNA-sequencing data processing and analysis Transposon-sequencing library preparation Only mice showing evidence of B-cell lymphoma at the time of death were analysed. Genomic
DNA was isolated from total mouse splenocytes using PureLink Genomic DNA kit (Invitrogen). 500ng of DNA was subsequently used for library preparation using NEBNext® Ultra™ II FS DNA
Library Prep Kit for Illumina® following manufacturer’s recommendations. Briefly, DNA was
fragmented for 15 minutes at 37 C. After end repair and A tailing steps, splinkerette adaptors
were ligated before PCR amplification using 2x KAPA HiFi HS ReadyMix kit. After purification
with SPRI beads, libraries were amplified and barcoded by PCR using TraDIS indexes. DNA
profiles were visually inspected using a Tape Station D5000. Final libraries were quantified by
qPCR using Library Quant Standards (Illumina) and pooled in equimolar amount for paired-
end 75bp sequencing on an Illumina MiSeq platform. Transposon-sequencing analysis and Common Insertion Sites identification
After sequencing, each sample existed across a set of 4 fastq files: One pair for "left handed"
and one pair for "right handed" data. The pairs refer to the forward and reverse sequenced
reads for each "handed" set. Files within a pair were processed together. Read pairs were
filtered out from the fastq files where the first read did not start with the expected junction
sequence or did contain the sequence of the non-mobilized transposon, Junction sequence
from the first read in each pair was trimmed for all reads kept. The processed reads were then
mapped (forward-reverse pairs combined) using bwa mem, followed by sorting and indexing
using samtools. Read pairs where the first read was not mapped were rejected. The start
location of each first read, indicating the exact site of an insertion, was retrieved. For further
processing both "handedness" start sites were combined: all reads starting at the exact same
base with the same mapping directionality compared to their "handedness" were treated
together, creating (up to) two stacks of reads per insertion site: combining Left-Forward and
Right-Reverse reads in one stack, and Left-Reverse and Right-Forward reads in the other stack,
making each read stack specific to the direction of an insertion at that location. Reads in each
stack were filtered based on mapping quality (>=20) and alignment score (>=30). Reads that
did not meet these thresholds, and reads where the second in pair was mapped on another
reference sequence or further than 1500 bp away, were removed. Prediction of murine immune cell type from RNA-sequencing data Prediction of murine immune cell type from RNA-sequencing data
Bulk RNA-sequencing profiles of murine haematopoietic cell populations of various lineages
and multiple differentiation trajectories were obtained from published datasets (accession
codes: GSE79672, GSE83436, GSE109125 and GSE133743) [26,27,28,29]. Original sequencing
runs were downloaded and processed as described above in the RNA-sequencing analysis
section, to produce raw sequencing counts per gene for all libraries. These reads were
subsequently normalised to fragments per kilobase of transcript per million mapped reads . CC-BY 4.0 International license
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bioRxiv preprint (FPKM). The FPKM-normalised read counts of each cell type were used as a reference dataset
to predict the differentiation status of our murine tumour cells using the R package SingleR
(version 1.10.0) [30]. The “SingleR” function was used to return the best-fitting annotation
for each mouse library in our contingent, using the labelled reference dataset with similarly-
normalised read counts of the same features (genes). (FPKM). The FPKM-normalised read counts of each cell type were used as a reference dataset
to predict the differentiation status of our murine tumour cells using the R package SingleR
(version 1.10.0) [30]. The “SingleR” function was used to return the best-fitting annotation
for each mouse library in our contingent, using the labelled reference dataset with similarly-
normalised read counts of the same features (genes). Early Crebbp loss cooperates with insertional mutagenesis to significantly accelerate the B-
cell lymphoma phenotype in a novel murine model To delineate the relative contribution of the haematopoietic compartment which initially
acquires Crebbp loss-of-function mutation and the effect of mutational synergy with Crebbp-
loss in lymphoma development, we generated a new DLBCL mouse model by introducing a
forward transpositional mutagenesis system into our existing Crebbp conditional knockout
mouse model [3]. Briefly, Mx1CrebbpFl/Fl mice were independently crossed with either Vk* PB
or GrOnc mice to generate the experimental Vk* PBTg/wt; GrOncTg/wt; Mx1CrebbpFl/Fl mice (here
after called IM-Mx1Crebbp) (Supp Fig 1A, B). Cre-mediated Crebbp excision initially occurs in
the HSPC compartment and is propagated in subsequent compartments, where it combines
with transposition of the GrOnc transposon in a B-cell lineage restricted manner. In keeping
with the requirement for further mutations to manifest full lymphoma transformation, this
new model considerably increased the penetrance of B-cell disease, where it accounted for
~80% of all deaths (Fig1A). In comparison with IM-Mx1Crebbp+/+ littermates that also had B-
cell restricted insertional mutagenesis, mice with early loss of Crebbp displayed a significantly
shorter latency (median survival 12.3 vs 17.8 weeks, p<0.05) and a slightly higher proportion
of B -cell diseases (Fig 1A, B). Macroscopically, mice from both genotypes presented with
hepatosplenomegaly and elevated white blood cell count (WCC) (Fig 1C, Supp Fig 1E, F)
although histological features were distinct for Crebbp deleted and WT cases. Lymphadenopathy was also observed in both models, however this was predominantly a
feature of the IM-Mx1Crebbp-/- mice (referred as ‘nodal’) (Fig 1D). Extranodal disease
infiltration was also seen in both genotypes but was more common in the IM-Mx1Crebpb+/+
mice (referred as ‘extranodal’, Fig 1D). Histological examination revealed disruption of normal
tissue architecture of the spleen and the lymph nodes and extension infiltration by neoplastic
cells. Extensive infiltration spread to other tissues including liver and kidneys (Fig 1D, E),
consistent with an aggressive DLBCL-like disease and previously described DLBCL mouse
models [33]. The lymphomas were most commonly classified as DLBCL for both genotypes,
with a higher proportion in the IM-Mx1Crebbp-/- cohort (64.5% vs 53.8% (Fig 1F) rising to
87.1% vs 69.2% when considering cases showing a mixture of DLBCL and FL). Conversely, the
incidence of BL was increased in the IM-Mx1-Crebbp+/+ cohort (19.2% vs 3.2%) (Fig 1F). These
results are consistent with observations in patients, where CREBBP mutations are more
frequently observed in DLBCL and FL, and are much less common in BL. Transposon-sequencing library preparation Each stack was given a
non-duplicate score (ndup), indicating how many reads after filtering had the same mapped
start position in the first read but a different mapped end position in the second read. This ndup count reduces the odds of double counting read duplicates and provides a
diminishing returns effect for sites with extremely high numbers of reads. For each sample,
the sum of all insertion sites' ndup scores on all chromosomes other than chromosome 19
(origin of the transposon) was calculated and a cutoff was set at 17.5% of this summed value. All chromosomal mapped insertion sites (including chromosome 19) in the sample were then
filtered, removing sites with an ndup value below the cutoff. This approach removes putative
insertion sites for which there is relatively little evidence while adjusting for sequencing depth
variations among samples. Next, the insertion sites of all samples were merged into a single . CC-BY 4.0 International license
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bioRxiv preprint file for further processing with Kernel Convolution Rule Based Method (KCRBM) [31,32]. We
altered KCRBM to work with BSgenome.Mmusculus.UCSC.mm10, use the PB insertion
system, enabled Local Hopping Correction (LHC) and used the ndup values as insertion depth
per insertion site. The insertion sites output was then further processed and visualised using
custom python scripts. file for further processing with Kernel Convolution Rule Based Method (KCRBM) [31,32]. We
altered KCRBM to work with BSgenome.Mmusculus.UCSC.mm10, use the PB insertion
system, enabled Local Hopping Correction (LHC) and used the ndup values as insertion depth
per insertion site. The insertion sites output was then further processed and visualised using
custom python scripts. An aberrant B220low/CD19+ population drives a DLBCL-like disease Early Crebbp loss cooperates with insertional mutagenesis to significantly accelerate the B-
cell lymphoma phenotype in a novel murine model Finally, the disease
efficiently transplanted to recipient mice (Fig 1G) and recapitulated features of the initial
disease, including hepatosplenomegaly (Supp Fig 1G, H). An aberrant B220low/CD19+ population drives a DLBCL-like disease . CC-BY 4.0 International license
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bioRxiv preprint To further characterise our new model, we analysed single cell suspensions from lymphoma
mouse spleens to determine their immunophenotype. These analyses revealed the presence
of an expanded and aberrant B220low/CD19+ population which was completely absent from
the spleen of control animals (Fig 2A). These neoplastic cells were also demonstrated across
all tissues analysed, consistent with the histological tissue infiltration (Fig 2B). The cells
expressed PNA, GL7 and, to a lesser extend, Fas, reminiscent of a germinal centre phenotype
(Fig 2C, Supp Fig 2A). Interestingly, they also demonstrated enhanced CD24 expression (Fig
2D, Supp Fig 2B). These cells were capable of reconstituting and giving rise to disease in
recipient mice after transplant, and the tumours that developed retained the same
immunophenotype as the primary DLBCL-like cells (Supp Fig 2C). Deep sequencing analysis of
B cell receptor repertoires was performed across lymphomas from IM-Mx1Crebbp-/-, IM-
Mx1Crebbp+/+, assessing B220low/CD19+ cells as tumour cells and compared to B220high/CD19+
cells from control mice. This revealed three main features. All tumours demonstrated BCR
VDJ rearrangements, with evidence of low levels of class switch recombination and modest
somatic hypermutation in keeping with their mature B cell origin (Fig 2E, F). Multiple
expanded clones per mouse were observed, however there was typically a single large
expansion within the IM-Mx1Crebbp-/- tumours, suggesting that the absence of Crebbp alters
clonal dynamics during lymphomagenesis (Fig 2G, H). Moreover, the consensus sequences of
the largest 3 clones per mouse were almost identical to the germline, supportive of antigen-
inexperienced B cells as precursors of these lymphomas (Supp Table 2). Early Crebbp loss cooperates with insertional mutagenesis to significantly accelerate the B-
cell lymphoma phenotype in a novel murine model Expanded clones were
predominantly IgHM with no differences in isotype usages between clones of different
genotypes, however, the relative proportion of class-switch recombination from IGHD/M to
IGHG2B and IGHA was significantly higher in IM-Mx1Crebbp-/- tumours than IM-Mx1Crebbp+/+
and controls (Fig 2H). Transcriptomic alterations in lymphoma tumours mimic changes in DLBCL patients To define similarities between our new mouse models and molecular features of human
DLBCL, we performed bulk RNA-sequencing on sorted B220low/CD19+ splenocytes from both
lymphoma models and compared them to control B220high/CD19+ splenocytes from wild-type
animals. Comparisons with the control revealed profound transcriptional changes in the
lymphoma cells with nearly 7000 differentially expressed genes (6885 in IM-Mx1Crebbp-/- vs
Ct and 6620 in IM-Mx1Crebbp+/+ vs Ct) (Fig 3A). Approximatively 60% were commonly
dysregulated in both models (Fig 3A). Amongst them the lymphoma-associated oncogenes
Ezh2 and Mycn [34] were up-regulated, alongside general proliferation genes including E2f1,
Cdk1, Cccnd3 and Mki67 reflective of an increased cell cycle status. Down-regulated genes
included several mediators of the immune response, such as Cd40, Cd74 and the TNF family
members Tnfsf14 and Tnfsf18. In addition, class II MHC genes Ciita, H2-Ab1, H2-Ob and H2-
Eb2 previously demonstrated to be down-regulated in Crebbp mutated lymphoma [14,35] (Fig
3B) were also reduced in their expression. GSEA analysis comparing both lymphoma models
found Crebbp-related signatures enriched in Crebbp replete mice, validating loss of Crebbp
transcriptional activity after Cre-mediated excision in our IM-Mx1Crebbp-/- mice (Fig 3C). Functional annotations showed that up-regulated genes in lymphoma vs control were
significantly enriched for biological processes related to cell proliferation and cell cycle,
whereas down-regulated genes were involved in immune response and other immune-
related processes (Fig 3D, E). Quantitative and qualitative differences were also observed
between lymphoma genotypes (Fig 3D). Using transcriptomic profiles available for normal . CC-BY 4.0 International license
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this version posted March 28, 2024. Transcriptomic alterations in lymphoma tumours mimic changes in DLBCL patients ;
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bioRxiv preprint mouse immune cell types [ImmGen Consortium,26,27,28,29], we were able to demonstrate
that the B220low/CD19+ neoplastic population from both models closely resembles germinal
centre cells at the transcriptomic level (Fig 3F). Loss of CREBBP later in B- cell development attenuates DLBCL-like disease Our results show that Crebbp loss in early hematopoietic progenitors in association with
forward mutagenesis drives an aggressive DLBCL-like phenotype. We have previously shown
that losing Crebbp function later during lymphoid development significantly decreased
lymphoma development [3] and thus we sought to test whether combination with insertional
mutagenesis would overcome this delayed latency. To test this hypothesis, we generated a
second mouse model that similarly allowed for B-cell transposition, where Crebbp excision
occurred in a committed B-cell progenitor (hereafter called IM-Cd19Crebbp-/-). In this new
combination, the majority of mice also develop B-cell lymphomas (75% vs 83.7% for IM-
Mx1Crebbp-/-), of which about half the cases displayed unequivocal features of DLBCL,
whereas the other half also demonstrated histological elements of FL (Fig 4A, B). The lower
penetrance of DLBCL and the increased proportion of FL suggested that Crebbp loss-of-
function within B-cell progenitors plus insertional mutagenesis leads to a less aggressive
disease, perhaps more in keeping with transformed FL. This was consistent with the longer
latency in IM-Cd19Crebbp-/- mice (median survival 12.3 IM-Mx1Crebbp-/- vs 51.9 weeks IM-
Cd19Crebbp-/-) (Fig 4C). However, at their terminal end point the IM-Cd19Crebbp-/- mice were
almost undistinguishable from their Mx1 counterparts. Apart from a reduced nodal
involvement, they presented with similarly enlarged spleens and livers, and elevated WCC (Fig
4D, E, Supp Fig 3A, B). At the cellular level, the neoplastic B220low/CD19+ population identified
in our initial model was also detected in multiple tissues including the spleen, the lymph nodes
and the liver (Fig 4F, Supp Fig 3C) and expressed the germinal centre markers PNA, GL7 and
Fas (Fig 4G, Supp Fig 3D). Finally, we could also demonstrate that IM-Cd19Crebbp-/- tumours
were transplantable and generated disease in recipient mice (Fig 4H, Supp Fig 3E-G). Transposon Common insertional Sites (CIS) identify known and putative lymphoma
oncogenes and tumour suppressor genes It contains both strong promoter and splice donor, acceptor and bi-
directional polyadenylation signals sequences. These can enhance gene expression when
landing upstream of a transcriptional start site or conversely insertions occurring in introns
can generate truncated proteins, leading to loss-of-function mutations (Supp Fig 1A) [38,37]. Knowing the genomic location of the insertions, it is therefore possible to predict their effect
on neighbouring genes. We mapped the location of all the insertions and experimentally
validated their effect on selected candidates. These included Flt3 and Nras, where insertions
occurred in a hotspot either upstream of a predicted transcriptional start site, or in the 5’
portion of the gene, leading to upregulation compared to tumours negative for those
particular insertions (Fig 5E, F). related to tyrosine kinase pathways (Flt3, Stat5b, Nras and Jak2) and known tumour
suppressors (Cdkn2a, Pten) (Fig 5B, Supp Table 3). Most of these were organised in a highly
connected network, suggesting their involvement in combinatorially coordinating the same
processes (Fig 5C). Functional annotations supported this finding, showing significant
enrichment of biological processes related to haematopoietic differentiation (interestingly
both lymphoid and myeloid) and signal transduction through tyrosine kinases (Fig 5D). Additionally, many of the genes, such as Nras, Flt3 and Sos1, are known for their contribution
to several cancers, including haematological malignancies (Supp Fig 4A). Because of its genetic design, the GrOnc transposon can alter gene expression through
multiple mechanisms. It contains both strong promoter and splice donor, acceptor and bi-
directional polyadenylation signals sequences. These can enhance gene expression when
landing upstream of a transcriptional start site or conversely insertions occurring in introns
can generate truncated proteins, leading to loss-of-function mutations (Supp Fig 1A) [38,37]. Knowing the genomic location of the insertions, it is therefore possible to predict their effect
on neighbouring genes. We mapped the location of all the insertions and experimentally
validated their effect on selected candidates. These included Flt3 and Nras, where insertions
occurred in a hotspot either upstream of a predicted transcriptional start site, or in the 5’
portion of the gene, leading to upregulation compared to tumours negative for those
particular insertions (Fig 5E, F). Transposon Common insertional Sites (CIS) identify known and putative lymphoma
oncogenes and tumour suppressor genes To assess the contribution of cooperating mutations to the development of DLBCL, our mouse
models utilised a transposition system that allows the cooperating lesions to be identified
through isolation of recurrent insertions. We then sequenced and analysed the insertion sites
in the lymphomas from 71 out of the 77 mice that presented with evidence of B cell
lymphoma at the time of death. The 6 remaining lymphoma samples failed to pass quality
control steps and were excluded from subsequent analysis. Ligation-mediated PCR followed
by next generation sequencing from individual tumours generated over 34 million reads
[36,37]. Of these reads, 91% were successfully paired and mapped to the mouse genome
(version mm39). Using a Kernel Convolution Rule Based Method (KCRBM), we identified 1615
unique genomic insertions across all tumours [31,32], of which the majority were genotype
specific (Fig 5A). When linked to the nearest gene, these insertions collapsed to 1264 genes,
of which 87 genes harboured at least 3 independent insertional events (Fig 5B, Suppl Table
3). Amongst the most common targeted genes we found major regulators of B-cell
development (Ebf1, Pax5, Ikzf1), alongside with well-known proto-oncogenes, particularly . CC-BY 4.0 International license
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bioRxiv preprint related to tyrosine kinase pathways (Flt3, Stat5b, Nras and Jak2) and known tumour
suppressors (Cdkn2a, Pten) (Fig 5B, Supp Table 3). Most of these were organised in a highly
connected network, suggesting their involvement in combinatorially coordinating the same
processes (Fig 5C). Functional annotations supported this finding, showing significant
enrichment of biological processes related to haematopoietic differentiation (interestingly
both lymphoid and myeloid) and signal transduction through tyrosine kinases (Fig 5D). Additionally, many of the genes, such as Nras, Flt3 and Sos1, are known for their contribution
to several cancers, including haematological malignancies (Supp Fig 4A). Because of its genetic design, the GrOnc transposon can alter gene expression through
multiple mechanisms. Discussion Non Hodgkin’s lymphomas (NHL) are the fifth most common cancer type and the most
common haematological malignancy. They comprise a range of genetically, phenotypically
and clinically heterogeneous diseases. Most of them derived from the malignant
transformation of germinal centre (GC) B cells, including DLBCL, the most common form of
lymphoma. CREBBP loss-of-function mutations have been identified as one of the main
genetic drivers involved in DLBCL [11,34]. Although ~60% of DLBCL patients are cured with
combined immnuno-chemotherapy [48], following relapse their outlook is poor with limited
therapeutic options. This disease therefore remains an unmet clinical need. To expand our
therapeutic arsenal and improve patient management, we must first obtain greater
understanding of the molecular mechanisms underlying lymphomagenesis. The development
of relevant DLBCL models which faithfully recapitulate human disease are also urgently
required to test novel therapeutics. Here, we refined our existing conditional Crebbp knock-out mouse models through
combination with a forward insertional transposition system. This allowed for both spatial
and temporal controls of Crebbp loss and the B-cell specific activation of the transposon
GrOnc. This powerful tool led us to assess the relative contribution of the cell of origin first
experiencing Crebbp loss and to investigate potential cooperative mutations. Crebbp loss
within the HSPC population resulted in a very aggressive DLBCL-like disease. Mice presented
with evidence of hepatosplenomegaly, as well as lymphadenopathy. Immunophenotypic
characterisation revealed an aberrant B220low/CD19+ population, which infiltrated across
multiple tissues. They expressed PNA and GL7 mainly, although Fas expression could also be
detected in some instances. Molecularly, they activated a transcriptional program with
enhanced cell cycle and cell proliferation, in part orchestrated by the up-regulation of the
transcription factor E2F1 and the cell cycle regulator, cyclin D3. These genes are known to be
preferentially expressed in GC B cells and contribute to the physiological clonal expansion
seen during the GC reaction [49]. In contrast, transcriptomic programs related to immune cell
activation, notably the CD40-CD40L signalling pathway, were silenced, preventing GC exit and
terminal differentiation. Collectively, these data reflect a global rewiring of key pathways
required during normal B cell maturation, most likely responsible for the malignant
transformation and the disease in mice. Although Crebbp loss from more committed B-cell progenitors also led to DLBCL, the latency
was significantly longer. This observation was consistent with another mouse model,
expressing EZH2Y641F mutation following Cre expression from the Cd19 locus, which develop
DLBCL after a year [50]. Positive correlation between murine models and human data ;
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bioRxiv preprint Positive correlation between murine models and human data Our new lymphoma mouse models faithfully recapitulated critical features of human DLBCL,
such as nodal involvement. They also identified cooperating mutations involving genes
important for normal B cell development such as Ebf1 and Pax5, as well as known oncogenes
and tumour suppressors such as Ezh2 and Cdkn2a, respectively, which may be relevant for
DLBCL progression. Analysis of gene-set libraries curated in the EnrichR tool [39,40] revealed
that our candidate list of DLBCL cooperating mutations highly correlated with proteomic
signatures of human lymphoid cell lines contained in CCLE proteomic database [41]. Out of
the 17 significantly enriched proteomic profiles, 7 (41%) were from lymphoid cell lines,
amongst which 3 were DLBCL lines (SUDHL4, OCI-Ly3, NUDHL1) (Fig 6A). We then investigated
whether the genes mutated in our DLBCL mouse models were also mutated in DLBCL patients
[42,43, 44,45]. 19/77 (~25%) of our candidate genes that had a human ortholog were found
to be mutated in DLBCL patients (Fig 6B). To get a more comprehensive view of the
commonalities between both data sets, we performed a comparative functional analysis
using Metascape [46]. We found a significant overlap between GO terms that were enriched
in each data set, as exemplified by the B cell receptor signalling pathway and the process of
leukocyte differentiation (Fig 6C, Supp Fig 5B). We also found a striking enrichment for gene-
disease signatures from the DisGeNET database [47] related to various B cell lymphomas,
amongst them diffuse large B-cell lymphoma and other lymphoid malignancies (Fig 6D). Given
the strong interconnection between both datasets, we integrated them in one single protein
interaction network, using Cytoscape. Interestingly this analysis highlighted several
interactions between the genes mutated in our mouse model and those mutated in DLBCL
patients such as JAK2 and NRAS (Fig 6E), suggesting that alterations of these signaling
pathways are important in DLBCL pathogenesis. . CC-BY 4.0 International license
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this version posted March 28, 2024. Discussion This suggests that the outcome of the disease varies according to the
cell of origin which first acquires Crebbp loss-of-function mutations. When acquired in the
haematopoietic stem and progenitor population, the intrinsic properties of this cell
compartment (i.e, self-renewal and proliferation) would provide a favourable cellular state
for transformation and potential effects on later B-cell differentiation. A later loss would also
provide similar proliferative and more limited self-renewal stimuli through the GC reaction
where more mature B cells will undergo active proliferation and somatic hypermutations. Alternatively, CREBBP mutations acquired early during B cell ontogeny would open a longer
temporal window for chromatin remodelling and cell re-programming to occur. Those signals
would be lacking when CREBBP mutations are acquired later which would explain the longer
latency and the seemingly less aggressive phenotype of the disease (consistent with FL) in our . CC-BY 4.0 International license
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bioRxiv preprint model. A potential caveat of our system, given the genetic design our IM-Cd19Crebbp-/-
model, where the Cre-recombinase is knocked into the Cd19 locus, is that CD19
haploinsufficiency may alter B-cell ontogeny, although we did not previously observe
significant dysfunction [3]. model. A potential caveat of our system, given the genetic design our IM-Cd19Crebbp-/-
model, where the Cre-recombinase is knocked into the Cd19 locus, is that CD19
haploinsufficiency may alter B-cell ontogeny, although we did not previously observe
significant dysfunction [3]. Immune evasion is a well-recognised hallmark in cancer that allows tumour cells to escape
immune surveillance and thus avoid macrophage-mediated phagocytosis. Discussion This phenomenon
is achieved via multiple ways, such as the loss of class I MHC expression at the surface of
malignant cells. Growing evidence shows that the lack of class II MHC also contributes to
immune evasion in DLBCL [51,35,52]. Our data reinforces this view with a significant
downregulation in the lymphoma cells of the master regulator of class II MHC gene expression
Ciita and the subsequent downregulation of Cd74, H2-Ab1, H2-Ob and H2-Eb2, which are
involved in antigen presentation and antigen processing [53]. Interestingly the
B220low/CD19+ neoplastic cells also enhanced CD24 expression on their cell surface. CD24
overexpression has been reported in several solid tumours [54,55,56] and haematological
malignancies [57]. In recent years, it has emerged as a novel ‘don’t eat me’ signal, which
modulates anti-tumour immunity through its binding to the inhibitory receptor Siglec-10
expressed by macrophages [56]. In line with this finding, two independent studies described
CD24high DLBCL as being ‘immune-cold’ with reduced expression of HLA genes and a lower
abundance of tumour-infiltrating immune cells, as determined by CIBERSORT [58,59]. These
data also suggest that our model may be of use in determining mechanisms of immune escape
and how these might be therapeutically targeted to improve outcomes in poor risk groups
such as the CD24high DLBCL patients [59]. The advent of NGS technologies has massively expanded our catalogue of somatic mutations
linked to human cancer. However, our understanding of the functional implications of such
mutations is still sparse and pinpointing true driver mutations remains challenging. Transposon-based screens implemented in mouse models have opened attractive avenues to
overcome these limitations. We, and others, have successfully used this approach in several
haematological malignancies and have identified known and novel genetic candidates
[36,15]. Similarly, by transferring this technology into the B cell context, we have uncovered
several common insertions sites (CIS) that have previously been associated with DLBCL and
other malignancies. Globally, these CIS were enriched for specific functional categories,
mainly ‘B cell development’, ‘Signalling’, ‘Cell cycle’ and ‘Transcription/Translation’. Interestingly, a similar functional observation was made from a genome-wide CRISPR screen
performed on human DLBCL cell lines [42]. This highlights that the corruption of several key
processes is a pre-requisite for the initiation and maintenance of transformation. Comparisons with human data also revealed significant functional overlaps, validating our
system. These promising results advocate for the investigation of novel CIS that demonstrated
functional relevance in our screen. rationale for prioritising candidates in mechanistic and therapeutic studies to improve
outcomes in DLBCL. rationale for prioritising candidates in mechanistic and therapeutic studies to improve
outcomes in DLBCL. Acknowledgements This study was carried out in the laboratory of B.J.P.H. with funding from Cancer Research UK
(C18680/A25508, C355/A26819 and DRCRPG-Nov22/100014), MRC (MR-R009708-1), the
Innovative Medicines Initiative (116026 and 945406) and the Cancer Research UK Cambridge
Major Centre (C49940/A25117). This research was supported by the NIHR Cambridge
Biomedical Research Centre (BRC-1215-20014), and was funded in part by the Wellcome
Trust, who supported the Wellcome – MRC Cambridge Stem Cell Institute (203151/Z/16/Z)
and Cambridge Institute for Medical Research (100140/Z/12/Z). The views expressed are
those of the authors and not necessarily those of the NIHR or the Department of Health and
Social Care. S.E.R. is supported by a Clinician Scientist Fellowship from Cancer Research UK
(C67279/A27957) and a Leukaemia UK John Goldman Fellowship (2022/JGF/004). This
research was supported by the CIMR Flow cytometry Core Facility. In particular, we wish to
thank Drs R. Schulte and G. Grondys-Kotarba for their advice and support in cell sorting. Conflict of interest The authors declare no competing financial interests. References 1
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made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
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The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint . CC-BY 4.0 International license
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
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bioinformatics analysis. Biosci Rep 42 (2022). https://doi.org/10.1042/BSR20212035 Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to significantly
accelerate the B-cell lymphoma phenotype in a novel murine model Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to significantly
accelerate the B-cell lymphoma phenotype in a novel murine model (A). Bar chart showing a high incidence of B-cell malignancies in both models. (B). Survival
curve demonstrating a significantly shorter survival in IM-Mx1Crebbp-/- mice (n=31)
comparted to IM-Mx1Crebbp+/+ littermates (n=26). Only mice with evidence of B-cell
malignancies were considered in this analysis. P<0.05, Log Rank test. (C). Violin plot showing
the spleen weight of lymphoma mice compared to control wild-type mice. Each dot
represents a mouse. (D). Pie chart comparing tissue infiltration between both lymphoma
models. Mice showing enlarged spleen and liver only were scored as “No lymph node”
(white), when they also had enlarged lymph nodes as “Nodal” (grey), when they had evidence
of additional tissue infiltration with or without lymph node involvement were scored as
“Extranodal” (dark grey). (E). Hematoxylin and eosin staining of mouse tissue sections
comparing healthy versus DLBCL-like disease. (F). Bar chart showing the distribution of B cell
lymphoma subtypes in both mouse models based on histological features. Mixed cases refer
to mice with evidence of DLBCL and FL. (G). Survival curve demonstrating an extremely short
survival after primary tumour transplant into recipient mice. Four primary tumours of
indicated genotypes (coloured lines) were transplanted into 7 recipient mice each. LN, lymph
node, V, hepatic vein, DLBCL, diffuse large B cell lymphoma, BL, Burkitt lymphoma, FL,
Follicular lymphoma. Figure 2: An aberrant B220low/CD19+ population drives a DLBCL-like disease Figure 2: An aberrant B220low/CD19+ population drives a DLBCL-like disease
(A). FACS plot showing B220 and CD19 cell surface marker expression in the spleen comparing
DLBCL-like lymphoma versus control. (B). FACS plot showing B220 and CD19 cell surface (A). FACS plot showing B220 and CD19 cell surface marker expression in the spleen comparing
DLBCL-like lymphoma versus control. (B). FACS plot showing B220 and CD19 cell surface . CC-BY 4.0 International license
made available under a
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bioRxiv preprint marker expression in DLBCL-like lymphoma mice across multiple tissues. (C). Heat map
showing the median expression of germinal centre markers in the neoplastic B220low/CD19+
population from both lymphoma models in comparison to control B220high/CD19+
splenocytes. (D). Boxplot showing the Mean Fluorescence Intensity (MFI) of CD24 in the
neoplastic B220low/CD19+ population from both lymphoma models in comparison to control
B220high/CD19+ splenocytes. (E). Graph showing the relative level of class-switch
recombination per indicated population. (F). Graph showing the mean number of somatic
hypermutations (SMH) in the three largest clones per indicated population. (G). Graphs
showing the clone size (as percentage) of the top largest clones per indicated population
isolated from control or lymphoma mice. (H) Representative BCR network plots of deep
sequenced PCR amplified immunoglobulin variable gene regions of indicated populations
isolated from control mice (n=7) or primary tumour samples (n=5-7). Figure 3: Transcriptomic alterations in lymphoma tumours mimic changes in DLBCL patients
(A). Venn diagram showing the overlap between the differentially expressed genes identified
from lymphoma versus control comparisons. The numbers indicate the number of
differentially expressed genes. (B). Volcano plot showing genes significantly dysregulated
(FDR 5% & Log2FC >|1|) in IM-Mx1Crebbp-/- vs control. (C). GSEA analysis showing two
independent Crebbp signatures enriched in IM-Mx1Crebbp+/+. (D). Dot plot showing biological
processes associated with up-regulated (top) and down-regulated (bottom) genes. (E). Selected heat maps illustrating the up-regulated and down-regulated processes. (F). Figure 2: An aberrant B220low/CD19+ population drives a DLBCL-like disease Heat
map showing the score of each sample analysed by RNA-sequencing for individual immune
cell type provided by the Immunological Genome Project (ImmGen) and additional studies.,
BM, bone marrow, CLP, Common lymphoid progenitor, DC, Dendritic cell, GC, germinal
centre, Mem, Memory. Figure 4: Loss of CREBBP in B-cell progenitors attenuates DLBCL-like disease Figure 4: Loss of CREBBP in B-cell progenitors attenuates DLBCL-like disease Figure 4: Loss of CREBBP in B cell progenitors attenuates DLBCL like disease
(A). Bar chart showing a high incidence of B-cell malignancies in both the IM-Cd19Crebbp-/-
and IM-Mx1 Crebbp-/- models. (B). Bar chart showing the distribution of B-cell lymphoma
subtypes in both mouse models based on histological features. Mixed cases refer to mice with
evidence of DLBCL and FL. (C). Survival curve demonstrating a significantly longer survival in
IM-Cd19Crebbp-/- mice (n=20) comparted with IM-Mx1Crebbp-/- mice (n=31). Only mice with
evidence of B-cell malignancies were considered in this analysis. P<0.001, Log Rank test. (D). Violin plot showing the spleen weight of mice with lymphoma compared to control wild-type
mice. Each dot represents a mouse. (E). Pie chart comparing tissue infiltration between both
lymphoma models. Mice showing enlarged spleen and liver only were scored as “No lymph
node” (white), when they also had enlarged lymph nodes as “Nodal” (grey), when they had
evidence of additional tissue infiltration with or without lymph node involvement as
“Extranodal” (dark grey). (F). FACS plot showing B220 and CD19 cell surface marker expression
across multiple tissues isolated from IM-Cd19Crebbpp-/-mice. (G). Heat map showing the
average expression of germinal centre markers in the neoplastic B220low/CD19+ population
from IM-Mx1Crebbp-/- and IM-Cd19Crebbp-/- lymphoma mice in comparison to control
B220high/CD19+ splenocytes from control mice. (H). Survival curve of recipient mice
transplanted with primary tumours of indicated genotype (n=5-7 recipients). DLBCL, diffuse
large B cell lymphoma, BL, Burkitt lymphoma, FL, Follicular lymphoma. Figure 5: Identification of transposon common integration sites (CIS) . CC-BY 4.0 International license
made available under a
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bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
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bioRxiv preprint (A). Venn diagram showing the distribution of individual insertions across genotypes. (B). Barplot showing the number of insertions for the top 20 most targeted genes. (C). String.db
network showing interactions between the genes harbouring at least three insertions. Single
nodes are not displayed. Interaction score confidence 0.6. (D). Figure 6: Positive correlation of murine models with human data (A). Functional annotations showing significant enrichment of insertionally targeted genes in
proteomic profiles of lymphoid cell lines contained in the CCLE proteomic database, using the
EnrichR tool. (B). Venn diagram showing the overlap between mouse candidates (human
orthologs) and the most recurrently mutated genes in DLBCL patients. (C). Heatmap showing
comparative enrichments of GO terms for the genes identified from our insertional mouse
screens and the most recurrently mutated genes in DLBCL patients. (D). Heatmap showing
comparative enrichments of gene-disease signatures from the DisGeNET database, as
designated in C. (E). Protein network showing interactions between genes as designated in C. Nodes are colour-coded according to their gene set of origin (blue for mouse, red for human,
green when found in both). Edges represent interactions between nodes (blue for
interactions between a mouse and a human gene, grey for any other combination). Figure 4: Loss of CREBBP in B-cell progenitors attenuates DLBCL-like disease Functional annotations
showing the Biological Processes for the genes insertionally targeted in mouse lymphoma
models. (E). Screen shots showing the genomic locations of insertions for Flt3 (top) and Nras
(bottom) genomic loci. (F). Boxplots showing the normalised read count for Flt3 (left) and
Nras (right) in control (Ct) and B lymphoma tissues in presence (Ins +) or absence (Ins -) of the
transposon GrOnc. Each dot represents an individual sample. Supplemental Figure Legend Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model significantly accelerate the B-cell lymphoma phenotype in a novel murine model
(A). Schematic representation of the genetic systems used in the study to model conditional
Crebbp loss upon Cre recombination and forward insertional mutagenesis based on genomic
insertions of the GrOnc transposon following the expression of the Piggy Bac transposase. (B). Breeding strategy used in the study to generate the experimental mice. Only genotypes of
interest are represented. (C). Schematic representation of the experimental design used in
the study. 6 to 12-weeks old mice were treated with poly(I) poly(C) (pIpC) to trigger the
expression of the Cre recombinase. After 6 weeks of recovery, they were challenged with
SRBC injections induce PB transposase expression. Blood samples were taken regularly. Mice
were culled when they showed evidence of illness. (D). Agarose gel showing Crebbp excision
(lower band) in IM-Mx1Crebbp-/- tissues after pIpC treatment. In contrast IM-Mx1Crebbp-/-
tissues harbour a full-length PCR product (higher band). (E). Violin plot showing the liver
weight of lymphoma mice compared to control wild-type mice. Each dot represents a mouse. (F). Violin plot showing the white blood cells count of lymphoma mice compared to control
wild-type mice. Each dot represents a mouse. (G-H). Violin plots showing the spleen (G) and
the liver (H) weight of recipient mice after transplantation of primary tumours of indicated
genotypes. Each dot represents a recipient mouse. Tum, tumour. Supplementary Figure 2: An aberrant B220low/CD19+ population drives a DLBCL-like disease
(A). Violin plot showing the percentage of positive cells for the germinal centre markers PNA,
GL7 and Fas assessed by flow cytometry. Cells were gated on B220high/CD19+ for the control . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
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doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
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bioRxiv preprint and B220low/CD19+ for both lymphoma models. Each dot represents a mouse. (B). Histogram
showing the Mean Fluorescence Intensity (MFI) of CD24 on B220high/CD19+ control cells and
B220low/CD19+ lymphoma cells of indicated genotypes. (C). Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model FACS plot showing the expression
of the germinal centre markers PNA, GL7 and Fas in B220low/CD19+ cells from recipient mice
after transplantation of primary tumour cells from total spleen. and B220low/CD19+ for both lymphoma models. Each dot represents a mouse. (B). Histogram
showing the Mean Fluorescence Intensity (MFI) of CD24 on B220high/CD19+ control cells and
B220low/CD19+ lymphoma cells of indicated genotypes. (C). FACS plot showing the expression
of the germinal centre markers PNA, GL7 and Fas in B220low/CD19+ cells from recipient mice
after transplantation of primary tumour cells from total spleen. Supplementary Figure 3: Loss of CREBBP in B-cell progenitors attenuates DLBCL-like disease
(A). Violin plot showing the liver weight of lymphoma mice of indicated genotypes compared
to control wild-type mice. Each dot represents a mouse. (B). Violin plot showing the white
blood cells count of lymphoma mice of indicated genotypes compared to control wild-type
mice. Each dot represents a mouse. (C). Hematoxylin and eosin staining on mouse tissue
sections from IM-Cd19Crebpp-/- lymphoma models. (D-F) Violin plots showing the spleen (D)
and liver (E) weight and the white blood cell count (F) of recipient mice after transplantation
of IM-Cd19Crebpp-/- primary tumours cells from total spleen. Tum, tumour. Supplementary Figure 4: Identification of transposon common integration sites (CIS)
(A). Functional annotations showing the KEGG pathways for the genes insertionally targeted
in mouse lymphoma models. Supplementary Figure 5: Positive correlation of murine models with human data
(A). Circos plot showing the overlap between ontology terms related to the most commonly
mutated genes in DLBCL patients and the commonly targeted genes in our DLBCL mouse
models. (B). Networks showing the genes involved the BCR signalling pathway and the process
of leukocyte differentiation. Genes are color-coded by species. 0
10
20
30
40
Cre-
Cre+
Figure 1
A
B
Number of cases
D
Spleen
Lymph Node
Liver
Kidney
Normal
DLBCL
x20
x2
x2
x2
x2
x2
x2
V
V
x10
x10
E
F
G
Survival (%)
50
100
25
75
Cre+
IM-Mx1
Crebbp-/-
IM-Mx1
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
Time post pIpC treatment (weeks)
20
40
60
0
Survival (%)
50
100
0
25
75
13
64
23
23
31
46
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
No LN
Nodal
Extranodal
Ct
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
0.1
0.3
1.0
3.0
C
Spleen weight (g)
Erythroid
B-cell
T-cell
Myeloid
Unknown
Solid tumour
IM-Mx1Crebbp-/- n=31
IM-Mx1Crebbp+/+ n=26
p < 0.05
. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this prepri
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint Figure 1 0
10
20
30
40
Cre-
Cre+
Figure
A
B
Number of cases
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
Time post pIpC treatment (weeks)
20
40
60
0
Survival (%)
50
100
0
25
75
Erythroid
B-cell
T-cell
Myeloid
Unknown
Solid tumour
IM-Mx1Crebbp-/- n=31
IM-Mx1Crebbp+/+ n=26
p < 0.05
. CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint B B
urvival (%)
50
100
75
IM-Mx1Crebbp-/- n=31
IM-Mx1Crebbp+/+ n=26
p < 0.05 A D
Spleen
Lymph Node
Liver
Kidney
Normal
DLBCL
x20
x2
x2
x2
x2
x2
x2
V
V
x10
x10
E
F
G
Time post transplant (Days)
10
20
30
0
Survival (%)
50
100
0
25
75
Percentage
Mixed (DLBCL+FL)
DLBCL
BL
FL
0%
20%
40%
60%
80%
100%
Cre-
Cre+
0
20
100
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
13
64
23
23
31
46
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
No LN
Nodal
Extranodal
40
60
80
Ct
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
0.1
0.3
1.0
3.0
C
Spleen weight (g)
IM-Mx1Crebbp+/+ - Tum 4 (n=7)
IM-Mx1Crebbp+/+ - Tum 3 (n=7)
IM-Mx1Crebbp-/- - Tum 2 (n=7)
IM-Mx1Crebbp-/- - Tum 1 (n=7) Ct
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
0.1
0.3
1.0
3.0
C
Spleen weight (g) D
13
64
23
23
31
46
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
No LN
Nodal
Extranodal 13
64
23
23
31
46
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
No LN
Nodal
Extranodal D C IM-Mx1
Crebbp+/+ IM-Mx1
Crebbp-/- 23 Ct Spleen
Lymph Node
Liver
Kidney
Normal
DLBCL
x20
x2
x2
x2
x2
x2
x2
V
V
x10
x10
E Spleen
Normal
DLBCL
x20
x2
x2
E Kidney
x10
x10 E Normal V DLBCL F
P
t
0%
20%
40%
60%
80%
100%
Cre-
Cre+
0
20
100
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
40
60
80 G G
Ti
t t
l
t (D
)
10
20
30
0
Survival (%)
50
100
0
25
75 G F Time post transplant (Days) Figure 2 Figure 2 LN
Lung
Liver
Control
DLBCL
A
B
C
DLBCL
Spleen
50
100
150
MFI CD24 (x100)
Ct
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
D
Ct
IM-Mx1
Crebbp+/+
IM-Mx1
Crebbp-/-
PNA
GL7
Fas
CD19
B220
CD19
B220
Figure 2
Expression
(%)
G
Clone Size (%)
Control
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
0
20
100
40
60
80
B220high/CD19+
Rank
2
4
6
8
B220low/CD19+
Rank
2
4
6
8
20
100
40
60
80
0
B220low/CD19+
Rank
2
4
6
8
20
100
40
60
80
0
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
Control
1
2
3
0
5
10
15
Mean SHM in largest
clones per sample (bp)
F
Rank
E
Relative Class-switch
recombination levels (arb. unit)
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
Control
Clone Size (%)
Clone Size (%)
. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
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doi:
bioRxiv preprint LN
Lung
Liver
DLBCL
B
CD19
B220
. CC-BY 4.0 International license
ilable under a
hor/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
3.25.586554 B B A A DLBCL Control
DLBCL
CD19
B220
made av
(which was not certified by peer review) is the au
https://doi.org/10.1101/2024. doi:
bioRxiv preprint C
50
100
150
MFI CD24 (x100)
Ct
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+
D
Ct
IM-Mx1
Crebbp+/+
IM-Mx1
Crebbp-/-
PNA
GL7
Fas
CD19
B220
CD19
B220
Expression
(%)
G
H
Clone Size (%)
Control
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
B220high/CD19+
B220low/CD19+
B220low/CD19+
Control
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
0
20
100
40
60
80
B220high/CD19+
Rank
2
4
6
8
B220low/CD19+
Rank
2
4
6
8
20
100
40
60
80
0
B220low/CD19+
Rank
2
4
6
8
20
100
40
60
80
0
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
Control
1
2
3
0
5
10
15
Mean SHM in largest
clones per sample (bp)
F
Rank
E
Relative Class-switch
recombination levels (arb. unit)
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
Control
Clone Size (%)
Clone Size (%)
. CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpe
The copyright holder for th
this version posted March 28, 2024. ;
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doi:
bioRxiv preprint CD19 C
D
Ct
IM-Mx1
Crebbp+/+
IM-Mx1
Crebbp-/-
PNA
GL7
Fas
Expression
(%) 50
100
150
MFI CD24 (x100)
Ct
IM-Mx1
Crebbp-/-
IM-Mx1
Crebbp+/+ D C 1
2
3
0
5
10
15
Mean SHM in largest
clones per sample (bp)
F
Rank E
Relative Class-switch
recombination levels (arb. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model unit)
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
Control F E E G
Clone Size (%)
Control
IM-Mx1Crebbp+/+
IM-Mx1Crebbp-/-
0
20
100
40
60
80
B220high/CD19+
Rank
2
4
6
8
B220low/CD19+
Rank
2
4
6
8
20
100
40
60
80
0
B220low/CD19+
Rank
2
4
6
8
20
100
40
60
80
0
Clone Size (%)
Clone Size (%) G
Clone Size (%)
Control
0
20
100
40
60
80
B220high/CD19+
Rank
2
4
6
8 G IM-Mx1Crebbp+/+
B220low/CD19+
Rank
2
4
6
8
20
100
40
60
80
0
Clone Size (%) IM-Mx1Crebbp-/-
B220low/CD19+
Rank
2
4
6
8
20
100
40
60
80
0
Clone Size (%) H
Control
B220high/CD19+ H IM-Mx1Crebbp+/+
B220low/CD19+ IM-Mx1Crebbp-/-
B220low/CD19+ Figure 3 A
951
3056
1085
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Lymphoma vs Ct
Up-regulated
680
1933
811
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Down-regulated
B
Figure 3
Down-regulated
Up-regulated
No change
Log10 adj. p-value
Log2 Fold Change
50
150
0
100
-10
-5
0
5
10
D
Hematopoietic or lymphoid organ development
Down-regulated
IM-Mx1Crebbp-/-
vs Ct
IM-Mx1Crebbp+/+
vs Ct
Regulation of leukocyte activation
Regulation of immune system process
Regulation of immune response
Regulation of cell activation
Lymphocyte activation
Leukocyte activation
Immune system development
Immune response-regulated signalling pathway
Immune response
Cell activation
Adaptive immune response
Positive regulation of immune system process
Up-regulated
Regulation of cell cycle progress
Plasma membrane bounded cell
Mitotic cell cycle
Mitotic cell cycle process
Nuclear division
Microtubule-based process
Microtubule cytoskeleton organization
Cytoskeleton organization
Cell projection organization
Cell division
Cell cycle process
Cell cycle
projection organization
Fold Enrichment
2.0 2.5 3.0 3.5
Gene Ratio
0.25
0.30
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
NES -1.82
qvalue 0.0
Core Crebbp Target Genes Zhang et a
Enrichment score (ES)
-0.2
0.0
-0.4
-0.5
NES -1.85
qvalue 0.02
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Enrichment score (ES)
-0.1
-0.2
0.0
-0.3
-0.4
-0.5
Mouse_shCrebbp.R2.DOWN_545_47
-0.3
-0.1
C
Signature Cell Cycle Cho&Whitfield
Signature CD40-Up1
Ct
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
2
1
0
-2
-1
Z-score
E
F
proB_FrBC
Mem_B_cells_IgM_C
Naïve_B_cells_C
GC_Light_Zone
GC_Gray_Zone
GC_Dark_Zone
T_cell_contaminant
NK_contaminant
Granulocyte contaminant
. CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprin
this version posted March 28, 2024. ;
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doi:
bioRxiv preprint B
adj. p-value
150
100 A
951
3056
1085
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Lymphoma vs Ct
Up-regulated
680
1933
811
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Down-regulated
B
Down-regulated
Up-regulated
No change
Log10 adj. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model p-value
Log2 Fold Change
50
150
0
100
-10
-5
0
5
10
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
NES -1.82
qvalue 0.0
Core Crebbp Target Genes Zhang et al
Enrichment score (ES)
-0.2
0.0
-0.4
-0.5
NES -1.85
qvalue 0.02
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Enrichment score (ES)
-0.1
-0.2
0.0
-0.3
-0.4
-0.5
Mouse_shCrebbp.R2.DOWN_545_47
-0.3
-0.1
C
. CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
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doi:
bioRxiv preprint A
951
3056
1085
Lymphoma vs Ct
Up-regulated
htt
doi:
bioRxiv preprint B IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
NES -1.82
qvalue 0.0
Core Crebbp Target Genes Zhang et al
Enrichment score (ES)
-0.2
0.0
-0.4
-0.5
-0.3
-0.1
C C A Lymphoma vs Ct Up-regulated NES -1.85
qvalue 0.02
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Enrichment score (ES)
-0.1
-0.2
0.0
-0.3
-0.4
-0.5
Mouse_shCrebbp.R2.DOWN_545_47
. onal license Mouse_shCrebbp.R2.DOWN_545_47
. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
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doi:
bioRxiv preprint Figure 4 Figure 4 B
IM-Cd19
Crebbp-/-
IM-Mx1
Crebbp-/-
Figure 4
CC BY 4 0 International license
nder a
der, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
6554 0
20
40
60
80
100
A
IM-Cd19
Crebbp-/-
IM-Mx1
Crebbp-/-
B
0
20
40
60
80
100
IM-Cd19
Crebbp-/-
IM-Mx1
Crebbp-/-
FL
Percentage (%)
Percentage (%)
Erythroid
B-cell
T-cell
Myeloid
Unknown
Solid tumour
Mixed (DLBCL+FL)
DLBCL
BL
Figure 4
. CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint B A A 0
20
40
60
80
100
Crebbp /
C
0
20
40
60
80
100
Crebbp-/-
FL
Percentage (%)
Cre+
12.9
64.5
22.6
IM-
Mx1Crebbp-/-
IM-
Cd19Crebbp-/-
23.8
47.7
28.5
Ct
IM-Mx1
Crebbp-/-
IM-Cd19
Crebbp-/-
Spleen weight (g)
D
E
F
Spleen
Lymph node
Liver
Time post pIpC treatment (weeks)
20
40
60
0
Survival (%)
50
100
0
25
75
80
IM-Mx1Crebbp-/- (n=31)
IM-Cd19Crebbp-/- (n=20)
p < 0.0001
Percentage (%)
Erythroid
B-cell
T-cell
Myeloid
Unknown
Solid tumour
Mixed (DLBCL+FL)
DLBCL
BL
0
1
2
3
No LN
Nodal
Extranodal
CD19
B220
G
H
Time post pIpC treatment (days)
20
40
60
0
Survival (%)
50
100
0
25
75
IM-Mx1Crebbp-/- - Tum 1 (n=7)
IM-Mx1Crebbp-/- - Tum 2 (n=7)
IM-Cd19Crebbp-/- - Tum 3 (n=5)
IM-Cd19Crebbp-/- - Tum 4 (n=5)
PNA
GL7
Fas
Expression
(%)
Ct
IM-Mx1
Crebbp-/-
IM-Cd19
Crebbp-/-
. CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model se 680
1933
811
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Down-regulated 680
1933
811
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
Down-r
Down-regulated
Up-regulated
No change
Log2 Fold Change
D
Hematopoietic or lymphoid organ development
Down-regulated
IM-Mx1Crebbp-/-
vs Ct
IM-Mx1Crebbp+/+
vs Ct
Regulation of leukocyte activation
Regulation of immune system process
Regulation of immune response
Regulation of cell activation
Lymphocyte activation
Leukocyte activation
Immune system development
Immune response-regulated signalling pathway
Immune response
Cell activation
Adaptive immune response
Positive regulation of immune system process
Up-regulated
Regulation of cell cycle progress
Plasma membrane bounded cell
Mitotic cell cycle
Mitotic cell cycle process
Nuclear division
Microtubule-based process
Microtubule cytoskeleton organization
Cytoskeleton organization
Cell projection organization
Cell division
Cell cycle process
Cell cycle
projection organization
Fold Enrichment
2.0 2.5 3.0 3.5
Gene Ratio
0.25
0.30
NES 1.85
qvalue 0.02
IM-Mx1Crebbp-/-
IM-Mx1
Enrichmen
-0.4
-0.5
Signature Cell Cycle Cho&Whit
Signature CD40-Up1
Ct
IM-Mx1Creb
IM-Mx1Creb
2
1
0
-2
-1
Z-score
E
Normal
Lymphoma
0.75
0.31
F
Score
proB_FrBC
Mem_B_cells_IgM_C
Naïve_B_cells_C
GC_Light_Zone
GC_Gray_Zone
GC_Dark_Zone
GC_IgG1_B
Naïve_B_cells_B
Mem_B_cells_IgG1_SP_B
Mem_B_cells_IgG1_DP_B
Mem_B_cells_IgG1_DN_B
T_cell_contaminant
NK_contaminant
Granulocyte_contaminant
DC_contaminant
Transitional_3
Plasma_cell
B1b
Follicular
Marginal_zone
Transitional_2
Fr_E
Plasma_cell_BM
Plasma_blast
Activated_B_cell_C
Mem_B_cell_IgG_C
Pro_B_FrA
ProB_CLP
Memory
Transitional_1 D
Up-regulated
Regulation of cell cycle progress
Plasma membrane bounded cell
Mitotic cell cycle
Mitotic cell cycle process
Nuclear division
Microtubule-based process
Microtubule cytoskeleton organization
Cytoskeleton organization
Cell projection organization
Cell division
Cell cycle process
Cell cycle
projection organization Signature Cell Cycle Cho&Whitfield
Signature CD40-Up1
Ct
IM-Mx1Crebbp-/-
IM-Mx1Crebbp+/+
2
1
0
-2
-1
Z-score
E E E D Signature Cell Cycle Cho&Whitfield
Si
t
CD40 U 1
E Signature Cell Cycle Cho&Whitfield He
Down-regulated
IM-Mx1Crebbp-/-
vs Ct
IM-Mx1Crebbp+/+
vs Ct
Re
Re
Re
Re
Lym
Le
Im
Im
Im
Ce
Ad
Po Signature CD40-Up1 IM-Mx1Crebbp-/-
vs Ct
IM-Mx1Crebbp+/+
vs Ct F Normal
Lymphoma
0.75
0.31
F
Score
proB_FrBC
Mem_B_cells_IgM_C
Naïve_B_cells_C
GC_Light_Zone
GC_Gray_Zone
GC_Dark_Zone
GC_IgG1_B
Naïve_B_cells_B
Mem_B_cells_IgG1_SP_B
Mem_B_cells_IgG1_DP_B
Mem_B_cells_IgG1_DN_B
T_cell_contaminant
NK_contaminant
Granulocyte_contaminant
DC_contaminant
Transitional_3
Plasma_cell
B1b
Follicular
Marginal_zone
Transitional_2
Fr_E
Plasma_cell_BM
Plasma_blast
Activated_B_cell_C
Mem_B_cell_IgG_C
Pro_B_FrA
ProB_CLP
Memory
Transitional_1 0
20
40
60
80
100
A
IM-Cd19
Crebbp-/-
IM-Mx1
Crebbp-/-
B
C
0
20
40
60
80
100
IM-Cd19
Crebbp-/-
IM-Mx1
Crebbp-/-
FL
Percentage (%)
Cre+
12.9
64.5
22.6
IM-
Mx1Crebbp-/-
IM-
Cd19Crebbp-/-
23.8
47.7
28.5
Ct
IM-Mx1
Crebbp-/-
IM-Cd19
Crebbp-/-
Spleen weight (g)
D
E
F
Spleen
Lymph node
Liver
Time post pIpC treatment (weeks)
20
40
60
0
Survival (%)
50
100
0
25
75
80
IM-Mx1Crebbp-/- (n=31)
IM-Cd19Crebbp-/- (n=20)
p < 0.0001
Percentage (%)
Erythroid
B-cell
T-cell
Myeloid
Unknown
Solid tumour
Mixed (DLBCL+FL)
DLBCL
BL
0
1
2
3
No LN
Nodal
Extranodal
CD19
B220
G
H
Survival (%)
50
100
25
75
Figure 4
PNA
GL7
Fas
Expression
(%)
. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint 0
20
40
60
80
100
FL
Percentage (%)
Mixed (DLBCL+FL)
DLBCL
BL C
Time post pIpC treatment (weeks)
20
40
60
0
Survival (%)
50
100
0
25
75
80
IM-Mx1Crebbp-/- (n=31)
IM-Cd19Crebbp-/- (n=20)
p < 0.0001 Ct
IM-Mx1
Crebbp-/-
IM-Cd19
Crebbp-/-
Spleen weight (g)
D
0
1
2
3 D C Survival (%) Time post pIpC treatment (weeks) Cre+
12.9
64.5
22.6
IM-
Mx1Crebbp-/-
Extranodal IM-
Cd19Crebbp-/-
23.8
47.7
28.5
No LN
Nodal F F
Spleen
Lymph node
Liver
CD19
B220 E G
PNA
GL7
Fas
Expression
(%)
Ct
IM-Mx1
Crebbp-/-
IM-Cd19
Crebbp-/- H H
Time post pIpC treatment (days)
20
40
60
0
Survival (%)
50
100
0
25
75 G Time post pIpC treatment (days) Figure 5 Figure 5
A
517
41
778
8
15
247
9
IM-Mx1Crebbp-/-
(n=30)
IM-Cd19Crebbp-/-
(n=17)
IM-Mx1Crebbp+/+
(n=24)
B
Regulation of protein tyrosine kinase activity
C
0
5
10
30
Ebf1
Flt3
Stat5b
Nras
Jak2
Cdkn2a
Pax5
Sos1
Rcl1
Galnt2l
Uhrf2
Prlr
Fto
Ric1
Zcchc7
Ak3
Celf2
Kdm2a
Glis3
Prkg1
Number of insertions
15
20
25
Regulation of monocyte differentiation
Regulation of Tumor Necrosis Factor
Lymphocyte differentiation
B cell differentiation
Negative regulation of protein kinase activity
Positive regulation of miRNA metabolic
B cell activation
Regulation of erythrocyte differentiation
Transmembrane receptor protein tyrosine
superfamily cytokine production
kinase signaling pathway
Gene Ratio
Biological Process
Process
Adj. Pval
0.03
0.02
0.01
Odds.Ratio
20 40 60
E
F
5.0
7.5
10.0
Normalized Read Count
12.5
15.0
-
B Lymphoma
Ct
-
+
Ins
Flt3
7.5
10.0
Normalized Read Count
12.5
15.0
-
B Lymphoma
Ct
-
+
Ins
Nras
D
Flt3
Nras
chr5:147,267,551-147,337,299
10 Kb
2 Kb
chr3:102,965,601-102,975,230
0.05 0.10 0.15 0.20 0.25
. CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint Figure 5
A
517
41
778
8
15
247
9
IM-Mx1Crebbp-/-
(n=30)
IM-Cd19Crebbp-/-
(n=17)
IM-Mx1Crebbp+/+
(n=24)
B
0
5
10
30
Ebf1
Flt3
Stat5b
Nras
Jak2
Cdkn2a
Pax5
Sos1
Rcl1
Galnt2l
Uhrf2
Prlr
Fto
Ric1
Zcchc7
Ak3
Celf2
Kdm2a
Glis3
Prkg1
Number of insertions
15
20
25
. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint Figure 6 Figure 6 Figure 6 g
A
Leukocyte activation involved in immune response
Cellular response to organonitrogen compound
Cytokine signaling in immune system
B cell receptor signaling pathway
Myeloid cell differentiation
IL4 signaling pathway
Pathways affected in adenoid cystic carcinoma
Chromatin organization
Leukocyte differentiation
Regulation of leukocyte proliferation
Regulation of alpha-beta T cell activation
Pathways in cancer
Positive regulation of cytokine production
Negative regulation of cell differentiation
Androgen receptor network in prostate cancer
Cell population proliferation
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Adaptative immune response
Chronic myeloid leukemia
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. CC-BY 4.0 International license
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
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significantly accelerate the B-cell lymphoma phenotype in a novel murine model CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. ;
https://doi.org/10.1101/2024.03.25.586554
doi:
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Cellular response to organonitrogen compound
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IL4 signaling pathway
Pathways affected in adenoid cystic carcinoma
Chromatin organization
Leukocyte differentiation
Regulation of leukocyte proliferation
Regulation of alpha-beta T cell activation
Pathways in cancer
Positive regulation of cytokine production
Negative regulation of cell differentiation
Androgen receptor network in prostate cancer
Cell population proliferation
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Adaptative immune response
Chronic myeloid leukemia
Cellular response to cytokine stimulus
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Mouse
T-cell lymphoma
Childhood Non-Hodgkin lymphoma
Follicular lymphoma
Mantle cell lymphoma
Classical Hodgkin’s lymphoma
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. CC-BY 4.0 International license
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model Pval
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CC-BY 4.0 Internat
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioR
this version posted
;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint A
697 Haematopoietic and Lymphoid Tissue TenPx30
HGC27 Stomach TenPx06
EOL1 Haematopoietic and Lymphoid Tissue TenPx33
SUDHL4 Haematopoietic and Lymphoid Tissue TenPx26
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288
Mouse
(n=77)
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58
. CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 28, 2024. Supplementary Figure 1: Early Crebbp loss cooperates with insertional mutagenesis to
significantly accelerate the B-cell lymphoma phenotype in a novel murine model ;
https://doi.org/10.1101/2024.03.25.586554
doi:
bioRxiv preprint Human
(n=307) Mouse
(n=77) Leukocyte activation involved in immune response
Cellular response to organonitrogen compound
Cytokine signaling in immune system
B cell receptor signaling pathway
Myeloid cell differentiation
IL4 signaling pathway
Pathways affected in adenoid cystic carcinoma
Chromatin organization
Leukocyte differentiation
Regulation of leukocyte proliferation
Regulation of alpha-beta T cell activation
Pathways in cancer
Positive regulation of cytokine production
Negative regulation of cell differentiation
Androgen receptor network in prostate cancer
Cell population proliferation
Epstein-Barr virus infection
Adaptative immune response
Chronic myeloid leukemia
Cellular response to cytokine stimulus
Human
Mouse
T-cell lymphoma
Childhood Non-Hodgkin lymphoma
Follicular lymphoma
Mantle cell lymphoma
Classical Hodgkin’s lymphoma
Childhood Burkitt lymphoma
Adult Non-Hodgkin lymphoma
Adult Burkitt lymphoma
Waldenstrom macroglobulinemia
T cell leukaemia
Adult T-cell lymphoma/leukaemia
Burkitt lymphoma
Precursor T-cell lymphoblastic lymphoma
Adult classical Hodgkin’s lymphoma
Adult diffuse Large B-cell lymphoma
Precursor B-cell lymphoblastic leukaemia
Human
Mouse
Acute leukaemia
Adult Hodgkin lymphoma
Non-Hodgkin lymphoma
Childhood Hodgkin lymphoma
C
D Leukocyte activation involved in immune resp
Cellular response to organonitrogen compou
Cytokine signaling in immune system
B cell receptor signaling pathway
Myeloid cell differentiation
IL4 signaling pathway
Pathways affected in adenoid cystic carcinom
Chromatin organization
Leukocyte differentiation
Regulation of leukocyte proliferation
Regulation of alpha-beta T cell activation
Pathways in cancer
Positive regulation of cytokine production
Negative regulation of cell differentiation
Androgen receptor network in prostate cance
Cell population proliferation
Epstein-Barr virus infection
Adaptative immune response
Chronic myeloid leukemia
Cellular response to cytokine stimulus
Human
Mouse
C C D T-cell lymphoma
Childhood Non-Hodgkin lymphoma
Follicular lymphoma
Mantle cell lymphoma
Classical Hodgkin’s lymphoma
Childhood Burkitt lymphoma
Adult Non-Hodgkin lymphoma
Adult Burkitt lymphoma
Waldenstrom macroglobulinemia
Adult Hodgkin lymphoma
Non-Hodgkin lymphoma
Childhood Hodgkin lymphoma Mouse Human Mouse SOS1
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PIP4K2A
CD47
CDK6
HDAC9
NCOA2
FLT3
JAK2
NRAS
MYC
HNRNPA2B1
PAX5
PRLR
ZFP423
EBF1
EP300
JAK1
SOCS1
PTEN
CD36
PTPRC
PTPN6
RB1
NCOR1
NR3C1
STAT3
CDKN2A
MET
JAK3
STAT5B
CBLC
GRB2
ETS1
NFKB1
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MYB
Mouse
Human
Both
Mouse-Human
Other
E
KRAS
NF1
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MAP2K1
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IL6
CCND1 SOS1
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PIP4K2A
CD47
CDK6
HDAC9
NCOA2
FLT3
JAK2
NRAS
MYC
HNRNPA2B1
PAX5
PRLR
ZFP423
EBF1
EP300
JAK1
SOCS1
PTEN
CD36
PTPRC
PTPN6
RB1
NCOR1
NR3C1
STAT3
CDKN2A
MET
JAK3
STAT5B
CBLC
GRB2
ETS1
NFKB1
BCL11A
MYB
Mouse
Human
Both
Mouse-Human
Other
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KRAS
NF1
RASA2
MAP2K1
PIK3CG
IL6
CCND1 E Mouse
|
https://openalex.org/W2807061776
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http://revistas.up.edu.pe/index.php/business/article/download/877/1056
|
English
| null |
How does Cultural Norms Influence Entrepreneurial Intention? A Cross Cultural Study
|
Journal of business
| 2,018
|
cc-by
| 8,431
|
Journal of Business, Universidad del Pacífico (Lima, Peru)
ISSN 2078‐9424
How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study
José Carlos Sánchez-García
jsanchez@usal.es
Universidad de Salamanca, Spain
Alexander Ward Mayens
alexander.ward@usal.es
Universidad de Salamanca, Spain
Gioconda Vargas Morúa
gvargasm@usal.es
Universidad de Salamanca, Spain
Jenny Linette Flórez Daza
jflorez@usal.es
Universidad de Salamanca, Spain
Brizeida Hernández Sánchez
brizeida@usal.es
Universidad de Salamanca, Spain
Abstract José Carlos Sánchez-García
jsanchez@usal.es
Universidad de Salamanca, Spain Alexander Ward Mayens
alexander.ward@usal.es
Universidad de Salamanca, Spain Gioconda Vargas Morúa
gvargasm@usal.es
Universidad de Salamanca, Spain Gioconda Vargas Morúa
gvargasm@usal.es
Universidad de Salamanca, Spain https://doi.org/10.21678/jb.2018.877
Paper received 06/09/2017 paper accepted 31/05/2018
Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Flórez Daza, J.L. & Hernández Sánchez, B. (2018). How does Cultural Norms
Influence Entrepreneurial Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69
Editor in Chief: Prof. Dr. Luis Camilo Ortigueira-Sánchez Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Flórez Daza, J.L. & Hernández Sánchez, B. (2018). How does Cultural Norms
Influence Entrepreneurial Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69
Editor in Chief: Prof. Dr. Luis Camilo Ortigueira-Sánchez https://doi.org/10.21678/jb.2018.877
Paper received 06/09/2017 paper accepted 31/05/2018 Introduction The benefits that entrepreneurship offers to society have become evident through
history, mainly due to its demand for innovation, development and creation of
employment opportunities, however, the role that entrepreneurship plays in improving
those conditions also acts in a reciprocal way: society plays in forming the conditions
that creates entrepreneurs. For this reason, it’s important to understand how the
environment is optimized to effectively promote entrepreneurial developement,
specifically, its intention. Entrepreneurial intention, although widely studied (eg, Carr &
Sequeira 2007, Kautonen, Luoto & Tornikoski 2010, Tornikoski, Erno & Kautonen 2009),
is still far from a complete understanding on how its dynamics work in different contexts
and countries. Bretones and Silva (2009), believe the study of culture and entrepreneurial behavior
includes that of social and economic aspects related with specific values and beliefs that
would influence behavior. This differentiates people from diverse backgrounds and
territories, including in the field of organizations. Hofstede (1980) defines culture as an
aggregation of shared values, beliefs, norms and expected behaviors; this study will be
heavily emphasized on norms. In the present study, entrepreneurial intention will be analyzed taking as reference a
sample of countries with different development contexts, considering each comprises a
different reality in their process of promoting and employing entrepreneurial values. For
example, India has high levels of poverty, approximately 350 million people (Bureau
2015), where of every three; one lacks basic needs, including employment and
education. In some cases, may even extend to the point of illiteracy (Shaw & de Bruin,
2013), however, it has a Total of Entrepreneurial Activity (TEA) of 10.6, matching
competitively with other countries of Asia and Oceania (GEM, 2016). Considered a
country with noticeable collectivist values, Tiwari, Bhat and Tikoria (2017) found a
significant relationship between its subjective norm and entrepreneurial intention. Similarly, a study by Iakovleva, Kolvereid, and Stephan (2011), investigated the
relationship of entrepreneurial intention between emerging and developed countries,
which India participated, demonstrating that the relationship of attitudes and perceived
control have less intensity in entrepreneurial intention within developing countries than
in developed ones. Contrasting among other emerging economies, Mexico leads among other Latin
American countries in entrepreneurial activity (GEM, 2016) and in 2015 showed a TEA
of 21% (GEM 2015). Abstract Entrepreneurial Intention is commonly attributed to the interaction of cultural values and
attitude; however, cultural values vary according to the context in which they are
developed. The following study intends to show how Subjective Norms affect intentions
of self-employment, as both, a direct factor and mediated through attitudinal values:
Perceived Behavioral Control and Desirability, within a cross-cultural framework. A
sample of 736 students from four different countries: Spain, Portugal, Mexico and India
were used. Results yielded a direct relationship between Subjective Norm and Intention
in all countries, except India. When mediated through attitude, the relation becomes
stronger, even more when it’s through Desirability, except in Portugal, which the
relationship is stronger directly through Social Norms. India does not show a relationship
between Perceived Behavioral Control and Intention. Conclusions show that the
influence of culture on intention, effectively, varies by context. Keywords: Entrepreneurship, Psychology, Intention, Culture, Subjective Norm. 52 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Introduction Despite this, Kantis, (2002) points financial restriction and
identification of regulatory obstacles (taxes, procedures, registration costs) as highlights
among difficulties for startups; while, showing institutional support and a tendency to rest
in close relationships (relatives, friends and acquaintances) as a strength. On the other hand, developed countries can also show different contexts that impact
entrepreneurial feasibility, pointing to different challenges. In the case of Portugal and
Spain, there is a comparable situation with respect to entrepreneurial activity, with a TEA
of 8.2% (GEM, 2013) and 5.7% (GEM, 2015) respectively, which places them below
other countries in Europe. Several studies have researched on how attitudes and
environment interact within these two countries in promoting entrepreneurship. Sampedro, Fernández-Laviada and Crespo, (2013) found that students in Spain show a
positive relationship in how they perceive social support towards entrepreneurial
behavior, as well as their willingness to endeavor within them. Also, results showed
students consider existing formation as acceptable, but access to funding limited. 53 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 At a comparative level, Portuguese university students are more prone to self-
employment than those in Spain (Sánchez & Yurrebaso, 2008), considering desirable
self-employment and perceiving favorable social support towards entrepreneurship. Another study found that students from Spain (Extremadura) recognize entrepreneurship
presently as more accessible than decades ago, while those in Portugal (Beira) consider
it more difficult nowadays. However, it is the students of Portugal (Beira) that often start
a business (Díaz-casero, Ferreira, Hernández Mogollón, & Barata Raposo, 2012). These arguments demonstrate the relevance of pursuing a deeper understanding of the
role played by prior exposure on the process of enterprising in a more systematic way,
specifically, in its intention, as well as a need to expand on their relationship by context:
how it can change by variables such as the individual and environment. Introduction The following study aims to answer these following questions: (1) How does a country's
culture influence the desirability and perceived control of students and their intention to
start a business?; (2) Is the effect of culture on entrepreneurial intention greater when
mediated by desirability and perceived control? (3); What is the difference between the
entrepreneurial intention of students from different socio-cultural backgrounds, when
perceived control and desirability act as mediating variables?; and (4) To what extent
does perceived control and desirability affect entrepreneurial intention in students, when
different socio-cultural backgrounds are taken into account? Based on these goals, the main objective of this research is to identify the effects of
subjective norm on entrepreneurial intention during the process, both directly and
indirectly, using as mediating variable desirability and perceived control. Participants in
this study correspond to a sample of university students from two developing countries
(Mexico and India), and two developed countries (Spain and Portugal). The analysis was
carried out with each country separately, then compared among them in order to contrast
the influence of each variable on entrepreneurial intention. Entrepreneurial Intention According to Krueger, (1993), entrepreneurial intention is a state of mind that people
have, opting for the creation of a new company or the creation of value within existing
organizations; the commitment to perform the necessary behavior to carry out an
entrepreneurial initiative. Authors such as Cabana-Villca, Cortes-Castillo, Plaza-Pasten,
Castillo-Vergara, Alvarez-Marin, (2013) and Krueger, (1993) consider intentions as the
best predictor of planned behavior, such as starting a business. The findings of several
studies have found a positive effect between intention and entrepreneurial behavior
(Ajzen, 1991, Fayolle, Gailly & Lassas-Clerc, 2006, Homer & Kahle, 1988, Jogiyanto,
2007, Kolvereid, 1996, Schwarz, Wdowiak, Almer- Jarz, Breitenecker, 2009; Tkachev &
Kolvereid, 1999). The following are two of the most researched models, which act as backbone of this
study: Theory of Planned Behavior and Shapero’s model of the Entrepreneurial Event,
which support the relationship between entrepreneurial behavior and entrepreneurial
intention, in order to answer the questions posed in the present study: 54 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 The Theory of Planned Behavior (TPB) (Ajzen, 1991) is based on intentions people
possess to carry out their behavior, and these can be predicted by the following
elements: attitudes towards behavior, subjective norms and perceived behavioral
control. Attitude towards behavior is defined as the degree in which a person has a
favorable or unfavorable evaluation or assessment of the behavior in question (Ajzen,
1991). The second, subjective norm, refers the perceived social pressure whether to
carry out the behavior or not (Ajzen, 1991), in other words, the opinion of how third parties
- such as family, friends or teachers - influences the individual. The third is the degree of
perceived behavioral control that, according to Ajzen (1991), is the viability of performing
a perceived behavior, since it is not always completely subject to the person’s will (Ajzen,
2002). Entrepreneurial Intention Ultimately, within the TPB (Ajzen, 1991), its core is the intention of people to
perform a certain behavior. Intentions, therefore, capture the motivational factors that
influence a behavior, as well as the amount of effort they are willing to exert to achieve
it (Ajzen, 1991). Shapero’s model of the Entrepreneurial Event (SEE) (Shapero & Sokol, 1982) introduces
two aspects that influence the creation of companies: desirability and feasibility. Both
aspects are products of the cultural and social environment that ultimately determine
entrepreneurial behavior. Shapero and Shokol (1982) examine the concept of desirability
using information about the family, circle of friends, ethnic group, among others. In terms
of feasibility, they point out the importance of the individual's ability to obtain resources
in a way that makes enterprising possible. Shapero and Shokol (1982), proposed three
phases: the first, in which a series of events trigger or predispose entrepreneurship to
happen; the second, which calls for desire, transforming a potential entrepreneur into a
possibility and the third, action, in which the individual finally decides to create its
business. Subjective Norm Subjective norm (NS) is the second predictor of intention, and refers to the perceived
social pressure to carry out or not a behavior (Ajzen, 1991); it corresponds to exogenous
variables, such as the expectations or opinions of others (Shapero & Shokol, 1982). Nilsson, Borgstede, and Biel (2004) claim that NS also accounts for a person's belief in
referring to the influence of others to behave in a certain way and meet their expectations. In other words, the tendency of an individual to consider the perceptions of others, such
as significant persons, families, friends or communities as a tool to guide them to
undertake or not decisions. Rhodes and Courneya (2003) consider that this construct is
also capable of mediating entrepreneurial behavior. Effectively, multiple studies have
shown a positive effect between subjective norm and entrepreneurial behavior (Armitage
& Conner, 2001; Miner, 2001; Rimal & Real, 2003), particularly, in entrepreneurial
intention (Ajzen, 1998, 1991, 2002; Fisbein; & Ajzen, 1975, Guido, Marcati & Peluso,
2011, Liñan & Chan 2009, Rhodes & Courneya, 2003, Shook & Britanu, 2008, Solesvick,
Westhead, Kolvereid & Matlay, 2012, Schwarz et al., 2009, Tkachev & Kolvereid, 1999;),
as well as effecting it indirectly (Paço, Ferreira, Raposo, Gouveia & Dinis, 2011). Although, contrasting results have been found as well, such as the meta-analysis carried
out by Armitage and Conner (2001), in which they conclude that NS is a weak predictor, 55 nchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
ention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 and in some cases, not significant at all (Autio, Keeley, Klofsten, Parker & Hay, 2001;
Krueger, Reilly, & Carsrud, 2000; Liñan, 2008). and in some cases, not significant at all (Autio, Keeley, Klofsten, Parker & Hay, 2001;
Krueger, Reilly, & Carsrud, 2000; Liñan, 2008). Evidence shows that self-employment outcomes are significantly affected by factors
such as individual skills, family background, employment status, liquidity constraints and
ethnic enclaves (Le, 1999). This indicates that entrepreneurs are influenced by the
closest environment that surrounds them, family being the only social institution in which
all entrepreneurs are rooted (Aldrich & Cliffb, 2003). Subjective Norm Some research indicates that the
experience of students with family businesses provides them with more developed
positive attitudes towards business (Harris, & Gibson, 2008, Mohamed, Rezai, Mad &
Muhammad, 2012), as well as greater influence and predisposition (Fayolle & Klandt,
2006). However, others suggest that this previous entrepreneurial exposure may also be
negative (Zhang et al., 2014). With respect to culture, Hofstede (1980) refers to it as "the collective programming of the
mind that distinguishes the members of one human group from another ... [e] includes
value systems" (p.25). Culture, as the underlying system of values of a specific group or
society, motivates the individuals of a certain society to engage in behaviors that may
not be evident in others (Mueller & Thomas, 2000) such as entrepreneurial behavior,
considering there are differences in values and cultural beliefs from one country to
another. In summary, literature shows the existence of influences of the subjective norm
(relatives, friends or close persons) towards the entrepreneurial intention, and that the
culture associated with the subjective norm in which a person develops can influence in
greater or lesser way entrepreneurial intention. Perceived Behavioral Control Perceived Behavioral Control is the third factor for predicting intention, and is the
feasibility of performing a perceived behavior, as factors concerning the success of its
execution may not be entirely up to the individual (Ajzen, 2002). Shapero and Shokol
(1982), on the other hand, indicate that business intentions are derived from the
perception of feasibility, as well as convenience, since people usually choose to adopt
behaviors that they think they will be able to control and dominate (Fayolle & Klandt,
2006), so that their behaviors are influenced by the confidence they have in their ability
to carry them out (Ajzen, 1991). Krueger and Carsrud (1993) state that "perceived self-efficacy or control for business
behaviors are influenced by the acquisition of management tools and exposure to
business situations" (page 326). In this case, beliefs of control are based on past
experiences of entrepreneurial behavior, as well as the influence that third-party
information has on it, such as the culture in which it was developed. Therefore, support of political, social and business leaders could be a fundamental factor
for the development of business activity (Krueger & Brazeal, 1994), since its support
provided through incentives encourages the formation of companies and makes more
likely that entrepreneurs perceive their chance of self-employment as something 56 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneu
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 possible. However, it is assumed that not all cultures behave in the same way and, that,
from one country to another, different situations and opportunities are offered, specific to
each. possible. However, it is assumed that not all cultures behave in the same way and, that,
from one country to another, different situations and opportunities are offered, specific to
each. Desirability Desirability refers to the desire and attraction that a person feels when initiating an
entrepreneurial activity, for which he makes important personal sacrifices and place it
before any other opportunity or activity that avoids its achievement (Sanchez, 2010). Peterman and Kennedy (2003) point out that the degree of change in the perception of
desirability and feasibility are positively related to previous experience, therefore, it is
considered that the environment in which it operates will be important for the
development of entrepreneurial intentions. According to the model of the Entrepreneurial
Event of Shapero and Shokol (1982), intentions are derived from the perceptions of
feasibility and desirability. That is, an individual who perceives a business opportunity
(which can be given by their environment or culture), will analyze it within the framework
of these two factors and, once confirmed, will lead to business creation. H4: The effect of Subjective Norm on Entrepreneurial Intention is greater when
influenced by Desirability and Perceived Behavioral Control. Sample A sample of 736 university students (from bachelor to doctorate), linked to various areas
of knowledge was used. Table 1 details samples by country Table 1. Sample Description
Age
Gender
Country
Total
Range
Mean
Male
Female
Spain
253
17-40
21,20
71
28,1%
182
71,9%
Portugal
104
17-60
25,98
41
39,4%
63
60,6%
México
254
18-59
21,97
111
43,7%
143
56,3%
India
125
18-51
21,83
112
89,6%
13
10,4%
Total
736
335
46%
401
54% Table 1. Sample Description The sample was collected through collaboration with teachers from universities of each
respective country, which administered a questionnaire designed to measure university
student’s potential (Sánchez, 2016) through both means, digital and by paper. Hypotheses for Study It’s important to stress that entrepreneurs can be influenced by the closest environment
that surrounds them, and the more favorable the expectation and social pressure
perceived, referring to NS, the greater the influence on behavior (Ajzen, 1991). It should
also be noted that the desire felt by the entrepreneur to carry out a behavior, and the
perception he feels of being able to carry out the activity, will ultimately influence his
intention. Shapero and Shokol (1982), suggest that there must be a trigger, which the desirability
and feasibility to act on opportunities derives from. For the purposes of this study, cultural
norms are proposed as one of these. Depending on the place and its conditions, an
entrepreneurial mindset will be either promoted or thwarted, potentially affecting how a
person will both value its wishes and the feasibility to start a business. Thus, while
cultural norms may possess a considerable influence, it could also be fundamental, and
with perhaps a stronger effect, how the person itself weights its desire and control on the
situation, forming ultimately his intention to act upon enterprising. The hypotheses
proposed for this study are as follows: H1: Subjective Norm directly influences Entrepreneurial Intention. H2: Subjective Norm influences Entrepreneurial Intention through Perceived Behavioral
Control. H3: Subjective Norm influences Entrepreneurial Intention through Desirability. H4: The effect of Subjective Norm on Entrepreneurial Intention is greater when
influenced by Desirability and Perceived Behavioral Control. 57 nchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
ention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Given the previous hypotheses, the following model is presented in Figure 1: Given the previous hypotheses, the following model is presented in Figure 1: Figure 1. Proposed model on the effect of Subjective Norm on the Entrepreneurial
Intention, with two mediating variables. Figure 1. Proposed model on the effect of Subjective Norm on the Entrepreneurial
Intention, with two mediating variables. Analysis and Procedure Analysis and Procedure For the analysis of the hypotheses according to the proposed model, the macro
PROCESS was used in SPSS 23 (Hayes, & Scharkow, 2013, Hayes & Preacher, 2014). Bootstrapping was used (Hayes, 2013) because of its facilitation of statistical inference
without raising assumptions about the normality of the data or requiring large samples,
since it constitutes a method of resampling (10,000 samples in this case) that is used to
approximate the distribution of a statistic (the coefficient of regression that estimates the
indirect effect) and builds the confidence intervals (IC) to make a statistical decision
about the significance of the observed effect (Hayes, 2013). It is considered that an
indirect effect (ab) is statistically significant if the established confidence interval (95%
IC) does not include the zero value. If the zero value is included in said confidence
interval, the null hypothesis that the indirect effect is equal to zero cannot be rejected,
that is, there is no association between the variables involved (Hayes, 2013). Therefore, the analytical strategy used was based on this procedure with the objective
of evaluating the effect of desirability and perceived control over entrepreneurial
intention, for which model 4 of Bootstrapping by Hayes (2013) was used, proposing a
model with two mediating variables. Instrument Four scales from the questionnaire (Sánchez, 2016) were used, specifically, containing
items that measure Intention (6), Subjective Norm (5), Perceived Behavioral Control (6) 58 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 and Desirability (8), for a total of 25 items. For its analysis, and to test the hypotheses of
the proposed model, the macro PROCESS in SPSS 23 (Hayes & Scharkow, 2013) was
used to analyze the direct and indirect effects between the variables. and Desirability (8), for a total of 25 items. For its analysis, and to test the hypotheses of
the proposed model, the macro PROCESS in SPSS 23 (Hayes & Scharkow, 2013) was
used to analyze the direct and indirect effects between the variables. The scales used to measure the variables are interval metrics (1-5 Likert scales), which
are detailed below: Entrepreneurial Intention- Corresponds to the dependent variable with six items. Questions such as "I am ready to do anything as an entrepreneur, or, I have thought very
seriously about starting a business" were asked. Subjective Norm- Corresponds to the independent variable. Five items are considered,
which ask if the family, friends, colleagues and culture view the business activity
favorably. Desirability- Corresponds to the first mediating variable; composed of eight items,
comprising of questions related to their desire and preference for business activity. Perceived Behavioral Control- Corresponds to the second mediating variable composed
of 6 items, in which questions are asked related to their ability to control the processes
of venture creation. Country- As control variable. Results Scale reliability was realized by country. Results obtained are shown in Table 2. Table 2. Scale Reliability analysis, by country
Cronbach’s α
Items
Spain
India
Mexico
Portugal
Entrepreneurial
Intention
6
0,949
0,941
0,940
0,934 Scale reliability was realized by country. Results obtained are shown in Table 2. Table 2. Scale Reliability analysis, by country 59 nchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
ention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Subjective Norm
5
0,640
0,734
0,791
0,762
Desirability
8
0,928
0,930
0,931
0,884
Perceived Control
6
0,876
0,815
0,909
0,936 This analysis concludes that each of the scales presents an adequate internal
consistency, and is capable of providing the required information. The most significant results of the study are presented below (Table 3) which are based
on the hypotheses and proposed model (see figure 1). The variables were assigned as: Independent Variable (X) Subjective Norm Independent Variable (X) Subjective Norm Dependent Variable (Y) Entrepreneurial Intention
Medidator 1 (M1) Desirability
Medidator 2 (M2) Perceived Control Table 3. Subjective Norm’s effect on Entrepreneurial Intention, including effect through
mediator variables
Inidrect Effect
Path
β Spain β Mexico β India
β
Portugal
Subjective Norm / Desirability
a
0,3684
0,3508
0,3033
0,2320
Desirability / Entrepreneurial Intention
b
0,7090
0,7157
0,8342
0,8404
Subjective Norm / Perceived Control
d
0,4364
0,4755
0,1126 x
0,4516
Perceived Control / Entrepreneurial
Intention
e
0,3208
0,2499
0,3503
0,1757
Direct Effect
c´
0,1589
0,1059 -0,0447 x
0,4067
Total Effect: Subjective Norm ->
Intention
ab+de+c´
0,5601
0,4758
0,2478
0,6810
Total Model Effect
R2
0,1001
0,1581
0,0383
0,2156
Sample
253
254
125
104
Notes: p<0,05 with exception of "x"; non-standarized coefficients; re-sampling number: 10000;
Confidence interval: 95%. Model 4 of Bootstrapping used (Hayes, 2013). e 3. Subjective Norm’s effect on Entrepreneurial Intention, including effect through
t
i bl Table 3. Subjective Norm’s effect on Entrepreneurial Intention, including effect through
mediator variables Table 3. As shown in Table 3, there is a relationship between culture and entrepreneurial
intention, and except for two results, all effects were statistically significant. Results Subjective Norm’s effect on Entrepreneurial Intention, including effect through
mediator variables
Inidrect Effect
Path
β Spain β Mexico β India
β
Portugal
Subjective Norm / Desirability
a
0,3684
0,3508
0,3033
0,2320
Desirability / Entrepreneurial Intention
b
0,7090
0,7157
0,8342
0,8404
Subjective Norm / Perceived Control
d
0,4364
0,4755
0,1126 x
0,4516
Perceived Control / Entrepreneurial
Intention
e
0,3208
0,2499
0,3503
0,1757
Direct Effect
c´
0,1589
0,1059 -0,0447 x
0,4067
Total Effect: Subjective Norm ->
Intention
ab+de+c´
0,5601
0,4758
0,2478
0,6810
Total Model Effect
R2
0,1001
0,1581
0,0383
0,2156
Sample
253
254
125
104
Notes: p<0,05 with exception of "x"; non-standarized coefficients; re-sampling number: 10000;
Confidence interval: 95%. Model 4 of Bootstrapping used (Hayes, 2013). Notes: p<0,05 with exception of "x"; non-standarized coefficients; re-sampling number: 10000;
Confidence interval: 95%. Model 4 of Bootstrapping used (Hayes, 2013). As shown in Table 3, there is a relationship between culture and entrepreneurial
intention, and except for two results, all effects were statistically significant. 60 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Indirect Effect: These occur when the mediating variables are used both for desirability
and for perceived behavioral control. In the countries of Spain, Mexico and Portugal, the
indirect effect of these mediating variables is statistically significant, given that the
confidence interval does not include the zero value, with a confidence index of 95%. In
the specific case of India, the mediation given by the desirability to start a business is
statistically significant, however, not when mediated through perceived behavioral
control, since the confidence interval includes the zero value, nullifying the hypothesis
(Ho: β = 0) therefore, cancels the mediational relationship. Results On the other hand, the sign of the beta coefficients is positive, so it can be concluded
that subjective norm produces a positive effect on students' desire to start a business
(M1), which in turn produces a positive effect on entrepreneurial intention. That is, cultural
norms can positively influence a student so that it shows a desire of self-employment,
which in turn motivates his intention to endeavor on this behavior. Similarly, but to a
lesser extent, cultural norms positively affects the perception of control that students have
over entrepreneurial activity, so it can be said that the indirect effect is partially explained
by the mediating variables of desirability and perception of control over entrepreneurial
activity. Direct effect. In the countries of Spain, Mexico and Portugal there is a direct and
statistically significant relationship between subjective norm and entrepreneurial
intention. These results, however, are not shared with India, where there was no
significant direct relationship (see table 3). When adding the indirect effects, - of desirability (ab) plus perceived control (of) -, and
comparing them with the direct effect, -subjective norm on the intention (c) -, is found that
the indirect effect is greater than the direct effect, with the exception of Portugal (See
table 4). This indicates that the desire and control that students feel to start a business is
a variable that partially mediates the relationship with intention. Thus, in all countries, except Portugal, subjective norm exerts an influence, on both, the
desire and the perceived control of students in their entrepreneurial intentions. This
indirect effect is greater than the effect subjective norm exerts directly on intention. The total effect. As observed in the results, the greatest total effect of subjective norms
on entrepreneurial intention, mediated by desirability and perceived control, is obtained
by Portugal, followed by Spain, Mexico, and finally India. The total effect multiplies each
part of the indirect effect, and then adds both: results obtained and direct effect. In the
specific case of India, one of the relationships is not statistically significant, making its
total effect lesser than the rest. In Table 4, the acceptance or rejection of the hypotheses presented is shown by country: Table 4, the acceptance or rejection of the hypotheses presented is shown by country: 61
Tabla 4. Hypothesis acceptance or rejection. Note: A: Hyptohesis Accepted; R: Hypothesis Rejected. As explained above, there is a direct relationship between subjective norm and
entrepreneurial intention in all countries, except India, where this effect is not statistically
significant, thus accepting H1 in all countries, except for India, where that hypothesis is
rejected. There is also an indirect effect between subjective norm and entrepreneurial intention
when mediated by the perception of the student's control of the behavior. This
relationship is observed in all countries, except for India, where it is not statistically
significant, thus, H2 is accepted in all countries except India. Regarding the effect
mediated by the desire of a student to start a business, this relationship is statistically
significant; therefore, H3 is accepted in all countries. Lastly, the indirect effect, mediated by desirability and perceived control, is greater than
the direct effect with the exception of Portugal, whereby H4 is accepted in all countries
with the exception of Portugal. Results Hypothesis
Spain
Mexico
India
Portugal
Direct Effect (c´)
H1
A
A
R
A
Indirect Effect
Mediator: Perceived Control
(de)
H2
A
A
R
A
Mediator: Desirability (ab)
H3
A
A
A
A 61 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneu
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 Total Indirect Effect > Direct
Effect
H4
A
A
A
R Note: A: Hyptohesis Accepted; R: Hypothesis Rejected. Note: A: Hyptohesis Accepted; R: Hypothesis Rejected. Conclusions and Discussion It is assumed that entrepreneurial behavior is given by many factors, and that these
change over time (Le, 1999). Among these, culture itself can be considered as a category
within subjective norm, since the community also exerts influence on people. Therefore,
according to this assumption, it was examined how culture affects entrepreneurial
intention, both directly and/or indirectly. It was to be expected that there would be differences between cultural norms between
the countries studied, and that those differences would affect, to a greater or lesser
extent, entrepreneurial intention. The results obtained show these differences, and the
magnitude of the effects are detailed below: The cultural norms of Spain, Mexico and Portugal, affect entrepreneurial intention in
students, both directly and indirectly. These results coincide with those of Ajzen,
(1998,1991,2002); Fisbein and Ajzen, (1975); Guido et al., (2011); Liñan and Chan
(2009); Rhodes and Courneya, (2003); Scholten, Kemp and Omta, (2004); Schwarz, et
al., (2009); Shook and Britanu, (2010); Solvesik et al., (2012); and Tkachev and
Kolvereid, (1999), where in their studies the positive effect between subjective norm and
entrepreneurial behavior is considered. However, this does not happen with India, adding
to the pool of other studies that have not found a significant relationship between
subjective norm and intention (Autio, et al., 2001; Krueger et al., 2000; & Liñán, 2008,
Tiwari, Bhat & Tikoria 2017). This study was also able to determine that the total indirect
effect is higher than the direct effect in the countries at study, with the exception of
Portugal. That is, cultural norms somehow affect students desire and perceived 62 Sánchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
Intention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 nchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
ention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 behavioral control on venture creation, and, in addition, the effect of these variables is
greater than if cultural norms acted by itself. behavioral control on venture creation, and, in addition, the effect of these variables
greater than if cultural norms acted by itself. Conclusions and Discussion India, despite being an emerging country, whose economy is currently growing the most
(OECE, p27), students thinking generally lean towards paid jobs, partly because of the
risk aversion attitude found in their society (Paul, Hermel & Srivastava, 2017),
diminishing their intentions of self-employment. Results show that Indian students have
positive views entrepreneurship, however, they will generally seek long-term, secure
employment, such as government jobs, as they do not receive substantial social security
benefits (Paul and Gupta 2014). Additionally, the political situation in India is conflictive
(OECE, 2016), so it is expected that students' desire for venture creation is statistically
significant, but because of this, the feeling of a possibility for starting a business not being
presently feasible (Paul & Gupta 2014 Paul, et al., 2017) as well. A possible line of future
research could be to explore the prospect of self-employment vs. paid job in India. Facing these results, authors such as Manolova, Eunni, and Gyoshev, (2008);
Hechavarria and Reynolds, (2009); and Williams, (2009), consider that entrepreneurial
behavior, in any emerging society, must evaluate their lack of information, developed
institutional structures and level of uncertainty. These factors could lead to a reduction
in entrepreneurial opportunities, given that business activity becomes more risky and
complex, ultimately affecting motivation for venture creation. In the case of Portugal, where the direct effect is greater than the indirect effect (culture
has a greater weight on entrepreneurial intention than the person’s attitude), could be
potentially explained by student’s low control perception, thus, feasibility, to engage in
entrepreneurial activity, opting for other activities that would give them greater outcomes
(Franco, Haase, & Lautenschläger, 2010). However, environment and family influence
have a chance of affecting desire to start a business. Additionally, the government
promotes a transformation of the educational model, so that students may perceive that
their culture favors entrepreneurial activity, although these results seem more ideological
than empirical. (Franco, et al., 2010). Results obtained in Spain and Mexico show a certain similarity, both in the direct and
indirect effect, on entrepreneurial intention, however, both countries show different
realities. One possible explanation in Spain is that enterprising is desired as a measure
to counteract the high levels of unemployment caused by the country's recent economic
crisis, along with a strong relationship of entrepreneurship with feelings of personal
enjoyment and satisfaction (Bosma, Acs, Autio, Coduras & Levie, 2008; Uslay, Teach, &
Schwartz, 2012). Conclusions and Discussion Although the country's high fear of failure (Bosma, et al., 2008) could
explain desirability’s stronger influence than perceived behavioral control, considerable
reduction of this fear over the years could explain at the same time how the social
valuation towards entrepreneurship has improved (GEM, 2015). Regarding Mexico, influence that stems from friends, colleagues and overall collective,
combined with governmental support on the promotion of business activities, gives them
a certain level of confidence and control over the achievement of venture creation. (Prieto, Wang, Hinrichs, & Aguirre-Milling, 2010). Likewise, their own beliefs that
becoming self-employed are signs ingeniousness and perseverance, along with few 63 nchez-García, J.C., Ward Mayens, A., Vargas Morúa, G., Hernández Sánchez, B. (2018). How does Cultural Norms Influence Entrepreneurial
ention? A Cross Cultural Study. Journal of Business, Universidad del Pacífico (Lima, Peru) Vol.10 (1): 52-69 decent employment alternatives in the labor market, intensify the desire to try to start a
business (Prieto, et al., 2010). These two ideas coincide with the results obtained in this
study, since both the desirability and the perceived behavioral control mediate the
relationship between culture and entrepreneurial intention. decent employment alternatives in the labor market, intensify the desire to try to start a
business (Prieto, et al., 2010). These two ideas coincide with the results obtained in this
study, since both the desirability and the perceived behavioral control mediate the
relationship between culture and entrepreneurial intention. The comparison of these four countries highlights Portugal as the country that has the
greatest total effect between cultural norms and entrepreneurial intention, mediated by
desirability and perceived behavioral control. Interestingly, although they do not have
high levels of perceived control to carry out self-employment, their high desire, coupled
with the support perceived by society, identify the students of Portugal with the greatest
entrepreneurial intentions. Future potential research would be to follow in a long-term
study Portuguese student’s success of venture creation, as well as exploring which
cultural aspects most influence their intention. In conclusion: There exists a direct relationship between subjective norm and entrepreneurial
intentions, especially in Mexico, Spain and Portugal. There exists an indirect relationship between subjective norms and entrepreneurial
intention, mediated by the desirability of venture creation, in all countries studied. There is an indirect relationship between subjective norm and entrepreneurial intention,
mediated by perceived control, in all countries studied, with the exception of India. Conclusions and Discussion The total indirect effect is greater than the direct effect on the relationship between
subjective norms and entrepreneurial intention, with the exception of Portugal, where the
direct effect is greater. It’s important to point that generalization of the results obtained should be cautious, due
to the limitations in its method. Only university students were used, and their job
preferences may change over time, as well as their interest in entrepreneurial behavior
accordingly. Additionally, there was no variability in terms of age and educational level,
as such, results may not be generalizable. For future research, it would be convenient to
consider a larger, stratified, sample, using different regions and variable characteristics. Likewise, longitudinal studies should be considered to further understand the evolution
of culture on entrepreneurial intention. As final word, it’s important to further explore aspects of the relationship that subjective
norm, through culture, has on entrepreneurial intention at a contextual level. As this study
showed, generalized models do not pertain to all specifics, and further research should
be done to deepen the understanding of their interactions with different variables,
including environment, to better assess the promotion and development of
entrepreneurship in each respective situation. 64 do Paço, A. M. F., Ferreira, J. M., Raposo, M., Gouveia, R. G., & Dinis, A. (2011).
Behaviours and entrepreneurial intention: Empirical findings about secondary students.
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http://dx.doi.org/10.1590/1678-4162-9630
Arq. Bras. Med. Vet. Zootec., v.70, n.4, p.1060-1068, 2018
Avaliação dos níveis plasmáticos do peptídeo natriurético NT-proBNP em cães da raça
Poodle em diferentes estágios da doença valvar crônica mitral
[Evaluation of plasma levels of NT-proBNP natriuretic peptide in Poodle dogs at different
stages of chronic mitral valve disease]
C.G.P. Santana1, J.P. Paiva2, C.J. Mucha3, N.X. Alencar4
Aluno de pós-graduação ˗ Universidade Federal Fluminense ˗ Niterói, RJ
2
Universidade Federal Rural do Rio de Janeiro ˗ Seropédica, RJ
3
Prática privada em Cardiologia Veterinária, Buenos Aires, ARG
4
Universidade Federal Fluminense ˗ Niterói, RJ
1
RESUMO
A doença valvar crônica mitral (DVCM) é comum em cães e pode não causar sintomas clínicos da
insuficiência cardíaca (IC) durante anos. O peptídeo natriurético tipo B (BNP) é armazenado nos miócitos
ventriculares e secretado para circulação com seu fragmento NT-proBNP, quando ocorre aumento. Este
estudo avaliou os níveis plasmáticos do peptídeo natriurético NT-proBNP em cães da raça Poodle em
diferentes estágios da DVCM, seguindo as diretrizes do American College of Veterinary Internal
Medicine (ACVIM, 2009). Amostras de sangue foram coletadas para determinação do biomarcador NTproBNP para comparação entre grupos. As medianas do NT-proBNP nos grupos estudados foram:
551pmol/L (controle), 302pmol/L (grupo B1), 1.033pmol/L (grupo B2), 954pmol/L (grupo C) e
5.541pmol/L (grupo D). Mediante o uso de um ponto de corte ideal de >709pmol/L, foi possível
identificar os cães com aumento cardíaco verdadeiro daqueles sem aumento cardíaco, com sensibilidade
de 75% e especificidade de 100%. O NT-proBNP aumentou de acordo com o avanço dos estágios da
DVCM, sendo os estágios B2, C e D aqueles com valores mais elevados desse biomarcador. Para o
estágio B2, a mensuração do NT-proBNP mostrou ser uma excelente ferramenta para diagnosticar
precocemente o aumento cardíaco em cães da raça Poodle.
Palavras-chave: patologia clínica, insuficiência cardíaca, biomarcador
ABSTRACT
Chronic mitral valve disease (CMVD) is common in dogs, it may not cause clinical symptoms of heart
failure (HF) for years. The type B natriuretic peptide (BNP) is stored in the ventricular myocytes and
secreted for circulation with its NT-proBNP fragment, when an increase occurs. This study evaluated the
plasma levels of the NT-proBNP natriuretic peptide in Poodles at different stages of CMVD, following the
guidelines of the American College of Veterinary Internal Medicine (ACVIM, 2009). Blood samples were
collected for determination of NT-proBNP biomarker for comparison between groups. This median NTproBNP in the studied groups were: 551pmol/L (Control), 302pmol/L (Group B1), 1,033pmol/L (Group
B2), 954pmol/L (Group C) and 5,541pmol/L (Group D). Using an ideal cutoff of > 709pmol/L it was
possible to identify dogs with true heart enlargement of those without a cardiac increase with sensitivity
of 75% and specificity of 100%. NT-proBNP increased according to the progress of the stages of CMVD,
being that stages B2, C and D, with the highest values of the biomarker. To stage B2, the NT-proBNP
measurement proved to be an excellent tool for early diagnosis of cardiac enlargement in Poodles.
Keywords: clinical pathology, heart failure, biomarker
Recebido em 22 de fevereiro de 2017
Aceito em 22 de novembro de 2017
E-mail: cicerocardiovet@hotmail.com
Avaliação dos níveis...
INTRODUÇÃO
A doença valvar crônica mitral (DVCM) é o
distúrbio cardíaco mais comum (Detweiler;
Patterson, 1965), constitui aproximadamente
75% dos casos vistos na prática veterinária de
cães idosos, atinge mais os machos do que as
fêmeas e é mais prevalente em cães de porte
pequeno (< 20kg) (Borgarelli; Haggstrom, 2010).
Muitas vezes o sopro cardíaco da insuficiência
da mitral e/ou tricúspide é um achado incidental.
Os sinais clínicos podem ficar ausentes durante
anos e nunca demonstrarem características da IC
em alguns cães. Naqueles que exibem os sinais
clínicos, estes estão geralmente relacionados à
diminuição de exercícios e intolerância a eles;
tosse e taquipneia durante esforço físico e
episódios de fraqueza transitória ou síncope
constituem as queixas comuns entre tutores de
cães (Côté et al., 2015).
A definição de biomarcadores diz que são
substâncias específicas de um órgão ou tecido
em investigação, as quais são sintetizadas e
secretadas proporcionalmente à lesão ou doença
que o está acometendo (Yonezawa et al., 2010).
Os biomarcadores são utilizados com o intuito de
auxiliar no diagnóstico clínico de animais com
doença cardíaca, com maior acurácia e em menor
tempo possível (Pant et al., 2012). Os peptídeos
natriuréticos constituem o padrão-ouro para os
biomarcadores cardíacos em seres humanos, e o
conhecimento sobre sua biologia e sua utilização
clínica tem aumentado rapidamente (Gaggin;
Januzzi, 2013).
São hormônios natriuréticos estruturalmente
semelhantes, bem como suas vias de degradação;
em conjunto, regulam o volume e a pressão
sanguínea, antagonizam o sistema reninaangiotensina-aldosterona e o sistema nervoso
simpático; provocam a broncodilatação e inibem
a proliferação de células musculares lisas. Ao
antagonizarem o sistema renina-angiotensinaaldosterona e o sistema nervoso simpático,
protegem o sistema cardiovascular da sobrecarga
de volume (Takemura et al., 2009). A oscilação
entre esses sistemas contribui para o
desenvolvimento de insuficiência cardíaca
congestiva (ICC). Os peptídeos natriuréticos são
importantes marcadores de hipertrofia cardíaca e
insuficiência cardíaca congestiva (Gaggin;
Januzzi, 2013).
Arq. Bras. Med. Vet. Zootec., v.70, n.4, p.1060-1068, 2018
O peptídeo natriurético tipo B (BNP) é
sintetizado a partir do seu precursor proBNP, que
é armazenado nos miócitos ventriculares. O
hormônio BNP ativo é clivado à porção
carboxiterminal da molécula precursora e
secretado para a circulação com seu respectivo
fragmento
aminoterminal
(NT-proBNP)
(MacDonald et al., 2003; Pernobian, 2011). Esse
biomarcador cardíaco pode auxiliar na
diferenciação de cães com doença cardíaca dos
hígidos, além de determinar que animais
possuam aumento cardíaco significativo e, ainda,
ajudar no diagnóstico diferencial de doenças
cardíacas e não cardíacas (Oyama et al., 2009).
Sua indicação clínica principal, em se tratanto de
pequenos animais, é a detecção precoce do
distúrbio cardíaco em animais assintomáticos, o
que ajuda a interceder antes do aparecimento dos
sinais clínicos. Ressalta-se que o teste do NTproBNP não substitui a auscultação cardíaca, um
método fácil, barato e altamente específico para a
doença valvular crônica em cães (Oyama, 2010).
Na medicina, ele é utilizado como auxiliar no
diagnóstico de pacientes com dispneia
ambulatorial e como suporte clínico na suspeita
de ICC; além disso, contribui para a avaliação
prognóstica e a orientação terapêutica dos
pacientes com ICC descompensada (Barretto et
al., 2013; Simons, Wauchope, 2015).
No entanto, variação considerável inter-racial na
concentração plasmática desses biomarcadores
pode ocorrer em cães saudáveis (Sjostrand et al.,
2014; Misbach et al., 2013), o que torna
fundamental outros estudos para a determinação
desses valores em diferentes raças. Considerando
que o padrão-ouro para o diagnóstico de
cardiopatias,
que
envolve
exames
eletrocardiográficos e ecocardiográficos, é
relativamente caro e requer experiência e
equipamentos (Oyama, 2010), a utilização de
biomarcadores pode ser útil na avaliação desses
pacientes. O presente estudo teve como objetivo
determinar os níveis plasmáticos do peptídeo
natriurético NT-proBNP em cães da raça Poodle
em diferentes estágios da DVCM.
MATERIAL E MÉTODOS
Em estudo transversal, foram incluídos 50 cães
da raça Poodle, entre machos e fêmeas sem
pedigree, de clientes encaminhados ao clínico
cardiologista do Hospital Universitário de
Medicina Veterinária (Huvet), divididos em
1061
Santana et al.
cinco grupos conforme achados clínicos
cardiológicos, seguindo as diretrizes do
American College of Veterinary Internal
Medicine (ACVIM, 2009) para o diagnóstico e o
tratamento da doença valvar crônica em cães.
Obesos com escore de condição corporal >5
(sendo entre 3 e 5 o escore ideal), portadores de
hemoparasitoses,
microfilaremia,
doenças
infecciosas sistêmicas e sinais clínicos
compatíveis com endocrinopatias, foram
excluídos deste estudo.
O estudo foi aprovado por um comitê de ética no
uso de animais. Todos os responsáveis dos
animais que participaram deste estudo foram
instruídos a assinarem o termo de consentimento
livre e esclarecido, com as descrições adequadas
e as informações essenciais ao entendimento da
proposta deste estudo. Em cada animal, foi
realizado
atendimento
clínico
seguindo
semiologia médica com especial atenção ao
sistema cardiovascular, de acordo com a
metodologia descrita por Fox et al. (1999), com
todos os parâmetros anexados ao prontuário
médico, contendo histórico, anamnese, exame
físico e resultados dos exames complementares.
A aferição da pressão arterial sistêmica não
invasiva se fez pelo método oscilométrico de alta
definição (Medidor de Pressão Arterial MDpro Beijing Choice Eletronic Tech Co. Ltda.).
Realizaram-se três aferições seguidas e, com
base na média destas, foi estabelecida a pressão
arterial sistólica (PAS), a diastólica (PAD) e a
média (PAM). A gravação do registro
eletrocardiográfico foi realizada durante dois
minutos, empregando-se o aparelho digital 12
Derivações ECGPC® – TEB (Tecnologia
Eletrônica Brasileira Ltda.). O ecocardiograma
foi realizado com uso do aparelho de ultrassom
Sonosite Titan® com transdutor multifrequencial
C11/5,0-8,0MHz. A obtenção dos parâmetros
ecocardiográficos foi feita conforme descrito por
Boon (2011), por meio de ecocardiografia
transtorácica, com avaliação qualitativa das
quatro câmaras cardíacas, válvulas mitral, aórtica
e tricúspide. A partir do modo-M, obteve-se
quantitativamente a relação entre o átrio
esquerdo e a aorta (AE:Ao), o diâmetro da
câmara ventricular esquerda (DVE), a parede
livre do ventrículo esquerdo em sístole e diástole
(PLVE) e a fração de encurtamento (FEC)
miocárdica; com o Doppler contínuo ou pulsátil
e em cores, obteve-se gradiente de pressão,
1062
velocidades e direções dos fluxos transvalvulares
e das vias de saída do coração, visando
identificar regurgitação valvar e reconhecer
possíveis disfunções sistólicas e diastólicas
causadas pela DVCM. A relação AE:Ao obtida
pela ecocardiografia foi utilizada como padrãoouro para determinar a existência de
remodelamento cardíaco.
As amostras para hemograma foram processadas
no Contador Hematológico Automatizado
Veterinário Sysmex® – Poch 100 iV; os
esfregaços sanguíneos foram confeccionados
para contagem diferencial de leucócitos, pesquisa
de hemoparasitas e avaliação hematoscópica. A
pesquisa de microfilárias ocorreu por meio de
duas técnicas: esfregaço sanguíneo e gota
espessa (Reagan et al., 2011). Na bioquímica
sérica, foram dosados a alanina aminotransferase
(ALT), o aspartato aminotransferase (AST), a
fosfatase
alcalina,
as
proteínas
totais
(albumina/globulinas/relação Alb:Glob.), a ureia
e a creatinina, seguindo-se as recomendações do
fabricante (Labtest Diagnóstica S.A.®). O
método de obtenção da urina foi a cistocentese
para realização da urinálise completa (EAS),
sendo realizada análise física (avaliação da cor,
odor, aspecto e densidade), química (química
seca por tiras reagentes para urina – Combur10
Test® M), sedimentoscopia, e a densidade
urinária foi determinada em refratômetro portátil
(Atago®).
Foi utilizado o kit Cardiopet proBNP® canino
para ensaio de imunoabsorção enzimática
(ELISA) do biomarcador cardíaco NT-proBNP,
o qual foi quantificado no Laboratório IDEXX®
Ltda. (Califórnia - EUA). As 50 amostras com,
no mínimo, 0,5mL de plasma sanguíneo em
tubos tipo Eppendorf®, sem inibidores de
proteases, ficaram armazenadas em duplicatas
por um período máximo de cinco meses, no
freezer, a -20ºC, até serem encaminhadas ao
laboratório IDEXX® (Califórnia - EUA), onde
foram processadas.
A análise estatística foi realizada por meio do
programa computacional BioEstat 5.3 (Ayres,
2000). Inicialmente foi feita a estatística
descritiva para resumir, descrever e compreender
os dados obtidos do biomarcador cardíaco NTproBNP de cada grupo separadamente. Foi
analisada pelo teste Kolmogorov-Smirnov a
normal distribuição das amostras. Em seguida,
Arq. Bras. Med. Vet. Zootec., v.70, n.4, p.1060-1068, 2018
Avaliação dos níveis...
para comparar a concentração plasmática do NTproBNP entre os estágios A, B1, B2, C e D
(ACVIM, 2009) causados pela DVCM, utilizouse o teste de Kruskal-Wallis para as amostras que
não obtiveram distribuições normais, a fim de
indicar se houve diferença significante. Valores
de P<0,05 foram considerados significativos.
Para avaliar a habilidade e a acurácia do teste
diagnóstico do NT-proBNP, utilizou-se a curva
ROC (Receiver Operating Characteristic) pelo
programa
computacional
estatístico
MEDCALC® (www.medcalc.org). Foi calculada
a área sob a curva ROC (AUC) e determinou-se
um valor de cut-off ideal, utilizando-se a análise
da curva ROC para discriminar os cães sem
aumento cardíaco (A, B1) dos cães com aumento
cardíaco (B2, C, D). Valores de P<0,05 foram
considerados significantes.
RESULTADOS
Os animais foram atendidos entre o meses de
janeiro e junho de 2016 (Huvet). Dos 50 cães
utilizados neste estudo, 31 eram fêmeas (62%) e
19 machos (38%), distribuídos entre os grupos
experimentais independentemente de sexo, idade
e estado reprodutivo. Os animais incluídos foram
classificados nos cinco grupos de acordo com as
diretrizes ACVIM (2009) para diagnóstico,
manejo e tratamento da doença valvular crônica
(DVC) em cães. A distribuição da população
estudada está listada na Tab. 1.
Tabela 1. Número de animais, distribuição de sexo, média de peso e faixa etária entre os grupos
ACVIM (2009)
Variáveis
A
B1
B2
C
D
Animais (n)
12
10
10
10
8
Sexo
5m/7f
2m/8f
3m/7f
5m/5f
5m/3f
5,2a
5,3a
5,6a
5,2a
5,7a
Peso (kg)
(± 2,5)
(± 2,7)
(± 1,9)
(± 1,3)
(± 1,9)
Idade (anos)
5a
9ab
12bc
12bc
15c
(± 2,7)
(± 1,7)
(± 2,4)
(± 3,1)
(± 1,0)
n= numero de animais; kg= quilogramas; m= machos; f= fêmeas; ± = desvio-padrão. Letras diferentes ao longo das
colunas (“a” “b” “c”) indicam variáveis que não foram significativas (P>0,05).
A mediana da concentração plasmática do NTproBNP do grupo controle (estágio A) foi de
551pmol/L; no grupo do estágio B1, a mediana
foi de 302, não apresentando diferença para o
grupo controle (P>0,05). Entretanto, nos grupos
dos estágios B2, C e D, a concentração
plasmática do NT-proBNP foi diferente (P<0,05)
em comparação ao grupo controle. No grupo do
estágio B2, a mediana foi de 1.033pmol/L; no
grupo do estágio C, a mediana foi de 954pmol/L
(primeiro quartil 618,25, terceiro quartil
2.554,75); e a mediana do grupo do estágio D foi
de 5.541pmol/L. Os resultados indicam um
aumento da concentração plasmática do peptídeo
natriurético NT-proBNP junto com o avanço da
DVCM em cães da raça Poodle (Tab. 2 e Fig. 1).
Tabela 2. Mediana, intervalo interquartil (IQ), valores mínimos e máximos da concentração plasmática do
NT-proBNP (pmol/L) dos cães Poodles, atribuídos a diferentes estágios da DVCM ACVIM (2009)
Mediana
Mín.
Máx.
Estágios
Intervalo IQ
(pmol/L)
(pmol/L)
(pmol/L)
A
551ᵃ
523,5 / 600,25
250
661
B1
302ᵃ
261 / 523
250
709
NT-proBNP
B2
1.033ᵇ
544,75 / 1.325,25
328
2.096
C
954ᵇ
618,25 / 2.554,75
394
3.178
D
5.541ͨ
3.086,25 / 5.991,25
2.265
8.872
Nos valores seguidos por diferentes letras (“a”, “b”, “c”), não houve alterações estatísticas significativas (P>0,05).
Arq. Bras. Med. Vet. Zootec., v.70, n.4, p.1060-1068, 2018
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Santana et al.
A
B1
B2
C
D
Figura 1. Concentração plasmática do NT-proBNP (pmol/L) de todos os cães do estudo, de acordo com
os estágios da DVCM (ACVIM, 2009). As linhas centrais dentro de cada bloco indicam a mediana do
grupo; as linhas superiores, o primeiro quartil; e as linhas inferiores dos blocos indicam o terceiro quartil.
Linhas verticais externas aos blocos são valores máximos e mínimos.
Após análise da curva ROC, houve determinação
do ideal cut-off (>709pmol/L), para discriminar
os cães verdadeiros positivos (aumento cardíaco)
dos verdadeiros negativos (ausência de aumento
cardíaco). O teste do biomarcador cardíaco NTproBNP obteve a sensibilidade de 75% e a
especificidade de 100%. A área sob a curva
(AUC) foi de 0.87 (Fig. 2).
NT-proBNP
100
Sensitivity
80
60
40
20
0
0
20
40
60
80
100-Specificity
100
Figura 2. Curva ROC representando a sensibilidade de 75% e a especificidade de 100% do teste NTproBNP em cães da raça Poodle com DVC. Área sob a curva (AUC) de 0.87.
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Arq. Bras. Med. Vet. Zootec., v.70, n.4, p.1060-1068, 2018
Avaliação dos níveis...
Não houve diferença significativa entre as
variáveis dos exames laboratoriais (P>0,05) em
relação ao grupo controle. A urinálise foi
utilizada como fonte simples complementar para
análise de distúrbios do trato urinário, combinada
com a avaliação bioquímica, em geral sem
variações significantes (P>0,05) entre todos os
grupos e o grupo controle (Tab. 3). Os resultados
dos exames cardiológicos estão apresentados na
Tab. 4.
Tabela 3. Médias e desvios-padrão referentes aos parâmetros hematológicos, bioquímicos séricos e
urinários dos cães Poodles separados de acordo com as diretrizes para doença valvular crônica do
ACVIM
A
B1
B2
C
D
Referência
VG (%)
47±5
48±5
45±6
46±7
44±6
37-55%
LG (x103/μL)
10.2±2.2
9.9±1.9
9.0±2.7
10.4±3.5
15.7±9.1
6.0-17.0
Ureia (mg/dL)
37±11
40±15
43±12
53±33
117±80
21-60
Creatinina (mg/dL)
0,8±0,2
0,8±0,2
0,8±0,3
0,8±0,2
1,1±0,8
0.5-1.5
AST (UI/L)
41±10
43±15
42±12
51±18
71±40
23-66
ALT (UI/L)
59±11
81±23
64±13
80±19
196±41
21-102
FA (UI/L)
46±24
58±23
51±21
48±32
88±73
20-156
Ptn totais (g/dL)
6.8±0.2
6.7±0.4
6.5±0.7
7.7±1.2
7.3±1.2
5.4-7.1
Rl: Alb/ptn
0.7±0.1
0,7±0.1
0.8±0.2
0.6±0.2
0.7±0.1
0.59-1.1
Ptn plasm (g/dL)
7.4±0.6
7.4±0.7
7.2±0.8
7.5±0.7
7.8±0.8
6-8
pH urinário
6±0
6±0.3
6±0.5
6±0.6
5±0.6
5,0-6,5
Densidade urinária
1.016
1.018
1.020
1.020
1.020
1.015-1.045
VG volume globular; LG leucometria global; AST aspartato transaminase; ALT alanina transaminase; FA fosfatase
alcalina; PTN TOTAIS proteínas totais; RL:Alb/ptn relação albumina/proteína; PTN PLASM proteína plasmática.
Tabela 4. Média, desvio-padrão, valores mínimos e máximos dos parâmetros clínico- cardiológicos dos
cães Poodles, separados pelos estágios da doença valvular crônica de acordo com as diretrizes do ACVIM
A
B1
B2
C
D
Referência
147±28
142±25
146±35
150±34
167±34
PAS (mmHg)
137
(103 - 181)
(109 - 183)
(86 - 194)
(109 - 206)
(97 - 206)
95±22
95±15
113±40
99±20
109±15
PAD (mmHg)
82
(45 - 127)
(79 - 134)
(57 - 182)
(63 - 131)
(83 - 131)
113±27
107±20
116±34
119±26
126±25
PAM (mmHg)
(82 - 168)
(88 - 149)
(65 - 178)
(76 - 157)
(87 - 157)
102
Freq. Cardíaca
133±15
124±24
124±16
144±24
147±32
60-180
(bpm)
(120 - 160)
(100 - 160)
(100 - 160)
(100 - 180)
(100 - 200)
1,1±0.1
1±0.1
1,7±0.3
1,7±0.1
1,9±0.3
AE:Ao
0,8-1,2
(1,0 - 1,5)
(0,8 - 1,2)
(1,1 - 2.5)
(1,5 - 2,0)
(1,6 - 2,8)
21.9±2.8
20.3±3.1
20.7±4.6
26.3±3.8
29.2±10.1
DVE-d (mm)
20.6-22,7
(17 - 26)
(13 - 45)
(12 - 26)
(21 - 32)
(15 - 25)
12.1±2.5
12.7±3.5
14.8±4.0
16.5±2.5
17.8±7.6
DVE-s (mm)
11,7-13,6
(5 - 14)
(9 - 20)
(8 - 21)
(10 - 19)
(7 - 31)
39±3.3
37±4.4
39±3.7
44±11.6
39±8.9
FEC (%)
28-44
(34 - 44)
(32 - 46)
(32 - 44)
(30 - 59)
(20 - 59)
1,5±0.2
1,5±0.3
1,5±0.4
1,5±0.3
2±1.1
E:A
1,9-2,7
(1,1 - 1,9)
(0,8 - 1,9)
(1,2 - 4,6)
(1,0 - 2,0)
(0,8 - 2,3)
PAS pressão arterial sistólica; PAD pressão arterial diastólica; PAM pressão arterial média; mmHg unidade
milímetro mercúrio; bpm batimentos por minutos; AE:Ao relação átrio esquerdo/aorta; DVE-d diâmetro do
ventrículo esquerdo em diástole; DVE-s diâmetro do ventrículo esquerdo em sístole; FEC fração de encurtamento;
E:A relação onda E/onda A. Fonte dos valores de referências (BOON, 2011).
DISCUSSÃO
O estudo em questão foi realizado com o
objetivo de mensurar o biomarcardor cardíaco
NT-proBNP somente em cães da raça Poodle
com DVCM, classificados segundo as diretrizes
do ACVIM (2009) para o diagnóstico e o
Arq. Bras. Med. Vet. Zootec., v.70, n.4, p.1060-1068, 2018
tratamento da DVC, e em um grupo controle.
Portanto, acredita-se que os resultados
apresentados representem um avanço nos estudos
para utilização de novas ferramentas para um
diagnóstico principalmente precoce e eficiente da
referida doença na clínica de pequenos animais.
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Santana et al.
De modo similar a estudos anteriores
(MacDonald et al., 2003; Chetboul et al., 2009;
Oyama et al., 2009; Ebisawa et al., 2012; Wolf
et al., 2012; Kanno et al., 2015), o presente
estudo evidenciou aumento significativo nas
concentrações plasmáticas do NT-proBNP, em
conjunto com o avanço dos sintomas e dos sinais
clínicos da DVCM. Entretanto, este estudo não
evidenciou relação entre os valores plasmáticos
de NT-proBNP e os critérios peso e sexo, tal qual
relatado por Misbach et al. (2013) e Sjostrand et
al. (2014). Conforme observado por Hezzell et
al. (2012), os animais mais idosos apresentaram
maior risco de insuficiência cardíaca. No
presente estudo, foi igualmente constatado que a
idade dos cães avançou acompanhando a
evolução da DVCM de acordo com os estágios
determinados pelas diretrizes do ACVIM (2009)
para essa enfermidade.
no resultado do biomarcador (Hezzell et al.,
2012). Além disso, em razão da influência das
diversas conformações corpóreas existentes nos
cães,
a
variabilidade
das
medidas
ecocardiográficas ocorre não somente entre as
espécies animais, mas também entre as raças
caninas (Yamato et al., 2006). Por outro lado,
cães do grupo B2 com níveis plasmáticos de 90L
até 1.500pmol/L são cães (n=3) com a DVCM,
porém sem sinais clínicos, sem chamar atenção
para a existência de estresse miocárdico e o
aumento cardíaco, o que indicou precocemente a
necessidade de acompanhamento clínico
cardiológico. Os cães (n=2) desse grupo B2 em
que os níveis plasmáticos foram acima de
1.501pmol/L foram considerados cães com
futuro risco de desenvolverem ICC e sintomas
clínicos graves da DVCM de forma aguda e até
mesmo falha cardíaca (Wolf et al., 2013).
Anterior ao presente estudo, Couto et al. (2015)
relatou, em cães Greyhound saudáveis, retirados
das corridas, um valor médio do NT-proBNP de
945pmol/L (512 – 2127pmol/L). Hezzell et al.
(2012), entretanto, em seu estudo, chegaram a
utilizar dois Poodles saudáveis misturados a
outras raças (n=30 cães) e obtiveram média de
324pmol/L (167 – 530pmol/L). Os valores
plasmáticos do NT-proBNP obtidos no grupo
controle do presente estudo (estágio A; ACVIM,
2009), em que foram utilizados 12 Poodles
saudáveis, podem colaborar futuramente para
comparações entre as próximas pesquisas com a
raça Poodle. A análise estatística da curva ROC
obteve um valor ideal de cut-off (>709pmol/L),
com 75% de sensibilidade e 100% de
especificidade, em que valores acima do valor
ideal de corte já indicavam aumento cardíaco e
estresse miocárdico comprovados pelos exames
ecocardiográficos. Para os cães do estágio B1,
não houve diferença (P>0,05) quando
comparados aos animais do grupo controle. A
mensuração dos níveis plasmáticos do NTproBNP nestes pacientes assintomáticos e sem
alterações hemodinâmicas, no presente estudo,
como afirmaram Sharma et al. (2015), mostrouse excelente auxiliar no diagnóstico e forneceu
resultados valiosos para discriminar os riscos
causados pela DVCM.
No grupo C, alguns cães (n=5) portadores da
DVCM, com sintomas discretos a moderados da
ICC e com valores do NT-proBNP baixos
(<900pmol/L), representaram a triagem que esse
biomarcador cardíaco faz entre distúrbios
crônicos do trato respiratório concomitantes com
a DVCM (Oyama et al., 2009); também pode ser
discutida a degradação da amostra no
armazenamento do biomarcador. Um cão (n=1)
desse grupo apresentou valor entre 90L e
1500pmol/L com DVCM e sintomatologia
clínica significativa; esse paciente respondeu
positivamente ao tratamento padrão (Wolf et al.,
2012). Já os cães desse grupo C com níveis
plasmáticos acima de 1800pmol/L (n=4)
correspondem a animais hospitalizados com
sintomas clínicos graves da ICC (ex.: síncopes e
angústias respiratórias), causados pela DVCM
com chance de falha cardíaca e necessidade do
tratamento terapêutico intensivo (Takemura et
al., 2009).
Os valores abaixo de 900pmol/L dos cães (n=4)
do grupo B2 podem ser explicados pela
degradação da amostra no tubo armazenado sem
inibidor de proteases, o que levou à diminuição
1066
Os cães do estágio D foram refratários ao
tratamento padrão da DVCM com sintomas
avançados da ICC e alguns com necessidades
urgentes de cuidados hospitalares. Neste estudo,
todos os animais desse grupo apresentaram
níveis plasmáticos do NT-proBNP muito acima
de 1800pmol/L, o que significa um alto
estiramento e estresse cardíaco, que ocasionaram
falência miocárdica (MacDonald et al., 2003). O
presente estudo utilizou os resultados básicos dos
testes laboratoriais para ajudar a identificar os
cães com a ICC descompensada e, assim, prestar
Arq. Bras. Med. Vet. Zootec., v.70, n.4, p.1060-1068, 2018
Avaliação dos níveis...
melhor assistência a esses cães hospitalizados, a
fim de minimizar o risco de morte hospitalar
(Ostrowska et al., 2016).
Este estudo encontrou limitações devido à falta
de tubos com inibidores de proteases para
conservação das amostras. Isso pode ter
favorecido a degradação do biomarcador NTproBNP antes do envio para a análise
laboratorial, sendo, desse modo, explicados os
resultados com valores abaixo do esperado.
Porém, com o auxílio diagnóstico da
ecocardiografia
e
de
exames
clínicos
complementares, as concentrações plasmáticas
do biomarcador foram consideradas
falsonegativas para aumento cardíaco. Vale ressaltar
que os valores de referência para o NT-proBNP
possuem variabilidades individuais de acordo
com o tipo de teste aplicado, a especificidade e o
manuseio das amostras, que são fatores que
devem ser considerados quando se faz
comparação com resultados de outros estudos.
CONCLUSÃO
Os níveis plasmáticos do peptídeo natriurético
NT-proBNP em cães da raça Poodle aumentam
com a evolução da DVCM, sendo os estágios B2,
C e D (ACVIM, 2009) aqueles em que se
observam os maiores valores desse biomarcador.
Além disso, utilizando-se o valor de corte de
>709pmol/L, foi possível identificar o
remodelamento cardíaco com sensibilidade de
75% e especificidade de 100%. Para os cães do
estágio B2 (ACVIM, 2009), a mensuração do
biomarcador cardíaco NT-proBNP mostrou ser
uma ótima ferramenta para diagnosticar, de
forma precoce, o aumento cardíaco antes da
sintomatologia clínica da DVCM.
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pro-B-type
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PERNOMIAN, L. Efeitos vasculares induzidos
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English
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Untargeted metabolomics unveil alterations of biomembranes permeability in human HaCaT keratinocytes upon 60 GHz millimeter-wave exposure
|
Scientific reports
| 2,019
|
cc-by
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|
Untargeted metabolomics unveil
alterations of biomembranes
permeability in human HaCaT
keratinocytes upon 60 GHz
millimeter-wave exposure
Pierre Le Pogam1, Yann Le Page2, Denis Habauzit 2, Mickael Doué1, Maxim Zhadobov1,
Ronan Sauleau1, Yves Le Dréan2 & David Rondeau 1,3 for Wireless Local/
Personal/Body Area Networks (WLAN, PAN, BAN) or in the context of the upcoming 5 G mobile standard1. These electromagnetic radiations correspond to frequencies ranging from 30 to 300 GHz (free-space wavelengths
spanning from 10 to 1 mm). Interestingly, MMW have been used for therapeutic purposes indicating that physi-
ological processes might be altered upon exposure to these radiations2. Accordingly, the assessment of the poten-
tial effects of MMW on human health is thus of paramount importance prior to the wide scale deployment of
technologies exploiting this band. MMW are known for their shallow penetration of human tissues (between a
few tenth of millimeter to a millimeter), making skin keratinocytes and free nerve endings the primary targets for
MMW3. It is assumed that some general effects of MMW may be initiated by the release of skin-secreted factors
into the blood stream or through the stimulation of superficial free nerve endings4,5. The shallow penetration depth of MMW in skin, results in elevated levels of Specific Absorption Rates (SAR)
compared to lower microwave frequencies with the same Incident Power Density (IPD). As a consequence, this
leads to a noticeable local heating for IPD exceeding typically 5 mW/cm2 6. Accordingly, one of the main safety
concerns regarding these frequencies is the local heating of skin caused by the absorption of MMW energy in the
human body7. Whether electromagnetic-field specific effects occur upon exposure to MMW8–12 or not13–17 has
been a matter of debate for a long period of time. Besides the warm-up effect, the mechanisms accounting for
1Univ Rennes, CNRS, IETR (Institut d’Électronique et de Télécommunication de Rennes), UMR 6164, F-35000,
Rennes, France. 2Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) –
UMR_S 1085, F-35000, Rennes, France. 3Département de Chimie, Université de Bretagne Occidentale, 6 avenue
Victor Le Gorgeu, 29238, Brest, Cedex, France. Correspondence and requests for materials should be addressed to
D.R. (email: david.rondeau@univ-rennes1.fr) Owing to the saturation of the lower part of the microwave spectrum and to the growing demand for higher data
rates, the millimeter waves (MMW) are increasingly used for wireless communications, i.e. for Wireless Local/
Personal/Body Area Networks (WLAN, PAN, BAN) or in the context of the upcoming 5 G mobile standard1. These electromagnetic radiations correspond to frequencies ranging from 30 to 300 GHz (free-space wavelengths
spanning from 10 to 1 mm). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 20 April 2018
Accepted: 30 May 2019
Published: xx xx xxxx Untargeted metabolomics unveil
alterations of biomembranes
permeability in human HaCaT
keratinocytes upon 60 GHz
millimeter-wave exposure
Pierre Le Pogam1, Yann Le Page2, Denis Habauzit 2, Mickael Doué1, Maxim Zhadobov1,
Ronan Sauleau1, Yves Le Dréan2 & David Rondeau 1,3 Received: 20 April 2018
Accepted: 30 May 2019
Published: xx xx xxxx Received: 20 April 2018
Accepted: 30 May 2019
Published: xx xx xxxx A joint metabolomic and lipidomic workflow is used to account for a potential effect of millimeter
waves (MMW) around 60 GHz on biological tissues. For this purpose, HaCaT human keratinocytes
were exposed at 60.4 GHz with an incident power density of 20 mW/cm², this value corresponding to
the upper local exposure limit for general public in the context of a wide scale deployment of MMW
technologies and devices. After a 24h-exposure, endo- and extracellular extracts were recovered to
be submitted to an integrative UPLC-Q-Exactive metabolomic and lipidomic workflow. R-XCMS data
processing and subsequent statistical treatment led to emphasize a limited number of altered features
in lipidomic sequences and in intracellular metabolomic analyses, whatever the ionization mode (i.e 0
to 6 dysregulated features). Conversely, important dysregulations could be reported in extracellular
metabolomic profiles with 111 and 99 frames being altered upon MMW exposure in positive and
negative polarities, respectively. This unexpected extent of modifications can hardly stem from the
mild changes that could be reported throughout transcriptomics studies, leading us to hypothesize that
MMW might alter the permeability of cell membranes, as reported elsewhere. 1
Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6
the upper local exposure limit for general public in the context of a wide scale deployment of MMW
technologies and devices. After a 24h-exposure, endo- and extracellular extracts were recovered to
be submitted to an integrative UPLC-Q-Exactive metabolomic and lipidomic workflow. R-XCMS data
processing and subsequent statistical treatment led to emphasize a limited number of altered features
in lipidomic sequences and in intracellular metabolomic analyses, whatever the ionization mode (i.e 0
to 6 dysregulated features). Conversely, important dysregulations could be reported in extracellular
metabolomic profiles with 111 and 99 frames being altered upon MMW exposure in positive and
negative polarities, respectively. This unexpected extent of modifications can hardly stem from the
mild changes that could be reported throughout transcriptomics studies, leading us to hypothesize that
MMW might alter the permeability of cell membranes, as reported elsewhere. Owing to the saturation of the lower part of the microwave spectrum and to the growing demand for higher data
rates, the millimeter waves (MMW) are increasingly used for wireless communications, i.e. Untargeted metabolomics unveil
alterations of biomembranes
permeability in human HaCaT
keratinocytes upon 60 GHz
millimeter-wave exposure
Pierre Le Pogam1, Yann Le Page2, Denis Habauzit 2, Mickael Doué1, Maxim Zhadobov1,
Ronan Sauleau1, Yves Le Dréan2 & David Rondeau 1,3 non-thermal effects occurring at low-power exposure have not been fully demonstrated yet so that their under-
standing remains an open challenge18. Consequently, the guidelines established by the International Commission
on Non Ionizing Radiation Protection (ICNIRP) aim at circumventing thermal hazards associated with MMW
exposure. This led to define two exposure scenarios. For far-field exposure, the IPD is limited to 1 mW/cm². For
body-centric wireless networks that are related to very limited exposition area, the IPD limit is set at 20 mW/cm²
(averaged over 1 cm²). While such exposure scenarios may trigger a heat-shock response, it is assumed that the
thermoregulation due to blood flow effect might be efficient to avoid any thermal damage.i non-thermal effects occurring at low-power exposure have not been fully demonstrated yet so that their under-
standing remains an open challenge18. Consequently, the guidelines established by the International Commission
on Non Ionizing Radiation Protection (ICNIRP) aim at circumventing thermal hazards associated with MMW
exposure. This led to define two exposure scenarios. For far-field exposure, the IPD is limited to 1 mW/cm². For
body-centric wireless networks that are related to very limited exposition area, the IPD limit is set at 20 mW/cm²
(averaged over 1 cm²). While such exposure scenarios may trigger a heat-shock response, it is assumed that the
thermoregulation due to blood flow effect might be efficient to avoid any thermal damage.i glf
gfi
y
g
Notwithstanding decades of investigations and a significant number of studies dedicated at unravelling the
effects of MMW on biosystems, the number of reliable and reproducible experimental data remains scarce,
most likely stemming for difficulties in dissociating thermal and electromagnetically pure effects. Our group
aimed at evaluating the biological effects of MMW in the 60 GHz band. Previous in vitro studies revealed that
MMW at 57–64 GHz exert no influence on protein homeostasis for IPD low enough to prevent any temperature
increase19–21. Moreover, transcriptomic studies highlighted no, or very weak, effect of MMW on keratinocyte gene
expression under athermal conditions22–24. By contrast, it must be mentioned that some authors have pointed out
the MMW effects on cell membranes, indicating that permeability changes could be induced by a direct effect on
membrane proteins or phospholipids domain organization25. Untargeted metabolomics unveil
alterations of biomembranes
permeability in human HaCaT
keratinocytes upon 60 GHz
millimeter-wave exposure
Pierre Le Pogam1, Yann Le Page2, Denis Habauzit 2, Mickael Doué1, Maxim Zhadobov1,
Ronan Sauleau1, Yves Le Dréan2 & David Rondeau 1,3 The possibility of permeation across the lipid bilayer
led us to consider the metabolomic profiling of human keratinocytes as a pertinent next step to better understand
the interactions between MMW radiation and cell membrane in the context of human body exposure to 60 GHz
waves. The goal of the present study is to apply an untargeted metabolomic strategy based on UHPLC-HRMS
assessment of metabolic changes appearing upon exposure to MMW in human HaCaT keratinocytes cell lines. Cells were exposed at 60.4 GHz with an IPD of 20 mW/cm² with the temperature being artificially maintained
constant between non-exposed and exposed samples26. As metabolites represent the sharp end of systems biology,
including multiple up-stream steps, the keratinocytes were exposed for 24 hours to enable possible changes. Then,
to afford as wide a coverage of HaCaT keratinocytes chemistry as possible, lipidomic and metabolomic profilings
were performed in both intra- and extracellular fractions in positive and negative polarities using a metabolomic
workflow that we had previously validated27. Untargeted metabolomics unveil
alterations of biomembranes
permeability in human HaCaT
keratinocytes upon 60 GHz
millimeter-wave exposure
Pierre Le Pogam1, Yann Le Page2, Denis Habauzit 2, Mickael Doué1, Maxim Zhadobov1,
Ronan Sauleau1, Yves Le Dréan2 & David Rondeau 1,3 Interestingly, MMW have been used for therapeutic purposes indicating that physi-
ological processes might be altered upon exposure to these radiations2. Accordingly, the assessment of the poten-
tial effects of MMW on human health is thus of paramount importance prior to the wide scale deployment of
technologies exploiting this band. MMW are known for their shallow penetration of human tissues (between a
few tenth of millimeter to a millimeter), making skin keratinocytes and free nerve endings the primary targets for
MMW3. It is assumed that some general effects of MMW may be initiated by the release of skin-secreted factors
into the blood stream or through the stimulation of superficial free nerve endings4,5.hi g
pi
g
The shallow penetration depth of MMW in skin, results in elevated levels of Specific Absorption Rates (SAR)
compared to lower microwave frequencies with the same Incident Power Density (IPD). As a consequence, this
leads to a noticeable local heating for IPD exceeding typically 5 mW/cm2 6. Accordingly, one of the main safety
concerns regarding these frequencies is the local heating of skin caused by the absorption of MMW energy in the
human body7. Whether electromagnetic-field specific effects occur upon exposure to MMW8–12 or not13–17 has
been a matter of debate for a long period of time. Besides the warm-up effect, the mechanisms accounting for 1Univ Rennes, CNRS, IETR (Institut d’Électronique et de Télécommunication de Rennes), UMR 6164, F-35000,
Rennes, France. 2Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) –
UMR_S 1085, F-35000, Rennes, France. 3Département de Chimie, Université de Bretagne Occidentale, 6 avenue
Victor Le Gorgeu, 29238, Brest, Cedex, France. Correspondence and requests for materials should be addressed to
D.R. (email: david.rondeau@univ-rennes1.fr) 1 www.nature.com/scientificreports/ Metabolome
Fraction
Ion mode
Ions
Ions with CV QC < 30%
(% of total ions)
Ions with both CV
QC < 30% and FC > 2
Exo
Lipido
Positive
3081
2161 (70.82)
289
Negative
1920
1126 (56.25)
126
Metabo
Positive
2750
2076 (75.42)
662
Negative
1632
1263 (77.37)
506
Endo
Lipido
Positive
4295
3920 (91.27)
540
Negative
2468
2164 (87.68)
264
Metabo
Positive
1910
1525 (79.84)
210
Negative
1017
843 (83.00)
58
Table 1. Validation of the generated data set using R-XCMS data processing. Table 1. Validation of the generated data set using R-XCMS data processing. Data quality assurance. Data quality assurance. The analytical strategy adopted herein was based on two different chromato-
graphic separation methods (HILIC and RPLC, see experimental section) coupled to detection in mass spectrom-
etry involving both positive and negative-ion modes. This strategy was applied by distinguishing the endo- and
exocellular fractions as well to deepen the coverage of the metabolome. As a result, this led to analyze each bio-
logical sample 8 times, in distinct sessions of analyses. g
p
y
Prior to any data processing or analysis, several quality checks were adopted (i.e column pressure checking,
variations of internal standards in QC/study samples and instrumental stability in terms of retention time and
accurate masses) along the whole batch. Accordingly, internal standards were spiked into each sample prior to
LC-HRMS analyses to assess retention time stability, the consistency of signal intensities and mass accuracy along
the whole batch. No significant drift in retention time could be evidenced. Likewise, standard accurate mass
measurements errors always remained below 5 ppm. For metabolomic analyses, the selected external standards
were leucine-d3 (RT 5.97 min), tryptophan-d3 (RT 6.10 min), indole acetic acid-d5 (RT, 1.81 min) and tetrade-
canedioic acid-d24 (RT 1.38 min). Creatine-d3 (RT 8.22 min) and L-lysine-d4 (RT 14.31 min) were selected as
internal standards. As to lipidomic sequences, phosphatidylcholine (15:0) (RT 8.37 min), lysophosphatidylcho-
line (15:0) (RT 1.75 min), ceramide (d18:1) (RT 17.95 min), C15:0 (RT 3.19 min) and C23:0 (RT 9.27 min) were
used as external standards, whereas C17:0 (RT 4.30 min) served as an internal standard. All these standards
revealed a coefficient of variation below 30% except in the case of C17:0 which displayed a slightly higher value in
negative-ion mode of the exo-lipidomics sequence (33.7%) (Tables S1 and S2).h g
The analysis of QC samples served as a further criterion to establish both the quality of the generated data and
the stability of the instrumental platform. For this purpose, it is highly recommended that QC peak tables should
pass pre-determined criteria, a widely admitted one being that a majority of features (i.e. 70%) shows a coefficient
of variation less than 30%28. With respect to this parameter, most sequences displayed satisfying values, with only
negative-ion mode exo-lipidomics sequence exhibiting a slightly lower value of 56.3% (Table 1). The stability of Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 2 www.nature.com/scientificreports/ Figure 1. Data quality assurance. PCA plots generated from endocellular lipidomic sequences from the features displaying both a Fold-
Change ≥2 and a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences
recorded in (A) Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with
j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the overall of the
quality control samples. Figure 1. PCA plots generated from endocellular lipidomic sequences from the features displaying both a Fold-
Change ≥2 and a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences
recorded in (A) Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with
j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the overall of the
quality control samples. he analytical instrumentation could also be assessed based on the clustering of the QC samples in a Principa
Component Analysis (PCA) score plot of all tested sequences. p
y
p
q
Altogether, these parameters underscore both the robustness of the LC-HRMS system operating conditions
nd the validity of the retrieved metabolomic data, paving the way for their statistical processing. p
y
p
q
Altogether, these parameters underscore both the robustness of the LC-HRMS system operating conditions
and the validity of the retrieved metabolomic data, paving the way for their statistical processing. Lipidomic RPLC-MS analyses. As to intracellular fractions, the filtered data subsets were represented by
540 and 264 features, for positive and negative ionization polarities respectively. Non-exposed and exposed sam-
ples clustered separately, being discriminated along PC1 component in these two sequences (Fig. 1).i In positive and negative polarities, univariate analyses respectively identified six and four masses as the
main discriminators between the two sample groups (Figs S1 and S2). Regarding positive-ion mode, tenta-
tive hits could be retrieved for two out of five species, which might correspond to ceramide derivatives and/or
N-palmitoylsphingosine derivatives (Table S3). Putative matches could be proposed for two ions appearing as
dysregulated in negative-ion mode, tentatively corresponding to a further ceramide derivative (Cer (d18:0/16:0))
and to a diglyceride derivative DG (36:3) (Table S4). Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 Data quality assurance. 60% for PC1 and ~ 9% for PC2). In these two groups, the samples could be discriminated in a straight-
forward manner along PC1 component (Fig. 4A,B). Regarding negative-ion mode, the PCA plot (engulfing 506
Figure 2. PCA plots yielded by exocellular lipidomic analyses from the ions having a Fold-Change ≥2 with
a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A)
Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are
related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control
samples. Figure 3. PCA plots obtained from endocellular metabolomic analyses from the features displaying both
a Fold-Change ≥2 and a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS
sequences recorded in (A) Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and
MMWj (with j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the
overall of the quality control samples. Figure 2. PCA plots yielded by exocellular lipidomic analyses from the ions having a Fold-Change ≥2 with
a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A)
Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are
related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control
samples. Figure 2. PCA plots yielded by exocellular lipidomic analyses from the ions having a Fold-Change ≥2 with
a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A)
Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are
related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control
samples. Figure 3. PCA plots obtained from endocellular metabolomic analyses from the features displaying both
a Fold-Change ≥2 and a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS
sequences recorded in (A) Positive-Ion Mode and (B) Negative-Ion Mode. Data quality assurance. Besides the very limited number of features appearing as
dysregulated, one should note that their corresponding Fold-Change indices display modest values remaining
below 5 for all features identified in the course of negative-ion mode and below 10 for those pinpointed through-
out positive-ion mode analyses. The only exception was M453T283 in this latter group, for which no putative
identification could be retrieved. i
Filtered RPLC datasets related to extracellular fractions consisted of 289 and 126 features in positive and
negative-ion modes, respectively. In both ionization modes, PCA plots accounted for roughly 60% of the total
variance between the two groups. However, whatever the considered ionization mode, these plots did not lead to
a clear-cut discrimination of the samples according to their exposure status (Fig. 2). g
g
While no discriminating feature could be highlighted during the course of negative-ion mode data process-
ing, two ions of interest could however be evidenced in positive polarity, i.e. M627T51 and M783T478 (Fig. S3). Putative hits could only be retrieved for this latter feature that displayed an important FC of 764, which corre-
sponds to a phosphatidylcholine derivative with a sum composition of (36:4) (Table S5). Metabolomic HILIC analyses. Endocellular data subset with features satisfying both FC > 2 and ANOVA
p-value < 0.05 were respectively represented by 210 and 58 features in positive and negative-ion modes. Generated PCA plots roughly represented 75% of the total variance and could discriminate between non-exposed
and exposed samples along PC2 component in both ion modes (Fig. 3).l Notwithstanding this discrimination, the statistical processing workflow did not lead to emphasize any rele-
vant biomarker in the negative-ion mode data set. Positive-ion mode endometabolomics only resulted in high-
lighting one biomarker with a moderate FC value of 2.8 for which two putative identifications could be retrieved
from HMDB database (i.e. either 2-aminoheptanedioic acid or N-carboxyethyl-γ-aminobutyric acid) (Fig. S4
and Table S6). )
PCA plots related to exometabolomic sequences revealed clear-cut discriminations according to millime-
ter wave exposure status in both polarities. Owing to the large number of features in the filtered data subset Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 3 www.nature.com/scientificreports/ www.nature.com/scientificreports
entificreports/
in positive-ion mode (662), the frames were split into two halves prior to generating the PCA plots after being
ordered by increasing masses. For both these data subsets, PCA plots roughly accounted for 70% of the total var-
iance (ca. Data quality assurance. Note that SHi (with i = 1 to 4) and
MMWj (with j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the
overall of the quality control samples. Figure 3. PCA plots obtained from endocellular metabolomic analyses from the features displaying both
a Fold-Change ≥2 and a p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS
sequences recorded in (A) Positive-Ion Mode and (B) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and
MMWj (with j = 1 to 4) are related to the non-exposed and exposed samples, respectively; QC represents the
overall of the quality control samples. in positive-ion mode (662), the frames were split into two halves prior to generating the PCA plots after being
ordered by increasing masses. For both these data subsets, PCA plots roughly accounted for 70% of the total var-
iance (ca. 60% for PC1 and ~ 9% for PC2). In these two groups, the samples could be discriminated in a straight-
forward manner along PC1 component (Fig. 4A,B). Regarding negative-ion mode, the PCA plot (engulfing 506
features) accounted for 74.1% of the total variance among the two groups, where PC1 and PC2 had respective
contributions of 62.7 and 8.4%. Control and exposed samples also exhibited an obvious separation along PC1
dimension (Fig. 4C).h g
The careful validation of both extracted ion chromatograms and box-and-whisker plot as described in the
Data Processing section led applied to retainment of 111 and 99 features as dysregulated in positive and negative
polarities, respectively (Figs S5 and S6). The current study was performed using a single HRMS analyser, so that Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 4 www.nature.com/scientificreports/ /
p
p
/
Figure 4. PCA plots of exocellular metabolomics sequences, selecting ions having both a FC > 2 and a
p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A,B)
Positive-Ion Mode and (C) Negative-Ion Mode. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are
related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control
samples. Figure 4. PCA plots of exocellular metabolomics sequences, selecting ions having both a FC > 2 and a
p-value < 0.05 upon R-XCMS data processing performed from UHPLC-HRMS sequences recorded in (A,B)
Positive-Ion Mode and (C) Negative-Ion Mode. Conclusion
A f
b As far as can be ascertained, this report represents the first metabolomic investigation focusing on the effects of
MMW. To get as wide an insight into cellular processes as possible, a joint metabolomic and lipidomic profiling
strategy was designed and the extra and intracellular contents were discriminated. It appeared that all lipid-
omic sequences and intracellular metabolomic profiles were slightly affected by MMW but drastic changes in
extracellular metabolomic sequences could be evidenced. During these experiments, we put great emphasis on
controlling cell culture parameters (temperature, pH, incubator humidity). Much attention has been paid to tem-
perature control, however, it cannot be ruled out that unexpected changes at subcellular scale have occurred
and could be responsible for the differences found. Moreover, by making the choice to put the non-exposed
control in the same place in our exposure system, we ensure that these cells are in the same growing conditions
that those that are exposed. Nevertheless, it should be noted that this strategy has the disadvantage of inducing
a shift of one day of culture between the control and the exposed samples. While the unusually high number
of dysregulated features makes of their unambiguous structural assignment an unrealistic purpose, the current
study enables drawing significant and unprecedented conclusions as to the effects of MMW exposure on cellular
systems, especially when combining them with former studies carried out by our group. Accordingly, as upstream
pan-transcriptomic studies in this cellular system did not led to emphasize any significant change upon 60-GHz
MMW exposure, it is reasonable to assume that the vast amount of dysregulations reported in these sequences do
not stem from alterations of gene expression but rather from alterations in membrane permeability, consistently
with previous reports on acellular phospholipidic systems. The tentative metabolites identified throughout the
current workflow might serve as a ground to focus on subsets of metabolites through the so-called target-based
metabolomics. For this purpose, multiplex LC-MS-MRM pipelines proved useful for the quantitative profiling of
some of the hundreds of expected metabolites in complex biological samples with no structural/identity ambi-
guity. Based on the current findings, such follow-up studies could be limited to the exocellular compartment. Finally, we can conclude that our model, purely in vitro, haven’t to be lead to a direct extrapolation of our results
at the organism level. Data quality assurance. Note that SHi (with i = 1 to 4) and MMWj (with j = 1 to 4) are
related to the non-exposed and exposed samples, respectively; QC represents the overall of the quality control
samples. the identification of the dysregulated features could only rely on exact mass data, including any limitation coming
along with it in terms of structural resolution. Resultantly, the structural assignments remain tentative and most
often do not lead to reach unambiguous metabolites. From a practical viewpoint, handling an array of references
covering the structural diversity of candidate biomarkers is not a realistic purpose in the frame of a metab-
olomic study pinpointing such an elevated number of features of interest. Despite this structural uncertainty,
these putative identifications reveal that dysregulated features encompass an array of structurally diverse metab-
olites (Tables S7 and S8). Emphasizing such a large extent of dysregulations, especially in the exo-metabolomic
sequences, appears as an intriguing outcome. Indeed, as upstream transcriptomics analyses did not highlight
significant alterations in gene expression23, such dramatic changes would not have been expected. This led us to
assume that these modifications might not be related to modifications of enzyme expression but rather stem from
alterations in membrane permeability. Cell membranes are regarded as major targets for the interactions between
millimeter waves and biological systems since a variety of bioeffects were reported upon exposure to these radi-
ofrequencies25. As an example, 60 GHz exposure with an incident power density of 0.9 mW/cm² (i.e. in the typ-
ical range of values expected from wireless communications) was proved to induce structural modifications of
artificial biomembranes. Consequently, MMW exposure was demonstrated to increase the lateral pressure of
phospholipid monolayers although not strongly enough to disturb phospholipid microdomain organization in
biomembranes29. Further biochemical processes could be evidenced such as the externalization of phosphati-
dylserine, even though the biological relevance of this event remains to be determined30. Likewise, 53-GHz Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 5 www.nature.com/scientificreports/ radiations and 130-GHz pulse modulated exposures were shown to alter the permeability of phospholipid ves-
icles31–33. MMW action on the orientation of charged and dipolar molecules in the region located between the
aqueous phase and the hydrocarbon interior of the membrane has been hypothesized to represent the driving
force to rearrange phospholipids bilayers, resulting in an increase of small molecules permeability across the
membrane33. Data quality assurance. This new organization of the bilayer presumably displays a higher curvature which would elicit
metabolites leakage. It can also be assumed that electromagnetic radiations of specific frequencies might excite
components of cell membrane, depending on their electric dipoles and possibly leading to form Bose-condensed
phonons34. Such assumptions are consistent with structural changes observed in cells upon MMW exposure, with
different deformations being reported in bacteria35. f
g
p
Likewise, it can be assumed that a similar phenomenon might trigger the leakage of intracellular metabo-
lites into the extracellular medium shown in this manuscript. This inference is further strengthened by the huge
majority of dysregulated features which are found to be upregulated in the treated group (107/111 and 98/99 in
positive and negative-ion modes, respectively. It is interesting to note that under different exposure conditions,
membrane permeabilization upon electric pulses of nanosecond duration were previously reported to occur in
mammalian cells36,37. Conclusion
A f
b In the future, further studies will be necessary to assess MMW bioeffects on animal models
and to investigate potential dysregulations induced by lower IPD values prior to the wide-scale deployment of
technologies based on these specific frequencies. Methods
Ch
i
l Briefly, it consists in powder reconstituted DMEM
medium, completed with fetal calf serum, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) and
antibiotics. Experimental setup for sample exposition. Sample irradiation was performed as previously reported24. A thorough description of the exposure system can be found in Zhadobov et al.19. Briefly, a 6-well culture
plate was positioned in a MEMMERT UE400 incubator to be exposed from the bottom by a standard pyram-
idal horn antenna. Cells were then irradiated or not by this antenna with an average incident power density
(IPD) of 20 mW/cm² for 24 hours, according to the guidelines established by the International Commission on
Non-Ionizing Radiation Protection (ICNIRP) for MMW with limited exposition area40. Both MMW-exposures
and non-exposed samples were performed inside the same incubator, on different days. To mitigate exposure
variability between sample groups, the non-exposed and MMW-Exposed cell samples were located in the same
position within the same incubator39. To avoid a thermal effect associated with MMW exposure, the temperature
increase was counteracted by lowering the incubator set point by the predicted increment. Consistently with
literature23, MMW exposure at such IPD resulted in an elevation of 8 °C leading to set the temperature of the
incubator at 28 °C in the MMW group to reach 36 °C, as in non-exposed cells. Assuming that thermal convection
currents occurring in the radiofrequency-exposed groups may lead to a differential condensation of the culture
medium, the consistency of the pH value was monitored in both non-exposed and exposed sample groups. An
identical pH value of 7.7 could be measured from the different culture media, irrespective of their exposure status. HaCaT keratinocytes cell viability was formerly reported to be of 100% for pH values spanning across the 7.0–8.2
range41. Metabolomics workflow. As MMW exposure might result in evidencing little to no biological effect, it was
very important to validate the ability of the retained metabolomic workflow to emphasize the dysregulations trig-
gered by a known interfering agent. This preliminary study, carried out using the cytotoxic drug 2-deoxyglucose,
demonstrated the adequacy of the proposed workflow for metabolomics purposes27. The whole sample prepara-
tion workflow is summarized in Fig. 5. l
g
Metabolomic analyses were carried out on 4 independent biological samples for both non-exposed and
MMW-exposed groups. As to extracellular profiling, a 100-µL aliquot of the culture medium was recovered for
further sample processing. Methods
Ch
i
l Chemicals, reagents and materials. LC-MS grade water, methanol (MeOH), methyl tert-butyl ether
(MTBE), chloroform (CHCl3), acetonitrile (MeCN), 2-propanol (IPA), ammonium acetate and acetic acid were
purchased from Sigma-Aldrich (St. Louis, MO, USA). The standard mixtures Calmix-positive (i.e. caffeine,
L-methionyl-arginyl-phenylalanylalanine acetate and Ultramark 1621) and Calmix-negative (i.e. acetic acid,
sodium dodecyl sulfate, taurocholic acid sodium salt hydrate and Ultramark 1621), were purchased from Thermo
Fisher Scientific (Waltham, MA, USA). i
(
)
1,2-Dipentadecanoyl-sn-glycero-3-phosphocholine (PC(15:0/15:0)), 1-pentadecanoyl-2
-hydroxy-sn-glycero-3-phosphocholine (Lyso PC(15:0)), 1,2-diheptadecanoyl-sn-glycero-3-p
hosphoethanolamine (PE(17:0/17:0)) and N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0))
were obtained from Coger (Paris, France). L-Tryptophan-2,3,3-d3 (Trypto-d3), indole-2,4,5,6,7-d5-3-acetic
acid (Ind-AA-d5), 1,14-tetradecanedioic-d24 acid (Tetra-A-d24) were purchased from Cluzeau Info Labo
(Sainte-Foy-La-Grande, France). 1,2,3-triheptadecanoyl-glycerol (TG (17:0)), pentadecanoic acid (C15:0), tri-
cosanoic acid (C23:0), heptadecanoic acid (C17:0), leucine-5,5,5-d3 (Leu-d3), creatine-(methyl-d3) (Crea-d3) and
lysine-4,4,5,5-d4 (Lys-d4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). y
4
y
4
p
g
Stock standard solutions (1 mg/L) were prepared in CHCl3 (for lipidic compounds) or in MeOH and kept at
−20 °C. The internal standard (IS) solution contains C17:0, Crea-d3 and Lys-d4 at 10 ng/µL in a MeOH/H2O mix-
ture (4/1). Lipidomic external standard (ES) solution containing (PC (15:0/15:0), Lyso PC (15:0), PE (17:0/17:0),
TG (17:0), Cer (d18:1/17:0), C15:0 and C23:0 at 0.5 ng/µL and metabolomic (ES) solution containing Leu-d3,
Trypto-d3, Ind-AA-d5 and Tetra-A-d24 at 1 ng/µL were subsequently prepared in CHCl3 (lipidomics ES) or in
MeOH (metabolomics ES). Cell culture. A human keratinocyte cell line (HaCaT) was cultured as described elsewhere22. In an attempt
to circumvent any senescence or drift of the cellular populations, keratinocytes were exposed at early passages
(between 10 and 16). To enable proper cross-sample comparison, the quantity of cellular material was estimated Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 6 www.nature.com/scientificreports/ Figure 5. Schematic workflow of the sample preparation procedure27. Figure 5. Schematic workflow of the sample preparation procedure27. through the evaluation of total ERK protein amount (Western Blot using anti ERK1 (K-23) antibody (Santa Cruz
Biotechnology, Dallas, Texas, USA))38. The exposure medium designed to keep pH buffering in the non-gassed
incubator of the exposure system as described elsewhere39. Briefly, it consists in powder reconstituted DMEM
medium, completed with fetal calf serum, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) and
antibiotics. through the evaluation of total ERK protein amount (Western Blot using anti ERK1 (K-23) antibody (Santa Cruz
Biotechnology, Dallas, Texas, USA))38. The exposure medium designed to keep pH buffering in the non-gassed
incubator of the exposure system as described elsewhere39. Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 Methods
Ch
i
l The column oven temperature was kept constant at 35 °C for metabolomic
HILIC runs and at 45 °C for lipidomic RPLC analyses. RPLC analyses were performed by gradient elution as
follows: T, 0 min, 40% B; 0–2 min, 50% B linear; 2–12 min, 70% B linear; 12–17 min, 99% B linear; 17–25 min,
99% B; 25–29 min, 40% B. HILIC acquisitions were obtained using the following gradient program: T, 0–2 min,
95% B; 2–5 min, 80% B; 5–12 min, 60% B linear; 12–14 min, 40% B linear; 14–16 min, 40% B; 16–26 min, 95% B.ll ;
,
;
,
;
,
;
,
;
,
ESI source conditions were set as follows: sheath gas flow, 55 Arbitrary Units (AU); auxiliary gas flow, 10 AU;
capillary temperature, 300 °C; spray voltage, either 3.5 kV (lipidomics) or 3.0 kV (metabolomics); S-lens radiofre-
quency, 50 AU. Mass spectra were either acquired over the m/z range 150–1500 (lipidomics) or 65–975 (metabo-
lomics) at a resolving power of 35000 Full Width Half Maximum (FWHM) measured at m/z 200. The Automatic
Gain Control (AGC target) was set at high dynamic range (5 × 105) with a maximum injection time of 100 ms. External calibrations of the MS instrument were performed using the Calmix-positive and Calmix-negative
standard solution for the positive and the negative ionization modes, respectively. Exact mass measurements did
not take into account the mass of the electron.h The analytical run was initiated by a number of injections of QC samples to ensure that LC and MS systems
had time to equilibrate and perform satisfactorily. Irrespective of their exposed or non-exposed sample status,
the study samples were randomized to limit the effect of time trends and thus minimize bias introduced by
non-biological parameters (e.g. instrumental drifts). Each sample was analyzed six times. Statistical processing. The statistical processing of the metabolomic data considered all exposed and unex-
posed samples independently of one another. LC/MS data were further processed by R package XCMS (ver-
sion 3.2). The preprocessing results generated a data matrix as a feature list table comprising their integrated
intensities (reconstructed ion chromatogram peak areas), along with the observed fold-changes and associated
p-values42. Applied peak picking parameters were prefilter = c(5,25), snthresh = 6, mzdiff = 0.01 and ppm = 15. Methods
Ch
i
l Regarding intracellular metabolite assessment, the cells were washed with 1 mL of
phosphate buffer solution after having discarded the remaining culture medium. The cells were subsequently
detached by scraping within 1250 µL of PBS solution. A 250 µL-aliquot was recovered to evaluate the amount of
total ERK, a ubiquitous protein, by western blotting. Both the endo- and exo-cellular fractions were spiked with
400 µL of Internal Standard solution dissolved in MeOH/H2O (4/1, v/v). Endocellular fractions were frozen at
−80 °C for 20 minutes to facilitate cell membrane disruption. Solutions were then added with 550 µL of MTBE
and vortexed three times for ten seconds (separated by one minute breaks in ice), 200 µL of cold water were
then added with the same vortexing sequence being repeated. After centrifugation of the solutions (12,000 g,
4 °C, 15 min), a 300 µL-aliquot of the upper organic phase was transferred to a vial to be spiked with 40 µL of the
lipidomics ES solution (0.5 ng/µL) in CHCl3 prior to being dried under N2 flux. The dry extract was dissolved in
200 µL of MeCN/IPA/H2O solution (65:30:5, v/v/v). Likewise, a 300 µL-aliquot of the lower aqueous phase was
recovered and centrifugally filtered through a Millipore 10 kDa cutoff filter (12,000 g, 4 °C, 20 min). The solution
was evaporated to dryness under N2 and later resuspended in 200 µL of a MeCN/H2O (9/1, v/v) solution. Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 7 www.nature.com/scientificreports/ QC samples were obtained by pooling 20 µL aliquots from the test samples to represent a bulk control sample. The samples were stored at −80 °C pending UHPLC-HRMS analysis. UHPLC/HRMS analyses. Analyses were performed using an ultrahigh performance liquid chromatogra-
phy (Waters Acquity), hyphenated with a Thermo QExactive mass spectrometer. Samples were injected (5 µL)
onto either an Acquity CSH C18 column (1.7 µm, 2.1 × 100 mm; Waters) for lipidomic sequences or a SeQuant
ZIC-HILIC column (3.5 µm, 2.1 × 100 mm; Merck) for metabolomic HILIC analyses. The standard mobile
phases for RPLC (lipidomic sequences) were A = MeCN/H2O/ammonium acetate 1 M/acetic acid (600/390/10/1,
v/v/v/v) and B = IPA/MeCN/H2O/ammonium acetate 1 M/acetic acid (880/100/10/10/1, v/v/v/v/v). For
HILIC conditions, the mobile phases were A = H2O/ammonium acetate 1 M/acetic acid (980/10/1, v/v/v) and
B = MeCN/solvent A (950/50, v/v). Methods
Ch
i
l Initial alignment (bw = 20, minfrac = 0.66, minsamp = 4, mzvid = 0.008) and retention time correction (standard
loess, plottype = c(deviation) were then applied. Further alignment steps were performed using the same process-
ing parameters with decreasing bw values (lowered to bw = 9 for the second round and to 5 for the final stage). Subsequently, R-package CAMERA was used for peak annotation after XCMS data processing43. Consistently
with reported guidelines, features found in less than 20% of the analyzed samples were removed according to the
so-called 80% rule44.fi As to univariate analyses, the coefficient of variation (CV) within QC samples was calculated by dividing the
standard deviation by the mean intensity of each feature, leading to a histogram of the resulting CV distribu-
tion. Subsequently, computation of the Fold-Change (FC, ratio of abundance between non-exposed and exposed
samples) along with the corresponding p-value (statistical significance from a Student t-test) streamlined the
selection of metabolites of interest. Features selection was based on the following criteria: CV QC < 30%, FC > 2
and ANOVA p-value < 0.05. Extracted Ion Chromatograms (EICs) were individually monitored to exclude
potential artifacts from the ion list. Later on, Box and Whiskers Plot related to all metabolites of potential inter-
est were also individually checked to retain the features for which no overlap existed between values obtained
from non-exposed and exposed samples. Such features were tentatively identified against databases as described
later on. The top and bottom of each box represent the 25th and 75th percentiles, the center line indicates the
median and the extent of the whiskers depicts the 5th and 95th percentiles. Regarding multivariate analyses,
Principal Component Analyses (PCA) were performed to get a general overview of the interrelationship between
study samples as well as QC. Statistical graphs were prepared using SigmaPlot 13.0® (Systat Software, Inc., USA). Filtered data sets related to each sequence (only retaining features displaying FC > 2 and p-value < 0.05) were
then analyzed by PCA to explore samples’ relationship and grouping. For this purpose, PCA retrieves a small
number of principal components that summarize the measured data to visualize trends and emphasize possible
outliers. Metabolite identification. [M + H]+, [M-H2O + H]+, [M + Na]+ and [M-H2O + Na]+ were selected
as possible adducts for positive polarity. Regarding negative-ion mode, [M-H]−, [M-H2O-H]− and
[M-H2O + HCOOH-H]− were considered. Methods
Ch
i
l Putative identifications were carried out against the freely available
database Human Metabolome Data Base (HMDB). Whenever possible, efforts were made to narrow down iden-
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567–574 (2006). Author Contributions P. Le Pogam performed experiments, analyzed data and mainly wrote the manuscript. Y. Le Page and D. Habauzit
performed experiments and contributed to the design of the biology-related aspects of the manuscript. M. Doué contributed to the technical validation of the metabolomic pipeline reported herein. M. Zhadobov and R. Sauleau supervised the radiofrequency exposure related aspects of the manuscript. Y. Le Dréan mainly designed
the biology-related research of the manuscript. D. Rondeau designed experiments, supervised the analytical
chemistry-related content of this investigation, and contributed to the analysis of the data and to the writing of
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Bioelectromagnetics 37, 444–454 (2016).i 40. Ahlbom, A. et al. Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz). Health Phys. 74, 494–521 (1998). Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 9 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-45662-6. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an
institutional affiliations. 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 Cre-
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material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted 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 license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports | (2019) 9:9343 | https://doi.org/10.1038/s41598-019-45662-6 10
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Reflections on the battle against COVID-19: The effects of emotional design factors on the communication of audio-visual art
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TYPE Original Research
PUBLISHED 07 November 2022
DOI 10.3389/fpsyg.2022.1032808 TYPE Original Research
PUBLISHED 07 November 2022
DOI 10.3389/fpsyg.2022.1032808 TYPE Original Research
PUBLISHED 07 November 2022
DOI 10.3389/fpsyg.2022.1032808 Reflections on the battle against
COVID-19: The effects of
emotional design factors on the
communication of audio-visual
art OPEN ACCESS
EDITED BY
Yongjin Chen,
Chongqing University, China
REVIEWED BY
Lijuan Guo,
Taishan College of Science
and Technology, China
Anguo Fu,
Hainan University, China
Hannah Palko,
University of North Carolina
at Charlotte, United States
Zhang Ailian,
Shandong University of Technology,
China REVIEWED BY
Lijuan Guo,
Taishan College of Science
and Technology, China
Anguo Fu,
Hainan University, China
Hannah Palko,
University of North Carolina
at Charlotte, United States
Zhang Ailian,
Shandong University of Technology,
China Wen-Ting Fang1*, Jian-Hua Sun2 and Qing-Dong Liang3* 1School of Art and Design, Shanghai Dianji University, Shanghai, China, 2Office of the CPC
Shandong University Committee, Shandong University, Jinan, China, 3School of Education, Jiangsu
University of Technology, Changzhou, China 1School of Art and Design, Shanghai Dianji University, Shanghai, China, 2Office of the CPC
Shandong University Committee, Shandong University, Jinan, China, 3School of Education, Jiangsu
University of Technology, Changzhou, China *CORRESPONDENCE
Wen-Ting Fang
f_wenting@163.com
Qing-Dong Liang
124753999@qq.com
SPECIALTY SECTION
This article was submitted to
Personality and Social Psychology,
a section of the journal
Frontiers in Psychology
RECEIVED 31 August 2022
ACCEPTED 14 October 2022
PUBLISHED 07 November 2022
CITATION
Fang W-T, Sun J-H and Liang Q-D
(2022) Reflections on the battle
against COVID-19: The effects
of emotional design factors on
the communication of audio-visual
art. Front. Psychol. 13:1032808. doi: 10.3389/fpsyg.2022.1032808 Fighting against the epidemic is an arduous and prolonged battle where
many artists hope to inspire people with the power of art through cultural
creativity. To explore the effects of emotional design factors on the
communication of audio-visual art and the audience’s perceptive experience,
this research takes the original anti-epidemic song and the film China Braves
Headwind as the research object. The research also uses such methods as
questionnaires, Structural Equation Models, and dependent samples t-tests
to conduct statistical analysis. The results are as follows: First, the emotional
design evaluation matrix based on the emotional communication model is
reasonable, and the scales of this research are feasible. Second, the emotional
design of audio-visual works can significantly affect the audience’s emotional
experience and further improve sharing intention. Third, Attribute A2 (Artistic
style, Thematic perception) and attribute C3 (Emotional resonance, Spiritual
sublimation) serve as common factors affecting the emotional experience in
terms of both musical works and film and television works. Fourth, compared
with musical works, film and television works are likely to resonate with the
audience. KEYWORDS
COVID-19,
emotional
design,
emotional
experience,
audio-visual
arts,
communication effects COVID-19,
emotional
design,
emotional
experience,
audio-visual
arts,
communication effects Introduction Therefore, cognitive engineering has been widely used in
the sectors of creative design in recent years. And cognitive
engineering has been combined with information dissemination
theory to help people deeply understand the communication
methods of artists and their audiences. All these shifts are
conducive for art to be closer to our life and humanity,
thus bridging the barriers and gaps caused by the creation
and expanding new possibilities of cultural consumption. Digital audio and video, animation games, webcasting, etc. are all important parts of the digital cultural industry based
on mobile smart terminals, mobile Internet technology, and
network big data. With audio and video works as the
medium, this research will study the effect of emotional
design factors on the communication of audio-visual art. The specific research purposes are as follows: to explore
how art creators influence the audience’s perception and
dissemination through audio-visual art; to understand the key
factors that audio-visual art affects the audience’s emotional
experience; to deeply analyze the differences in the audience’s
perception of the emotional design of musical works and film &
television works. The COVID-19 pandemic at the start of 2020 has brought
heavy damage to countries worldwide in the aspect of economy,
politics, and culture (World Health Organization, 2020). Individuals with a high fear of COVID-19 may experience
negative emotions and cognitions (Brooks et al., 2020; Rettie
and Daniels, 2020). Meanwhile, fighting against the epidemic
is an arduous and prolonged battle where many artists hope
to inspire people with the power of art through cultural
creativity (Larissa et al., 2021; Lydia, 2022). The studies suggest
a potential role for mixed emotions in pandemic-related
outcomes, which can promote complex thought processes and
eudaimonic well-being (Berrios et al., 2018; Oh and Tong,
2021). Some findings emphasize the importance of emotional
experience and emotion regulation for self-efficacy, subjective
wellbeing, and positive coping during the pandemic (Cattelino
et al., 2021; ˙Ime and Ümmet, 2022). The results showed
optimism as a protective factor against the psychological impact
of the COVID-19 pandemic which can increase confidence,
motivate individuals to achieve goals, and increase positive
affect and well-being through its effects on perceived stress
and infection stress anticipation (Brosschot et al., 2005; Puig-
Perez et al., 2022). Art language is composed of codes,
which is a well-organized and understandable information
system. Introduction their desires and fantasies can be realized. In turn, the
audience who enjoy these works can release their inner
depression and can feel a sense of pleasure (Winner, 1982). The
dissemination of art can relieve stress and negative emotions,
which has a significant effect on fighting against COVID-
19 (Lydia, 2022; Niels et al., 2022). Therefore, it is an issue
worth exploring how artists express their creative ideas and
inner feelings, and how viewers can grasp the essence of
creativity and gain emotional experience during the COVID-19
pandemic. As many countries around the world went into a frenzy
of cultural creativity at the beginning of the 21st century,
humanistic aesthetics has been placed high on the agenda in
the design community. Developing an “aesthetic economy”
with cultural characteristics has been an inevitable trend. In
terms of creative cultural design, the key lies in extracting
cultural elements and fostering a taste of lifestyle to finally
transfer a wholly new aesthetic meaning to the audience through
emotional content (Hsu and Lin, 2011; Yeh et al., 2011; Yang
et al., 2022). Norman (2004) pointed out that emotional factors
were the key to designing. A successful cultural and creative
product can definitely arouse the inner desire of its audience. A high-quality design thus must be creative, culture-rooted, and
human nature-centered from stem to stern. During the epidemic, China’s economy has been hit
severely, and the people’s normal life and production have
been disrupted. The COVID-19 pandemic has caused new
patterns of behaviors that differ from previous ones in terms
of responses and emotions to external stimuli (D’Uggento
et al., 2022). Online access to cultural activities could sustain
the educational and entertaining demands of diverse groups
during mass confinement (Samaroudi et al., 2020; Ginzarly
and Srourb, 2022). Cultural and artistic activities online, such
as virtual museums, virtual Concerts, art galleries, and live
theaters, created more opportunities for people to experience
the arts and achieve artistic consumption (Choi et al., 2020). Studies found a remarkable increase in the consumption
of digital cultural offerings (e.g., music, film, literature, and
theater) during COVID-19-related restrictions (Gotthardt et al.,
2022). Internet-based digital cultural consumption has seen
its main consumers shifting from offline to online, which has
become an important factor in driving economic recovery. Some scholars have pointed out that cultural and creative
products should focus on consumers’ inner feelings and
spiritual experiences (Gao et al., 2018; Radermecker, 2021). Reflections on the battle against
COVID-19: The effects of
emotional design factors on the
communication of audio-visual
art The combination of music and visual sensation can help open
up the conception of artistic works and convey their meanings to viewers. Therefore, it’s necessary to explore the emotional communication mode
between audio-visual artists and the audience. It helps artists think about
how to create works innovatively and is conducive to marketizing works and
stimulating cultural consumption demand. Fang W-T, Sun J-H and Liang Q-D
(2022) Reflections on the battle
against COVID-19: The effects
of emotional design factors on
the communication of audio-visual
art. Front. Psychol. 13:1032808. doi: 10.3389/fpsyg.2022.1032808 © 2022 Fang, Sun and Liang. 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. Frontiers in Psychology 01 frontiersin.org 10.3389/fpsyg.2022.1032808 Fang et al. Frontiers in Psychology The emotional design and emotional
communication mode of audio-visual
art Audio-visual artistic works can stimulate the audience’s
perceptual system through certain design factors, thereby
triggering
emotional
responses,
generating
emotional
connections,
and
arousing
their
inner
desires. People’s
heartstrings are touched mainly by such works’ attributes as
artistic styles, story plots, audio-visual structures, and thematic
connotations (Schmetkamp, 2017). With the help of the fantasy
world expressed by the screen, the audience can gain pleasant
sensations intertwined with curiosity, desires, and overlapping
emotions
aroused
by
dramatic
conflicts,
thus
obtaining
some spiritual comfort (Mulvey, 1975). Bourdieu (2017) also
mentioned that an artistic work would be meaningful to viewers
with a cultural sensibility and arouse their interest, and people’s
emotional projection would be manifested through the color,
line, rhythm, style, form, or purpose of works. As coders of
interpretation of artistic works, audio-visual artists are good at
establishing an interdependent relationship between themselves
and their works by integrating emotions into works. In this
case, emotional design refers to the design in audio-visual art
that can mobilize the audience’s perceptual system and generate
their emotional interaction (Nicoló et al., 2022). Nowadays,
with the rapid development of science and technology, design
has gone beyond the realization of functional purposes, into a
reflection of emotional and cultural values (Flores and Roldo,
2012). Emotional involvement is also central to the immersive
experience, especially driven by auditory and visual stimuli
in a virtual environment (Sangkyun, 2012; Jan et al., 2020;
Salselas et al., 2021). Studies by some scholars indicated that
the use of standardized visual and auditory emotional stimuli
could induce different emotional states, thereby affecting the
interaction process between the audience and the works, both
on physiological and psychological levels (Ashby and Johnson,
2003; Zhou et al., 2013; Ding, 2021). The emotional design of art
can finally penetrate the spiritual world of its audience, excavate
their purest emotion, and empower them to understand the
works’ deepest meaning, by which the audience can reach
a higher realm of realizing the sublimation from sensory
appreciation to spiritual reflection. Speaking of the emotional communication of audio-visual
art, it holds creative connotation based on the emotional design
of the artists and reflects the audience’s perceptive feelings with
the aim of their emotional experience, both of which work to
build a bridge of communication connecting artists and the
audience. Literature review For example, Norman (2004)
believed that high-quality design must take into account both
aesthetics and usability, which could be evaluated through
such attributes as aesthetics, attractiveness, fun, and usability. Research launched by Khalid and Helander (2004) indicated that
the information value or utility, functionality and semantics,
familiarity, and usability of cultural and creative products would
affect the demands and preferences of the audience. These
factors showed that the function of their abilities played an
important role in emotional perception. Creativity comes from
the yearning for a certain lifestyle, from which the symbolic
meaning is extracted, transformed into a visual consumption
symbol, and finally integrated into artistic works (Hsu and Lin,
2011). Works with the added value of cultural creativity, such
as image code, pattern type, and modeling composition, can
satisfy the emotional demands of the audience from external
signs to connotation. From what has been suggested above,
artists are good at integrating the three dimensions of beauty,
functionality, and creativity, as well as conveying potential
or implied emotions and meanings, which is also of vital
importance in the construction of emotional design. Literature review (Izard, 2013; Sakhaei et al., 2022). Mcluhan (2000) proposed
that the medium was an extension of human beings, and audio-
visual media had a strong influence on people’s touch, and
thus made people’s perception a three-dimensional structure. Langer (1957) once said that any kind of art was an external
manifestation of inner essence, an objective representation of
subjective reality. In other words, the measurement standard
of emotional design in audio-visual art includes the objective
scope, and the latter’s internal structure can be reflected
through correlation hierarchy. In the past few decades, many
scholars have proposed evaluation criteria that affect the
audience’s emotional perception. For example, Norman (2004)
believed that high-quality design must take into account both
aesthetics and usability, which could be evaluated through
such attributes as aesthetics, attractiveness, fun, and usability. Research launched by Khalid and Helander (2004) indicated that
the information value or utility, functionality and semantics,
familiarity, and usability of cultural and creative products would
affect the demands and preferences of the audience. These
factors showed that the function of their abilities played an
important role in emotional perception. Creativity comes from
the yearning for a certain lifestyle, from which the symbolic
meaning is extracted, transformed into a visual consumption
symbol, and finally integrated into artistic works (Hsu and Lin,
2011). Works with the added value of cultural creativity, such
as image code, pattern type, and modeling composition, can
satisfy the emotional demands of the audience from external
signs to connotation. From what has been suggested above,
artists are good at integrating the three dimensions of beauty,
functionality, and creativity, as well as conveying potential
or implied emotions and meanings, which is also of vital
importance in the construction of emotional design. (Izard, 2013; Sakhaei et al., 2022). Mcluhan (2000) proposed
that the medium was an extension of human beings, and audio-
visual media had a strong influence on people’s touch, and
thus made people’s perception a three-dimensional structure. Langer (1957) once said that any kind of art was an external
manifestation of inner essence, an objective representation of
subjective reality. In other words, the measurement standard
of emotional design in audio-visual art includes the objective
scope, and the latter’s internal structure can be reflected
through correlation hierarchy. In the past few decades, many
scholars have proposed evaluation criteria that affect the
audience’s emotional perception. Introduction When artists have the same code system and code
perception as their audience, the truth that their artistic
creations can be deeply perceived makes art an emotion
transmitter without frontiers. Philosopher Dewey (1964) also
put forward that in a shared world full of divides and walls
that limit people’s experience, artistic works serve as the only
means of communication between people, completely without
barriers. As a branch of the cultural and creative industries,
audio-visual art has a unique form of artistic expression
and is also an important way of emotional expression. Art
creators, through their understanding, design situations and
artistic conceptions for emotional communication. Artists with
creative ideas intrinsically create artistic works, by which Frontiers in Psychology 02 frontiersin.org Fang et al. 10.3389/fpsyg.2022.1032808 Literature review Frontiers in Psychology The emotional design and emotional
communication mode of audio-visual
art Given that, the sense of beauty is more like insight
beyond agreeable sensations; Harmonious coexistence means
that all elements integrate subtly as one to create an aura
of graceful harmony. So these two attributes can reflect the
inside beauty of the artwork through the subtle organization
with the measurement reference of beauty enjoyment. At the
effectiveness level, Artistic charm (C1-1) and Aesthetic value
(C1-2) can be extracted from the aesthetic dimension. Artistic
charm actually reshapes the relationship between artists and
their works, both of which exist because of art, the original third
party that gives people a unique perceptive feeling of artistic
events (Heidegger, 2011); Aesthetic value contains imagination,
expression, emotion, motivation, transformation and many
ways to realize the beauty of an artistic work, and it is a
reproduction of beauty of the aesthetic creation (Langer, 1957). according to the original meaning (Lin and Li, 2015; Fang
et al., 2018). The audience’s emotional experience of an artistic
work involves their decoding process. At the level of the body’s
instinct, the audience will be attracted by the external senses
of an artistic work; at the level of the mind’s behavior, they
will understand and feel the meaning beyond the perception
of artistic works. Eventually, they will return to the level of
spiritual reflection where the audience will be touched deeply
in their hearts and the artistic work will be evocative of their
memories of emotion in their lives. Given that, this research
combines the relevant communication and cognitive theories to
give a conclusion about the emotional communication mode of
audio-visual art, as shown in Figure 1. The emotional design and emotional
communication mode of audio-visual
art As the composer Cage John (2013) said, art was
not the act of an artist to create well-connected works, but
a place for the audience to interact with artistic works (a.k.a. art medium). In this case, artistic works become an interactive
medium that the artists encode and the audience decodes. The
procedural school among communication theories believes a
successful encoding needs the artist to take account of three
aspects, namely the technical level, the semantic level, and the
effectiveness level (Jakobson, 1987; Craig, 1999; Fiske, 2010). The technical level means that the code creator sends messages
that convey his intention so that artistic work can be perceived
by the code recipient graphically; the semantic level means that
the code sender expresses his meaning of an artistic work, which
can be well understood by the code recipient authentically; the
effectiveness level means that the code sender has an effective
influence on the code recipient on his expectant behaviors The audience’s perception and appreciation of audio-visual
works represent the first stage of emotional communication
between people and art objects. In this process, the audience
is required to combine their knowledge, experience, and
perception to decipher and decode (Formilan and Stark,
2021). Evoking attention by showing audio-visual artistic works
activates the motor system and embodiment as the emotional–
motivational state is linked with cognitive processes in human–
environment interactions from a sensory-cognitive structure Frontiers in Psychology 03 frontiersin.org 10.3389/fpsyg.2022.1032808 Fang et al. dimension. Expression techniques refer to the application of
techniques, such as film composition, shooting techniques, lens
using, tune arrangement, and singing styles, which are the
most basic design elements for expressing beauty; Audio-visual
language can be generalized as an effect being represented
by the internal organizational structure of an artistic work,
which specifically includes the fluency of scenes switching, the
richness of melody, and the vividness of the singing. Both
the above attributes emphasized the technical methods that
artists use to convey aesthetic feelings with the measurement
reference of beauty representation. At the semantic level, the
aesthetic dimension includes Rhythm and melody (B1-1) and
Harmonious coexistence (B1-2). Rhythm and melody have an
overlapping or interlaced effect which is similar to a sparse or
dense arrangement of characters according to different meaning
rules. Emotional design evaluation matrix of
audio-visual art On the theoretical basis of the emotional communication
mode of audio-visual art, this research establishes the emotional
design evaluation matrix of audio-visual art to better understand
the classification of evaluation attributes and provide a reference
for the measurement of subsequent research structures, as
shown in Table 1. The aesthetic dimension created by the
artist using logical conception can condense the audience’s
aesthetic experience into a profound aesthetic experience. To
resonate with the audience in such three dimensions as beauty,
function, and creativity, artists should make endeavors in these
three aspects: technique, meaning, and effect. At the technical
level, Expression techniques (A1-1) and Audio-visual language
(A1-2) can be summarized as two attributes of the aesthetic FIGURE 1
The emotional communication mode of audio-visual art. FIGURE 1
The emotional communication mode of audio-visual art. FIGURE 1
The emotional communication mode of audio-visual art. 04 04 Frontiers in Psychology frontiersin.org Fang et al. 10.3389/fpsyg.2022.1032808 TABLE 1 Emotional design evaluation matrix of audio-visual art. TABLE 1 Emotional design evaluation matrix of audio-visual art. Artist
Code\Measurement indicators
Beauty
Function
Creativity
Technical Level
(See?)
A1-1
Expression techniques
A1-2
Audio-visual language
A2-1
Artistic style
A2-2
Thematic perception
A3-1
Content conception
A3-2
Scene creation
Physical
(Attraction)
Semantic Level
(Understand?)
B1-1
Rhythm and melody
B1-2
Harmonious coexistence
B2-1
National culture
B2-2
Spirit of times
B3-1
Emotion presupposition
B3-2
Imagination stimulation
Mental
(Accuracy)
Effectiveness Level
(Touched?)
C1-1
Artistic charm
C1-2
Aesthetic value
C2-1
Implication and connotation
C2-2
Thought promoting
C3-1
Emotional resonance
C3-2
Spiritual sublimation
Spiritual
(Affecting)
Aesthetic experience
Meaningful experience
Artistic conception
Emotional experience\Decode
Audience In general, these two attributes are the objective expression of
the audience’s inner essence of aesthetic experience. Implication and connotation are likely to be a unity of artistic
works’ artistry and ideology, and an implication of possibilities
of being understood deeply; On the other hand, an artistic work
will lead people to think based on recognizing its meaning,
which is called Thought promoting effect. Just as a literary
critic Bryson (1983) put forward, art was a composition of
various cultural symbols whose symbiosis relationship between
connotation and denotation was highlighted by art in turn. Art
tends to represent universal truths about things, not only in
the way as it was and as it is thought to be, but also as it
ought to be (Aristotle, 1989). Emotional design evaluation matrix of
audio-visual art So the functional dimension of
audio-visual artistic works is to realize the expected effect of
the works’ meaning as they ought to be, based on the technical
and meaning-express levels, as well as to deliver the true content
of art. The functional value of audio-visual artistic works is to
create emotions through pure forms of expression, such as
virtual images or musical notes, to draw forth the audience’s
certain sentiments and make them taste the deepest connotation
of artistic works. Therefore, in terms of the functional
dimension, different attributes are based on such levels as
technique, meaning, and effect. At the technical level, the
functional dimension can be divided into Artistic style (A2-
1) and Thematic perception (A2-2). Artistic style is an
exceptionally expressive way of indicating the interconnection
of space, time, and events in the audio-visual world; Thematic
perception means that an artistic work profoundly expresses
its purpose and meaning through external forms and thus
reveals its theme. As Goodman (1990) said in Languages of
Art, creating an artistic work was a process of building its
structure where existed the abstraction and expression of its
style, the establishment and modification of its purposes, and the
distinction and connection of its thematic elaborating methods. At the semantic level, two main attributes of the functional
dimension are National culture (B2-1) and Spirit of times (B2-2). The National culture in a video embodies national feelings and
cultural deposits, basically, with the use of symbolism, regional
customs, linguistic structure, etc.; the social environment where
art attaches and maintains its status empowers artistic works
with the Spirit of times, which can be described as a core value
philosophy under specific circumstances. These two attributes
provide the audience with a deep interpretation of audio-visual
artistic works at the functional level. At the effectiveness level,
two main features are Implication and connotation (C2-1) and
Thought promoting (C2-2). Art is a reflection of people’s desire
for knowledge, and researches on its production, nature, and
audience are of better help for people to understand its potential
order and value (Grosse, 1987). Therefore, on the one hand, The essence of art is artists’ sentimental illusion, a reaction
to some ideas, and the audience will obtain artistic conceptions
beyond the outside from the independent existence of this kind
of illusion and the space with certain forms (Langer, 1957). Frontiers in Psychology Audio-visual emotional design and sharing
intention From the perspective of sociology, Arnold (1987) believed
that art could not realize its value without sharing functions,
or it could only be defined as some simple words, symbols,
or pictures. True art is a form of communication more than
expression. As far as the semantic school of communication
theory is concerned, an artistic work successfully expressed
through a symbolic system must embrace three functions:
signification, impression, and communication (Barthes, 1967;
Jakobson, 1987). Art can satisfy people’s spiritual needs. To fully
convey emotional expressions, art language needs to convey
emotional meaning through an independent structure based on
the functions of signification and impression. As a result, the
communication function will be achieved by experience sharing
between artistic works and the audience. In this process, how
emotions are organized in art becomes the key to satisfying the
audience’s emotional needs and is also an important factor for
information dissemination. Given that, the motivation of audio-
visual emotional design is based on the audience’s demands,
and the main approach of its spread is the audience’s sharing
intention. Besides, artists’ creation of emotional design aims at
great communication. Emotional design evaluation matrix of
audio-visual art At
the technical level of the creative dimension, the two attributes
of Content conception (A3-1) and Scene creation (A3-2) are
summarized. Content conception means that the ingenious
structure of artistic works makes people feel touched and then
internalizes this feeling into an almighty tension. In other words,
the diversity, complexity, and infinity inside artistic works will
create an invisible force to be attractive and intriguing; Scene
creation refers to the unique scenes and atmosphere of artistic
works where the audience can be personally on the scene and
immerse themselves in the emotions. Given that, both two
attributes are objective conditions for creative performances of
audio-visual artistic works. In the semantic level of the creative
dimension, the two attributes are Emotion presupposition (B3-
1) and Imagination stimulation (B3-2). To realize the design
of artistic conception conforming to normal logic, Emotion
presupposition will be used to combine the creative rules of 05 Frontiers in Psychology frontiersin.org Fang et al. 10.3389/fpsyg.2022.1032808 feelings and a certain emotion, so the emotional design is highly
related to emotional experience. artistic works with their texts; Imagination stimulation becomes
the key to developing the audience’s spiritual feeling. Under
the narrative structure, the audience will have associations
with individuals, society, and culture and imagine an infinite
space with the symbols of music and scenes. These two
attributes are semantic-level strategies artists use to give play
to their creativity. In the effectiveness level of the creative
dimension, there are also two attributes, which are Emotional
resonance (C3-1) and Spiritual sublimation (C3-2). To imagine
an artistic work is to imagine the way of life it works. Only
by reconstructing the relevant system of meaning like an artist
can people grasp the emotions conveyed by art (Danto, 2007). That is why Emotional resonance becomes an inner purpose of
realizing aesthetic creativity; At the same time, what becomes
the result is Spiritual sublimation, which arouses the audience’s
latent desires after their perception of art. As Nietzsche (1872)
stated in The Birth of Tragedy, that the art could save people
could not be put down to its moral connotations but could be
put down to the Dionysian spirit. People would have an eternal
spiritual bailment after the Dionysian spirit making them realize
the pain and cruelty of life. It can be seen from this that the
essence of creative audio-visual art is the emotional comfort and
the purification of the soul through aesthetic forms. Emotional experience and sharing intention The audience, or consumers of audio-visual artistic works,
can be seen that their inner feelings and potential needs have
a positive influence on their actions and decisions. In the
process of enjoyment of artistic works, they play an active
role, which is that they gain an experience of interaction when
they have a perception during the conversation with artistic
works. This process indicates that the audience participates in
the creation and subsequent sharing of emotional experience
they obtain from artistic works is exactly the behavioral
result. This is so in everyday life where perception, emotion,
action, cognition, and the world tend to fuse (Crippen, 2021). Fictional emotions evoked by art can motivate actions of
various kinds (Werner, 2020). As art is complete only after
being accepted by people, the connection between the audience
and audio-visual artistic works illustrates the completion of
appreciation activities. Barthes (2002) stated that the birth of
readers must be at the expense of the author’s death. Once
the audience obtained the ability of independent thinking
about art, they could fill in the blanks of the artistic works
themselves, only leaving an opportunity for the production
of some kind of relationship between artistic works and the
audience. The study finds that emotional experience has a
significant and positive effect experience symbolic consumption
(Tangsupwattana and Liu, 2018). A better understanding of
the interrelations between the emotional experience and the Frontiers in Psychology Research structures and
presuppositions This research explores the audience’s emotional experience
and sharing intention of audio-visual artistic works from
the perspective of emotional design. The emotional design
consists
of
three
measurement
dimensions,
namely
the
aesthetic
dimension,
the
functional
dimension,
and
the
creative dimension. The structure of this research is also
designed
with
the
use
of
relevant
theories,
document
analysis
(Figure
2),
and
emotional
experience
as
an
intermediate variable. The purpose of this design is to
discuss whether emotional design can affect the audience’s
sharing intention. According to the NO.1 research purpose
listed in chapter one, the research come up with the
following
presupposition:
Ha1
(Emotional
design
has
a
significant influence on emotional experience in musical
works);
Ha2
(Emotional
experience
has
a
significant
influence on sharing intention in musical works); Ha3
(Emotional design has a significant influence on sharing
intention in musical works); Ha-1 (Emotional experience
has
an
intermediate
effect
on
emotional
design
and
sharing
intention
in
musical
works);
Ha-2
(Emotional
design has an impact on emotional experience and then
promote
people’s
sharing
intention
in
musical
works);
Hb1
(Emotional
design
has
a
significant
influence
on
emotional experience in film & television works); Hb2
(Emotional experience has a significant influence on sharing
intention in film & television works); Hb3 (Emotional
design has a significant influence on sharing intention in Research objects In order to verify the evaluation matrix and the research
structure of emotional experience in audio-visual artistic works,
researchers make evaluations with two original works created
by themselves as objects, which are an anti-epidemic song and a
short film China Braves Headwind. The details of the lyrics are
shown in Table 2. Audio-visual emotional design and emotional
experience Audio-visual art needs to take the audience’s emotional
feelings as the priority. Empathy is one of the design factors
that artists take into account and only artistic works with
the ability to reflect this kind of empathy can remind the
audience of their life experience, thus realizing a sense of
identity and empathy. Arnheim (2003) proposed that people’s
psychology, including feelings, perception, and memory, could
be conceptualized as a hierarchical feedback network by which
everyone could have the same reference from a different branch
of the network. In other words, the emotional design provides a
basis for this reference, establishing emotional tone with abstract
audio-visual language, and then constructing a psychological
situation from various dimensions to achieve the goal of
emotional description. As a psychological phenomenon, the
emotional experience usually includes joy, passion, movement,
excitement, happiness, etc. An event (i.e., a stimulus) triggers
one of several stereotyped responses in the brain and body
(Wilson-Mendenhall et al., 2013). The emotions are experienced
primarily as structures of feeling which give meaning to
relational experience (Burkitt, 2014). Given that, a high-quality
design, as a stimulus, must be able to arouse the audience’s inner Frontiers in Psychology 06 frontiersin.org 10.3389/fpsyg.2022.1032808 Fang et al. film & television works); Hb-1 (Emotional experience has
an intermediate effect on emotional design and sharing
intention in film & television works); Hb-2 (Emotional
design has an impact on emotional experience and then
promote people’s sharing intention in film & television works). According to NO.2 research purpose, the research comes
up with the following presupposition: there are different
key factors affecting the emotional experience of musical
works as well as film & television works. According to NO.3
research purpose, the research comes up with the following
presupposition: Significant differences appear in the audience’s
evaluation of music, films, and television in the level of
emotional design. sharing intention can become an important reference for the
audience’s art consumption. Meanwhile, artists can activate
the audience’s unconscious and in-depth emotions by using
the orientation, methods, and strategies of creation and can
influence communication behavior and life orientation. Questionnaire design The questionnaire is divided into two parts. The first part
is the basic information of the subjects, including gender,
age, academic degree, and educational background; The
second part lists 22 questions on the evaluation matrix of
emotional experience in the audio-visual artistic works,
using the Likert scale (1 represents the lowest degree
of
agreement
and
5
represents
the
highest
degree
of
agreement). With the Likert scale, subjects are asked to
rate the two objects, the music, and the short film both
named China Braves Headwind. Questions 1 to 6 cover
categories of aesthetics, which are based on six factors:
expression
techniques,
audio-visual
language,
rhythm FIGURE 2
Research structure. Frontiers in Psychology
07
frontiersin.org 07 Frontiers in Psychology frontiersin.org Fang et al. 10.3389/fpsyg.2022.1032808 TABLE 2 The music China Braves Headwind. Mom says always remember
This year’s extraordinary winter
People of all ethnic groups
We brave headwinds together
Lyric
Don’t need to be scared of troubles
In the way of the national revival
As long as we stick as one
Success will come to all
Medical workers are already in the field
They are Nightingales of the new era
We ride the waves freely with no fear
Under the shelters of their invincible will and melody, harmonious coexistence, artistic charm, and
aesthetic value; Questions 7 to 12 cover categories of
function, which are based on six factors: artistic style,
thematic perception, national culture, the spirit of times,
implication
and
connotation,
and
thought
promoting;
Question 13 to 18 cover categories of creativity, which are
based on six factors: content conception, Scene creation,
emotion presupposition, imagination stimulation, emotional
resonance, and spiritual sublimation. Questions 19 to 21
cover categories of emotional experience which are based
on three factors: preference degree, moving degree, and
inspiring
degree;
Question
22
covers
the
category
of
sharing intention which is based on the extent people are
likely to share artistic works with others. Questionnaire
star platform is used to produce such questions and the
websites
are
https://www.wjx.cn/jq/65312477.aspx
and
https://www.wjx.cn/jq/65057119.aspx. questionnaires with the same scores for too many questions
are eliminated. Finally, 374 valid questionnaires for the
music test and 326 for the film test are collected, with
subjects 17 times more than the data. Questionnaire design In the Structural
Equation Modeling analysis, some scholars’ research show
that the demand for the number of samples is related
to the length of the research scale, and the longer the
research scale is, the more samples will be required. The
ideal number of samples is at least five times more than the
number of measurement variables, for which the number
of
samples
should
be
between
200
and
400
(Ghiselli
et al., 1981; Gorsuch, 1983; Hair et al., 1998; Wu and
Tu, 2010). Therefore, the samples taken in this research
meet the needs of the number of samples for research
and analysis. The questionnaire was subject to reliability and validity
tests,
regression
analysis
and
dependent
samples
t-test
with statistical tests using SPSS 22.0, as well as structural
equation modeling (SEM) in AMOS 22.0. Whether the
measurement scale is feasible can be confirmed by verification
factors. And then through the path diagram of SEM, the
presuppositions
can
be
verified
followed
by
multiple
regressive
analyses
to
explore
the
key
factors
affecting
the audience’s emotional experience. Finally, the research
will study the audience’s differences in emotional design Experimental design The research samples are teachers and students from
various
colleges
and
universities,
ranging
in
age
from
19 to 40 years. Among them, subjects participating in
the questionnaire about music include 90 men and 284
women,
and
subjects
participating
in
the
questionnaire
about films include 73 men and 253 women, who have
undergraduate and postgraduate degrees and have various
learning backgrounds, such as art, science and engineering,
humanities, and social science, as is shown in Table 3. This
research conforms to ethical requirements and respects
the privacy of the subjects. There is no sex discrimination,
racial discrimination, and other issues. The questionnaires
are mainly distributed online where researchers ask for the
subjects’ agreement to fulfill the questionnaires. Researchers
convene the subjects through Tencent Conference software as
an online laboratory. At first, researchers elaborate on their
purpose and the instructions for answering the questions
in detail before playing the music China Braves Headwind. After the play of music, subjects are allowed to fulfill the
first questionnaire in 10 min. Then, they have 10 min to
fulfill the second questionnaire after watching the short
film China Braves Headwind. After the questionnaires are
submitted, researchers discard samples that cannot meet
the conditions after reviewing them. The standard is that TABLE 3 Profile of the respondents. TABLE 3 Profile of the respondents. Characteristics
Levels
Film and television
works
Musical
works
Number
Number
Gender
Male
73
90
Female
253
284
Age
19–30
315
363
31–40
11
11
Education
Undergraduate
306
350
Postgraduate
20
24
Academic degree
Arts
62
78
Science and
engineering
93
121
Humanities and
social science
171
175 TABLE 3 Profile of the respondents. Characteristics
Levels
Film and television
works
Musical
works Frontiers in Psychology 08 Fang et al. 10.3389/fpsyg.2022.1032808 Convergent validity and discriminant validity Convergent validity and discriminant validity in the music and the short film, using the dependent
samples t-test. At this stage, the research uses the method of measurement
model analysis. Based on valid questionnaires on the music
China Braves Headwind, the research launches the model
construction analysis and deals with the structural relationship
between the observed variables and latent variables through
confirmatory factor analysis (CFA). And then, convergent
validity and discriminant validity in the measurement model
are tested. According to the findings through CFA, in terms
of factor loadings, the beauty dimension ranges from 0.772 Frontiers in Psychology frontiersin.org The analysis of structural model
construction TABLE 4 The research model confirmatory factor analysis table. Dimension
Items
Skewness Kurtosis
Parameter saliency estimation
Factor
loadings
Question
reliability
Composite
reliability
Convergent
validity
SK
KU
Unstd. S.E. t-value
P
Std. SMC
CR
AVE
Beauty
A1-1
−0.502
0.081
1.000
0.800
0.640
0.919
0.653
A1-2
−0.527
−0.213
0.996
0.058
17.095
***
0.798
0.637
B1-1
−0.685
0.007
1.056
0.060
17.538
***
0.814
0.663
B1-2
−0.486
−0.420
1.045
0.059
17.704
***
0.820
0.672
C1-1
−0.402
−0.426
1.193
0.065
18.397
***
0.844
0.712
C1-2
−0.348
−0.387
1.020
0.062
16.366
***
0.772
0.596
Function
A2-1
-0.737
0.087
1.000
0.683
0.466
0.904
0.612
A2-2
−1.121
1.011
0.979
0.075
13.105
***
0.747
0.558
B2-1
−0.942
0.480
0.949
0.071
13.318
***
0.761
0.579
B2-2
−0.886
0.357
1.024
0.071
14.488
***
0.838
0.702
C2-1
−0.760
0.150
1.151
0.079
14.553
***
0.843
0.711
C2-2
−0.941
0.898
1.012
0.072
14.049
***
0.808
0.653
Creativity
A3-1
−0.628
0.101
1.000
0.735
0.540
0.925
0.675
A3-2
−0.499
−0.485
1.245
0.074
16.874
***
0.866
0.750
B3-1
−0.615
−0.242
1.176
0.072
16.316
***
0.839
0.704
B3-2
−0.321
−0.668
1.252
0.079
15.856
***
0.817
0.667
C3-1
−0.863
0.342
1.208
0.073
16.486
***
0.847
0.717
C3-2
−0.679
0.079
1.115
0.070
15.902
***
0.819
0.671
Emotional
experience
D
−0.367
−0.377
1.000
0.831
0.691
0.897
0.745
F
−0.458
−0.237
1.071
0.053
20.372
***
0.922
0.850
G
−0.596
0.052
0.923
0.049
18.897
***
0.833
0.694
Emotional
design
Beauty
/
/
1.000
0.924
0.854
0.956
0.879
Function
/
/
0.896
0.062
14.548
***
0.910
0.828
Creativity
/
/
1.117
0.072
15.498
***
0.978
0.956
***p < 0.001. TABLE 5 Second-order model confirmatory factor analysis table. Second-order CFA
χ2 value
Degree of freedom (df)
χ2/df
GFI
AGFI
CFI
RMSEA
1. Model 1: First-order Three-factor
Model (There are correlates among these
factors)
397.503
132.000
3.011
0.896
0.865
0.951
0.073
2. Model 2: Second-order Factor Model
397.503
132.000
3.011
0.896
0.865
0.951
0.073
The Advised Value
The smaller, the better
The larger,the better
<5
>0.8
>0.8
>0.9
<0.08 ABLE 5 Second-order model confirmatory factor analysis table. Second-order CFA
χ2 value
Degree of freedom (df)
χ2/df
GFI
AGFI
CFI
RMSEA
1. Model 1: First-order Three-factor
Model (There are correlates among these
factors)
397.503
132.000
3.011
0.896
0.865
0.951
0.073
2. Model 2: Second-order Factor Model
397.503
132.000
3.011
0.896
0.865
0.951
0.073
The Advised Value
The smaller, the better
The larger,the better
<5
>0.8
>0.8
>0.9
<0.08 09 Frontiers in Psychology Fang et al. The overall model fitness test The structural equation model (SEM) assumed in this
research tests its multivariate normality in two aspects. One
is the normality of observed variables, and the other is the
multivariate normality after observed variables integration. Some scholars put forward that when the absolute values of the
skew coefficient and kurtosis coefficient of observed variables
are less than 2, it can be determined that it has normality
(Bollen and Long, 1993; Qiu, 2006; Yen, 2018). From Table 4,
the research can figure out that the absolute values of the skew
coefficient and kurtosis coefficient of all observed variables in
the structural equation model in the research are less than 2. Therefore, the research can safely conclude that the observed
variables have normality. What’s more, the tested multivariate
value is 42.448 > 5, which does not meet the multivariate
normality standard (Barbara, 2016). When the data of SEM
does not have multivariate normality, Bollen–Stine bootstrap
can be used to correct standard errors and the statistics of
fitness (Bollen and Stine, 1992; Enders, 2005; Fisher and King, The analysis of structural model
construction 10.3389/fpsyg.2022.1032808 to 0.844; the function dimension from 0.683 to 0.843; the
creativity from 0.735 to 0.866; emotional experience dimension
from 0.831 to 0.922; emotional design dimension from 0.910
to 0.978. All factor loadings are more than 0.5, showing that
the measurement model meets the standard, as shown in
Table 4. What’s more, the composite reliability (CR) of all
dimensions in the research ranges between 0.897 and 0.956, and
the average variance extracted (AVE) varies between 0.612 and
0.879. Both of them conform to the values 0.60 and 0.50 —
advised ones in related research (Fornell and Larcker, 1981;
Bagozzi and Yi, 1988; Hair et al., 1998). The data indicate
the internal consistency in the model is acceptable and has
convergent validity. When it comes to the verification analysis
of discriminant validity, The value of the square root of AVE
for every dimension in the research is 75% more than the
proportion of the total number of correlation coefficients for
every dimension of the total compared number. It shows there is
desirable discriminant validity among the variables (Hair et al.,
1998). In the research, the research designs and compares the first-
order three-factor model (there are correlates among these
factors) and the second-order factor model (Figure 3). The
result shows χ2(Model1)/χ2(Model2) = 1 and the target factor
of the model is 1 (Table 5). Therefore, the research uses the result
of the second-order CFA to analyze the model construction
(Lai et al., 2010). The study indicates that there are correlates
among three factors, such as beauty, function, and creativity. They are key factors in emotional design. It is reasonable to use
the second-order CFA in the research. Analysis of second-order confirmatory factor
analysis model fitness The research can decide whether to use the first-order CFA
or the second-order CFA based on the concept of the targeted
factor (Marsh and Hocevar, 1985). According to the fitness
related to the first-order model, this target factor is removed by
the fitness of the second-order model. If the result is closer to 1,
that indicates that the second-order model is more streamlined,
and the second-order model can represent the first-order model. FIGURE 3
First-order three-factor model (There are correlates among these factors) and second-order factor model. 10 10 Frontiers in Psychology frontiersin.org 10.3389/fpsyg.2022.1032808 Fang et al. path diagram of musical works structure model analysis are
shown in Figure 4 and Table 6 — Ha1: emotional design in
musical works has a notable influence on emotional experience;
Ha2: emotional experience in musical works has a notable
influence on sharing intention; Ha3: emotional design in
musical works has a notable influence on sharing intention. Therefore, the above three hypotheses are true. Results of
the path diagram of film & television works structure model
analysis are shown in Figure 5 and Table 6 — Hb1: emotional
design in film & television works has a notable influence on
emotional experience; Hb2: emotional experience in film &
television works has a notable influence on sharing intention;
Hb3: emotional design in film & television works has a notable
influence on sharing intention. Therefore, the above three
hypotheses are true. 2010). After Chi-square correction, the research tests the model
fitness. The results show that the ratio of chi-square value
to degree of freedom range from 1 to 3 (χ2/DF = 1.35),
GFI = 0.962 > 0.9, AGFI = 0.95 > 0.9, RMSEA = 0.031 < 0.08,
RMR = 0.041 < 0.08, TLI(NNFI) = 0.989 > 0.9, CFI = 0.99 > 0.9,
IFI = 0.99 > 0.9, and Hoelter’s N(CN) = 277.528 > 200. From
what has been suggested above, the overall index of this model
is acceptable (Doll et al., 1994; Baumgartner and Homburg,
1996; Hair et al., 2010; Chen and Wang, 2011). In the study,
the theoretical structure of the overall structural model is well-
matched with the empirical data and the model has desirable
construct validity. Model research hypothesis testing The research has shown that emotional design, emotional
experience, and sharing the intention of audio-visual works
are influenced by each other. The research can conclude
that the hypotheses are true. Ha-1: in the musical works, In the research, the SEM is used to test the validation of
research hypotheses. After the research and analysis, there are
six dimensions and 22 measurement variables. Results of the FIGURE 4
Path diagram of musical works structure model in the research. FIGURE 4
Path diagram of musical works structure model in the research. FIGURE 4
Path diagram of musical works structure model in the research. TABLE 6 Path coefficient of structural statistical model. Type
Path
Coefficient (Variant)
Standardized
path coefficient
t-value C.R. P
Decision
Musical works
Ha1
Emotional Design
->
Emotional Experience
0.930
17.384
***
Supported
Ha2
Emotional Experience
->
Sharing Intention
0.150
3.342
***
Supported
Ha3
Emotional Design
->
Sharing Intention
0.679
17.384
***
Supported
Film & Television works
Hb1
Emotional Design
->
Emotional Experience
0.875
14.299
***
Supported
Hb2
Emotional Experience
->
Sharing Intention
0.187
3.897
***
Supported
Hb3
Emotional Design
->
Sharing Intention
0.630
14.299
***
Supported
***p < 0.001. TABLE 6 Path coefficient of structural statistical model. 11 Frontiers in Psychology Fang et al. 10.3389/fpsyg.2022.1032808 FIGURE 5
Path diagram of film & television works structure model in the research. FIGURE 5
Path diagram of film & television works structure model in the research. prove the hypothesis Ha-1: in the musical works, emotional
experience plays a mediating role between emotional design
and sharing intention. From the standardized path coefficient
in the model, the research can know that the influence path
coefficient of emotional design on sharing intention (direct
effect) is 0.68; the path coefficient of emotional design on sharing
intention (indirect effect) is 0.140 (0.93∗0.15); the total effects
are 0.93∗0.15 + 0.68 = 0.82 > 0.68 (total effects > direct effect). All data can prove hypothesis Ha-2: in the musical works,
the emotional design will impact emotional experience, further
improving sharing intention. emotional experience plays a mediating role between emotional
design and sharing intention; Ha-2: in the musical works, the
emotional design will impact emotional experience, further
improving sharing intention. Hb-1: in the film & television
works, emotional experience acts as an intermediary between
emotional design and sharing intention; Hb-2: in the film &
television works, the emotional design will impact emotional
experience, further improving sharing intention. Model research hypothesis testing To test the indirect effect of emotional design on sharing
intention and the mediating effect of emotional experience in
musical works, the research uses the Sobel-Goodman tests. The results show that the Z value is 3.264, more than the
standard value of 1.96, indicating the mediating effect is
remarkable (Sobel, 1982, 1986). And then, The research uses
the Bootstrap techniques for error estimation to continue the
hypotheses test and re-estimate the confidence level, standard
error, standardized coefficient, and significant level (Z value)
of indirect effect (MacKinnon et al., 2007; MacKinnon, 2008;
Taylor et al., 2008). The 95% confidence interval of the indirect
effect path of emotional design on sharing intention does
not contain zero. Its Bias-corrected (0.115, 0.337), percentile
(0.110, 0.333), and p < 0.001 indicate the significance and the
direct effect is not zero; the Z value of the indirect effect is
3.911, more than 1.96 and the results show the significance
and there is the mediating effect, as shown in Table 7. In
addition, the Z value of the direct effect is 16.400, more than
1.96, and the results show significance, indicating that the
emotional design does have a direct effect on sharing intention
(Table 7). Therefore, all results show there is a mediating
effect and it plays the role of partial mediation, and they can To test the indirect effect of emotional design on sharing
intention and the mediating effect of emotional experience in
film & television works, the research uses the Sobel-Goodman
tests. The results show that the Z value is 3.7332, more than
the standard value of 1.96, indicating the mediating effect is
remarkable. And then, The research uses Bootstrap techniques
for error estimation to continue the hypotheses test and re-
estimate the confidence level, standard error, standardized
coefficient, and significant level (Z value) of indirect effect. The
95% confidence interval of the indirect effect path of emotional
design on sharing intention does not contain zero. Its Bias-
corrected (0.102, 0.421), percentile (0.100, 0.420), and p < 0.001
indicate the significance and the direct effect is not zero; the
Z value of the indirect effect is 3.229, more than 1.96 and
the results show the significance and there is the mediating
effect, as shown in Table 7. Frontiers in Psychology Model research hypothesis testing In addition, the Z value of the
direct effect is 12.165, more than 1.96, and the results show
significance, indicating that the emotional design does have a
direct effect on sharing intention. Therefore, all results show Frontiers in Psychology 12 frontiersin.org Fang et al. 10.3389/fpsyg.2022.1032808 rdized indirect, direct, and total mediating effects in the musical works structural model. Variants
Point estimation
value
Product of coefficients Bias-corrected 95% CI
Percentile 95% CI
Two-tailed
significance
SE
Z
Lower
Upper
Lower
Upper
Musical works
Standardized total effects
Emotional design->
Sharing intention
1.285
0.067
19.179
1.169
1.428
1.165
1.426
0.000***
Standardized indirect effect
Emotional design->
Sharing intention
0.219
0.056
3.911
0.115
0.337
0.110
0.333
0.000***
Standardized direct effect
Emotional design->
Sharing intention
1.066
0.065
16.400
0.948
1.201
0.950
1.202
0.000***
Film and television works
Standardized total effects
Emotional design->
Sharing intention
1.302
0.090
14.467
1.131
1.502
1.130
1.501
0.000***
Standardized indirect effect
Emotional design->
Sharing intention
0.268
0.083
3.229
0.102
0.421
0.100
0.420
0.000***
Standardized direct effect
Emotional design->
Sharing intention
1.034
0.085
12.165
0.869
1.201
0.878
1.211
0.000***
***p < 0.001. indicators and predictive variables of emotional design in the
works. Through these efforts, the research will carry on the
multivariate regression analysis on the factors affecting the
audience’s emotional experience and further explore the impacts
of nine factors on emotional experience. there is a mediating effect and it plays the role of partial
mediation, and they can prove the hypothesis Hb-1: in the
film & television works, emotional experience plays a mediating
role between emotional design and sharing intention. From the
standardized path coefficient in the model, the research can
know that the influence path coefficient of emotional design on
sharing intention (direct effect) is 0.63; the path coefficient of
emotional design on sharing intention (indirect effect) is 0.167
(0.88∗0.19); the total effects are 0.88∗0.19 + 0.63 = 0.797 > 0.63
(total effects > direct effect). All data can prove hypothesis Hb-2:
in the film & television works, the emotional design will impact
emotional experience, further improving sharing intention. As is shown in Table 8, from all predictive variables
and the correlation between the audience and the intensity
of emotional experience in the musical works, the research
can know nine predictive variables and their correlation
coefficients are 0.703, 0.740, 0.737, 0.720, 0.720, 0.602, 0.650,
0.721, 0.766, 0.820, meeting the significant level 0.001. Model research hypothesis testing From
the multivariate regression analysis in Table 8, the research
can find that the correlation coefficient R between overall
prediction variables and dependent variables is 0.869. And
the explanatory variance of nine predictive variables on the
intensity of the emotional experience is 75.6% and the F
value is 73.291, meeting the significant level, 0.000. According
to the results, there are significant correlations between the
nine attributes and the intensity of emotional experience,
and these nine attributes have a considerable degree of joint
explanatory power for the intensity of the emotional experience. Among these attributes, the more prominent ones in order
are A1 (Expression techniques, Audio-visual language), B1
(Rhythm and melody, Harmonious coexistence), A2 (Artistic
style, Thematic perception), B2 (National culture, Spirit of
times), B3 (Emotion presupposition, Imagination stimulation),
and C3 (Emotional resonance, Spiritual sublimation). All Frontiers in Psychology Analysis of difference in perception of
emotional design these attributes represent key factors affecting the intensity of
emotional experience in musical works. these attributes represent key factors affecting the intensity of
emotional experience in musical works. From all predictive variables and the correlation between the
audience and the intensity of emotional experience in the film &
television works, the research can know nine predictive variables
and their correlation coefficients are 0.613, 0.687, 0.643, 0.664,
0.522, 0.608, 0.721, 0.711, 0.743, meeting the significant level,
0.001. From the multivariate regression analysis in Table 8,
the research can find that the correlation coefficient R between
overall prediction variables and dependent variables is 0.822. And the explanatory variance of nine predictive variables on
the intensity of “emotional experience” is 67.6% and the F
value is 73.291, meeting the significant level, 0.000. According
to the results, there are significant correlations between the
nine attributes and the intensity of emotional experience,
and these nine attributes have a considerable degree of joint
explanatory power for the intensity of the emotional experience. Among these attributes, the more prominent ones in order
are A2 (Artistic style, Thematic perception), A3 (Content
conception, Scene creation), and C3 (Emotional resonance,
Spiritual sublimation). All these attributes represent key factors
affecting the intensity of emotional experience in film &
television works. At this stage, to explore the audience’s perception of
evaluation attributes in different types of works with the same
theme, the research employed the dependent sample t-test to
test the evaluation factors of musical works and film & television
works. The analysis is shown in Table 9. Among nine evaluation
factors, significant differences between musical works and film &
television works are seen in attribute C1, attribute C2, affection
level, touch level, inspiration level, and sharing intention. The
comparative analysis of the average score of these evaluation
factors shows that the average score of the film & television
works is higher than that of the musical works. The analysis of key factors affecting
emotional experience with mediating
effect All the above research has proven that the emotional
design both in musical works and film & television works
has a significant effect on the audience’s emotional experience. And, emotional experience with mediating effect serves as the
emotional bond between the artists’ creation and the audience’s
perceptions and their sharing intention. At this stage, the
research will take the audience’s emotional experience as a
dependent variable and nine evaluation factors listed in the
Emotional Design Evaluation Matrix of Audio-visual Art as 13 Frontiers in Psychology frontiersin.org Fang et al. 10.3389/fpsyg.2022.1032808 TABLE 8 The regression analysis of emotional experience. Type
Dependent
variables
Predictive
variables
Simple
correlation
Regression
coefficient
Standardized
T value
Significance
level
regression
coefficient
Musical works
The Intensity of
“Emotional
Experience”
(N = 374)
A1
0.703***
0.117
0.106
2.432*
0.015
B1
0.740***
0.159
0.147
3.015**
0.003
C1
0.737***
0.063
0.062
1.236
0.217
A2
0.720***
0.130
0.116
2.577*
0.010
B2
0.602***
–0.116
−0.093
−2.013*
0.045
C2
0.650***
0.049
0.042
0.899
0.369
A3
0.721***
0.053
0.050
1.003
0.316
B3
0.766***
0.128
0.129
2.548*
0.011
C3
0.820***
0.425
0.410
8.240***
0.000
Invariable
–0.237
R = 0.869
Rsq = 0.756
F = 125.175
SigmfF = 0.000
Film and Television
works
The Intensity of
“Emotional
Experience”
(N = 326)
A1
0.613***
0.073
0.068
1.341
0.181
B1
0.687***
0.113
0.108
1.899
0.058
C1
0.643***
0.016
0.017
0.312
0.755
A2
0.664***
0.153
0.119
2.253*
0.025
B2
0.522***
–0.071
−0.056
−1.169
0.243
C2
0.608***
0.066
0.060
1.141
0.255
A3
0.721***
0.208
0.199
3.308**
0.001
B3
0.711***
0.114
0.110
1.837
0.067
C3
0.743***
0.350
0.325
6.102***
0.000
Invariable
–0.220
R = 0.822
Rsq = 0.676
F = 73.291
SigmfF = 0.000
*p < 0.05, **p < 0.01, ***p < 0.001. *p < 0.05, **p < 0.01, ***p < 0.001. Analysis of difference in perception of
emotional design Number
M
SD
t value
Significance level
Difference comparison in aesthetic experience, perceived autonomy, and relatedness
(Koehler and Neubauer, 2020; Gotthardt et al., 2022). As
two kinds of creative activities, musical and film & television
works have their unique text processing modes. Barthes (2005)
puts forward that artistic works include extension information
and connotation information. The emotional design hides in
connotation information through coding, which requires the
audience to interpret it imaginatively. This process promotes
emotion to occur. Kant (2002) believes the appreciation
judgment is perceptual judgment. Through imagination, the
audience will produce pleasant or unpleasant emotional
connections with the subject of art. The emotional design in
artistic works is the prerequisite for the audience to perceive
and judge. Musical works create auditory sensual pleasure
through musical notes, while film & television works stimulate
emotional experience through the combination of pictures
and sound. Therefore, the purpose and method of emotional
design influence the audience’s experience of artistic works. Furthermore, the audience’s emotional experiences including
such emotions as love, pleasure, satisfaction, excitement, and
moving from the audio-visual works will positively drive their
behavior and then impact how the works are communicated
(Wieck et al., 2022). Sharing and communicating online during
the lockdown promoted person-to-person virtual interaction
which contributed to potentially social contact and cross-
cultural communication (Fraser et al., 2021). After getting the Frontiers in Psychology Analysis of difference in perception of
emotional design Specifically,
there is a notable difference in the two types in attribute A1
and attribute B1 (P < 0.05), with the significance level of
0.012 and 0.028, respectively; there is a notable difference in
the two types in attribute B2, attribute A3 and attribute B3
(P < 0.01), with the significance level of 0.003, 0.005, and
0.006; there is a well-significant difference in in the two types
in attribute A2, attribute C3, element D, element F, element
G, and element H (P < 0.001), with the significance level
of 0.000. Frontiers in Psychology 14 frontiersin.org Fang et al. 10.3389/fpsyg.2022.1032808 TABLE 9 The table of analysis of differences in testers’ perception of emotional design. TABLE 9 The table of analysis of differences in testers perception of emotional design. Items
Type
Number
M
SD
t value
Significance level
Difference comparison
A1
Music
F & T
374
326
3.930
4.075
0.7890
0.7123
−2.531*
0.012
Music <
F & T
B1
Music
F & T
374
326
4.003
4.132
0.8080
0.7345
−2.202*
0.028
Music <
F & T
C1
Music
F & T
374
326
3.822
3.914
0.8585
0.7925
−1.464
0.144
Music <
F & T
A2
Music
F & T
374
326
4.186
4.416
0.7703
0.5947
−4.446***
0.000
Music <
F & T
B2
Music
F & T
374
326
4.326
4.474
0.6951
0.5962
−3.027**
0.003
Music <
F & T
C2
Music
F & T
374
326
4.218
4.271
0.7476
0.6911
−0.979
0.328
Music <
F & T
A3
Music
F & T
374
326
3.957
4.124
0.8265
0.7279
−2.818**
0.005
Music <
F & T
B3
Music
F & T
374
326
3.890
4.058
0.8779
0.7388
−2.747**
0.006
Music <
F & T
C3
Music
F & T
374
326
4.068
4.288
0.8389
0.7101
−3.718***
0.000
Music <
F & T
D: Affection level
Music
F & T
374
326
3.626
3.975
0.9927
0.8482
−5.027***
0.000
Music <
F & T
F: Touch level
Music
F & T
374
326
3.786
4.058
0.9588
0.8663
−3.945***
0.000
Music <
F & T
G: Inspiration level
Music
F & T
374
326
3.939
4.169
0.9140
0.7878
−3.579***
0.000
Music <
F & T
H: Sharing intention
Music
F & T
374
326
3.703
3.975
1.0837
0.9279
−3.581***
0.000
Music <
F & T
F & T refers to the film & television works, *p < 0.05, **p < 0.01, ***p < 0.001. Discussion The model construction analysis in the research shows that
the construct validity is desirable, the scales of the research are
feasible, and the Emotional Design Evaluation Matrix of Audio-
visual Art is reasonable. The research explained the emotional
design and demonstrated the way of coding by artists from
three dimensions, such as beauty, function, and creativity. There
are nine evaluation factors in three dimensions, measuring the
effects and experience of these dimensions from the levels of
techniques, semantics, and effects. Through these efforts, the
research has found that the emotional design will attract the
audience, help them perceive the creation, and touch them. According to the structural equation model survey, the
emotional design of musical and film & television works will
have an impact on the audience’s sharing intention. Specifically,
the emotional design has a significant positive effect on the
emotional experience; the emotional experience has a significant
positive effect on the sharing intention; the emotional design
has a significant positive effect on the sharing intention; the
emotional experience serves as a partial mediator between
emotional design and sharing intention. The emotional design
of both musical and film & television works has a significant
effect on the audience’s emotional experience, further improving
their sharing intention. It has been suggested digital cultural
offerings increased optimism indirectly through an increase 15 frontiersin.org 10.3389/fpsyg.2022.1032808 Fang et al. development from the COVID-19 pandemic (Niels et al., 2022). Therefore, this attribute serves as the most important factor in
evaluating the emotional experience in audio-visual works. emotional experience, the audience will connect works with
their values and judgments so that the way of sharing and
communication will be decided by the perception and feeling
of the audience. Through the dependent sample t-test, the research has
found that there is a significant difference in seven attributes,
emotional experience, and sharing intention among the
audience in the musical and film & television works with the
same subject. And the average score of film & television works
is higher than that of musical works. Therefore, compared with
musical works, film & television works are likely to resonate
with the audience. Although there are differences in artistic
forms between the two types of art, both of them boasts
endless artistic attraction and aesthetic value. Discussion The short film
China Braves Headwind is good at combining pictures and
sounds, creating a virtual-and-real situation and narrative text,
enhancing the interest of the audience, and making them enter
a broad space for imagination. Especially, visual representation,
with which meaning is constructed through the structuring of
these elements (Cano-Martínez et al., 2022), is a recreation
of social, cultural, and ideological mediation (Desai, 2000;
Knochel, 2013). The music China Braves Headwind is more like
the art of poetry. The image it outlines requires the audience
to have a higher aesthetic taste to mobilize the emotional
experience of life. Breaking the cultural barrier, the combination
of music, lyrics, and videos amid the epidemic played a critical
role in maintaining relationships and promoting emotional
communication (Lydia, 2022). As a result, the emotional
resonance relieved the sorrow and bitterness of individuals and
society. Therefore, film & television works with high-quality
musical works can help open up the conception of artistic works
and convey their meanings to viewers, further promoting their
dissemination. As Eco (2005) believes, the artistic works by
artists remain to be finished, which will be done by recipients
through their deductive dialogs. Film & television works are
more open than musical works. Their multivariate structures
mobilize the sense organs of the audience. Therefore, their
awakening of self-consciousness and their interpretation after
art decoding serve as important factors in art communication. The research, through multivariate regression analysis,
explores the key factors impacting the emotional experience in
musical works and film & television works. The results indicate
key factors affecting the emotional experience in musical works
represent the level of techniques (A1, A2) and the level of
semantics (B1, B2, B3) in the Emotional Design Evaluation
Matrix of Audio-visual Art. And key factors affecting the
emotional experience in film & television works represent
the level of techniques (A2, A3) only. Therefore, attribute
A2 and attribute C3 serve as common factors affecting the
emotional experience in both types of art. The music China
Braves Headwind uses exquisite artistic expression techniques in
composition, arrangement, and singing, and has a rich melody,
reasonable tune, vivid singing, and graceful harmony. The style
of the music is infectious and the main idea of anti-epidemic
conveyed is clear. From what has been suggested above,
musicians must take the audience’s physical and psychological
feelings into account during the COVID-19 pandemic. Only
in this way, can the audience resonate with works. Discussion To present
the emotional context in works, apart from the attributes
in the level of techniques, creators must convey profound
meanings to consumers in ways that resonate with them. During
the pandemic, emotional resonance brought by music had a
positive effect on the sense of happiness (Cabedo-Mas et al.,
2021). As Langer (1986) said, music represents the highest
level of reflection of life, that is, the symbolic expression of
human emotional activities. In the short film China Braves
Headwind, the application of various techniques like scene
settings, plot arrangements, and styles produces a kind of
fictional experience. They will touch the audience and make
them emotional through the power of visual sense. The reason
why attribute A2 (Artistic style, Thematic perception) serves as
a key factor affecting the emotional experience in both musical
works and film & television works is that it demonstrates
works’ features from the outside to the inside. The artistic
style reflects the multivariate artistic phenomenon and abstract
symbols represent the connotations of works, which meets
the viewers’ inner needs and further produces the empathetic
effect. As for themes in works, only a clear intention can
convey the creators’ purpose and significance. Therefore, only
when valid information both in musical works and film &
television works is conveyed, will the audience get the emotional
experience. C3 (Emotional resonance, Spiritual sublimation)
at the level of effects will reach deep into the soul of the
audience. The attribute shows that musical works and film &
television works deeply probe into the essence of life, which
gives viewers spiritual support and gives birth to some emotion. In this case, the emotional experience of social belonging,
empathy, and kindness have been described as critical factors for
b
tin
i l
h i n
nd f
t rin r ili n
r
r
nd Frontiers in Psychology Conclusion As the mediums and resources of communication update
at a high speed, the audience has transformed from passive
recipients to experiencers and even participants and producers
that will select medium consumption actively and their demands
for audio-visual works are improving. So it is of great
significance to meet individuals’ emotional needs during the
pandemic. Based on the emotional communication mode
of audio-visual art theoretically, the research explores the
emotional design factors in the communication of audio-visual
art and the audience’s perceptive experience. The results of the
research can offer some useful suggestions for the long-term 16 frontiersin.org Fang et al. 10.3389/fpsyg.2022.1032808 Funding The practical significances of the research are as follows:
first, in terms of the design of audio-visual arts, having a
deep understanding of the audience’s psychological perception
and emotional engagement with the art will help the artists
think about how to create works innovatively and meet the
marketization needs. Second, focusing on the needs of the
audience’s artistic perception is the prerequisite for forming
an aesthetic economic climate. The endeavor will improve the
cultural images of the audio-visual art industry, make it more
competitive, and stimulate new cultural consumption demands
during the COVID-19 pandemic. This study was supported by the education reform project
of Shanghai Dianji University, Research on the Teaching
Mode of Cultural and Creative Design Courses based on
“Taoist, Implements, Transformation and Practice” in the
Context of New Liberal Arts (Project No. G2-20-7201-003-
05-050-20). Limitations and prospects However, there are still some deficiencies in this study. First,
as almost every area was hit by COVID-19 for a long time, the
impact on testers of different ages varies greatly. Therefore, it
should be discussed further evaluation factors constructed in the
research and the difference in the perception of artistic works. Second, the research has not analyzed deeply the current audio-
visual arts sharing and communication path. Follow-up research
can be based on this study to explore the impact of audience
participation behavior-related factors, and initiate an in-depth
discussion on the culture, market, brand strategy, and other
aspects of audio-visual art. 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. 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. Author contributions W-TF identified research ideas, designed and facilitated
this
research,
wrote
the
draft,
and
made
substantial
revisions to this work. J-HS collected the data. Q-DL
contributed to the conception and design of the study. All authors contributed to the article and approved the
submitted version. Ethics statement development of audio-visual art. The conclusions are as follows:
(1) The emotional design evaluation matrix based on the
emotional communication model is reasonable, and the scales
of this research are feasible. The measurement tools in the
research integrate emotional factors into the creation of audio-
visual works and further analyze the audience’s emotional
experience and sharing intention. (2) The emotional design
of audio-visual works can significantly affect the audience’s
emotional experience and further improve sharing intention. As
a result, the emotional experience is essential to the connection
between audio-visual works, artists, and the audience. Only
when works stimulate viewers to produce special emotions,
can their added value beyond their forms be seen and spread
widely. (3) Attribute A2 (artistic style, thematic perception)
and attribute C3 (emotional resonance, spiritual sublimation)
serve as common factors affecting the emotional experience in
terms of both musical works and film & television works. (4)
Compared with musical works, film & television works are likely
to resonate with the audience. The combination of music and
visual sensation can help open up the conception of artistic
works and convey their meanings to viewers. The
studies
involving
human
participants
were
reviewed
and
approved
by
Shanghai
Dianji
University. The patients/participants provided their written informed
consent to participate in this study. Frontiers in Psychology References Aristotle (1989). On Poetry and Style. Indianapolis: Hackett Publishing
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included
in
the
article/Supplementary material,
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Light-Induced Stomatal Opening Is Affected by the Guard Cell Protein Kinase APK1b
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PloS one
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Introduction The activity of several protein kinases has been shown to be
important in mediating stomatal responses to environmental
stimuli, including light intensity, pathogens, atmospheric carbon
dioxide concentration and the drought hormone abscisic acid
(ABA). Well-characterised Arabidopsis guard cell kinases include
HIGH LEAF TEMPERATURE (HT1), OPEN STOMATA 1
(OST1), phototropins PHOT1 PHOT2, PKS5, BLUE LIGHT
SIGNALLING 1 (BLUS1) and calcium-dependent protein kinases
CPK 3, 6 21 and 23, which are required for stomatal closure
responses to atmospheric CO2 concentration [1,2] and abscisic
acid (ABA) [3,4,5] and for the stomatal opening response to blue
light [6,7,8]. Additionally a lectin type receptor kinase (LecRK-
V.5) is important for control of stomatal aperture in response to
pathogens [9]. In
this
study
we
demonstrate
that
APK1b
is
expressed
predominantly in guard cells. We report experiments with two
Arabidopsis T-DNA insertion lines lacking the APK1b gene
transcript, which indicate that this protein kinase is required for
full light-induced stomatal opening. The Arabidopsis genes Arabidopsis protein kinase 1a (APK1a;
AT1g07570) and APK1b (At2g28930) encode protein kinases with
peptide sequences that are 90% identical. The encoded proteins
phosphorylate tyrosine, serine and threonine residues in vitro [10]
but the nature of their in vivo substrate(s) have not been
investigated. Beyond their
kinase activity their
function
is
unknown, although transcriptomics experiments suggest that
APK1a and APK1b are predominantly expressed in guard cells
[11] (Figure S1). Homology searching suggests that these genes
are members of the receptor-like cytoplasmic kinase VII subfamily
[12]. The role of this subfamily of kinases has not previously been
investigated in stomatal responses. The best characterised member
of the VII subfamily is the Brassica, M locus protein kinase Abstract * E-mail: j.e.gray@sheffield.ac.uk * E-mail: j.e.gray@sheffield.ac.uk (MLPK) which acts in cells of the stigma, downstream of the S
receptor kinase to mediate rejection of self-incompatible pollen
[13]. MLPK has sequence similarities with receptor-like kinases
but like APK1a and APK1b, it has no apparent signal sequence or
transmembrane domain and is believed to attach to the plasma
membrane via N-myristoylation [12]. There are 46 members of
the kinase VII subfamily encoded in the Arabidopsis genome. APK1a and APK1b have closest homology to Botrytis Induced Kinase
1 (BIK1), lack of which results in severe susceptibility to
necrotrophic fungal pathogens but does not impair responses to
the bacterial pathogen Pseudomonas syringae [14]. Nagat S. Elhaddad, Lee Hunt, Jennifer Sloan, Julie E. Gray* Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom Abstract Guard cells allow land plants to survive under restricted or fluctuating water availability. They control the exchange of gases
between the external environment and the interior of the plant by regulating the aperture of stomatal pores in response to
environmental stimuli such as light intensity, and are important regulators of plant productivity. Their turgor driven
movements are under the control of a signalling network that is not yet fully characterised. A reporter gene fusion
confirmed that the Arabidopsis APK1b protein kinase gene is predominantly expressed in guard cells. Infrared gas analysis
and stomatal aperture measurements indicated that plants lacking APK1b are impaired in their ability to open their stomata
on exposure to light, but retain the ability to adjust their stomatal apertures in response to darkness, abscisic acid or lack of
carbon dioxide. Stomatal opening was not specifically impaired in response to either red or blue light as both of these
stimuli caused some increase in stomatal conductance. Consistent with the reduction in maximum stomatal conductance,
the relative water content of plants lacking APK1b was significantly increased under both well-watered and drought
conditions. We conclude that APK1b is required for full stomatal opening in the light but is not required for stomatal
closure. Citation: Elhaddad NS, Hunt L, Sloan J, Gray JE (2014) Light-Induced Stomatal Opening Is Affected by the Guard Cell Protein Kinase APK1b. PLoS ONE 9(5):
e97161. doi:10.1371/journal.pone.0097161 Editor: Carl Ng, University College Dublin, Ireland Received January 3, 2014; Accepted April 15, 2014; Published May 14, 2014 Received January 3, 2014; Accepted April 15, 2014; Published May 14, 2014 Copyright: 2014 Elhaddad 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. Copyright: 2014 Elhaddad et al. This is an open-access article distributed under the terms of the Creative Comm
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a studentship from the Libyan government and Biotechnology and Biological Sciences Research Council grant BB/I002154/1. 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. Competing Interests: The authors have declared that no competing interests exist. Results APK1b Promoter Directs Expression to Guard Cells
To determine the cellular expression pattern, PAPK1b: GUS-GFP
seedlings were histochemically stained. GUS expression was
strongly localized to guard cells (Figure 1). GUS staining was
particularly apparent in mature stomata of leaves with a lesser
amount
of
GFP
fluorescence
detectable
in
some
stomatal
precursor cells (meristemoids or guard mother cells Figures
S2A–C). Expression of PAPK1b: GUS-GFP was also associated with
the mature cauline leaves and sepals (Figure S2). May 2014 | Volume 9 | Issue 5 | e97161 PLOS ONE | www.plosone.org 1 Guard Cell Kinase APK1b k1b
nes, apk1b-1
st exon and
CR used to
nt in either
A) was used
n leaves of
investigate
aken under
nutes, then
onductance
plants had
experiment
differences
appeared to
h genotypes
n darkness,
atal closure. plants were
easing light
nishing with
creased by
the guard
cted by the
dlings. Scale
Figure 2. Stomatal conductances of apk1b-1 are lower th
control plants. Mean stomatal conductances of apk1b-1 plant lea
compared to Col-0 control leaves. A: Leaves of plants exposed
saturating light for 30 min, then exposed to darkness, arrow indica
when light was switched off. B: Leaves exposed to increasing li
intensities ranging from 0 to 2000 mmol photon m22 s21. C: Leaves Figure 1. APK1b promoter direct GUS expression to the guard
cells. GUS histochemical localisation of expression directed by the
APK1b upstream gene promoter in leaves of 3 week old seedlings. Scale
bar = 50 mm. doi:10.1371/journal.pone.0097161.g001 Figure 2. Stomatal conductances of apk1b-1 are lower than
control plants. Mean stomatal conductances of apk1b-1 plant leaves
compared to Col-0 control leaves. A: Leaves of plants exposed to
saturating light for 30 min, then exposed to darkness, arrow indicates
when light was switched off. B: Leaves exposed to increasing light
intensities ranging from 0 to 2000 mmol photon m22 s21. C: Leaves of
plants exposed to red light for 60 min, then exposed to blue and red
light, arrow indicates when blue light was switched on. Error bars
represent the standard error. Values were statistically tested using
unpaired t-tests and significant differences from Col-0 control are
indicated (* = p,0.05, ** = p,0.01). doi:10.1371/journal.pone.0097161.g002 Figure 1. APK1b promoter direct GUS expression to the guard
cells. GUS histochemical localisation of expression directed by the
APK1b upstream gene promoter in leaves of 3 week old seedlings. Scale
bar = 50 mm. doi:10.1371/journal.pone.0097161.g001 The Effect of Changes in Light Intensity on apk1b
Stomatal Conductance Two independent Arabidopsis T-DNA insertion lines, apk1b-1
and apk1b-2, that disrupt the APK1b gene within the first exon and
the first intron respectively, were identified and RT-PCR used to
confirm that the wild-type gene transcript was not present in either
plant genotype (Figure S3). Infrared gas analysis (IRGA) was used
to investigate alterations in stomatal conductance in leaves of
apk1b-1 plants in response to light and dark stimuli. To investigate
the effect of darkness on apk1b-1, IRGA readings were taken under
saturating light (1500 mmol photon m22 s21) for 30 minutes, then
the light source was turned off and stomatal conductance
measured every five minutes in darkness. Mutant plants had
lower mean conductances than wild-type plants in this experiment
following exposure to saturating light but none of the differences
observed were significant (Figure 2A). Conductances appeared to
reduce at a similar rate in the absence of light and both genotypes
had reached minimum conductances after 30 minutes in darkness,
suggesting that the lack of APK1b does not affect stomatal closure. Figure 2. Stomatal conductances of apk1b-1 are lower than
control plants. Mean stomatal conductances of apk1b-1 plant leaves
compared to Col-0 control leaves. A: Leaves of plants exposed to
saturating light for 30 min, then exposed to darkness, arrow indicates
when light was switched off. B: Leaves exposed to increasing light
intensities ranging from 0 to 2000 mmol photon m22 s21. C: Leaves of
plants exposed to red light for 60 min, then exposed to blue and red
light, arrow indicates when blue light was switched on. Error bars
represent the standard error. Values were statistically tested using
unpaired t-tests and significant differences from Col-0 control are
indicated (* = p,0.05, ** = p,0.01). doi:10.1371/journal.pone.0097161.g002 To investigate the effect of changing light intensity, plants were
taken during the photoperiod and exposed to increasing light
intensities in the IRGA chamber starting with 0 and finishing with
2000 mmol photon m22 s21
. Light intensity was increased by
50 mmol photon m22 s21 every 5 minutes. At the start of the
experiment, in darkness, the difference between apk1b-1 mutant
and control mean stomatal conductances was not significant. Both
increased at higher light intensities but mean apk1b-1 conductance
remained lower than controls throughout the experiment. The
differences in conductance observed were significant between 50
and 2000 mmol photon m22 s21 (Figure 2B). The Effect of Changes in Light Intensity on apk1b
Stomatal Conductance Thus apk1b-1 leaf
stomatal conductance was lower than control plants over a range
of light intensities but was not significantly different in dark-
adapted plants, suggesting that the stomata of apk1b-1 may be
impaired in their ability to open in response to increasing light, but
not in their ability to close in the dark. Both photosynthetic rate
and intracellular CO2 were recorded in the IRGA chamber at the
same time as stomatal conductance but neither were significantly
different at any time point except for the response to 500 mmol
photon m22 s21 light (Figure S4) which was lower in apk1b-1
than controls. stabilised, blue light was also applied. apk1b-1 mean stomatal
conductance at the start of the experiment (in the dark) was similar
to controls but after thirty minutes exposure to red light, was
consistently lower than controls, in line with the results presented
above (in Figure 2A and B). However, as the difference in
conductance
between
mutant
and
control
plants
was
not
significant at any time point, and apk1b-1 stomatal conductances
increased in response to both red light and blue light, the stomatal
opening response to neither of these light wavelengths appeared to
be specifically impaired. b Mutants have Normal Stomatal Developmen Stomata of plants lacking APK1b cannot open as
wide in response to light but can close in response to ABA or
CO2. The effect of A: light, B: ABA and C: CO2 on stomatal aperture
measurements from apk1b-1, apk1b-2 and control plants. Apertures
were measured from detached leaf abaxial peels following treatment
with stimuli as indicated. For the response to light shown in A. plants
were dark-adapted and peels prepared in dim light at the start of the
experiment. Significant differences from Col-0 control are indicated
(* = p,0.05). doi:10.1371/journal.pone.0097161.g004 Figure 4. Stomata of plants lacking APK1b cannot open as
wide in response to light but can close in response to ABA or
CO2. The effect of A: light, B: ABA and C: CO2 on stomatal aperture
measurements from apk1b-1, apk1b-2 and control plants. Apertures
were measured from detached leaf abaxial peels following treatment
with stimuli as indicated. For the response to light shown in A. plants
were dark-adapted and peels prepared in dim light at the start of the
experiment. Significant differences from Col-0 control are indicated
(* = p,0.05). doi:10.1371/journal.pone.0097161.g004 there were no significant differences between genotypes (Figure 3). Nor were there any obvious differences in stomatal size or
appearance. Mean stomatal aperture lengths of apk1b-1, apk1b-2
and Col-0 were measured from epidermal peels and found to be
4.64, 4.84 and 4.77 mm respectively. These results suggest that the
altered stomatal conductances described above may be due to
defective stomatal aperture control in response to light, rather than
defective stomatal development. there were no significant differences between genotypes (Figure 3). Nor were there any obvious differences in stomatal size or
appearance. Mean stomatal aperture lengths of apk1b-1, apk1b-2
and Col-0 were measured from epidermal peels and found to be
4.64, 4.84 and 4.77 mm respectively. These results suggest that the
altered stomatal conductances described above may be due to
defective stomatal aperture control in response to light, rather than
defective stomatal development. The Effect of Light, ABA and CO2 on Stomatal Apertures
To determine whether reduced stomatal conductance could be
due
to
an
alteration
in
stomatal
aperture
control,
direct
measurement of apk1b-1 and apk1b-2 stomatal apertures was
carried out following treatment of epidermal peels with differing
environmental stimuli. Aperture changes in response to light, ABA
and CO2 treatments were measured by microscopy. b Mutants have Normal Stomatal Developmen As stomata open in response to both blue and red light signals,
we carried out IRGA to investigate whether either of these
responses was specifically affected by the lack of APK1b. In the
experiment shown in Figure 2C leaves were initially exposed to
red light then after an hour, when stomatal conductances had Epidermal cell counts were carried out to investigate whether
apk1b-1 leaves could display lower stomatal conductance as a result
of reduced stomatal number. However, the stomatal index and
density were similar for wild-type, apk1b-1 and apk1b-2 plants and May 2014 | Volume 9 | Issue 5 | e97161 PLOS ONE | www.plosone.org 2 Guard Cell Kinase APK1b there were no significant differences between genotypes (Figure 3). Nor were there any obvious differences in stomatal size or
appearance. Mean stomatal aperture lengths of apk1b-1, apk1b-2
and Col-0 were measured from epidermal peels and found to be
4.64, 4.84 and 4.77 mm respectively. These results suggest that the
altered stomatal conductances described above may be due to
defective stomatal aperture control in response to light, rather than
defective stomatal development. The Effect of Light, ABA and CO2 on Stomatal Apertures
To determine whether reduced stomatal conductance could be
due
to
an
alteration
in
stomatal
aperture
control,
direct
measurement of apk1b-1 and apk1b-2 stomatal apertures was
carried out following treatment of epidermal peels with differing
environmental stimuli. Aperture changes in response to light, ABA
and CO2 treatments were measured by microscopy. To investigate
stomatal opening in response to light, plants were kept in the dark
overnight; epidermal peels were taken from each genotype in dim-
light and kept in resting buffer for an hour before being transferred
to opening buffer in the light. The mean apertures of apk1b
stomata were similar to controls in the dark-adapted samples at the
start of the experiment. The apertures of wild-type and both apk1b-
1 and apkb-2 genotypes increased during exposure to light but
apk1b-1 and apk1b-2 stomata did not open as wide as controls. The
differences observed between control and apk1b-1 and apk1b-2
mean stomatal apertures were significant after 180 minutes
exposure to light (p,0.05) (Figure 4A). This result is consistent
with the apk1b-1 reduced stomatal conductance in the light
observed by IRGA (Figure 2) and further suggests that the
stomata of apk1b mutant plants are defective in light induced
Figure 4. b Mutants have Normal Stomatal Developmen apk1b-1 and apk1b-2 have reduced levels of transpi-
ration and increased relative water content in comparison to
control plants. A: Temperature of the leaf surface following drought
imposition for the time indicated was measured by infra-red
thermography. The onset of watering is indicated by an arrow. Significant differences are indicated (* = p,0.05). B: Relative water was added and following incubation with 1 or 10 mM ABA
(Figure 4B). However, apk1b-1 and apk1b-2 stomata appeared to
have similar sensitivity to ABA as controls at these ABA
concentrations. The response of stomatal apertures to CO2 concentration was
studied by incubating epidermal peels for two hours in the light in
opening buffer supplied (and pre-equilibrated) with either CO2
free air or ambient CO2 air before apertures were measured. Again apk1b-1 and apk1b-2 mean stomatal apertures were
significantly smaller than control apertures under both conditions
(Figure 4C). However, the magnitude of response to CO2-
concentration was similar for all genotypes and the response of
apk1b stomata to CO2 was not impaired. Together these results
indicate that apkb1 stomata are able to adjust their apertures to at
least the same extent as control stomata in response to a variety of
closure stimuli (darkness, exogenous ABA, and elevated CO2) but
are unable to open as to the same extent in response to light. Figure 5. apk1b-1 and apk1b-2 have reduced levels of transpi-
ration and increased relative water content in comparison to
control plants. A: Temperature of the leaf surface following drought
imposition for the time indicated was measured by infra-red
thermography. The onset of watering is indicated by an arrow. Significant differences are indicated (* = p,0.05). B: Relative water
content was calculated from measurements of well-watered plants and
plants subjected to 7 days of drought. Letters indicate significance
groups (p,0.05). doi:10.1371/journal.pone.0097161.g005 apk1b Plants have Reduced Transpiration and Increased
Water Content We investigated whether the reduced stomatal opening pheno-
type of the apk1b plants that we describe above, could have an
effect on plant transpiration and plant water status by examining
leaf temperature, and relative water content. Infra-red thermal
imaging indicated that both apkb1-1 and apkb1-2 plants had higher
mean day-time leaf temperatures (suggesting reduced evaporative
cooling from transpiration) following severe drought treatment
and subsequent re-watering. 2 days after re-watering following a
period without water the mutant plant lines were both approx-
imately 0.8uC hotter than control plants indicating a reduced level
of transpiration (Figure 5A). Both apk1b-1 and apk1b-2 plants had significantly higher RWC
than controls under either a normal watering regime or following
seven days without watering, confirming that apk1b-1 and apk1b-2
are
better
able
to
conserve
water
than
wild-type
plants
(Figure 5B). The increased leaf temperature and water content
are both consistent with apk1 stomata opening less in the light
(Figures 2 and 4). b Mutants have Normal Stomatal Developmen To investigate
stomatal opening in response to light, plants were kept in the dark
overnight; epidermal peels were taken from each genotype in dim-
light and kept in resting buffer for an hour before being transferred
to opening buffer in the light. The mean apertures of apk1b
stomata were similar to controls in the dark-adapted samples at the
start of the experiment. The apertures of wild-type and both apk1b-
1 and apkb-2 genotypes increased during exposure to light but
apk1b-1 and apk1b-2 stomata did not open as wide as controls. The
differences observed between control and apk1b-1 and apk1b-2
mean stomatal apertures were significant after 180 minutes
exposure to light (p,0.05) (Figure 4A). This result is consistent Figure 3. Stomatal density and index of apk1b-1and apk1b-2 are
similar to those of control plants. A: Stomatal density and B:
stomatal index were calculated from the abaxial surface of fully
expanded leaves. Error bars represent the standard error. doi:10.1371/journal.pone.0097161.g003 Figure 4. Stomata of plants lacking APK1b cannot open as
wide in response to light but can close in response to ABA or
CO2. The effect of A: light, B: ABA and C: CO2 on stomatal aperture
measurements from apk1b-1, apk1b-2 and control plants. Apertures
were measured from detached leaf abaxial peels following treatment
with stimuli as indicated. For the response to light shown in A. plants
were dark-adapted and peels prepared in dim light at the start of the
experiment. Significant differences from Col-0 control are indicated
(* = p,0.05). with the apk1b-1 reduced stomatal conductance in the light
observed by IRGA (Figure 2) and further suggests that the
stomata of apk1b mutant plants are defective in light-induced
opening. To investigate the inhibition of stomatal opening by ABA
epidermal peels were incubated two hours in the light in opening
buffer with differing concentrations of ABA and then stomatal
apertures were measured. Both apk1b mutant lines had signifi-
cantly lower mean stomatal apertures than controls when no ABA Figure 3. Stomatal density and index of apk1b-1and apk1b-2 are
similar to those of control plants. A: Stomatal density and B:
stomatal index were calculated from the abaxial surface of fully
expanded leaves. Error bars represent the standard error. doi:10.1371/journal.pone.0097161.g003 May 2014 | Volume 9 | Issue 5 | e97161 May 2014 | Volume 9 | Issue 5 | e97161 PLOS ONE | www.plosone.org 3 Guard Cell Kinase APK1b Figure 5. Discussion A number of transcriptomics experiments have suggested that
the protein kinase encoded by APK1b is highly expressed in guard
cells relative to other cell types [11,15,16]. In the current study it
was demonstrated that the upstream promoter region of the APK1b
gene directs expression predominantly in guard cells, and the
potential role that APK1b may play in the control of stomatal
apertures was explored. Gas exchange analysis revealed that plants
lacking APK1b expression (apk1b-1) had significantly lower stomatal
conductance levels than controls during stomatal opening in
response to increasing light intensity. However apk1b-1 conduc-
tance levels were not significantly lower during dark-induced
stomatal closure (Figure 2). This finding was consistent with the
results of stomatal aperture measurement experiments showing
that stomata of apk1b-1 and apk1b-2 were both less open than
control stomata in the light, and also in the presence of other
stimuli such as ABA and CO2 in the light (Figure 4). These
stomatal aperture measurements suggested that guard cell ABA
and CO2 signaling pathways are not affected in apk1b-1 and apk1b-
2 as both mutant genotypes adjust their stomata to a similar extent
in response to these stimuli. Both mutants also closed their stomata
to the same extent as controls in the dark. In addition, lack of
APK1b had no effect on stomatal size, stomatal density or stomatal Figure 5. apk1b-1 and apk1b-2 have reduced levels of transpi-
ration and increased relative water content in comparison to
control plants. A: Temperature of the leaf surface following drought
imposition for the time indicated was measured by infra-red
thermography. The onset of watering is indicated by an arrow. Significant differences are indicated (* = p,0.05). B: Relative water
content was calculated from measurements of well-watered plants and
plants subjected to 7 days of drought. Letters indicate significance
groups (p,0.05). Figure 5. apk1b-1 and apk1b-2 have reduced levels of transpi-
ration and increased relative water content in comparison to
control plants. A: Temperature of the leaf surface following drought
imposition for the time indicated was measured by infra-red
thermography. The onset of watering is indicated by an arrow. Significant differences are indicated (* = p,0.05). B: Relative water
content was calculated from measurements of well-watered plants and
plants subjected to 7 days of drought. Letters indicate significance
groups (p,0.05). g
p
p
doi:10.1371/journal.pone.0097161.g005 index (Figure 3) indicating that APK1b is not required for
stomatal development. index (Figure 3) indicating that APK1b is not required for
stomatal development. The smaller stomatal apertures that we observed in apk1b
mutants in the light suggest that these plants would be expected to
lose water less readily, and indeed their leaf temperatures were
increased following drought stress indicating reduced transpira- May 2014 | Volume 9 | Issue 5 | e97161 May 2014 | Volume 9 | Issue 5 | e97161 PLOS ONE | www.plosone.org 4 Guard Cell Kinase APK1b ferrocyanide, 1 mM potassium ferricyanide, 0.2% Triton X-100,
2 mM
5-bromo-4-chloro-3-indolyl-b-d-glucuronic
acid,
and
10 mM EDTA after vacuum infiltration at 37uC. Leaves were
decolorized overnight with 70% (v/v) ethanol, and washed in 10%
glycerol. Images were captured with an Olympus BX51 micro-
scope connected to a DP51 digital camera. Expression pattern
shown was typical of several independently transformed lines. tion. In addition, the relative water content of both abk1b
genotypes was found to be significantly higher than control plants
when plants were either well-watered or following one week of
drought (Figure 5). Thus, apk1b leaves have an enhanced ability
to retain water over controls, and therefore might be expected to
have increased water use efficiency and tolerance to drought
conditions. Taken together the results discussed above indicate that APK1b
protein kinase is involved in mediating stomatal opening in the
light. Stomata are responsive to red and blue light wavelengths. They open gradually in response to red light, but rapidly and
strongly
in
response
to
a
blue
light
signal
[17]. Protein
phosphorylation is known to be important in the blue light-
induced stomatal opening pathway. Blue light perception by
phototropins causes the multiple phosphorylation of phototropins
themselves, the phosphorylation of BLUS1 kinase [6] and
ultimately the phosphorylation and activation of the guard cell
plasma membrane H+-ATPase. The pumping of protons out of
guard cells causes membrane hyperpolarization, activating plasma
membrane channels that take up potassium ions (K+
in channels),
thus bringing about an increase in turgor pressure that results in
stomatal opening. The identity of the kinase that phosphorylates
and activates the H+-ATPase is not known although its activity has
been detected in plasma membrane fractions [18]. We investigated
the possibility that the APK1b protein kinase may affect the
phosphorylation cascade that regulates the activity of the H+-
ATPase triggering blue light-induced stomatal opening but we
could not identify a specific impairment in apk1b blue light-
induced opening. Stomatal Aperture Analysis The stomatal of abaxial leaf epidermal strips were analysed
using a method described [22]. Strips of epidermis were taken
from leaves of five to six week-old plants (3–5 leaves of each
genotype) using tweezers and then floated on resting buffer
(10 mM MES, pH 6.2). Strips were transferred to opening buffer
(10 mM MES, 50 mM KCL, pH 6.2) in the light (300 mmol
m22 s21), aerated with CO2-free air and maintained at 20uC. To
investigate the effect of ABA on stomatal aperture, epidermal peels
were incubated on resting buffer for 10 minutes and then
transferred to opening buffer supplemented with ABA concentra-
tions as indicated, and exposed to light and CO2 free air for 2
hours. To investigate the effect of CO2, epidermal peels were
exposed to CO2-free or ambient air in light for two hours. Digital
images were recorded for stomatal aperture width (and length)
measurements by light microscopy (Olympus BX51; Tokyo) using
a fitted camera and graticule. For each time point or treatment
and genotype, 40 pore apertures were measured. To avoid
experimenter bias, measurements were performed without the
researcher being aware of the sample identity. Each experiment Characterisation of Homozygous Knockout Plants y
Homozygous plants were identified using primers APK1b-1F:
59-TCTGAGTCGTGTAAACGAGCC-39
APK1b-1R:
59-
CCTTTATCTTGGACTCTCCGG-39,
APK1b-2F:
59-
TCTGAGTCGTGTAAACGAGCC-39and
APK1b-2R:
59-
CCTTTATCTTGGACTCTCCGG-39. Primers APK1b-1F and
APK1b-1R were used to amplify the cDNA of apk1b-1 and apk1b-
2. RNA was extracted from seedlings using a Qiagen RNAeasy kit,
and RNA reverse transcribed into cDNA using oligo dT primer
and SuperscriptII (Invitrogen). UBQ10 amplification was used as a
loading control. No DNA amplification was detected with APK1b
specific primers after 35 thermocycles. Stomatal Density and Index Dental resin (Coltene Whaledent, Switzerland) was applied to
leaf adaxial surfaces. Nail varnish peels were taken from set resin
and mounted on microscope slides. Cell counts were taken from
the widest area of 3 leaves each from 3 plants of each genotype. Three different areas from each impression were examined under
the light microscope with an eyepiece grid. The number of
stomata and epidermal cells per square millimeter were obtained
to calculate stomatal density, and stomatal index using the
equation stomatal index = stomatal number/(stomatal number +
epidermal cell number) 6100. Plant Growth Arabidopsis thaliana mutants were identified from the SALK
collection [19] and seed obtained from the Nottingham Arabi-
dopsis Stock Centre (Salk_001115 and Salk_104202 respectively). Columbia (Col-0) background ecotype and mutant seeds were
stratified on M3 compost for 72 hours in the dark at 4uC before
transfer to a growth room with 10 hours photoperiod, 140 mmol
m22 s21 light intensity, 20uC/16uC day/night temperature,
relative humidity 60%. Plants were grown in 8 cm diameter pots
and sub-irrigated every three days, until rosettes were mature but
flowering had not commenced. Fully expanded leaves were used
for IRGA, relative water content and stomatal measurement
analyses. index (Figure 3) indicating that APK1b is not required for
stomatal development. It therefore appears likely that APK1b acts
elsewhere in the light-induced stomatal opening pathway, or
perhaps has a more general effect on the mechanism of stomatal
opening. Gas Exchange Analysis Infra-red gas analysis was applied to investigate carbon
assimilation,
intercellular
CO2
concentration,
and
stomatal
conductance using a Li-COR 6400 system (Lincoln, NE, US). Measurements were taken from four leaves (attached to the plant)
from four separate plants of each genotype. The leaf chamber
(2 cm22) was maintained at 20uC. Flow rate was 500 mmol
m22 s21 and humidity (58%–63%). Supporting Information Figure S1
APK1B shows similar expression patterns to
the know guard cell gene OST1, and is enriched in guard
cell preparations. Raw expression values were extracted from
the
manually
dissected
guard
cells
(A;S1),
or
protoplasts
(B,C;S2,S3). OST1 is expressed in guard cells in leaves (S4)
whereas STOMAGEN (STOM) is mesophyll specific (S5). (TIF) References 1. Hashimoto M, Negi J, Young J, Israelsson M, Schroeder JI, et al. (2006)
Arabidopsis HT1 kinase controls stomatal movements in response to CO2. Nat
Cell Biol 8: 391–397. 11. Leonhardt N, Kwak JM, Robert N, Waner D, Leonhardt G, et al. (2004)
Microarray expression analyses of Arabidopsis guard cells and isolation of a
recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell
16: 596–615. 2. Li XM, Zhang LH, Ma LJ, Li YY (2011) Elevated Carbon Dioxide and/or
Ozone Concentrations Induce Hormonal Changes in Pinus tabulaeformis. J Chem
Eco 37: 779–784. 12. Shiu SH, Bleecker B (2001) Receptor-like kinases from Arabidopsis form a
monophyletic gene family related to animal receptor kinases. Proc Natl Acad Sci
USA 98: 10763–10768. 3. Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J (2002) Arabidopsis
OST1 protein kinase mediates the regulation of stomatal aperture by abscisic
acid and acts upstream of reactive oxygen species production. Plant Cell 14:
3089–3099. 13. Goring DR, Walker JC (2004) Plant sciences Self-Rejection-a New Kinase
Connection. Science 303: 1474–1475. 14. Veronese P, Nakagami H, Bluhm B, Abuqamar S, Chen X, et al. (2006) The
membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in
Arabidopsis resistance to necrotrophic and biotrophic pathogens. Plant Cell 18:
257–273. 4. Geiger D, Scherzer S, Mumm P, Marten I, Ache P, et al. (2010) Guard cell
anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+
affinities. Proc Natl Acad Sci USA 107: 8023–8028. 5. Mori IC, Murata Y, Yang Y, Munemasa S, Wang YF, et al. (2006) CDPKs
CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and
Ca(2+)-permeable channels and stomatal closure. PLoS Biol 4: e327. 15. Yang Y, Costa A, Leonhardt N, Siegel RS, Schroeder JI (2008) Isolation of a
strong Arabidopsis guard cell promoter and its potential as a research tool. Plant
Methods 4: 6. 6. Takemiya A, Sugiyama N, Fujimoto H, Tsutsumi T, Yamauchi S, et al. (2013)
Phosphorylation of BLUS1 kinase by phototropins is a primary step in stomatal
opening. Nature Communications 4: 2094. doi:10.1038/ncomms3094. 16. Pandey S, Wang RS, Wilson L, Li S, Zhao Z, et al. (2010) Boolean modeling of
transcriptome data reveals novel modes of heterotrimeric G-protein action. Mol
Syst Biol 6: 372. opening. Nature Communications 4: 2094. doi:10.1038/ncomms30 17. Shimazaki K, Doi M, Assmann SM, Kinoshita T (2007) Light regulation of
stomatal movement. Annu Rev Plant Biol 58: 219–247. 7. Relative Water Content 8–10
mature
leaves
were
excised
from
well-watered
or
droughted plants (water withheld for 7 days) and their fresh
weight measured immediately. Leaves were floated on water at
4uC overnight and weighed to record the hydrated weight. They
were oven-dried at 70uC overnight and weighed to obtain their
dry weight; the RWC was calculated using the following formula
[23]: RWC = (fresh weight2dry weight)/(hydrated weight2dry
weight). Figure S3
apk1b-1 and apk1b-2 are null mutants. cDNA
was synthesised from RNA extracted from seedlings of the T-DNA
disruption mutant apk1b-1 and apkb1-2 and Col-0 control and
amplified with gene specific primers, for 35 cycles before
electrophoresis. No wild-type APK1b transcript was amplified from
either mutant. (TIF) PAPK1b: GUS Analysis A region approximately 1200 bp upstream of the ATG
promoter translational start codon in APK1b was amplified from
genomic DNA using forward 59-CACCTGCTTTTACTTTT-
CAGGTGCCTA-39 and reverse primers 59-GGAGTGAAT-
TAAACCAAACACCA-39 and KOD DNA polymerase, inserted
into the pENTR-D-TOPO entry vector (Invitrogen), transformed
into E. coli competent cells and recombined with the pHGWFS7
destination vector [20], before transfer into Agrobacterium tumefaciens
cells and transformation into Col-0 Arabidopsis by floral dip [21]. Seeds were selected on 20 mgml21 hygromycin and insertion
confirmed by PCR using forward APK1b specific primer and GUS
gene reverse primer (59-TGCTCAGGTAGTGGTTGTCG-39). Histochemical staining for GUS activity was carried out on leaves
of T2 seedlings in 50 mM potassium phosphate, 1 mM potassium May 2014 | Volume 9 | Issue 5 | e97161 PLOS ONE | www.plosone.org 5 Guard Cell Kinase APK1b was then repeated on three separate days using fresh plants
(n = 120). seedlings. A Abaxial fluorescence, showing mature guard cells with
fainter fluorescence in developing guard cells. Bar = 50 m B
Adaxial fluorescence, showing mature guard cells in developing
leaf. Bar = 1 mm C Adaxial fluorescence, showing mature guard
cells in developing leaf Bar = 100 m. D. GUS expression in cauline
leaves, flowers and stem. Bar = 2 cm. (TIF) Statistical Analyses Values were statistically tested using unpaired t-tests (Figures 2,
3, 4, 5A, S4) or ANOVA (Figure 5B). Values were statistically tested using unpaired t-tests (Figures 2,
3, 4, 5A, S4) or ANOVA (Figure 5B). Author Contributions Conceived and designed the experiments: NE LH JS JEG. Performed the
experiments: NE LH JS. Analyzed the data: NE LH JS JEG. Wrote the
paper: NE LH JS JEG. Conceived and designed the experiments: NE LH JS JEG. Performed the
experiments: NE LH JS. Analyzed the data: NE LH JS JEG. Wrote the
paper: NE LH JS JEG. Thermal Imaging 8 week old well-watered plants were subjected to drought for 12
days; plants were then watered normally for a further 6 days. Plants were imaged every 2 or 3 days between 1 and 2 pm with a
FLIR SC660 thermal imaging camera. Images were analysed
using ThermaCAM Researcher Professional 2.9. For each image,
the mean temperature from spots in the centre of 3 representative
leaves was calculated. Figure
S4
Photosynthesis
and
intracellular
CO2
in
apk1b-1 mutants. IRGA photosynthetic parameters measured
by IRGA of leaves. A, C & E: Assimilation rate; B, D & F:
intercellular CO2 levels. A & B: Leaves of plants exposed to
saturating light for 30 min, then exposed to darkness, arrow
indicates when light was switched off. C & D: Leaves exposed to
increasing light intensities ranging from 0 to 2000 mmol photon
m22 s21. E & F: Leaves of plants exposed to red light for 60 min,
then exposed to blue and red light, arrow indicates when blue light
is switched on. Error bars represent the standard errors. Values
were statistically tested using unpaired t-tests and significant
differences are indicated (** = p,0.01). (TIF) Figure
S4
Photosynthesis
and
intracellular
CO2
in
apk1b-1 mutants. IRGA photosynthetic parameters measured
by IRGA of leaves. A, C & E: Assimilation rate; B, D & F:
intercellular CO2 levels. A & B: Leaves of plants exposed to
saturating light for 30 min, then exposed to darkness, arrow
indicates when light was switched off. C & D: Leaves exposed to
increasing light intensities ranging from 0 to 2000 mmol photon
m22 s21. E & F: Leaves of plants exposed to red light for 60 min,
then exposed to blue and red light, arrow indicates when blue light
is switched on. Error bars represent the standard errors. Values
were statistically tested using unpaired t-tests and significant
differences are indicated (** = p,0.01). (TIF) 22. Webb AA, Hetherington AM (1997) Convergence of the abscisic acid, CO2, and
extracellular calcium signal transduction pathways in stomatal guard cells. Plant
Physiol 114: 1557–1560. 23. Gaxiola RA, Li J, Undurraga S, Dang LM, Allen GJ, et al. (2001) Drought- and
salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc Natl
Acad Sci USA 98: 11444–11449. References Fuglsang AT, Guo Y, Cuin TA, Qiu Q, Song C, et al. (2007) Arabidopsis
protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing
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ATPase in Stomatal Guard Cells. Plant and Cell Physiology 52: 1238–1248. 19. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, et al. (2003) Genome-
Wide Insertional Mutagenesis of Arabidopsis thaliana. Science 302: 653–657. 9. Desclos-Theveniau M, Arnaud D, Huang TY, Lin GJ, Chen WY, et al. (2012)
The Arabidopsis lectin receptor kinase LecRK-V.5 represses stomatal immunity
induced by Pseudomonas syringae pv. tomato DC3000. PLoS Pathogens 8: e1002513. 20. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-
mediated plant transformation. Trends Plant Sci 7: 193–195. 10. Hirayama T, Oka A (1992) Novel protein kinase of Arabidopsis thaliana (APK1)
that phosphorylates tyrosine, serine and threonine. Plant Molecular Biology 20:
653–662. 21. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-
mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743. PLOS ONE | www.plosone.org 6 May 2014 | Volume 9 | Issue 5 | e97161 Guard Cell Kinase APK1b May 2014 | Volume 9 | Issue 5 | e97161 PLOS ONE | www.plosone.org 7 PLOS ONE | www.plosone.org 7
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Studying a 2 millennia old bronze kettle using easily accessible characterization techniques
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Introduction Objects made of copper and its alloys are often covered
by layers of corrosion products called patina. The char-
acteristic green-blue patina colour is caused by the Cu(II)
ion in compounds forming the outer patina layer, such as
brochantite (Cu4SO4(OH)6), antlerite (Cu3(SO4)(OH)4) or
atacamite (Cu2Cl(OH)3). The Cu(II) ion acts as a colour
forming ion, and is part of all compounds in the outer
patina layer. Patina can be darkened by presence of cop-
per sulphides or lead, lightened by lead carbonate or tin-
oxide and reddened by an underlying layer of cuprite, *Correspondence: kmarusic@irb.hr
4 Ruđer Bošković Institute, Division of Materials Chemistry, Radiation
Chemistry and Dosimetry Laboratory, Bijenička c. 54, Zagreb HR‑10000,
Croatia
Full list of author information is available at the end of the article © The Author(s) 2021. 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 A bronze kettle dating from the 1st to 2nd century was found in a riverbed of the Kupa river in Croatia. After excava-
tion it spent another 50 years in a depot of a museum in atmospheric conditions prior to starting the conservation
treatment and our studies. A study on the surface layers development was performed to determine the whereabouts
of the object through its history. This study is a demonstration of how such analysis can be used to reconstruct what
the object went through during its life span. Samples taken from the kettle were observed by optical and scanning
electron microscopy (SEM), and analysed by X-ray fluorescence (XRF), X-ray energy dispersive spectroscopy (EDS) and
Fourier transform infrared spectroscopy (FTIR). It was determined that the kettle is made of low-tin bronze, with low amounts of iron, aluminium, calcium and nickel. While being in the riverbed malachite formed on the kettle. After longer exposure to the river Si-oxides and CaCO3
formed on the surface of the kettle, over malachite. It was shown that the kettle probably had a ferrous alloy handle
which degraded and disappeared in time. After excavation, the kettle came again in an oxygen-enriched atmosphere
and formed new surface layers over the malachite layer. As the museum the kettle has been kept in since excavation
is set in a highly industrial area sulphur compounds have been formed on the surface. Keywords: Ancient bronze, Corrosion products, Patina, Surface layers Cu2O [1]. It is hard to distinguish different compounds
by mere observation of the patina colour [2]. A large
number of studies have shed light on different conditions
and effects influencing formation of a variety of copper
and bronze patinas [1, 3–5]. For example, when copper
corrosion occurs during burial usually first an inner layer
of copper(I) compounds forms next to the metal surface
and on top of it outer layers of green or blue copper(II)
compounds, consisting of one or more copper(II) salts. Presence of carbon dioxide in humid closed spaces can
lead to formation of copper(II) carbonate hydroxides like
malachite or azurite [6]. It is also known that after sud-
den or drastic changes in environment, e.g. after exca-
vation corrosion is restarted or accelerated on bronze
objects which leads to formation of new layers of corro-
sion products [7]. In museums or museum depots harm-
ful gaseous pollutants can be present, forming a variety of Kotlar et al. Herit Sci (2021) 9:10
https://doi.org/10.1186/s40494-021-00484-6 Kotlar et al. Herit Sci (2021) 9:10
https://doi.org/10.1186/s40494-021-00484-6 Kotlar et al. Herit Sci (2021) 9:10
https://doi.org/10.1186/s40494-021-00484-6 Open Access Studying a 2 millennia old bronze kettle
using easily accessible characterization
techniques Marta Kotlar1, Nives Matijaković Mlinarić2, Vladan Desnica3 and Katarina Marušić4* © The Author(s) 2021. 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. *Correspondence: kmarusic@irb.hr
4 Ruđer Bošković Institute, Division of Materials Chemistry, Radiation
Chemistry and Dosimetry Laboratory, Bijenička c. 54, Zagreb HR‑10000,
Croatia
Full list of author information is available at the end of the article Kotlar et al. Herit Sci (2021) 9:10 Page 2 of 13 Kotlar et al. Herit Sci (2021) 9:10 corrosion products such as nitrates, sulphates, sulphides,
oxides, formates, acetates, and salts of higher carboxylic
acids [8]. If one understands the relationship and inter-
action between environments metals are exposed to and
the formation and stability fields of corrosion products
on metals, one may be able to reconstruct environmental
conditions surrounding the object in the past. This infor-
mation should provide investigators with the possibility
of writing the biography of ancient metal artefacts [5], i.e. by studying the corrosion products layering and compo-
sition it is possible to reconstruct the whereabouts of an
object through its history. The conclusions obtained from
such studies are valuable both for the person performing
the conservation treatment as well as for art historians.h very diverse surface layers were very interesting, not
only for their different structure and colours, but also
for their complexity. We intended to study which cor-
rosion products had been created on the surface of the
object, considering the environment in which the object
was exposed to, as well as the chronological order of the
corrosion products formation. In order to do so differ-
ent analysis and instrumentation were used on a selected
number of samples. Materials and methods
Materials In the 1960s during dredging of the Kupa river in Sisak,
an industrial city located in central Croatia, a large group
of objects was found, among which was the object of our
investigations. A bronze kettle 22 cm high and 22 cm
wide dating from the 1st to 2nd century, Roman Empire
period, probably from the Roman Empire city Siscia
spent over a millennium in the riverbed. After exca-
vation, the kettle was stored in the depot of the Sisak
Municipal Museum and was not processed until 2018
when it was sent for conservation-restoration treatment
to the Art and Restoration Department of University of
Dubrovnik, Workshop for conservation and restoration
of metal. Prior to performing the complete conservation
treatment different surface layers were visually distin-
guished and sampled. The investigations were performed
on these samples. There are numerous objects made of copper alloys that
were found with no known history. The problem is that
metals do not possess physical properties that can be
expressed as a function of time and thus metallic artefacts
cannot be directly dated. There are different approaches
for authentication of ancient metals [9], but still the his-
tory of the artefacts is often not clear. When working with
objects of historical or cultural importance it is possible
to obtain samples of corrosion products from the artefact
during the restauration treatment, but it is not possible
to do studies that would use any destructive methods on
the object itself. There are standard techniques used for
analysing corrosion products like X-ray radiography, or
X-ray diffraction which is useful for validating hypotheti-
cal presence of unusual products. Yet, many objects are
kept in small museums where limited instrumentation is
available. Also, often the owners of an object of interest
do not permit large interventions. This is the reason why
conservators are frequently left to their own resourceful-
ness upon examination of an interesting object to obtain
the necessary data through an alternative approach and
limited resources. This work presents an effort of a joined
collaboration of conservators and scientists to character-
ize surface layers and corrosion products of an ancient
bronze artefact using only elemental analysis in combi-
nation with Fourier Transform Infrared Spectroscopy. It
shows how it is still possible to obtain very useful data
despite common limitations if an appropriate collabora-
tion of curators and scientists is established. X‑ray fluorescence (XRF) analysish yl
y
The samples were investigated using a portable XRF
instrument, which was developed at the Laboratory
of the Academy of fine arts in Zagreb. It consists of a
50-kV Rh transmission excitation tube (Moxtek, USA)
and a Peltier cooled silicon drift detector (Amptek,
USA) with energy resolution of 145 eV at the Mn Kα
excitation line. Depending on the analytical needs, the
device can provide either mili or micro X-ray beam for
sample excitation, by employing a motorized collima-
tor interchanger to switch between a pinhole collima-
tor (spot size ca. 1.5 mm) and a polycapillary lens (IfG,
Germany, spot size ca. 65 µm). The operating param-
eters for tube voltage and anode current during the
measurements were set to 40 keV and 150 µA, respec-
tively, and the acquisition real time was 100 s. For these
measurements the device was used in a micro mode
and the diameter of the beam was set to ca. 65 µm. For
easy and reproducible alignment of the sample in front
of the X-ray source and the detector, a two-laser beam Observation by optical microscopy Once the samples were collected from the kettle they
were all observed by a digital microscope Dino-Lite
AM4113ZT under 60x and 240x magnification, prior to
any analyses. Fourier transform infrared spectroscopy (FTIR) analysis
Fourier transform infrared spectroscopy (FTIR) was per-
formed using Bruker’s Tensor II spectrometer (Bruker
Optik GmbH, Ettlingen, Germany) equipped with Atten-
uated total reflection (ATR) module for checking pres-
ence of functional groups on the surface of the samples. FTIR spectra were recorded at room temperature in fre-
quency range 500–4000 cm− 1 at 4 cm− 1 scan step and
total 16 scans per measurement. Sampling
h
h When the kettle was received at the Workshop for res-
toration treatment it was in a very bad condition and
covered with various corrosion products and remains
of sand trapped in the corrosion products. According
to the museum inventory form the kettle is made by the
embossing technique. No X-ray radiography or cross-
sectional study was possible, thus after visual examina-
tion, six samples were taken from the kettle for further
analysis. Two samples were fragments of the kettle which
broke off while manipulating the object due to its poor
condition and metal fatigue of the thin bronze sheet. Since both samples were observed as metal core, they
could be used for determining the composition of the
alloy the object is made from. They were covered with
corrosion products and analysed as Samples 1 and 2. Four samples (named Samples 3 to 6) were taken from
the surface of the object as presented in Fig. 1. Each sam-
ple seemed to have a different surface layer based on the
difference in colour and structure. These samples were
scratched from the surface using scalpel during mechani-
cal cleaning in conservation interventions. A bronze kettle dating from the 1st to 2nd century,
Roman Empire period was found in a riverbed in the
place of the Roman Empire city Siscia in Croatia. After
excavation it spent another 60 years in a depot of a
museum in highly polluted atmospheric conditions prior
to starting the conservation treatment and our studies. The item was received in a very bad condition and was
covered with various corrosion products and remains of
sand trapped in the corrosion products. Upon observ-
ing the exact state of the object, it was obvious that these Page 3 of 13 Kotlar et al. Herit Sci (2021) 9:10 Kotlar et al. Herit Sci Front side
Back side
Sample 4
Sample 3
Sample 5
Sample 6
Fig. 1 Image of the bronze kettle and the surface layers’ sampling positions Fig. 1 Image of the bronze kettle and the surface layers’ sampling positions system is used. The lasers are aligned in such a way that
the point of their beam intersection coincides with the
cross-point of the X-ray tube and detector axes. On
every sample two measurements were made and the
results are a combination of the obtained data. sual and optical observation of the surface layers sual and optical observation of the surface layers p
y
Figure 2 presents the images of the studied surface sam-
ples taken by a digital microscope. The most preva-
lent corrosion product was greenish in colour. It was
observed on various samples, most expressed on Sample
3. Also, on the surface of the object, some other impuri-
ties have been trapped in corrosion products and accu-
mulation of materials like sludge stones was observed. Other black, brown, white-yellow and blue layers were
also visible on the surface. Some of them covered the
surface of the green areas, while some were close to the
surface of the metal and degraded the metal until they
made holes in the material itself. During removal of the
layers, it was observed that the black layers (Sample 6)
were partially diluted in distilled water and 96 % ethanol. They were easily removed by mechanical cleaning with a
scalpel. Below the black layers was a layer of green cor-
rosion products. The brown products (Samples 4 and 5)
strongly adhered to the substrate itself. Under the brown
layers, green products existed only in traces and did not
have such a uniformly covered surface as was the case
under the black layers. The yellow-white layers observed
on Sample 4 were powdery and below them there was a
layer of partially formed green corrosion products, while Scanning electron microscopy and X‑ray energy dispersive
spectroscopy (SEM/EDS) analysish The samples were also inspected using the JEOL JSM-
7000F field emission scanning electron microscope (FE-
SEM) equipped with the Oxford Instruments INCA-350
X-ray energy dispersive spectrometer microanalysis
system (EDS) at an acceleration voltage of 10 or 15 kV
depending on the sample. Computer program ImageJ
was used in processing of FE–SEM sample images to
obtain particle size distributions. Kotlar et al. Herit Sci (2021) 9:10 Kotlar et al. Herit Sci (2021) 9:10 Page 4 of 13 in other places a very thin layer of reddish-brown layers
was observed. The elemental composition of the samples determined
by XRF In order to distinguish the alloying elements from the
elements embedded from other sources into the corro-
sion products X-ray fluorescence measurements were
performed on all samples. The results are presented in
Table 1. The results consist of three groups of elements:
main elements are the principal elements comprising Surface layer analysis the bulk of the material with weight concentrations of up
to 90 %, other elements exhibit concentrations of 0.5 to
10 %, while trace elements are present in concentrations
below 0.5 %. As the XRF analysis was carried out using a
portable device in air, with no reference materials and on
corroded samples of unequal sizes and shapes, the meas-
urements yielded only semi-quantitative results. the bulk of the material with weight concentrations of up
to 90 %, other elements exhibit concentrations of 0.5 to
10 %, while trace elements are present in concentrations
below 0.5 %. As the XRF analysis was carried out using a
portable device in air, with no reference materials and on
corroded samples of unequal sizes and shapes, the meas-
urements yielded only semi-quantitative results. IR spectra provide valuable information on the pat-
ina composition because IR spectroscopy senses the
low crystalline and amorphous fraction and it has an
extremely high detection limit for some species com-
monly found in the patina [11]. FTIR-ATR measurements
were performed on all six samples and the results are pre-
sented in Fig. 3. The first observation is that the spectra
obtained on Samples 2 and 3 are very similar, just as the
spectra obtained on Samples 5 and 6. The same observa-
tion can be made when looking at the samples with an
optical microscope presented in Table 1. All the samples
were also analysed with SEM and EDS. The individual
results of the samples are presented grouped according to
the similarities of the results. One of the fragments of bronze found discarded in the
kettle (Sample 2) was used for analysing the bronze alloy. After performing measurements on the layers covering
the surface of the sample, it was cleaned manually in mul-
tiple steps. After each cleaning step XRF was performed
until a repetitive spectrum was obtained. Upon analysing
the obtained spectrum, the approximate concentrations
of the present elements were determined: 89.0 % Cu,
9.5 % Sn with traces of Fe, Al and Ca (below 0.5 %).h The kettle was found in Kupa which is a karst river. This would explain the presence of calcium from calcium
carbonate, CaCO3, in all samples, although calcium was
obviously also one of the alloying elements. Table 1 XRF results obtained on the investigated samples
of surface layers Table 1 XRF results obtained on the investigated samples
of surface layers Table 1 XRF results obtained on the investigated samples
of surface layers
Sample
Main elements
Other elements
Trace elements
Sample 1
Fe Cu, Sn
-
Si, S, Ca, Ni
Sample 2
Fe, Cu, Sn
-
Al, Si, S, Ti, Pb
Sample 2
cleaned
Cu
Sn
Al, Ca, Ni
Sample 3
Fe, Cu, Sn
Ca
Si, S, Ti, Mn
Sample 4
Ca, Fe, Cu
Si, S, K, Ti
Mn
Sample 5
Fe, Cu
Si, K, Ca, Sn
Al, K, Ti, Mn, Zn, As
Sample 6
Fe, Cu
Ca
Si, P, S, K, As Fig. 2 Images of the examined samples taken by a digital microscope: Samples 1 and 2 which are fragments under 60x and the rest of the samples
under 240x magnification Fig. 2 Images of the examined samples taken by a digital microscope: Samples 1 and 2 which are fragments under 60x and the rest of the samples
under 240x magnification examined samples taken by a digital microscope: Samples 1 and 2 which are fragments under 60x and the rest of the samples
tion Kotlar et al. Herit Sci (2021) 9:10 Kotlar et al. Herit Sci (2021) 9:10 Page 5 of 13 Page 5 of 13 Kotlar et al. Herit Sci (2021) 9:10 Surface layer analysis According to
Bilinski [10] the lower part of Kupa river where the ket-
tle was found consist primarily of SiO2 (81 %), while the
other compounds present in Kupa’s sediments are Al2O3,
CaO, Fe2O3, K2O, Na2O and MgO and in very small
amounts TiO2, P2O5 and MnO. Silicon is present either as
one of the other elements or as trace elements in all cases. The presence of silicon in large quantities was firstly
thought to be an indication of alloying with silicon, but
results on the cleaned Sample 2 have shown that silicon
is not present in the alloy. Thus, silicon obviously comes
from the riverside sediment that has been deposited on
the object during the long period of time when the kettle
was buried in the river mud. Sample 1
S
l 1 Sample 1 shows a predominantly greenish surface as can
be seen in Fig. 2. SEM analysis of Sample 1 showed a uni-
fied structure, while EDS showed a unified composition
presented in Fig. 4. The EDS results show a significant amount of carbon. As explained earlier presence of a form of calcium car-
bonate is expected. Also presence of malachite, a green
carbonate-based corrosion product on copper-based
objects buried in a riverbed is common because the envi-
ronment may be rich in oxygen as well as carbonate/
bicarbonate anions. Carbonate is presented by four nor-
mal modes of vibration peaks in FTIR spectra: ν1 sym-
metric C-O stretching, ν2 CO3 out-of-plane bending, ν3
asymmetric C-O stretching mode, and ν4 O-C-O bend-
ing (in-plane deformation) mode [4, 10, 12]. In the FTIR
spectrum obtained on Sample 1 the bands characteristic Fig. 3 FTIR-ATR results on the investigated samples of corrosion products Fig. 3 FTIR-ATR results on the investigated samples of corrosion products Page 6 of 13 Kotlar et al. Herit Sci (2021) 9:10 Kotlar et al. Herit Sci Fig. 4 a SEM image, b FTIR-ATR spectrum and c elemental composition obtained by EDS on Sample 1 Fig. 4 a SEM image, b FTIR-ATR spectrum and c elemental composition obtained by EDS on Sample 1 ig. 4 a SEM image, b FTIR-ATR spectrum and c elemental composition obtained by EDS on Sample 1 because of a large overlap observed for different compo-
nents in the spectra of different compounds [17]. for carbonate that are easiest to observe are the two
broad bands in the region around 1500 − 1300 cm− 1. The peak at 1488 cm− 1 indicates that calcium carbon-
ate is present in the form of vaterite on this sample,
since it has been reported that different calcium carbon-
ate phases can be identified using FTIR [13–15]. This
is confirmed by the presence of peaks at 743 cm− 1 and
879 cm− 1 [13]. Presence of malachite, Cu2CO3(OH)2, is
also observed through various peaks. The bands at ~ 1097
and ~ 1034 cm− 1 are assigned to the CO symmetric
and asymmetric stretching vibrations. The two bands at
879 and 816 cm− 1 are attributed to out of phase and in
phase bending modes of carbonate in malachite [16]. Sample 1
S
l 1 Both
of these peaks can be observed in the spectrum in Fig. 3b. carbonate in malachite [16]. The two bands clearly pre-
sent at ~ 3403 and ~ 3319 cm− 1 are assigned to O–H
groups bound to intramolecular H-links [22], but as said
earlier they cannot be used for distinguishing presence of
different copper patinas. EDS showed clear presence of Si in some form in
the sample. Inorganic silicates have Si-O bands at
1050 − 1000 cm− 1 (strong) and they also absorb at
540 − 440 cm− 1 (medium). Silica absorbs near 1100 cm− 1
(strong), near 800 cm− 1 (weak) and near 475 cm− 1
(medium). The silica bands overlapped by other peaks are
present in the spectra, as well as the band at ~ 419 cm− 1
which is related to the (O–Si–O) deformation mode [21,
22]. f
EDS and XRF analysis showed presence of Si in these
samples. The peak at ~ 1097 cm− 1 which is also attrib-
uted to malachite in combination with the rest of the
peaks explained earlier indicated presence of Si-oxides. Presence of calcium carbonate in the form of vaterite is
again presented by peaks at 1489, 879 and 743 cm− 1 on
Sample 3, while in the spectrum of Sample 2 the peak
at 712 cm− 1 represents calcite [13]. SnO2 is presented
by 533 and 480 cm− 1. These samples showed presence
of iron in the form of Fe2O3 with very small peaks at
786 cm− 1 and 462 cm− 1. Both XRF and EDS analysis showed a significant
amount of iron in this sample. Fe2O3 is expected to be
present on the surface, if not formed on the bronze, then
at least deposited from the river’s sediments as explained
earlier. The two bands at 533 and 480 cm− 1 that are
attributed to SnO2 are overlapping bands of Fe–O which
indicate presence of Fe2O3 [23]. The broad peak at 1097 cm− 1 could also be attributed
to a sulphate compound. Although by EDS analysis sul-
phur was not found, XRF indicated its presence on both
Samples 2 and 3. Upon observing the FTIR spectra it can
be seen that the main peaks that present sulphate cop-
per corrosion products are present [17, 24–26]. Samples 2 and 3 Sample 3 was the sample with the most expressed green
colour with small presence of light blue hues. The surface
layers used for the analysis of Sample 3 were prevalent
in green. Sample 2 was also green, although its colour
was uniform. The SEM image of the surface of Sample
3 showed two different structures presented in Fig. 5. The main difference between the two structures is that
the composition of the second structure shows presence
of tin, while the first composition does not. Sample 2
showed presence of only one composition that is similar
to the composition in Point 1 with slightly higher content
of calcium and lower content of aluminium and magne-
sium, indicating that the oxides composition is the only
difference. Sample 1
S
l 1 They
show the internal vibrational modes of sulphate ions at
1097 and 1034 cm− 1 (ν3), 879 cm− 1 (ν1), 605 cm− 1 (ν4),
the signals below 500 cm− 1 are assigned to the ν2 inter-
nal mode of SO2−
4 and to Cu-O vibrations, while the peaks
at 3403 and 3319 cm− 1 are attributed to ν(OH). Samples
2 and 3 were the only samples were sulphate patina was
detected, and considering the intensity of the peaks com-
pared to the peaks of malachite it was not the main type
of patina on the samples. It is hard to distinguish which
sulphate copper patina is present on bronze, brochantite,
Cu4SO4(OH)6, antlerite, Cu3(SO4)(OH)4, or posnjak-
ite, Cu4SO4(OH)6·H20, using FTIR, but posnjakite is not
normally found on archaeological bronzes. On the other
hand, brochantite mainly forms in exposed areas and it
is a common corrosion product on bronze sculptures
located in urban areas where atmospheric sulphur diox-
ide is present [27]. On bronze objects exposed to fresh-
water antlerite is most commonly found [5]. The presence
of sulphate patina does not exclude presence of inorganic
sulphates that were found on Sample 1. EDS and XRF analysis also showed that sulphur is pre-
sent on the surface of Sample 1. The peak that is attrib-
uted to sulphates is around 1100 cm− 1. The peak at
1097 cm− 1 exists, but the peaks that would represent typ-
ical sulphate copper corrosion products like brochantite,
Cu4SO4(OH)6, antlerite, Cu3(SO4)(OH)4, or posnjakite,
Cu4SO4(OH)6·H20, are not present in the spectra. So, it
can be assumed that sulphate is present in an inorganic
form on Sample 1 [24, 25]. The peak attributed to cuprite, Cu2O, at around
625 cm− 1 [24] is not observed on this sample or any of
the other samples. Sample 1
S
l 1 The
spectrum also shows peaks in the OH stretching mode
region, but although the different basic copper(II) salts
developed on copper during corrosion may be identified
by IR reflectance spectroscopy, the OH stretching mode
region (above approximately 3000 cm− 1) cannot be used for carbonate that are easiest to observe are the two
broad bands in the region around 1500 − 1300 cm− 1. The peak at 1488 cm− 1 indicates that calcium carbon-
ate is present in the form of vaterite on this sample,
since it has been reported that different calcium carbon-
ate phases can be identified using FTIR [13–15]. This
is confirmed by the presence of peaks at 743 cm− 1 and
879 cm− 1 [13]. Presence of malachite, Cu2CO3(OH)2, is
also observed through various peaks. The bands at ~ 1097
and ~ 1034 cm− 1 are assigned to the CO symmetric
and asymmetric stretching vibrations. The two bands at
879 and 816 cm− 1 are attributed to out of phase and in
phase bending modes of carbonate in malachite [16]. The
spectrum also shows peaks in the OH stretching mode
region, but although the different basic copper(II) salts
developed on copper during corrosion may be identified
by IR reflectance spectroscopy, the OH stretching mode
region (above approximately 3000 cm− 1) cannot be used f
According to both XRF and EDS analysis Sample 1 con-
tains a high amount of tin, actually the highest among the
analysed surface samples (21 %). Tin as a corrosion prod-
uct on antient metals is usually found in the tin(IV)oxide
form, i.e. cassiterite, SnO2. Due to its characteristics cas-
siterite is very difficult to characterize as corrosion prod-
uct. It is also usually covered by other corrosion products
and thus the peaks attributed to SnO2 in a FTIR spectrum
are hard to distinguish. The FTIR spectrum obtained on
Sample 1 showed different peaks in the fingerprint region
presented in Fig. 3b. The spectrum showed a peak at
816 cm− 1 which corresponds to the metal-oxide stretch-
ing vibrations [18], in this case both to Sn-O and Cu-O
stretching. The peaks at 533 and 480 cm− 1 present the
bending vibrations [18]. The mineral cassiterite is usu-
ally identified in a FTIR spectrum with a peak at around Kotlar et al. Herit Sci (2021) 9:10 Page 7 of 13 670 cm− 1 and a peak at around 550 cm− 1 [19, 20]. Sample 4 Sample 4 has a blackish colour, as can be seen in Fig. 2. The main colour is not green/blue pigmented, so it is
expected that this sample is not a sample where copper
corrosion products prevail. The SEM image obtained
on Sample 4 is presented in Fig. 7. It shows presence of
two different structures pointed by numbers 1 and 2. The structure in Point 1 has a rougher structure, while
the structure in Point 2 is smoother. EDS analysis were
performed in these two points and the results are also f
The FTIR spectra obtained on Samples 2 and 3 were
very similar as can be seen in Fig. 6. Presence of mala-
chite, Cu2CO3(OH)2, was observed through various
peaks. The bands at ~ 1097 and ~ 1034 cm− 1 are assigned
to the CO symmetric and asymmetric stretching vibra-
tion. The two bands at ~ 879 and ~ 821 cm− 1 are attrib-
uted to the out of phase and in phase bending modes of Kotlar et al. Herit Sci (2021) 9:10 Page 8 of 13 Kotlar et al. Herit Sci Fig. 5 SEM image of two different positions on Sample 3 and elemental composition in atomic-% obtained on the two positions by point analysis presents silica is not present on this sample, but the
bands at 1050 − 1000 cm− 1 (strong) and at 540–440 cm− 1
(medium) presenting inorganic silicates are present in
the spectra. presented in Fig. 7. It is clear that in Point 1 iron is a
dominant element, while in point 2 silicon is. FTIR results obtained on Sample 4 show clear peaks
only in the region up to 1500 cm− 1 presented on Fig. 8. Metal oxides of various types have bands from the
metal-O-metal group that can absorb throughout the
800 − 200 cm− 1 region [21]. As stated earlier, various
metal-oxides are expected to be present according to the
composition of the traces of river Kupa. According to the
EDS analysis presence of Al2O3, MgO and NaO is most
likely and the other peaks in this region are most likely
attributed to their presence. In the spectrum of Sample
4 the bands representing Fe2O3 are observed as on the
other samples with peaks at 796 and 468 cm− 1. Sample 4 The broad wide peak at ~ 1001 cm− 1 is much more
expressed in this spectrum compared to the spectra
of Samples 1–3 and it again indicates presence of SO4
vibrations. As in the case of Sample 1 there are no
peaks indicating copper sulphate patina, so it can be
assumed that the inorganic sulphates are present here
as well. The spectrum of Sample 4, unlike the spectra of Sam-
ple 1 indicates that calcium carbonate is present in
the form of calcite rather than vaterite. This is due to
only one band at ν3–418, ν2 –77 and the peak at ν4–
695 cm− 1 which is specific for calcite [13]. EDS showed significant amount of Si in the
sample. The band at around ~ 1000 cm− 1 that i
Sample 4 was the only sample that did not show pres-
ence of tin on its surface. Page 9 of 13 Kotlar et al. Herit Sci (2021) 9:10 Kotlar et al. Herit Sci analysis (Table 2) on the two samples showed quite dif-
ferent results. It seemed each measured point had a dif-
ferent composition as can be seen in Table 2. Sample 5
showed a significant amount of iron and copper while
Sample 6 showed higher content of tin and silicon,
depending on the position. Also, the content of carbon in
Sample 6 is significantly higher than on the other exam-
ined samples. Sample 6 also showed very small content of
copper indicating that just like in the case of Sample 4 in
this sample copper corrosion products do not prevail. As
for Sample 5 the copper content is somewhat higher. Fig. 6 FTIR spectra observed on Samples 2 and 3 The FTIR spectra in the metal-oxide region, i.e. at
lower wavenumbers, show very many peaks that are close
to each other. It can be assumed that some of the peaks
representing different metal-oxides are overlapping. When we look at EDS results, we can assume presence
of different metal oxides. The peak at 1002 cm− 1 pre-
sents Si-O bands, while the peak at 796 cm− 1 represents
quartz, i.e. SiO2. The peak at 796 cm− 1overlaps with the
peak of SnO2 which is also indicated by peaks at 527 and
465 cm− 1. The peak at 527 is also presenting Fe2O3. Sample 4 The
two peaks at around 1571 and 1375 cm− 1 indicate pres-
ence of carbonate in some form. The peaks at 796, 750
and 1375 cm− 1 indicate presence of malachite, although
it is clear from the size of the peaks that its quantity is
small as on sample 4. The peak at 778 cm− 1 presents
overlapping peaks presenting both quartz and malachite. Discussionh Samples 5 and 6 seem to have a similar appearance by the
bare eye and this can be seen from the images in Fig. 2. They are both multi-coloured. The SEM results showed
a similar structure as can be seen in Fig. 9a and the FTIR
spectra obtained on the two samples are also very sim-
ilar as can be seen on Fig. 9b. On the other hand, EDS The results have shown that the kettle alloy contains
approximately 9.5 % of tin. Thus, it is made of a low-tin
bronze (containing up to 14 wt% tin). Alloys that contain
more than about 14 wt% tin (high-tin bronzes) are brittle
and hard. On the other hand, low-tin bronzes are usually Fig. 7 SEM image of Sample 4 and elemental composition in atomic-% obtained by EDS analysis on the two points marked on the SEM image Fig. 7 SEM image of Sample 4 and elemental composition in atomic-% obtained by EDS analysis on the two points marked on the SEM image Kotlar et al. Herit Sci (2021) 9:10 Page 10 of 13 Page 10 of 13 Kotlar et al. Herit Sci 1400 1200 1000
800
600
400
Wavenumber (cm-1)
). u
. a
(
e
c
n
a
b
r
o
s
b
A
1001
~800
468
1418
877
695
527
Fig. 8 FTIR spectrum obtained on Sample 4 to Utrect in The Netherlands and reported that in the
majority of the artefacts above 5 % of iron was present. Iron in the alloy that the kettle was made from may origi-
nate from impurities of the raw material for making the
object. Also, it is possible that the same furnace was used
for different metal alloys so impurities can originate from
there as well. The kettle was an object for everyday use, and the dam-
age began to form while it was still in function. After that,
it was buried for almost 2000 years, which led to creation
of various aerobic corrosion products and production of
beautiful patina which was intermittently interrupted by
active products of corrosion. After excavation, drastic
and sudden changes in environmental conditions, espe-
cially increase of oxygen concentration, induced restart-
ing and accelerating corrosion of the bronze substrate
[4, 7] and new layers of corrosion products were created
on the kettle. Discussionh There were no interventions on the kettle
after it was excavated until the moment it arrived at the
Workshop, which means it had been waiting for restora-
tion while being exposed to atmospheric conditions of
the Museum’s depot for more than 50 years. Table 3 presents the different compounds that were
found present on the examined samples. The characteris-
tic green-blue appearance of the patina layer is due to the
colour forming Cu(II)-ion in the outer patina layer which
needs to be 12 ± 2 µm to fully cover the inner oxide layer
[2]. The patina on the kettle was identified as malachite,
Cu2CO3(OH)2. It was formed over most parts of the
object’s surface, but it is not everywhere in equal amount
and thickness. Figure 10 presents the presence of mala-
chite on the kettle. The schematic image was made by
isolating only the colour of the malachite from the image
of the kettle. It is the corrosion product that was created
on the surface of the kettle while the object was buried
in the mud of the Kupa River. It was the first corrosion single-phase alloys and can be shaped by hammering and
thus they are suitable for the embossing technique which
was used to make the kettle [6]. Aluminium and nickel
were added to the alloy in order to reinforce the struc-
ture of the alloy, while calcium was added to reduce the
melting point which greatly facilitates the processing and
serves as a deoxidizer for copper [6]. Presence of iron in a copper alloy is not common since
pure iron is generally not added to bronze, yet it is occa-
sionally found. For example, Pronti et al. [28] reported
that 0.2–0.4 wt-% of iron was found in Greek and Roman
coins from Pompei or Fernandes et al. [29] investigated
44 Roman copper-alloyed artefacts from a location close pectra observed on the samples Fig. 9 a Typical SEM image of Samples 5 and 6, and b FTIR spectra observed on the samples Fig. 9 a Typical SEM image of Samples 5 and 6, and b FTIR spectra observed on the samples Page 11 of 13 Kotlar et al. Discussionh Herit Sci (2021) 9:10 Page 11 of 13 Table 2 Elemental composition in atomic-% obtained
by EDS analysis on Samples 5 and 6 in various positions
Element
Sample 5
Sample 6
Position 1
Position 2
Position 1
Position 2
Position 3
C
22.0
23.7
47.9
52.8
66.1
O
58.5
55.5
42.8
39.3
29.7
Cu
14.7
8.0
0.6
1.7
2.1
Fe
1.3
9.6
0.3
0.7
0.5
Sn
–
0.6
–
4.1
–
Ca
0.4
0.3
0.7
0.8
0.9
Si
1.9
1.5
4.4
0.3
0.4
Al
0.8
–
2.3
–
0.2
Mg
0.4
–
0.6
–
–
S
0.1
0.4
0.2
0.2
0.1
P
–
0.6
–
0.2
– Table 2 Elemental composition in atomic-% obtained
by EDS analysis on Samples 5 and 6 in various positions 0.8 ± 0.2 µm [2]. We were not able to identify cuprite on
the kettle. This was either because the layer was well cov-
ered by other compounds or because SnO2 was present
in a large amount. 0.8 ± 0.2 µm [2]. We were not able to identify cuprite on
the kettle. This was either because the layer was well cov-
ered by other compounds or because SnO2 was present
in a large amount. Si-OH, SiO2 and SiO, as well as CaCO3, were also
observed on most samples. We can conclude that they
are sediments from the river Kupa. These layers were
present over malachite and they are a result of longer
exposure to the river environment. These Si and Ca based
layers have a large volume and are porous, thus sediment
like soil and stones located in the riverbed in the proxim-
ity of the object were captured in the corrosion products. All other trace elements that were found can also origi-
nate from the structure of the sediment in Kupa River
or be part of the corrosion products produced while the
object was buried [10]. The three samples (Samples 4, 5 and 6) which were
not predominant in green colour, but rather brown/
blackish showed presence of a higher quantity of fer-
ric oxide, Fe2O3. Iron does not have to originate from
the bronze alloy. The assumption is that the bronze
kettle had a ferrous alloy rim and a handle which was
attached while forging, so there was no need to solder it product that covered the surface during this period. Conclusionsh The kettle is made of low-tin bronze. The alloy has 9.5 %
tin with addition of aluminium, calcium, nickel and iron
in concentrations bellow 0.5 %. Such low-tin bronzes
were used for manufacturing by embossing because
of their resistance to mechanical and corrosion resist-
ance. Addition of calcium reduced the melting point of
the alloy, while addition of aluminium and nickel can be
attributed to the reinforcement of the bronze.h The layers of corrosion products were formed accord-
ing to the conditions the object was exposed to. The ket-
tle was probably used for preparing food and in this case
exposed to fire. Thus, the first product that would have
formed on the surface would be tin(II) oxide, SnO2. SnO2
is insoluble in water and the oxides stayed on the surface
during its exposure to water in the Kupa river. The ket-
tle was buried for almost two millennia in the mud of the
Kupa River. Since no chlorides are present in such waters,
the subject has remained fairly well preserved. The lim-
ited oxygen presence and very humid conditions in which
the object was found almost completely covered it with a
malachite layer that gave it a greenish-blue colour. Sur-
face layers, such as iron oxides most likely formed by
dissolving the iron rim and handle which have not been
preserved until today, as they have completely degraded,
probably while the kettle was still underground.h pp
y p
Below two samples a layer of malachite was found:
Sample 5 is brown in colour and Sample 6 is black. Analyses have shown that these are Fe, Sn and Si
oxides. The brown sample (Sample 5) showed a greater
content of iron, and it can be concluded that it consists
mainly of iron oxides. The black surface layers (Sample
6) indicated presence of tin oxides, as well the pres-
ence of SiO. The areas on the kettle where Sample 5
was taken had brown surface layers. The layer of mala-
chite beneath the brown surface layers is slightly thin-
ner, i.e. on every part of the metal surface on which a
stable surface layer is not formed, has a possibility of
later oxidation to occur when exposed to air, and the
result is the creation of new corrosion products after
excavation of the object. On Sample 6 tin oxide prevails
which explains the black colour of the surface layers. Abbreviations
XRF X R
Fl Abbreviations
XRF: X-Ray Fluorescence; FTIR: Fourier transform infrared spectroscopy; ATR
: Attenuated total reflection; SEM: Scanning electron microscopy; FE-SEM:
Field emission scanning electron microscope; EDS: X-ray energy dispersive
spectroscopy. Discussionh Part
of the Fe2O3 creation may have been caused by the deg-
radation of the ferrous alloy handle. As ferrous alloy
degraded, ferrous corrosion products were created
locally. Vicinity of ferrous objects such as a rim or some
other object can also be source of Fe oxides forma-
tion, but there was no evidence if other ferrous objects
were found in such vicinity of the kettle itself. This
would explain the inability to form malachite below
this layer of corrosion products, since galvanic corro-
sion occurred and the iron dilution rapidly accelerated,
while in these areas copper was cathodically protected. to the kettle. The sample was taken at the top of the ket-
tle, where the handle would have been located. Unfor-
tunately, no part of the handle was preserved, however,
most of the kettles found in that period had one. Part
of the Fe2O3 creation may have been caused by the deg-
radation of the ferrous alloy handle. As ferrous alloy
degraded, ferrous corrosion products were created
locally. Vicinity of ferrous objects such as a rim or some
other object can also be source of Fe oxides forma-
tion, but there was no evidence if other ferrous objects
were found in such vicinity of the kettle itself. This
would explain the inability to form malachite below
this layer of corrosion products, since galvanic corro-
sion occurred and the iron dilution rapidly accelerated,
while in these areas copper was cathodically protected. Acknowledgements The authors would like to thank Sisak Municipal Museum for providing the
object of this matter for investigation of surface layers and corrosion products
development as well as for the conservation-restoration treatment. The
authors are very grateful to dr. sc. Damir Kralj for access to FTIR. Conclusionsh In
this case these oxides were also formed after the for-
mation of malachite, since by removing the powdered
black coloured product underneath the already formed
green malachite layer was found. On the same sample,
compounds such as Fe2O3 were found and the presence
of carbonates was detected. The item was excavated in the 1960s and stored in a
museum depot. After excavation, the object came again
in an oxygen-enriched and polluted atmosphere and it
developed additional layers of corrosion products over
the malachite layer. The analysed samples showed that
the upper layers are mainly iron oxides and tin oxides,
which are brown and black. Also, calcium carbonate and
silicon oxides are present on the kettle. They are com-
pounds that remained trapped in the corrosion products
from the mud of the Kupa river. Sulphur presence was
detected on all samples, mostly in the form of sulphates,
both inorganic and as patina. These compounds were
also most likely formed last during exposure to the pol-
luted atmosphere in the city of Sisak. Sulphur compounds exist in highest amount in Sam-
ple 4 (XRF results), and in somewhat smaller amounts
in Samples 1, 2 and 3, and smallest amounts are found
in Samples 5 and 6. The peak representing sulphate (ca. 1100 cm− 1) is clearly observable on Samples 1–3 while
on sample 4 it is observable as a shoulder peak partially
overlapped by the very strong silica peak (ca. 1000 cm− 1). We assume that sulphur presence on the surface of
bronze is from a newer date since the city Sisak was one
of the main industrial cities in Croatia with very high air
pollution in the recent centuries. Thus, these surface lay-
ers were created after excavation, on parts of the kettle’s
surface which was not completely protected by other sur-
face layers. Discussionh When copper alloys are buried in damp places, they tend
to form green corrosion products such as georgite, which
forms before malachite. Also, the blue-coloured mineral
azurite may appear, however, in little less humid places,
but it is also prone to creation under buried conditions
[6]. Neither georgite or azurite were found in our case.h Fig. 10 Prevalence of malachite on the bronze kettle The samples had several other compounds present
on the surface. Presence of SnO2 which is black in col-
our in the form of cassiterite was observed on most of
the samples. Tin oxide is a common corrosion product
in all tin bronzes in various environments. Among oth-
ers, it forms when tin is exposed to fire in presence of air
[30]. Since this type of kettle was used for cooking part
of the present SnO2 was formed while it was used to pre-
pare food on open fire. Thus, we can assume that some
of the SnO2 was created before the kettle was buried in
the river mud and remained on the kettle as it is not solu-
ble in water. We assumed that beneath malachite a layer
of cuprite, Cu2O, is present. Such a layer when formed in
atmospheric conditions is fully attained at thickness of Fig. 10 Prevalence of malachite on the bronze kettle Table 3 Overview of the different compound’s presence on the examined samples
H high content, L low content
Sample
1
2
3
4
5
6
Malachite, Cu2(CO3)(OH)2
H
H
H
L
L
L
Cassiterite, SnO2
H
H
H
-
-
L
Calcium carbonate, CaCO3
H
H
H
L
-
-
Ferric oxide, Fe2O3
L
L
L
H
H
H
Inorganic sulphates
L
L
L
H
-
-
Sulphate patina
-
L
L
-
-
-
SiO2
L
-
-
-
H
H
Si-oxides
-
L
L
H
-
- Table 3 Overview of the different compound’s presence on the examined samples Kotlar et al. Herit Sci (2021) 9:10 Kotlar et al. Herit Sci (2021) 9:10 Page 12 of 13 Page 12 of 13 to the kettle. The sample was taken at the top of the ket-
tle, where the handle would have been located. Unfor-
tunately, no part of the handle was preserved, however,
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lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. 11. Nunez L, Reguera E, Corvo F, Gonzalez E, Vazquez C. Corrosion of copper
in seawater and its aerosols in a tropical island. Corr Sci. 2005;47:461–84. https://doi.org/10.1016/j.corsci.2004.05.015.
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Mapping the Glocal Turn: Literature Streams, Scholarship Clusters and Debates
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Glocalism
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MAPPING THE GLOCAL TURN:
LITERATURE STREAMS, SCHOLARSHIP
CLUSTERS AND DEBATES VICTOR ROUDOMETOF
Department of Social and Political Sciences
University of Cyprus
roudomet@ucy.ac.cy Abstract: Based on a bibliographical survey, this article presents evidence of a silent glocal turn in 21st
century academia. Several terms compete for describing the newfound situations of hybridity and fusion
in the world, and glocalization is a new term that offers a high level of precision in comparison to other
contenders. Three specific clusters of inter-disciplinary scholarship are identified as cutting edge areas of
research: the study of consumer culture, the field of urban studies and the study of management and/or
organizations. Within these areas, glocalization is employed in varied and often contested ways according
to specific research agendas. Glocalization thus has become a contested term. The article identifies and
describes three debates that involve contrasting appropriations of glocalization. First, there is a contrast
between geographical and social interpretations of glocalization, which in turn are based on contrasting
definitions of space (geographical versus social). Second, there is a debate over the extent to which glo-
calization is sufficiently incorporated into global studies, or whether glocal studies should be defined
separately from global studies. Third, there is a contrast between homogenization versus hybridization
advocates in cross-cultural management and the social sciences. Although often cast as a conflict between
proponents of globalization versus proponents of glocalization, this particular debate might be trans-
cended in favor of more inclusive perspectives that suggest a “both/end” solution over an “either/or”
interpretation of the opposing views. Glocalization is a recent addition to the vocabulary of 21st century
humanities and social sciences. Its employment is also part of a broader wave of interest in the glocal that
is not contained within these fields but, rather, extends further into information-communication tech-
nology (ICT), medicine and environmental science. To mention one such example, it is not accidental
that the glocal has been invoked in the context of discussions about the “participatory web” or “Web
2.0” (Boyd 2005). It is obviously impossible to address all the twists and turns within the multitude of
fields that have employed the terms glocal and glocalization in the course of a single discussion. Inevita-
bly, a full treatment is reserved for a lengthier and more in-depth discussion elsewhere (Roudometof
Forthcoming). In the following, then, I restrict myself to an overview of the employment of the glocal. MAPPING THE GLOCAL TURN:
LITERATURE STREAMS, SCHOLARSHIP
CLUSTERS AND DEBATES Although I briefly touch on the employment of glocalization in business, I nonetheless concentrate on
the humanities and social sciences. The goal is to present an overview of the various bodies of literature,
to identify and discuss clusters of scholarship where glocalization is among the important research foci
and to offer a brief overview of emerging debates within this nascent field of study. As I have stated else-
where (Roudometof 2015) the glocal is a new word whose origins most likely lie somewhere in the early
1990s. It stands in sharp contrast to the global, the appearance of which dates back to the late 19th or
early 20th centuries. The emergence of the glocal in scholarly discourse is a feature of the post-1989 era,
and its rise has been ubiquitous after the turn of the millennium. Keywords: glocalisation, geographical, social, hybridization, homogenization. THE SILENT GLOCAL TURN Unlike cosmopolitanism and globalization, two concepts that
have become extremely popular among academic audiences and,
since the fall of communism, have saturated literature in a variety ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved VICTOR ROUDOMETOF 2 of fields, the terms glocal and glocalization have been far less pop-
ular as an explicit object of academic preoccupation. For example,
in the Routledge International Handbook of Globalization Studies
(Turner 2010), glocalization does not even appear in the volume’s
index. And yet, a headline in the Financial Post (Shaw 2011) de-
clared that “Glocalization Rules the World”, and in an article in
Time magazine, the post-crisis economy is described as “going glo-
cal” (Foroohar 2012)1. It is precisely this important disjuncture
between the practical employment of glocal in research and also in
the popular press, on the one hand, and the relative under-
theorization of the glocal among the academic community, on the
other hand, that gives rise to the realization that there is a glocal
turn in academic research. That turn has been somewhat muted or
to be more precise it has been a silent one: while using the term,
many researchers do not engage with it theoretically. But as this
discussion shows, glocalization has gained a prominent place in
intellectual discussions in several disciplines or fields of study. p
y
After all, the disjuncture between the growing academic pop-
ularity of glocalization and the explicit engagement with glocaliza-
tion in its own right has created the conceptual room for the crea-
tion of this journal. Evidence of the glocal’s growing usage is in-
controvertible. Based on a survey of several databases from EBSCO
Host there were a total of 4,079 entries using the word glocal in
text2. There is a clear rise in the use of the word with 511 entries
reported between 1996 and 2003 and over 3,000 entries appear-
ing between 2004 and 2014. Although the glocal is increasingly
employed in academic discourse, it is not always consistently used
and the concept of glocalization is not always uniformly interpret-
ed. That is by no means surprising, as different and sometimes
contrasting research agendas pursue different interpretations. ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved THE SILENT GLOCAL TURN If one adopts a broader view of the literature and a less literal
approach – in other words, if the criterion used is whether people
use a notion akin to the glocal – it is clear that the general notion
of the glocal is quite extensive (for such an approach, see Robert-
son 2013). Such an approach though tends to blend the glocal
alongside some other popular terms: hybrid, syncretism, trans-
cultural, mestizae and creole (for an overview, see Burke 2009: 34-
65; for an example, see Ritzer 2011). Of course, the idea of fusion
or cultural hybridity is among the most widely diffused notions in
21st century academia. The popularity of this idea is partly related ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved MAPPING THE GLOCAL TURN 3 to the very biographies of individual authors – as several intellec-
tuals (Edward Said, Stuart Hall, Homi Bhabha, Paul Gilroy and
Roland Robertson, to name just a short list of them) have incar-
nated such hybrid existence. y
The contribution of Latin American scholarship to the revi-
talization of these ideas is considerable. It is from within that re-
gion that the terms transculturalism, mestizae and creole emerged
and eventually transferred into the discourses of North Americans
and Europeans. The idea that intercultural mixed peoples (mé-
tissage) offer the key in legitimizing Latin American identity is a
notion originally put forth in 1891 by José Marti in an article en-
titled, “Nuestra America”. Métissage was viewed as a distinctive
trait of a culture founded upon a mixture of the native population
with different immigrant groups. In the 1940s Fernando Ortiz
developed this notion further in order to articulate the concept of
transculturalism. Calcini (1995) has furthered Ortiz’s ideas and
expanded them into the idea of cultural hybridity. Eventually, the
concept of transculturalism gained a foothold not only in the
fields of literary studies but also in the social sciences (for exam-
ples, see Antor et al. 2010; Stockhammer 2012). In Ortiz’s initial formulation, transculturalism entails a syn-
thesis of two simultaneous phases: a de-culturing of the past and a
métissage of the present (Cuccioletta 2001/2002). American cul-
ture is thus conceived as a new common culture based on the
meeting and intermingling of different peoples and cultures. ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved THE SILENT GLOCAL TURN Commonly referred to in Latin American countries as creole cul-
ture, this form of hybridity has come to characterize the national
cultures of several nations in that region (Cohen 2007; Burke
2009: 61-65). There are of course important vicissitudes stem-
ming from the term creole, which has a deep historical connection
to racial issues and nation-building in Latin America. As such, the
term creole is perhaps too specific for generalization. But the broader idea, that of a third hybrid culture, quickly
left the confines of its original Latin American milieu and became
a portable notion, especially thanks to the work of Homi Bhabba
(1994). In the 21st century, glocal hybridity has been used to de-
scribe the formation of new third cultures and has become a factor
influencing higher education worldwide. As Patel and Lynch
(2013) noted, glocalization in higher education offers an alterna-
tive to the conventional strategy of internationalization. It em- ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved 4 VICTOR ROUDOMETOF braces third culture building, thereby promoting global communi-
ty building. It thus offers a strategy that encourages the enhance-
ment of the learners’ glocal experience through a critical academic
and cultural exchange of global and local socio-economic and po-
litical issues. This realization is not theoretical, as glocal students
have already been identified as a target group – and universities are
eager to capitalize on this new potential target market. The above discussion has made it quite clear that, instead of
conflating the glocal with other related terms (hybrid or transcul-
tural or creole), it is far wiser to adopt a more literal approach. If
one’s attention is focused on the terms glocal and glocalization
and not on the aforementioned related terms, there is still an im-
pressive list of fields of study where these terms are used. The very
act of their employment is an apt demonstration that researchers
have found them to be terms that are of particular relevance and
utility to their own work. These researchers come from an extensive range of fields and
areas of study: the study of popular music and musical cultures
and subcultures (Chang and Amam 2012; Kim and Shin 2010;
Seago 2004), education (Caena 2014), social work (Hong et al. THE SILENT GLOCAL TURN 2010), language and translation (Riemenschneider 2005; Tong
and Cheung 2011), the sociology of sport (Giulianotti and Rob-
ertson 2007; Jijon 2013; Weedon 2012), literary criticism
(Langwald 2011), religion (Beyer 2007; Robertson and Garrett
1991; Roudometof 2013, 2014a, 2014c), theology (Pearson
2007), geography (Short 2001; Swyngedouw 2004), environmen-
tal science (Gupta et al. 2007), urban studies (Lin and Ke 2010;
Sassen 2004), European studies (Robertson 2014), global studies
(Pieterse 2013), consumer culture (Lam 2010; Matusitz 2011;
Ritzer 2003a, 2003b; Smith and Hu 2013), social movements’ re-
search (Harsin 2014; Urkidi 2010; Waisanen 2013), methodology
(Gobo 2011; Salazar 2010), art and culture (Cheung 2014; de
Duve 2007), mass communication (Dowd and Janssen 2011; Mo-
ran 2009), international marketing (Sinclair and Wilken 2009;
Sutikno and Cheng 2012), organizations (Czarniawska 2010;
Drori et al. 2013a), criminology (Heeres 2011) and terrorism
(Marret 2008). The above listing comprises only a partial thematic
account of different areas of study and is not an attempt at an ex-
haustive listing of the various publications. Its goal is to provide a ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved 5 MAPPING THE GLOCAL TURN general idea of the wide range of topics to which the glocal has al-
ready been applied. CLUSTERS OF SCHOLARSHIP The popularity of glocalization among diverse fields contrib-
utes to the emergence of divergent research agendas. These offer
the opportunity to intersect the glocal into specific areas of schol-
arship. It is possible though to identify some specific clusters that
feature the growing relevance of the glocal and of glocalization for
the scholarly community. First, the glocal is widely employed in the study of consumer
culture, a vibrant area of inquiry with contributions from not only
sociologists and anthropologists but also scholars from business
and management. The notion of glocalization has been employed
in the context of debates on the role, significance and impact of
consumption upon cultures and societies around the globe. This
debate is generally polarized between proponents and critics, and
this polarization reflects at least two distinct approaches to the
study of cultural economies. On the one hand, it is possible to explore the socio-economic
facets of various organizations and trace their social and cultural
implications. Social scientists most often adopt this strategy in or-
der to articulate a critique of the organizational logic of capitalist
enterprises. From within these lenses a variety of terms have been
developed to describe this logic: these terms range from grobaliza-
tion (Ritzer 2003a) to McDonaldization (Ritzer 1993/2000;
American Behavioral Scientist 2003) to Americanization (Beck et
al. 2003) and Disneyization (Bryman 2004). In most cases, the
analysis focuses in outlining the organizational logic of firms and
tracing its repercussions for cultures and societies. On the other hand, it is possible to explore the cultural ap-
propriation or context-specific tailoring of various products, goods
and services. In this line of analysis, the focus of analysis lies in
specifying the manner in which local distinctiveness blends or in-
tertwines with global blueprints. The emphasis is squarely on the
people’s ability or the enterprise’s willingness to adapt, shift or
modify their commercial products in order to make goods and
services relevant to diverse cultural contexts. In this second line of ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved VICTOR ROUDOMETOF 6 interpretation, the focus lies not on the management of organiza-
tions or the cultural logic of the capitalist enterprises but, rather,
on the varied, multiple and at times subversive appropriations of
the same commercial object in diverse cultures. CLUSTERS OF SCHOLARSHIP Jenkins’s (2006,
2013) notions of participatory culture and his interpretation of
active audiences as textual poachers are two widely cited reference
points, but in fact, they represent an entire line of research in mass
communication3. This approach rejects the cultural doping of audiences and in-
stead adopts de Certeau’s (1984) argument that the notion of con-
sumption itself obscures the users’ active role – and that one needs
to understand what users actually do with commercial goods. Hence, researchers focus on the meanings constructed and pro-
jected by content users – consumers are seen as prosumer-oriented
audiences. Originally Toffler (1980) defined prosumers as people
who produce some of the goods and services entering their own
consumption – when people produce for use, production and con-
sumption are united in the same person. When they produce for
exchange, then production and consumption are separated. In sev-
eral instances – ranging from music fans to hackers – people who
originally began as prosumers, working in a DIY mode, ended up
constructing the foundations for commercial products. The list of
such names includes icons of contemporary technology, such as
Steve Jobs – but also cultural icons, such as Jonny Rotten. These
examples vindicate Simmel’s insight that in modern and even
post-modern societies, culture becomes a source of value – which
in turn is commercialized and eventually evaluated in money. Start-up companies are based on this principle, e.g., transforming
passion into business. Perhaps the most widely known cases of
such audiences include the varied musical scenes across the globe
and the glocal youth culture; both form indispensable components
of the global entertainment scene (Kjeldgaard and Askeraard
2006; Seago 2004). Second, glocalization is used in the cross or inter-disciplinary
area of urban studies, an area that combines contributions from
geography, sociology, urban planning and related fields4. In these
fields the spatial component is an important focus of inquiry, and
the micro-level forces are viewed not solely as passive recipients of
large-scale macro-processes but also as active agencies. ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved 7 MAPPING THE GLOCAL TURN MAPPING THE GLOCAL TURN In particular, geographers have examined the spatial dynamics
of cities – and the relationship between glocalization and urban
life. They are not the only ones who have highlighted the signifi-
cance of the urban context for the study of the glocal. For Bauman
(2013: 4) the urban space – the “middle level” or the “level of
one’s own society” – operates like a laboratory “inside which fu-
ture modes of human cohabitation, made indispensable by global-
ization and enabled to emerge by the “glocalization” form it took,
are designed and tested”. The urban context offers the opportuni-
ty to their dwellers to learn how to apply new modes of human
cohabitation in the practice of shared life: The word glocal implies the bridging of a hiatus from the particular
to the general, a conceptual jump across a discontinuity formulated in
geo-political terms: the city, the world (...). The glocal ethos, we might
argue, adapts cosmopolitanism to the needs of our time. (de Duve 2007:
683) Cities get involved in international activities as a reaction to
socio-economic processes and serve as nodal points in the new in-
formation and network economy5. As a result, cities can become
disembedded from the national territorial context because their
fates depend more on their international contacts than on their
national ones. In his If Mayors Ruled the World, Benjamin Barber
(2013) offers a brilliant example of glocalism applied in urban
studies and public policy. He argues that, in the 21st century, na-
tions have become increasingly dysfunctional in their efforts to re-
structure society and to address a range of contemporary social
problems – from environmental issues to terrorism or gun control: If mayors ruled the world, the more than 3.5 billion people (over
half of the world’s population), who are urban dwellers and the many
more in the exurban neighborhoods beyond could participate locally and
cooperate globally at the same time – a miracle of civic “glocality” prom-
ising pragmatism instead of politics, innovations rather than ideology
and solutions in place of sovereignty. (Barber 2013: 5) Barber argues that cities have been the original locus of crea-
tivity, immigration and thus civilization – but were overtaken by
states due to questions of scale, which they were unable to address. ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved MAPPING THE GLOCAL TURN Today, though, the interconnectedness of the world means that ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved 8 VICTOR ROUDOMETOF scale becomes an insoluble obstacle to states (Barber 2013: 23). While states feel compelled to protect and safeguard their cher-
ished sovereignty, cities, not having sovereignty, are able to apply
soft power and soft governance models. “Nation-states cannot ad-
dress the cross-border challenges of an interdependent world” [and
as a result] “the forward to cosmopolis may demand of us a jour-
ney back to the polis” (Barber 2013: 77). Instead of nations, it is cities that offer the most suitable ter-
rain for global restructuring. “Glocality strengthens local citizen-
ship and then piggybacks global citizenship on it” (Barber 2013:
23). It is not prime ministers but mayors who count; successful
mayors approach problem solving pragmatically and cross over
partisan party lines. Barber offers extensive documentation of the
spawning network of urban municipalities that crisscross the
world and connect thousands of cities into networks of coopera-
tion. Third, the glocal has gained the interest of management
scholars through the realization that management needs to be
aligned with global trends toward sustainability, ethical responsi-
bility and local accountability. One of the first books in cross-
cultural management issues containing the word “glocal” was pub-
lished in Malaysia (Abdullah 1996) by a Malay corporate consult-
ant. Her emphasis throughout the book is to find a blend between
Malay cultural roots or akar and the demands of the modern
business workplace. Hilb (2009) published the first international
textbook focusing on glocal management of human resources6. g
g
g
Under the auspices of the UN’s Principles for Responsible
Management Education (PRIME) network, 500 educational insti-
tutions collaborated, leading to the publication of a management
textbook that aims to incorporate these dimensions into the field
(Conaway and Laasch 2015). In the fields of organizations and
management, the growth of research on glocalization has taken
place as an extension of the world society perspective into these
fields (Drori et al. 2013a, 2013b). MAPPING THE GLOCAL TURN This line of inquiry extends
conventional foci of the world society perspective (such as loose
coupling, incomplete diffusion and disjuncture) into the cross-
cultural and international study of firms and organizations. For
Drori et al. (2013b: 10), glocalization “involves translation – as in
order to adjust ideas, structures and models to new and different
social and cultural domains”. While the world society perspective’s ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved MAPPING THE GLOCAL TURN 9 notion of theorization “emphasizes top-down influence” in the
process of global diffusion, “the dynamic nature of transcendental
glocalization is a rebound effect [...] where locally enacted ideas
and models influence the globally theorized schemes” (Drori et al. 2013b: 10). This turn of events demonstrates the significance of trans-
disciplinary cross-fertilization and illustrates the establishment of
connections that prompted the initial formulation of the glocal in
the early 1990s. Unlike the conventional narrative concerning the
emergence of glocal, the fields of business and management were
not in fact forerunners in the use of glocal in scholarship. Rather,
the employment of glocalization in these fields has mirrored its
growing popularity across diverse fields of study. THREE DEBATES Within the contours of the material surveyed in this discus-
sion, it is now time to turn to some of the emerging debates with-
in glocalization scholarship. These debates reflect the growing at-
tention that the glocal has recently received among the scholarly
community and also register the interest of scholars to locate glo-
calization within specific schemes of interpretation or research
programs. This brief primer is meant basically as a means of orien-
tation and is certainly far from exhaustive. First, there is a tension between the geographical and the so-
cial interpretation of glocalization. Geographers have argued that
glocalization is something more than the mere juxtaposition or in-
terplay and interpenetration of the local and the global. It involves
relationships among the sub-national (or local), the national and
supranational (or global). Perhaps the most straightforward con-
ceptualization of the glocal concerns the spatial understanding of
the term (Swyngedouw 1997, 2004; Swyngedouw and Kaıka
2003). Accordingly, space forms a nested scalar hierarchy running
all the way from the global to the regional, national and local. This image is reminiscent of the Russian dolls (matryoshka dolls)
that fit one inside the other. This conception represents a scalar
understanding of the glocal: global, local and glocal are concepts
that indicate the sheer scale of a specific process or social phenom-
enon. ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
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Some rights reserved ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved 10 VICTOR ROUDOMETOF Although the nested hierarchy or scalar approach to glocaliza-
tion can thus offer tools for interrogating urban strategies and con-
tentious politics of scale, it also raises important questions about
space. These pertain to the broader issue of understanding glocali-
zation as such. By far the best way to make sense of the geogra-
phers’ engagement with the glocal is to understand the central im-
portance of the nature of space or any other spatial term (territory,
place or network). Space can be interpreted quite differently de-
pending on whether it is seen as absolute space or as relative space. Absolute space refers to units that can be measured numerically (in
terms of miles, kilometers and so on). THREE DEBATES Absolute space is ontologi-
cally given – that is, it exists independently of the way it is per-
ceived. This space is “real” in a realist sense. Absolute space is an
external given that in turn has neutral discursive meaning. In con-
trast, relative space refers to space as it is perceived by humans. It
does not correspond to a fixed unit and is not measurable; rather it
is the humans’ “sense of space” that matters. Relative space varies
according to the specifics of human culture, available technology
and resources. Both sociologists and anthropologists have argued that the
glocal is a metaphor for a collectively imagined space – or a social
space. The local and the global should not be seen as binary oppo-
sites, as the local is constructed in contradictory ways and always
has been, at least partly, the product of outside influences (Appa-
durai 1995). Such an interpretation inherently dovetails with the
notion of the glocal. Salazar (2010) suggested the notion of glocal
ethnography, and Holton (2008) adopted a somewhat similar posi-
tion through his use of the phrase methodological glocalism. Glocal
ethnography, however, does not employ the model of nested hier-
archy that is the characteristic of global ethnography; that is, it
does not conceive of the global, national and local as nested con-
centric spaces. It is plain to see that, depending upon whether
space is viewed as relative (social) or absolute (geographical), radi-
cally different interpretations of glocalization can emerge. Second, there is debate over whether glocal studies and global
studies form or should become distinct fields of study (Pieterse
2013; Roudometof 2015). In particular, Pieterse (2013) has sug-
gested that global studies emerges as a consequence of global-level
data, e.g., data that are about the world as a whole. To make the
point more explicit, the various international social survey pro- ISSN 2283-7949
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Some rights reserved ISSN 2283-7949
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2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved MAPPING THE GLOCAL TURN 11 grams [EVS (European Values Study), ISSP (International Social
Science Program), WVS (World Values Survey), ESS (European
Social Survey)] deliver new objects of inquiry that make it possible
to study social relations in a manner hitherto impossible. THREE DEBATES The
emergence of such “new objects of study” (Pieterse 2013: 5) is
partly the result of greater interconnectivity (greatly facilitated by
ICTs) as well as multiple and increasing interactions of different
actors upon each other. Pieterse’s perspective inevitably stresses the integral notion of
the global – and not the idea of globalization as a self-limiting pro-
cess. This latter viewpoint has been endorsed by Robertson
(2013); it is a viewpoint that adopts Turner’s (2007) “enclave so-
ciety” thesis and suggests that globalization involves not only the
construction of new models or units of integration but also the
systematic fragmentation of pre-existing units and the construc-
tion of new units and groups that exist behind new barriers to un-
restricted communication and movement. Increasingly, in the af-
termath of the 2008 global economic crisis, walls were erected to
obstruct the free flows of trade, money and people, as govern-
ments adopted a selective approach concerning trade partnerships,
foreign capital investment and immigration policies (Samuelson,
2013). In light of this new entrenched reality (the “new normal”),
globalization is not viewed as the deliverer of a new singularity;
instead, it produces a multitude of fragmentation – hence, it is in
effect glocalization (see Steger 2013: 775-776). It is rather evident
that this interpretation directly clashes with Pieterse’s interpreta-
tion. Of course, commentators (Juergensmeyer 2013; Khondker
2013; Steger 2013) have suggested that conceptual and empirical
opportunities exist for inserting glocalization into the practice of
global studies. There is much to be gained from maintaining an
inclusive strategy in global studies and avoiding further fragmenta-
tion through the creation of global studies and glocal studies. Still,
as I have argued elsewhere (Roudometof 2015), one cannot ex-
clude the possibility that one of the possible outcomes is that the
entire debate on globalization, or what used to be called “globali-
zation studies”, might eventually settle into four partly overlap-
ping but relatively coherent networks or groups of like-minded
scholars: global studies; glocal studies; transnational studies; and ISSN 2283-7949
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Some rights reserved 12 VICTOR ROUDOMETOF cosmopolitan studies (for overviews of the last two fields, see
Levitt and Khagram 2007; Delanty 2012). ISSN 2283-7949
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2015, 3, DOI: 10.12893/gjcpi.2015.3.1
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Some rights reserved THREE DEBATES Third, within business studies (e.g., international manage-
ment and cross-cultural marketing) there is what might be called
the standardization versus heterogenization debate – although of
course different words can be used to convey this general idea (for
example: globalization versus localization or indigenization). This
particular debate has an extensive spillover effect into debates
within sociology and anthropology. In business, the origins of this
debate lie with Levitt’s (1983) classic work about the globalization
of markets. Levitt, who historically has been credited among busi-
ness’ scholars as the very inventor of “globalization”, translated in-
ternationalization into standardization; he argued that the latter
would bring forth economies of scale and make the former a lucra-
tive business opportunity. For a period of time, it seemed that this
was indeed the appropriate economic logic for TNCs (transna-
tional companies) and MNCs (multinational companies). p
p
However, in due course of time – through the practical con-
frontation with the realities of cultural differences – it became evi-
dent that such a strategy was not necessarily a uniform guide for
all firms but also might not be a suitable strategy for TNCs and
MNCs. Instead, firms chose to glocalize their marketing, and that
has been documented through analyses of the commercial strate-
gies of Coca-Cola, McDonalds, Procter & Gamble (Sinclair and
Wilken 2009), Starbucks (Maguire and Hu 2013) and Nike (Ko-
bayashi 2012). This corporate glocalization is particularly pro-
nounced in Asia, reflecting both the global importance of the re-
gion as well as the necessity for tailoring into specific cultural con-
texts. As a result, the choices among standardization versus locali-
zation or a blend between the two have all become available op-
tions that managers can choose – a choice that largely depends
upon the specific situation of a particular firm, its constraints and
its available options (see Mareck 2014). A similar divide also has resurfaced within the social sciences,
albeit in a different format. It concerns the well-known division
between the proponents of hybridization (Pieterse 1995) or glocal-
ization (Robertson 1995) versus the proponents of McDonaldiza-
tion (Ritzer 1993/2000), grobalization (Ritzer 2003a, 2003b) or
Americanization – or for that matter, any other conceivable term
that registers cultural homogenization. THREE DEBATES This divide reflects con- ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
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Some rights reserved MAPPING THE GLOCAL TURN 13 trasting scholarly orientations. Glocalization scholarship in general
highlights the extent to which people are seen as active and crea-
tive agents who construct new forms of authenticity out of the
commercial items that are at their disposal. In contrast, critical
consumer-culture scholarship highlights the extent to which cor-
porations, firms, nations or other large-scale organizations super-
impose their will upon geographical locations, thereby turning
people into servants of their will to profit and eroding the substan-
tive foundations of cultural meaning in society. It is instructive
that, building on George Romero’s sci-fi cult classic, Ritzer
(2003c) has referred to McDonaldized systems as “islands of the
living dead”; although there is much life on these “islands”, they
also are in many senses “dead”. The zombie analogy is of course
highly revealing. g y
g
It should be immediately observed that the latter line of re-
search carries out a rather explicit critique of contemporary society
and culture. It is therefore perhaps convenient – but extremely
misleading – to suggest that the former research agenda is more
conservative whereas the latter is more progressive. In fact though,
glocalization has been evoked in a variety of contexts and situa-
tions of protest or conflict against corporate interests. Examples
include analyses of social movements against gold mining and po-
litical struggles over the organization of workers in the informal
economy in Latin American countries (Lindell 2009; Urkidi
2010) or anti-authoritarian movements in Serbia (Waisanen
2013) and protests in France (Harsin 2014). In a study of the
emergence of local organizations and social movements in Ecuador
and Peru, Bebbington (2001) argues that these are shaped by the
constraints and possibilities that occur within the local move-
ments’ relationships with wider transnational development net-
works. Forms of global entanglement vary greatly across sites. Be-
cause the effects of globalization on livelihoods and landscapes
vary widely, Bebbington suggests historically situated studies of
glocalization to capture real-life effects of globalization into specif-
ic contexts. THREE DEBATES Furthermore, Fasenfest (2010) has highlighted the theoretical
significance of the extent to which oppositional politics themselves
can be “glocal”; this means that instead of the popular academic
stereotype of glocalization as a gimmick employed by TNCs and
MNCs (see for example Thornton 2000) people have in fact ISSN 2283-7949
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2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved 14 VICTOR ROUDOMETOF seized glocalization in order to develop suitable blueprints for
popular mobilization. The Occupy Wall Street movement offers a
highly relevant contemporary example; since its original 2011
launch, it has spread to over 100 US cities with actions in over
1,500 cities globally7. As the aforementioned examples make clear, both research
agendas can be appropriated quite differently to provide a means
for challenging global or transnational capitalism. However, the
question should not be posed in this manner; instead, the appro-
priate question concerns the extent to which these agendas offer
persuasive and relevant descriptions of contemporary life. Given
that both of them feature prominently across the social sciences, it
is important to realize that most researchers increasingly approach
them strategically, whereby the methodologically relevant question
is to select those aspects of their overall gaze that offer useful heu-
ristics for the analysis of specific contexts, cases and research sites. CONCLUSIONS In this article, I have sought to present a thematic overview of
the rise of glocalization across several scholarly fields of study. The
central objectives are: to offer a thematic overview of the literature
streams that glocalization has been employed; to highlight and de-
scribe the clusters of scholarship where glocalization represents a
particularly significant topic for researchers; and finally, to outline
scholarly debates concerning glocalization, its use and divergent
interpretations among researchers. p
g
Of course, it is not possible to exhaustively cover the diverse
fields of study; and hence, the focus of attention was thematic,
while extra attention was paid to social-scientific and business lit-
erature. It is hoped though that the inter- or trans-disciplinary
character of this survey allows readers to gain a better understand-
ing of glocalization. There are similarities and differences among
disciplines and fields of study. That is normal and reflects differ-
ences in research foci. In this overview, I have noted the close
similarity between the glocal and the varied terminology on cul-
tural hybridity – but I also noted the differences among the vari-
ous concepts. My choice was to adopt a literal approach, as it pre-
serves greater specificity. Even with such scope restrictions, among ISSN 2283-7949
GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
Published online by “Globus et Locus” at www.glocalismjournal.net
Some rights reserved MAPPING THE GLOCAL TURN 15 diverse scholarly streams the evidence of a glocal turn is incontro-
vertible. This glocal turn is not accompanied by the flurry of aca-
demic attention that has been reserved for other concepts such as
cosmopolitanism or globalization; but nonetheless, this relative
absence of extreme popularity might be a positive factor. It allows
researchers to work in more careful manner and build foundations
that might last longer. g
g
There are three clusters of scholarship in which glocalization
has been the object of particular scholarly interest: the area of con-
sumer culture (conceived as an inter-disciplinary field), the equally
inter-disciplinary area of urban studies and the fields of business
and management studies. In these areas, there are marked diver-
gences in the employment of glocalization. The study of consumer
culture raises questions over the meaning of the division between
production and consumption, and in this regard glocalization is
far more connected with perspectives that stress the pro-active role
of audiences and users. CONCLUSIONS In urban studies, the glocal has been iden-
tified as a particular configuration of urban space, and in certain
formulations it is seen as a promising new terrain that could lead
to urban rejuvenation and prosperity. In the areas of business, or-
ganization and management studies there has been a similar inter-
est in engagement with glocalization. However, these fields are not
necessarily the forerunners of glocalization as it is often naively as-
sumed; but their engagement with glocalization is often the result
of anthropological or sociological ideas that filter into their prac-
tices. Finally, three inter- or cross-disciplinary debates were pre-
sented. Of course, these by no means exhaust the conceptual ter-
rain. For example, glocalization has been interjected into the
scholarly debate concerning the relationship between globalization
and nationalism. Although traditionally nations and nationalism
were seen as mere extensions of modernization, some contempo-
rary scholarship has come to realize the dependency of nation-state
formation upon broader social processes and institutions (see for
example, Hutchinson 2011; Walby 2003). This theme has be-
come more pronounced in recent discussions (see Halikiopoulou
and Vasilopoulou 2011; Roudometof 2014b). The intersection of
glocalization into the discourse of nationalism offers a means to
further explore the cultural hybridity of nations – a theme that
features prominently in Latin American scholarship (Calcini 1995; ISSN 2283-7949
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Some rights reserved 16 VICTOR ROUDOMETOF Cohen 2007). Moreover, glocalization remains an area of active
research within the study of religion and theology (Pearson 2007;
Roudometof 2013, 2014a, 2014c) as well as in social linguistics
(Coupland 2010). p
While acknowledging the existence of vibrant debates con-
cerning glocalization among several disciplines and inter-
disciplinary fields, I have sought to capitalize on just three of them
in order to demonstrate the growing sophistication of scholarship
in its continuing engagement with glocalization. First, in urban
studies, geographers have suggested the nested hierarchy model of
geographical space as a means of incorporating the idea of the glo-
cal into their analyses. This interpretation departs significantly
from the social-scientific understanding of social or relative space,
whereby the glocal is produced by human action. It is necessary to
point out that these different understandings of space spearhead
different definitions of glocalization. MAPPING THE GLOCAL TURN 17 NOTES 1 Foroohar’s (2012) new rules of the post-2008 economy’s are: hometown bankers
know best, manufacturing matters, blue collar jobs go high tech, closer is faster and faster is
good, and local leadership must step up. The above reflect the growing significance of local
knowledge for an entire array of business – from banking to old-fashioned manufacturing. Thi
id
ti
f l
l ti
ti
t
i i “ l b li ti
” good, and local leadership must step up. The above reflect the growing significance of local
knowledge for an entire array of business – from banking to old-fashioned manufacturing. This reconsideration of local ties comes as a correction to pre-crisis “globalization” excesses. g
y
g
g
This reconsideration of local ties comes as a correction to pre-crisis “globalization” excesses. 2 Search results from February 20, 2014. The databases searched were: Academic
Search Complete, Business Source Complete, Communication & Mass Media Complete,
ERIC, GreenFILE, Humanities International Complete, Psychology and Behavioral Sci-
ences Collection, Humanities Abstracts (H.W. Wilson), EconLit, MLA International Bib-
liography, Political Science Complete &, eBook Academic Collection (EBSCOhost). g
3 Although Jenkins’s (2006, 2013) work is mainly about the US context, in their
cross-national study on the impact of mass communication within national studies, Norris
and Inglehart (2009) have confirmed that the “national filter” remains an important factor
shaping the impact of global cross-cultural communication. 4 For an excellent overview of the 1990s debates in urban studies see Roman (2006),
who offers lucid summaries of the perspectives developed by Neil Brenner and Erik
Swyngedouw. Brenner’s (1998, 1999) interpretation is too closely tied to conventional
arguments about capitalism, whereas Swyngedouw’s (2004) ideas are far more relevant. 5 It might be suggested that this debate concerns only a handful of “global cities”. However, none other than Sassen (2011) has highlighted the extent to which glocal ties can
offer valuable stimulus to Latin American urban contexts. 6 For additional perspectives on glocalization and management, see Svensson (2001),
Svensson and Anderson (2009), and the individual chapters in Drori, Höllerer & Walgen-
bach (2013). 7 For an analysis, see Castells (2012). For additional and more updated information
on the Occupy Wall Street movement, see their websites http://occupywallst.org/about/
and http://www.occupytogether.org/aboutoccupy. CONCLUSIONS Second, in global studies, the glocal represents a new notion
that, to date, represents a rather unexplored conceptual territory. To the extent that global studies privileges totalities over the glocal
fragmentation of practices, blueprints and ideas, it might lead to
the formation of glocal studies as a different inter-disciplinary
field. Regardless of this eventuality, it is clear that glocalization has
earned a place within the networks of scholars working on various
facets of globalization. g
Third, in the cross-disciplinary study of consumer culture,
there is well-known division among proponents of homogeniza-
tion versus proponents of heterogeneity. This division is observed
in management and business studies, but it also has migrated into
the social sciences. In this formulation, glocalization is often set
against Americanization, McDonaldization or grobalization. I
have argued however that, in all these disciplines, researchers in-
creasingly realize that one-sided accounts are oversimplifying exist-
ing social complexity. As a result, these theoretical perspectives are
not an issue of an “either/or” choice, but they offer analytically
construed alternatives that researchers can self-reflexively select on
the basis of the cases and goals they explore. ISSN 2283-7949
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2015, 3, DOI: 10.12893/gjcpi.2015.3.1
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Some rights reserved MAPPING THE GLOCAL TURN VICTOR ROUDOMETOF 18 VICTOR ROUDOMETOF N. Brenner (1998), Global Cities, Glocal States: Global City Formation and State Ter-
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GLOCALISM: JOURNAL OF CULTURE, POLITICS AND INNOVATION
2015, 3, DOI: 10.12893/gjcpi.2015.3.1
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Impact of a Heat Shock Protein Impurity on the Immunogenicity of Biotherapeutic Monoclonal Antibodies
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Pharm Res (2019) 36: 51
https://doi.org/10.1007/s11095-019-2586-7 RESEARCH PAPER ABSTRACT These data suggest that Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11095-019-2586-7) contains supplementary
material, which is available to authorized users. Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11095-019-2586-7) contains supplementary
material, which is available to authorized users. * Jeremy P. Derrick
Jeremy.Derrick@manchester.ac.uk
1
School of Biological Sciences, Faculty of Biology Medicine and Health
Manchester Academic Health Science Centre, The University of
Manchester, Michael Smith Building
Oxford Road, Manchester M13 9PT, UK
2
Medimmune Ltd, Granta Park, Cambridge CB21 6GH, UK
3
Medimmune, 1 Medimmune way, Gaithersburg, Maryland 20878, USA * Jeremy P. Derrick
Jeremy.Derrick@manchester.ac.uk * Jeremy P. Derrick
Jeremy.Derrick@manchester.ac.uk Impact of a Heat Shock Protein Impurity on the Immunogenicity
of Biotherapeutic Monoclonal Antibodies
Shraddha S. Rane1 & Rebecca J. Dearman 1 & Ian Kimber 1 & Shahid Uddin2 & Stephen Bishop3 &
Maryam Shah1 & Adrian Podmore2 & Alain Pluen1 & Jeremy P. Derrick1 Received: 2 November 2018 /Accepted: 5 February 2019 /Published online: 15 February 2019
# The Author(s) 2019 Received: 2 November 2018 /Accepted: 5 February 2019 /Published online: 15 February 2019
# The Author(s) 2019 * Jeremy P. Derrick
Jeremy.Derrick@manchester.ac.uk ABSTRACT heat shock protein impurities can selectively accumulate by
binding to mAb aggregates and thus influence immunogenic
responses to therapeutic proteins. Purpose Anti-drug antibodies can impair the efficacy of ther-
apeutic proteins and, in some circumstances, induce adverse
health effects. Immunogenicity can be promoted by aggrega-
tion; here we examined the ability of recombinant mouse heat
shock protein 70 (rmHSP70) - a common host cell impurity -
to modulate the immune responses to aggregates of two ther-
apeutic mAbs in mice. KEY WORDS adaptive immunity . aggregation . host cell
impurity . immunogenicity . monoclonal antibody KEY WORDS adaptive immunity . aggregation . host cell
impurity . immunogenicity . monoclonal antibody ABBREVIATIONS
ADA
Anti-drug antibody
AF4
Asymmetric flow field flow fractionation
APCs
Antigen presenting cells
BMDC
Bone marrow-derived dendritic cells
BSA
Bovine serum albumin
CHO
Chinese hamster ovary
DLS
Dynamic light scattering
HCP
Host cell protein
HRP
Horseradish peroxidase
IFNβ-1a
Interferon beta-1a
I.P. Intraperitoneal
mAb
Monoclonal antibody
NMS
Normal mouse serum
PBS
Phosphate buffered saline
RICS
Raster image correlation spectroscopy
rmHSP70
Recombinant mouse heat shock protein 70
ROI
Region of interest
scFv
Single chain variable fragment ABBREVIATIONS
ADA
Anti-drug antibody
AF4
Asymmetric flow field flow fractionation
APCs
Antigen presenting cells
BMDC
Bone marrow-derived dendritic cells
BSA
Bovine serum albumin
CHO
Chinese hamster ovary
DLS
Dynamic light scattering
HCP
Host cell protein
HRP
Horseradish peroxidase
IFNβ-1a
Interferon beta-1a
I.P. Intraperitoneal
mAb
Monoclonal antibody
NMS
Normal mouse serum
PBS
Phosphate buffered saline
RICS
Raster image correlation spectroscopy
rmHSP70
Recombinant mouse heat shock protein 70
ROI
Region of interest
scFv
Single chain variable fragment Methods Heat and shaking stress methods were used to gen-
erate aggregates in the sub-micron size range from two human
mAbs, and immunogenicity assessed by intraperitoneal expo-
sure in BALB/c mice. Results rmHSP70 was shown to bind preferentially to aggre-
gates of both mAbs, but not to the native, monomeric pro-
teins. Aggregates supplemented with 0.1% rmHSP70 induced
significantly enhanced IgG2a antibody responses compared
with aggregates alone but the effect was not observed for mo-
nomeric mAbs. Dendritic cells pulsed with mAb aggregate
showed enhanced IFNγ production on co-culture with T cells
in the presence of rmHSP70. Conclusion The results indicate a Th1-skewing of the im-
mune response by aggregates and show that murine
rmHSP70 selectively modulates the immune response to
mAb aggregates, but not monomer. INTRODUCTION For example, aggregate per-
centage and the extent of denaturation of interferon beta-1a
(IFNβ-1a) have been shown to influence the ability of aggre-
gates to break tolerance in transgenic mice [9]. Only aggre-
gates that retained native epitopes were able to stimulate a
transient immune response and their removal prevented the
breakdown of tolerance [9]. Aggregation is thought to be ini-
tiated by association of partially unfolded conformational
states which can occur at any stage of bioprocessing and stor-
age. They range in size and dimensions in the 0.1-10 μm
range have been identified as being the most immunogenic
[10]. Characteristics of aggregates which may contribute to
immunogenic potential include the formation of neo-epitopes,
multiple valency, post-translational modifications, concentra-
tion and size [11–13]. Despite the wealth of evidence demon-
strating that aggregation results in enhanced immunogenicity,
the underlying mechanisms are incompletely understood [14]. shown, for example, that HSP-peptide complexes successfully
elicited MHC class I restricted cytotoxic T lymphocyte re-
sponses, whereas HSP or peptide alone were not immunogen-
ic, establishing the tumor-derived HSP gp96 as the first ad-
juvant of mammalian origin [24]. HSP-based cancer vaccines,
such as artificially reconstituted HSP peptide antigen com-
plexes, have been widely exploited [25]. In addition, the ad-
juvant property of mycobacterial Heat Shock Protein 70 fu-
sion protein has been demonstrated using a variety of model
antigens [26]. Our laboratory has recently shown that low
levels of an E. coli HSP, DnaK, were able to enhance the
immunogenicity of a recombinant 25 kDa human single chain
variable fragment (scFv) following immunization of BALB/c
strain mice [27]. HSPs therefore have the potential to function
as adjuvants. The principal aim of the current investigation was to estab-
lish whether this adjuvant-like effect could also be observed
with aggregated human biotherapeutic mAbs and a cognate
mammalian HSP, similar to that found in CHO cells. To this
end, we used recombinant mouse HSP70 (rmHSP70), an
ortholog of E. coli DnaK which is 98% identical to CHO
HSP70 [27]. We show that rmHSP70 binds preferentially to
aggregates and is able to exert an adjuvant-like effect on im-
mune responses in a BALB/c mouse model. The implications
for the contribution of HCPs to the immunogenicity of ther-
apeutic protein aggregates are discussed. The removal of host cell protein (HCP) impurities is an
important problem in the isolation of biologics for clinical
use [15]. INTRODUCTION HCPs originate from the expression host, most com-
monly Chinese Hamster Ovary (CHO) cells for monoclonal
antibodies. Measurement of total HCP content is commonly
conducted by ELISA: null cell line isolates are used to immu-
nize mice and obtain polyclonal antisera. This method is lim-
ited, however, in that it is dependent on the total average
response to a crude mixture of HCPs which will, individually,
have differential abilities to stimulate antibody production
[15]. HCPs have been shown to influence immunogenicity
as antigens or adjuvants [16,17], through HCP-induced pro-
tease activity [18] or direct biological activity [17], highlight-
ing the need for HCP identification and individual
quantification. Animals Female BALB/c strain mice (8–12 weeks old) were used for
these experiments (Envigo, Bicester, UK). Mice were housed
on sterilized wood bedding with materials provided for envi-
ronmental enrichment. Food (Beekay Rat and Mouse Diet
No1 pellets; B&K Universal, Hull, UK) and water were avail-
able ad libitum. The ambient temperature was maintained at
21 ± 2°C and relative humidity was 55 ± 10% with a 12 h
light/dark cycle. All procedures were carried out in accor-
dance with the Animals (Scientific Procedures) Act 1986,
and approved by Home Office licence. Heat shock proteins (HSPs) are a common HCP impurity
[19]; they participate in housekeeping functions and are part
of responses to environmental stresses [20,21]. Their chaper-
one activity in macromolecular complex assembly, protein
transport and degradation acts to stabilize and correct the
folding of nascent polypeptides de novo, dissociating aggre-
gates, and re-folding stress-denatured proteins [22]. Stresses
can exacerbate protein conformational problems, exceeding
the capacity of chaperone systems to prevent aggregation. In
addition, the combined use of HSPs has been explored as a
strategy for enhancing vaccine potency [23]. Studies have INTRODUCTION Monoclonal antibodies (mAbs) are the fastest growing sector
of the global pharmaceutical industry, particularly for the
treatment of cancer and inflammatory diseases [1,2]. Unwanted immunogenicity and the formation of anti-drug
antibodies (ADAs) is a significant challenge for the use of
biotherapeutics, even for those that have been humanized 51
Page 2 of 14 Pharm Res (2019) 36: 51 51 [3], impacting safety and efficacy. A variety of factors can
influence immunogenicity: these include product-related fac-
tors such as protein conformation or impurities, patient-
related variables such as immune and genetic background,
disease status and treatment-related factors such as route
and duration of exposure [4, 5]. Aggregation is an important
factor which has been implicated in immunogenicity and its
likelihood is increased with the use of mAbs at high concen-
trations [6–8]. The link between aggregation and enhanced
immunogenic responses is well established in mouse models
(including transgenic animals). For example, aggregate per-
centage and the extent of denaturation of interferon beta-1a
(IFNβ-1a) have been shown to influence the ability of aggre-
gates to break tolerance in transgenic mice [9]. Only aggre-
gates that retained native epitopes were able to stimulate a
transient immune response and their removal prevented the
breakdown of tolerance [9]. Aggregation is thought to be ini-
tiated by association of partially unfolded conformational
states which can occur at any stage of bioprocessing and stor-
age. They range in size and dimensions in the 0.1-10 μm
range have been identified as being the most immunogenic
[10]. Characteristics of aggregates which may contribute to
immunogenic potential include the formation of neo-epitopes,
multiple valency, post-translational modifications, concentra-
tion and size [11–13]. Despite the wealth of evidence demon-
strating that aggregation results in enhanced immunogenicity,
the underlying mechanisms are incompletely understood [14]. [3], impacting safety and efficacy. A variety of factors can
influence immunogenicity: these include product-related fac-
tors such as protein conformation or impurities, patient-
related variables such as immune and genetic background,
disease status and treatment-related factors such as route
and duration of exposure [4, 5]. Aggregation is an important
factor which has been implicated in immunogenicity and its
likelihood is increased with the use of mAbs at high concen-
trations [6–8]. The link between aggregation and enhanced
immunogenic responses is well established in mouse models
(including transgenic animals). Monoclonal Antibodies Two human monoclonal antibodies were used for the current
study, hereafter referred to as mAb1 and mAb2. mAb1 has a
theoretical molecular mass of 148 kDa and an experimentally
measured pI of 7.6. mAb2 (a bispecific antibody) has a theo-
retical molecular mass of 204 kDa and an experimentally
measured pI of 9.1. Both the mAbs were provided by
MedImmune (Cambridge, UK). Page 3 of 14
51 Pharm Res (2019) 36: 51 Fluorescence Microscopy rmHSP70 (low endotoxin) was purchased and was obtained
commercially labelled with BODIPY FL by Life
Technologies, USA. BODIPY FL labelled rmHSP70 (0.1%)
was added to the thermal stressed mAb1 and shaking stressed
mAb2 aggregates. 2.5x SYPRO® Red dye was added to the
aggregated mAb immediately before analyzing the sample
using a Zeiss 510 Confocor 2 microscope. Asymmetric Flow Field Flow Fractionation (AF4) Purified mAbs were diluted into 1 mg/mL in Dulbecco’s
phosphate buffered saline (DPBS) without Ca+2 or Mg+2
(Sigma-Aldrich, St Louis, Missouri). In order to form aggre-
gates of mAb1 by thermal stress, it was treated at 60°C for
25 min. To generate mAb1 aggregates using shaking stress,
the solution at 1 mg/mL was shaken in a bench top shaker at
3000 rpm for 12 h at 22°C. mAb2 aggregates were formed by
shaking stress in the same way, but at 1500 rpm for 4 h at
22°C. rmHSP70-aggregate complex samples were prepared
by addition of rmHSP70 (Enzo Life Sciences, UK) to 0.1% by
mass into the mAb aggregate within 5 min of mAb aggrega-
tion. The aggregates formed were stable and did not dissociate
into monomers when the temperature was subsequently de-
creased by refrigeration, or after storage at −80°C. Asymmetric flow field-flow fractionation (AF4) is used to
measure aggregate content and molecular mass distribution,
as an orthogonal method to size exclusion chromatography or
analytical ultracentrifugation. In the current study, aggregates
of mAb1 (generated by thermal stress) and mAb2 (generated
by shaking stress) in the presence and absence of rnHSP70
were prepared as described above. Separation was achieved
by a liquid cross-flow which takes place in a narrow, ribbon-
like channel of trapezoidal geometry, which is built up by a
spacer, between a porous and a nonporous plate. The porous
plate is covered by a membrane, which acts as accumulation
wall and allows the eluent to pass the membrane, while the
particles/macromolecules are retained [30]. Water was used
as solvent for the method at 25°C, with 30 min analysis time
and 0.294 s sampling time. The total area of the peaks and the
molecular mass of the rmHSP70-aggregates were measured. Corresponding monomer mAbs (with and without rmHSP70)
were used for comparison. The ASTRA chromatography
software (Wyatt Technology) was used to analyse the data. Dynamic Light Scattering (DLS) Measurements of DLS were performed using a Malvern
Zetasizer Nano ZS ZEN3600 (Malvern, Herrenberg,
Germany), equipped with a 633 nm laser. Each sample
(70 μL) was measured in a Suprasil® quartz cuvette (Hellma
GmbH, Muellheim, Germany) with a path length of 3 mm
and 200–2500 nm spectral range. Monomeric and stressed
samples at 1 mg/mL were measured at 25°C to determine
the volume-based average protein particle diameter in
solution. Raster Image Correlation Spectroscopy (RICS) SYPRO® Red (Molecular Probes, Oregon) was prepared as a
50x stock solution in pre-filtered histidine-sucrose buffer and
diluted to a final working concentration of 2.5x for fluores-
cence studies immediately prior to use (all solutions were pre-
pared on the day of use) [28]. SYPRO® Red was added
15 min prior to visualization with confocal microscopy. A
Zeiss 510 Confocor 2 (Zeiss, Jena, Germany) confocal micro-
scope equipped with a c-Apochromat 40×/1.2NA water-
immersion objective was used for image acquisition. Imaging
was carried out by exciting the dye with a Helium-Neon laser
at 543 nm and the emitted fluorescence collected above
585 nm (LP585 filter set). A confocal image time series of
1024 × 1024 pixel resolution was captured over 100 frames
with a corresponding pixel dwell time of 6.4 μs. In-house
RICS software (ManICS) was applied to analysis of images
acquired using confocal microscopy. A full description of the
RICS algorithm has been described elsewhere [28,29]. The
image time series were sub-divided into 32 × 32 pixel sub-
regions and the diffusion coefficients (D) of each region of
interest (ROI) was generated. The method is described in
greater detail previously [28]. SDS Page Protein samples were diluted in SDS-PAGE sample buffer
(Bio-Rad, Berkley, CA, USA) containing 1% (v/v) 2-
mercaptoethanol and heated for 5 min at 90°C. Samples were
resolved on a pre-cast NuPAGE 4–12% Bis-Tris Protein gels
(Invitrogen™) and stained using InstantBlue™Coomassie
protein stain (Expedeon, Swavesey, UK). Protein samples were diluted in SDS-PAGE sample buffer
(Bio-Rad, Berkley, CA, USA) containing 1% (v/v) 2-
mercaptoethanol and heated for 5 min at 90°C. Samples were
resolved on a pre-cast NuPAGE 4–12% Bis-Tris Protein gels
(Invitrogen™) and stained using InstantBlue™Coomassie
protein stain (Expedeon, Swavesey, UK). Immunizations Mice (n = 3 or 5 per group) were immunized by intraperito-
neal (I.P.) injection on days 0, 7 and 14 and were exsangui-
nated on day 21. Animals were immunized with 250 μL of
1 mg/mL of mAb1 and 150 μL of 1 mg/mL of mAb2 (mo-
nomeric or aggregated) in PBS with or without rmHSP70 at a
ratio of 1 in 1000 (0.1% by mass) relative to the immunizing
mAb immediately after aggregate formation. Individual se-
rum samples were prepared and stored at −80°C until
analysis. Western Blot Protein samples were resolved on pre-cast 4–12% acrylamide
gel at various concentrations (0.1, 0.01, 0.001, 0.005 and
0.05 μg/mL in PBS), transferred onto nitrocellulose mem-
brane and detected using anti-HSP70 horseradish peroxidase
(HRP) conjugated antibody (StressMarq Bioscience Inc.,
Victoria, British Columbia, Canada) diluted at 1:2000. Proteins on the blots were visualized using enhanced chemi-
luminescence reagents (Thermo Scientific). Monomer and aggregate samples were prepared as de-
scribed previously. rmHSP70 was added at 1:1000 ratio by 51
Page 4 of 14 Pharm Res (2019) 36: 51 51 titre (log2) calculated as the lowest serum dilution at which a
3x serum blank OD450nm reading was reached. mass to monomer and aggregated mAbs. For monomeric
samples, where pellets were not observed, 30 μl PBS was
added to the tube to dissolve any sedimented material. Protein samples were resolved on a pre-cast 4–12% acrylam-
ide gel and transferred onto a nitrocellulose membrane. The
presence of rmHSP70 was detected using anti-HSP70 horse-
radish peroxidase (HRP) conjugated antibody and blots were
visualized using enhanced chemiluminescence reagents, as de-
scribed above. [3H]Thymidine Splenocyte Proliferation Assay A single cell suspension of splenocytes from immunized mice
was prepared using mechanical disaggregation. Splenocytes
were stimulated in vitro with respective protein samples for
7 days. 5 × 104 cells/well splenocytes were co-cultured with
5 × 103 bone marrow derived dendritic cells (BMDCs) pulsed
with the protein immunogen or no protein (for control) in
quadruplicate in round-bottom 96-well plates [31]. [3H]thymidine incorporation proliferation assay method is
described elsewhere [14]. Cultures were incubated for ap-
proximately 60 h at 37°C, 5% CO2 and [3H]thymidine
(3HTdR) (PerkinElmer, Waltham, MA, USA) was added at
37 kBq/well for the final 18 h, plates were harvested onto glass
fibre filter mats with a multichannel semi-automated harvest-
ing device (Titertek, Skatron AS, Lierbyen, Norway) and
quantified with β scintillation counting. Results are presented
as counts per min (cpm) as means of quadruplicates as de-
scribed previously [31]. Measurement of IFNγ Secretion Using ELISpot Assay To analyse the serum from mAb immunized mice, plastic
Maxisorb® plates (Nunc, Copenhagen, Denmark) were coat-
ed with 0.1 mg/mL monomer or 0.05 mg/mL aggregate
mAbs in PBS overnight at 4°C. Protein-coated plates were
blocked with 2% (w/v) bovine serum albumin (BSA)/PBS
(Sigma Aldrich) at 37°C for 30 min. Doubling dilutions of
serum samples were added (starting dilution 1:140 for IgG,
1:1120 for IgG1, 1:35 for IgG2a and 1:70 for IgM) prepared
in 1% BSA/PBS and plates incubated for 3 h at 4°C. Negative control naïve mouse serum (NMS) or PBS sham
control mouse serum samples were analyzed concurrently. Plates were incubated for 2 h at 4°C with HRP labelled sheep
anti-mouse IgG diluted 1:4000, sheep anti-mouse IgG1 dilut-
ed 1:2000 (both Bio-Rad) or goat anti-mouse IgM diluted 1:
6000 (Invitrogen, Paisley, UK), diluted in 1% BSA/ PBS. For
IgG2a ELISAs, MCA1588P rat anti mouse IgG2a HRP-
heavy chain antibody (Bio-Rad) was used for mAb1 and
STAR133P goat anti mouse IgG2a HRP antibody (Bio-
Rad) was used for mAb2 (both at 1:1000 dilution). Plates were
washed between incubations with 0.05% Tween 20 in PBS. For color development, plates were incubated with substrate
(1.6 mg/mL o-phenylenediamine and 0.4 mg/mL urea hy-
drogen peroxide in 0.5 M citrate phosphate buffer (pH 5)),
100 μL/well, for 15 min in the dark and reactions were
stopped with 50 μL/well of 0.5 M citric acid. Absorbance
was read at 450 nm using an automated reader (ELx800;
BioTek Instruments, Inc., Winooski, US), using the Gen 5
1.10 software. Data are displayed with respect to antibody Splenocytes from immunized mice were cultured ex vivo
using mAb1 or mAb2. BMDCs prepared as described
previously were used as Antigen Presenting Cells (APCs)
[31]. ELISpot assays were performed according to the
manufacturer’s protocol (Mabtec, Nacka Strand,
Sweden). Aliquots of 5 × 104 cells/well were assayed in
triplicate. The plates were developed after 48 h with
BCIP (5-bromo-4-chloro-3-indoyl phosphate p-toluidine
salt) and NBT (p-nitro blue tetrazolium chloride) color
development solution (Bio-Rad) for 30–45 min and
plates were rinsed with tap water. Spots were quantitat-
ed with an ELISpot reader (Cellular Technology
Limited, Bonn, Germany). An animal was scored as
positive when the response in the peptide-containing
well was at least twice that of control wells, as described
previously [32]. RESULTS both mAb1 and mAb2 (Fig. 1a, solid line). For mAb1,
different stress conditions (thermal stress, shaking stress,
and stir stress) were found to give rise to different size
populations of aggregate species, as analysed by DLS
(Supplementary Fig. 1A). The mAb1 and mAb2 aggregates
generated were irreversible and did not dissociate on dilu-
tion. In contrast to mAb1, mAb2 appeared more stable to
different stresses applied and demonstrated different ag-
gregation kinetics. Agitation stress was also applied to both
mAbs using a tube rotator for up to 24 h, but failed to
generate detectable aggregates (Supplementary Fig. 1). To characterize the aggregate populations more compre-
hensively, they were analyzed by Raster Image Correlation
Spectroscopy (RICS). RICS provided a quantitative mea-
surement of aggregate numbers (particles/fL) for four dif-
ferent size ranges, and for both mAbs (Fig. 1b). The results
confirm that shaking stress caused aggregates to form with-
in the 0.05–0.5 μm and 0.5–5 μm size ranges, a shift to
larger size populations compared with the results of ther-
mal stress induction on mAb1. These aggregate prepara-
tions were then used for an analysis of murine recombinant
HSP70 (rmHSP70) binding and immunological responses. both mAb1 and mAb2 (Fig. 1a, solid line). For mAb1,
different stress conditions (thermal stress, shaking stress,
and stir stress) were found to give rise to different size
populations of aggregate species, as analysed by DLS
(Supplementary Fig. 1A). The mAb1 and mAb2 aggregates
generated were irreversible and did not dissociate on dilu-
tion. In contrast to mAb1, mAb2 appeared more stable to
different stresses applied and demonstrated different ag-
gregation kinetics. Agitation stress was also applied to both
mAbs using a tube rotator for up to 24 h, but failed to
generate detectable aggregates (Supplementary Fig. 1). To characterize the aggregate populations more compre-
hensively, they were analyzed by Raster Image Correlation
Spectroscopy (RICS). RICS provided a quantitative mea-
surement of aggregate numbers (particles/fL) for four dif-
ferent size ranges, and for both mAbs (Fig. 1b). The results
confirm that shaking stress caused aggregates to form with-
in the 0.05–0.5 μm and 0.5–5 μm size ranges, a shift to
larger size populations compared with the results of ther-
mal stress induction on mAb1. These aggregate prepara-
tions were then used for an analysis of murine recombinant
HSP70 (rmHSP70) binding and immunological responses. STATISTICAL ANALYSES Statistical analyses were performed using Graphpad Prism 7. Analysis of variance (ANOVA) was used to determine statisti-
cal significance of differences between groups. Experiments
were analyzed by non-parametric one way or two way
ANOVA followed by Tukey’s post hoc test (*p < 0.05,
**p < 0.01, ***p < 0.001). Page 5 of 14
51 Pharm Res (2019) 36: 51 51 Characterization of Therapeutic mAbs in Native
and Aggregated States This investigation sought to investigate whether the
adjuvant-like effect, observed previously using bacterial
DnaK with aggregates of a scFv fragment [27], could also
be recorded using intact monoclonal antibodies and a
mammalian HCP. Consequently, two human IgG1
mAbs, mAb1 and mAb2, were used in the current study. Both mAbs were prepared at 1 mg/mL in PBS and aggre-
gates generated by application of thermal or shaking stress. mAb2 showed no aggregation in response to thermal stress
(at 45, 50 or 60°C) but aggregates of mAb1 were generated
by both methods. The sizes of the generated mAb1 and
mAb2 aggregates were analyzed by DLS (Fig. 1a). Both
mAb monomers showed a narrow size distribution at
~10 nm, as anticipated (dashed line). Application of ther-
mal stress to mAb1 generated an aggregate population
within the sub-visible size range. Aggregate sizes were
much larger (~1 μm) when formed by shaking stress for Fig. 1 Characterization of mAb1 and mAb2 aggregates by DLS and RICS. (a) Aggregate particle size measured using DLS. Dashed line, monomer
mAb; solid line, shaking stress induced aggregated mAb; dotted line, thermal stress induced aggregated mAb. (b) Analysis of aggregate species using RICS. Horizontal and vertical axes represent particle counts per mL. Data were separated into size ranges of <0.05, 0.05–0.5, 0.5–5 and > 5 μm. Values represent
means ± SD (n = 3). Fig. 1 Characterization of mAb1 and mAb2 aggregates by DLS and RICS. (a) Aggregate particle size measured using DLS. Dashed line, monomer
mAb; solid line, shaking stress induced aggregated mAb; dotted line, thermal stress induced aggregated mAb. (b) Analysis of aggregate species using RICS. Horizontal and vertical axes represent particle counts per mL. Data were separated into size ranges of <0.05, 0.05–0.5, 0.5–5 and > 5 μm. Values represent
means ± SD (n = 3). Fig. 1 Characterization of mAb1 and mAb2 aggregates by DLS and RICS. (a) Aggregate particle size measured using DLS. Dashed line, monomer
mAb; solid line, shaking stress induced aggregated mAb; dotted line, thermal stress induced aggregated mAb. (b) Analysis of aggregate species using RICS. Horizontal and vertical axes represent particle counts per mL. Data were separated into size ranges of <0.05, 0.05–0.5, 0.5–5 and > 5 μm. Values represent
means ± SD (n = 3). Fig. 1 Characterization of mAb1 and mAb2 aggregates by DLS and RICS. (a) Aggregate particle size measured using DLS. Characterization of Therapeutic mAbs in Native
and Aggregated States Dashed line, monomer
mAb; solid line, shaking stress induced aggregated mAb; dotted line, thermal stress induced aggregated mAb. (b) Analysis of aggregate species using RICS. Horizontal and vertical axes represent particle counts per mL. Data were separated into size ranges of <0.05, 0.05–0.5, 0.5–5 and > 5 μm. Values represent
means ± SD (n = 3). 51
Page 6 of 14 Pharm Res (2019) 36: 51 Binding of rmHSP70 to mAb1 and mAb2 Aggregates and b). The partition of rmHSP70 was compared in the pres-
ence of monomeric mAb (right panels of Fig. 2a, b) or aggre-
gated mAb (left panels of Fig. 2a, b). rmHSP70 alone migrated
as a monomer (~75 kDa) or dimer (~150 kDa). For both mAb1
and mAb2, rmHSP70 selectively accumulated into the aggre-
gated pellet fractions; this was not the case for monomeric
mAb1 and mAb2, where rmHSP70 was exclusively found in
the supernatant. We conclude that rmHSP70 selectively binds
to mAb1 or mAb2 aggregates, rather than monomer. Our previous work has shown that the HSP DnaK was able to
bind to scFv aggregates, a property which could contribute to its
stimulation of immune response to aggregates [27]. We there-
fore conducted similar experiments to examine whether
rmHSP70 was able to bind to aggregates of mAb1 and
mAb2. rmHSP70 was added at 0.1% by mass with respect to
mAb concentration to monomer or aggregated mAb1 and
mAb2, insoluble aggregates were separated from monomer by
centrifugation, and supernatant and pellet fractions were ana-
lyzed by western blotting using anti-rmHSP70 antibody (Fig. 2;
SDS PAGE gel images are shown in Supplementary Fig. 2a Asymmetric Flow Field Flow Fractionation (AF4) was used
as an additional method to examine rmHSP70 binding to the
aggregated mAb preparations. Migration profiles of mAb1
and mAb2 monomers and aggregates were compared with 2 Partition of rmHSP between aggregated and monomer fractions analyzed using western blot. mAb1 and mAb2 were aggregated u
mal and shaking stresses, respectively. rmHSP70 was added at 0.1% by mass to monomer and aggregated mAbs immediately after aggregation. Sam
e centrifuged at 12000 rpm for 30 min to sediment aggregated species; pellet and supernatants were then harvested. Blots were incubated with
ugated anti rmHSP70 antibody and developed by chemiluminescence. Lanes are labelled as follows, M, monomer; A, aggregate; Sup, supernatant; Pel, p g. 2 Partition of rmHSP between aggregated and monomer fractions analyzed using western blot. mAb1 and mAb2 were aggregated usin
ermal and shaking stresses, respectively. rmHSP70 was added at 0.1% by mass to monomer and aggregated mAbs immediately after aggregation. Sample Fig. 2 Partition of rmHSP between aggregated and monomer fractions analyzed using western blot. mAb1 and mAb2 were aggregated using
thermal and shaking stresses, respectively. rmHSP70 was added at 0.1% by mass to monomer and aggregated mAbs immediately after aggregation. Binding of rmHSP70 to mAb1 and mAb2 Aggregates This is understandable, given that rmHSP70 was only added
to 0.1% of mAb content by mass. A summary of each run with
the peaks for mAb1 and mAb2 monomer and aggregated
proteins (with/without rmHSP70) are shown in
Supplementary Fig. 3. The results provide additional support
for the specific interaction of rmHSP70 with mAb aggregates. 4 Effect of rmHSP70 on Immune Responses to mAb1
and mAb2 in BALB/c Model The effect of rmHSP70 on the AF4 mAb aggregate profiles
prompted us to investigate the interaction further by micros-
copy. rmHSP70 was labelled with the green fluorescence dye
BODIPY and its binding to mAb1 and mAb2 aggregates In vivo approaches have been usefully adopted to compare
immune responses elicited by aggregated proteins and their Binding of rmHSP70 to mAb1 and mAb2 Aggregates Samples
were centrifuged at 12000 rpm for 30 min to sediment aggregated species; pellet and supernatants were then harvested. Blots were incubated with HRP
conjugated anti rmHSP70 antibody and developed by chemiluminescence. Lanes are labelled as follows, M, monomer; A, aggregate; Sup, supernatant; Pel, pellet. Fig. 2 Partition of rmHSP between aggregated and monomer fractions analyzed using western blot. mAb1 and mAb2 were aggregated using
thermal and shaking stresses, respectively. rmHSP70 was added at 0.1% by mass to monomer and aggregated mAbs immediately after aggregation. Samples
were centrifuged at 12000 rpm for 30 min to sediment aggregated species; pellet and supernatants were then harvested. Blots were incubated with HRP
conjugated anti rmHSP70 antibody and developed by chemiluminescence. Lanes are labelled as follows, M, monomer; A, aggregate; Sup, supernatant; Pel, pellet. Pharm Res (2019) 36: 51 Page 7 of 14
51 51 studied. SYPRO® Red dye was added to mAb1 and mAb2
aggregates which were clearly visible by red fluorescence
(Fig. 4a). Visualization of the BODIPY dye, for both mAb1
and mAb2, showed distinct concentration of the fluorophore
in the presence of aggregates (Fig. 4b) but not in the presence
of monomer (Fig. 4c) or absence of mAbs (rmHSP70-
BODIPY alone control, Fig. 4d). It should be noted that
mAb1 was used in a more diluted form than mAb2 in order
to achieve optimal conditions for imaging, leading to the low-
er frequency of BODIPY-labelled rmHSP70-mAb1 aggre-
gates observed in Fig. 4b. We infer that BODIPY-rmHSP70
bound to mAb aggregates, forming clumps which could be
more readily visualized than when dispersed in bulk solution. These observations are therefore also consistent with specific
adhesion of rmHSP70 to aggregates of mAb1 and mAb2. and without rmHSP70 addition (Fig. 3). 50 μL of mAb1 and
10 μL of mAb2 samples were loaded to obtain optimum elu-
tion profiles; the heights, peak areas and elution times are
shown in Table I. Addition of rmHSP70 did not significantly
alter the elution times of monomers or aggregates but in-
creased the peak area, by 22% for mAb1 aggregate and, re-
markably, about 300% for mAb2 aggregate (Table I). No
such effects were seen for addition of rmHSP70 to mAb
monomers. We propose that these effects are caused by
changes to aggregate properties, such as shape, rather than
overall mass, and therefore indirectly influence peak area. Fig. 3 Assessment of rmHSP70 binding to aggregated mAb using AF4. rmHSP70 was added to the aggregated mAbs immediately after aggregation
and binding was assessed by AF4. Chromatograms for mAb1 and mAb2 monomer (with and without rmHSP70) and aggregates (with and without rmHSP70) are
overlaid as shown. The peak profiles were used to compile the data summarized in Table I (representative data from a single experiment). 4 Fig. 3 Assessment of rmHSP70 binding to aggregated mAb using AF4. rmHSP70 was added to the aggregated mAbs immediately after aggregation
and binding was assessed by AF4. Chromatograms for mAb1 and mAb2 monomer (with and without rmHSP70) and aggregates (with and without rmHSP70) are
overlaid as shown. The peak profiles were used to compile the data summarized in Table I (representative data from a single experiment). 51
Page 8 of 14 Pharm Res (2019) 36: 51 Table I
Asymmetric Flow Field Flow Fractionation (AF4)1
#
#
Height2
Area
Elution time (min)
mAb1
Monomer
5
368.2
9.7
rmHSP70-Monomer
4.9
347.8
9.7
Aggregate
6
1152.8
21.2
rmHSP70-aggregate
7.7
1413.6
21.5
mAb2
Monomer
4.4
288.3
10.3
rmHSP70-Monomer
4.2
281.3
10.3
Aggregate
4.4
217.8
6.7
rmHSP70-aggregate
21
864.4
6.5 euthanized on day 21. Figure 5a shows the serum anti-
mAb1 IgM, IgG and selected subclasses (IgG1, IgG2a) anti-
body responses following administration of thermal and shak-
ing stress-induced aggregates. Levels of anti-mAb1 IgG anti-
body were elevated in the mice immunized with aggregate
from thermal-stress and rmHSP70-aggregate mAb1 immu-
nized mice, compared to those immunized with monomer
mAb1, rmHSP70-monomer mAb1 or the PBS immunized
control group (Fig. 5a). Similar patterns were recorded with
anti-mAb1 IgG1 antibody responses but, due to the sensitivity
of the anti-IgG1 detection antibody, these achieved consider-
ably higher titres than those obtained for total IgG. Importantly, aggregation of mAb1enhanced the IgG2a anti-
body response compared to monomer and addition of
rmHSP70 further enhanced IgG2a for the aggregate but not
the monomeric species. These observations are indicative of a
Th1-type skewing of the immune responses and consistent
with those observed previously for an antibody fragment
scFv [27]. The ability of mAb1 aggregates generated using
shaking stress (1 μm) to induce immune responses in the
BALB/c model was also examined. A very similar pattern
was observed to the thermal stressed aggregates, with aggre-
gate enhancing IgG and IgG1 antibody production and with
rmHSP70 addition having a more profound effect on monomeric counterparts. A common approach is to measure
antibody responses provoked in mice by immunizations with
the monomeric or aggregated protein [33,34]. A BALB/c
mouse model was therefore used in the current study to assess
the immune responses generated following intraperitoneal
(I.P.) immunization with monomer, rmHSP70-monomer, ag-
gregated or rmHSP70-aggregated human mAbs [27]. mAb1 (250 μg) was administered as monomer or aggre-
gate, both in the presence or absence of rmHSP70. Naïve
mice or PBS only immunization was used as a sham control. Mice were boosted twice at 7 day intervals and were Fig. 4 Assessment of rmHSP70 binding to aggregated mAb using fluorescence imaging by RICS. (a) SYPRO® Red dye stained aggre
samples of mAb1 and mAb2. (b) BODIPY FL tagged rmHSP70 (green) added to mAb1 and mAb2 post aggregation (0.1% by mass). (c) Monomer mAbs
rmHSP70-BODIPY FL. (d) rmHSP70-BODIPY FL alone. Data were obtained from a typical experiment using RICS mode and a 40x water immersion obje
with slow scanning (pixel size 40 nm and scanning speed 6 4 microseconds per pixel Fig. 4 Assessment of rmHSP70 binding to aggregated mAb using fluorescence imaging by RICS. (a) SYPRO® Red dye stained aggregated
samples of mAb1 and mAb2. (b) BODIPY FL tagged rmHSP70 (green) added to mAb1 and mAb2 post aggregation (0.1% by mass). (c) Monomer mAbs with
rmHSP70-BODIPY FL. (d) rmHSP70-BODIPY FL alone. Data were obtained from a typical experiment using RICS mode and a 40x water immersion objective
with slow scanning (pixel size 40 nm and scanning speed 6.4 microseconds per pixel. Fig. 4 Assessment of rmHSP70 binding to aggregated mAb using fluorescence imaging by RICS. (a) SYPRO® Red dye stained aggregated
samples of mAb1 and mAb2. (b) BODIPY FL tagged rmHSP70 (green) added to mAb1 and mAb2 post aggregation (0.1% by mass). (c) Monomer mAbs with
rmHSP70-BODIPY FL. (d) rmHSP70-BODIPY FL alone. Data were obtained from a typical experiment using RICS mode and a 40x water immersion objective
with slow scanning (pixel size 40 nm and scanning speed 6.4 microseconds per pixel. Page 9 of 14
51 Pharm Res (2019) 36: 51 Fig. 5 Antibody responses to aggregated mAbs. (a) mAb1 thermal stress-induced aggregates (b) mAb1 shaking stress-induced aggregates (c) mAb2
shaking stress-induced aggregates. BALB/c mice received monomer or aggregated protein injections (I.P.) with or without rmHSP70 addition. Mice received
booster injections on day 7 and day 14, and were euthanized on day 21. Doubling dilutions (in 1% BSA/PBS) of serum samples from monomer, aggregate-
immunized animals, PBS immunized or naïve negative control serum samples were analyzed against monomer-coated plates by ELISA for IgG, IgG1, IgG2a and
IgM. Data are representative of two independent experiments for mAb1 (n = 8), and mAb2 (n = 5). ELISA titres for negative control mice (naïve or PBS) were
invariably lower than the protein immunization groups (p < 001), symbols not shown. Differences in antibody serum titres between all sera groups against each
substrate were analysed using one way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). Pharm Res (2019) 36: 51
Page 9 of 14
51 Fig. 5 Antibody responses to aggregated mAbs. (a) mAb1 thermal stress-induced aggregates (b) mAb1 shaking stress-induced aggregates (c) mAb2
shaking stress-induced aggregates. BALB/c mice received monomer or aggregated protein injections (I.P.) with or without rmHSP70 addition. Mice received
booster injections on day 7 and day 14, and were euthanized on day 21. Doubling dilutions (in 1% BSA/PBS) of serum samples from monomer, aggregate-
immunized animals, PBS immunized or naïve negative control serum samples were analyzed against monomer-coated plates by ELISA for IgG, IgG1, IgG2a and
IgM. Data are representative of two independent experiments for mAb1 (n = 8), and mAb2 (n = 5). ELISA titres for negative control mice (naïve or PBS) were
invariably lower than the protein immunization groups (p < 001), symbols not shown. Differences in antibody serum titres between all sera groups against each
substrate were analysed using one way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). Mice immunized with shaking stress-induced mAb2 aggre-
gates demonstrated increased IgG, IgG1, and IgG2a re-
sponses compared to immunization with monomer (Fig. 5c)
and, as recorded for mAb1, rmHSP70 further enhanced
IgG2a antibody production. A specific anti-mAb2 IgM anti-
body response was recorded for aggregate-treated mice, but
not monomer. Similar patterns of antibody responses were
observed in ELISAs for both monomer and aggregate-coated
plates of mAb1 and mAb2. enhancing IgG2a antibody production than did aggregation
alone (Fig. 5b). There was no detectable anti-mAb1-induced
IgM antibody in response to mAb1 immunization (Fig. 5a, b). enhancing IgG2a antibody production than did aggregation
alone (Fig. 5b). There was no detectable anti-mAb1-induced
IgM antibody in response to mAb1 immunization (Fig. 5a, b). 51
Page 10 of 14 Pharm Res (2019) 36: 51 In order to further characterize adaptive immune re-
sponses induced in response to mAb immunization,
splenocyte proliferation assays were conducted. Spleens were
harvested on day 21 post-immunization and cultured ex vivo
with addition of mAb1 or mAb2, with or without rmHSP70. The splenocytes were co-cultured with dendritic cells (DCs)
from naïve BALB/c mice and mAb1 or mAb2; proliferation
was measured with [3H]-thymidine incorporation. Statistically significant increases in proliferation were observed
in the groups of mice that were immunized with rmHSP70-
aggregate protein compared to mAb1 or mAb2 aggregate
alone (Fig. 6a). IFNγ secretion ELISpot assays were per-
formed by co-culturing in vitro protein-stimulated splenocytes
with protein pulsed-DCs. For mAb1 (both thermal and shak-
ing stress induced aggregates), a significantly higher frequency
of IFNγ secreting splenocytes was observed in the group of
mice immunized with rmHSP70-aggregate compared with
aggregate alone. For mAb2, however, we did not observe
any effect of rmHSP70 addition (Fig. 6b). Nevertheless, the
cellular responses obtained were generally consistent with the
antibody measurements, indicative of a Th1 enhancement in
the groups immunized with aggregated mAbs, and provided
further evidence for a stimulatory effect of rmHSP70 on im-
mune responses to aggregated mAbs. proteins which are retained for ubiquitination and subsequent
targeting to the proteasome for degradation [20,43]. Using
co-sedimentation and western blotting, we have shown that
rmHSP70 bound to aggregates of both mAbs. Similar phe-
nomena were observed when aggregates were analyzed using
AF4 where the presence of rmHSP70 had an effect on the
migration profile of each mAb. Supporting these observations,
microscopy demonstrated that fluorescently labelled
rmHSP70 bound to the aggregated mAbs. Although the level
of rmHSP70 used in these studies is high by industry standards
(1:1000), our results provide evidence that HSP70 impurities
can be selectively accumulated by adhesion to aggregates. This opens up the possibility that HSPs in general can selec-
tively accumulate by adhesion to aggregated material, even
though the overall levels of HSPs are low. g
Our observations on IgG subclass specificity are consistent
with those made previously by Ratanji et al. [27], which
showed a stimulation of IgG2a levels characteristic of Th1-
skewing of the immune response (Fig. 5). Interestingly, no
increases in IgM antibody levels above PBS controls were
detected for the immunizations of mAb1. 51
Page 10 of 14 This observation
could be due to the immunogenic potential of mAb1, the
serum IgM repertoire and the lifespan of IgM in the in vivo
system [44]. Additional evidence for the stimulatory effects of
rmHSP70 was obtained from cellular proliferation assays,
using splenocytes cultured with antigen-primed DCs. Splenocyte proliferation was elevated in the group of mice
immunized with rmHSP70-aggregated protein, compared
with those immunized with aggregate alone, demonstrating
an enhanced CD4-mediated T cell response. Stimulation of
IFNγ secretion was particularly pronounced for mAb1 aggre-
gates in splenocytes from the mice which received rmHSP70-
aggregate, compared with aggregate alone. We think that the
enhancement of mAb immunogenicity by rmHSP70 which
we observe is unlikely to be attributable to its immunogenicity,
for several reasons. First, the levels of rmHSP70 used were
very low (0.1% of total protein inoculated). It is remarkable
that such a low level of impurity can drive significant responses
to IgG2a (Fig. 5) and splenocyte proliferation (Fig. 6a). Second, the Cricetulus griseus (Chinese Hamster) HSP70 is
98% identical to the mouse HSP70 sequence. Third, we do
not observe an enhancement of IgG2a levels for monomer
compared with rmHSP70-monomer, only for aggregate com-
pared with rmHSP70-aggregate (Fig. 5). In order to investigate whether these observations were
particular to mAb1, mAb2 was investigated in a similar man-
ner. Preliminary studies performed using mAb2 indicated that
lower doses were needed to obtain measurable serum anti-
body responses in immunized mice (data not shown), and a
dose of 150 μg mAb2 was used for the ensuing experiments. In order to investigate whether these observations were
particular to mAb1, mAb2 was investigated in a similar man-
ner. Preliminary studies performed using mAb2 indicated that
lower doses were needed to obtain measurable serum anti-
body responses in immunized mice (data not shown), and a
dose of 150 μg mAb2 was used for the ensuing experiments. 51
Page 10 of 14 DISCUSSION Protein aggregation can be described as the self-association of
monomers in their native or partially unfolded forms [35,36],
and is a common phenomenon observed in biopharmaceuti-
cal preparations. There is evidence that aggregates can stim-
ulate an anti-drug immune response which may impair drug
efficacy. However, the mechanisms through which immuno-
genicity is enhanced or conferred on proteins are only poorly
understood [37]. Recently we reported that IgG2a antibody
responses to an aggregated model scFv were stimulated in a
mouse model by addition of a low (0.1%) level of a bacterial
HSP, DnaK [27]. This observation led us to investigate
whether this effect was also observed with human mAbs and
a murine HSP. Aggregates that may be present in protein
products can range from dimers to subvisible or visible parti-
cles. They can be formed during different stages of produc-
tion, transport or delivery to the patient, in response to diverse
stresses [35,38]. Aggregates formed in biotherapeutic mono-
clonal antibodies under the influence of various stresses have
been characterized by various techniques on the basis of their
sizes, ranging from nm to micron dimensions [39–42]. The
mAb aggregates employed in this study fall within this range
and can therefore be regarded as typical, at least in terms of
size, compared with those studied previously. Previous studies using mouse models have shown that
HSP70 can be used as an adjuvant in cancer vaccine devel-
opment strategies [45,46]. HSPs have therefore been
harnessed as adjuvants of vaccines against cancers and infec-
tious diseases [47]: examples include Phase I clinical trials for
Glioblastoma (HSP70), colon carcinoma, Phase I-II studies
for Non-small cell lung carcinoma (HSP70-activated NK
cells) and a Phase I HIV vaccine study [25]. The adjuvant-
like activity of HSP70 has been demonstrated in vitro by It is well established that HSP family members recognize
exposed hydrophobic residues of misfolded or denatured oupling to superparamagnetic iron oxide nanoparticles
SPIONs) to form HSP70 SPION conjugates HSP70
peptides from tumor lysates to DCs, stimulating a tumor-s
cific CD8+ cytotoxic T cell response in experimental glio
Fig. 6 Assessment of cellular responses in BALB/c mice immunized with aggregated mAbs in the presence or absence of rmHSP70
3H] thymidine incorporation proliferation assay for aggregated mAb1 (thermal stress) and mAb2 (shaking stress). Splenocytes were isolated from immunized m
nd stimulated ex vivo with protein. DISCUSSION [3H] thymidine incorporation proliferation assays were performed by co-culturing in vitro protein-stimulated splenocytes f
mmunized mice with protein-pulsed DCs. A PBS only immunization was used as sham control. (b) IFNγ ELISpot assays. The IFNγ ELISpot assay was perform
nder the same culture conditions as (A) to assess IFNγ secretion in response to co-culture. The experiments were performed on two separate occasions, n =
or thermal and shaking stress aggregates from mAb1) and n = 5 for mAb2. Comparisons of the means (± SEM) between groups are made. Statistical significa
f the results obtained was calculated using one way ANOVA. *p < 0.05,**p < 0.01,***p < 0.001. harm Res (2019) 36: 51
Page 11 of 14 Page 11 of 14
51 Pharm Res (2019) 36: 51 Fig. 6 Assessment of cellular responses in BALB/c mice immunized with aggregated mAbs in the presence or absence of rmHSP70. (a)
[3H] thymidine incorporation proliferation assay for aggregated mAb1 (thermal stress) and mAb2 (shaking stress). Splenocytes were isolated from immunized mice
and stimulated ex vivo with protein. [3H] thymidine incorporation proliferation assays were performed by co-culturing in vitro protein-stimulated splenocytes from
immunized mice with protein-pulsed DCs. A PBS only immunization was used as sham control. (b) IFNγ ELISpot assays. The IFNγ ELISpot assay was performed
under the same culture conditions as (A) to assess IFNγ secretion in response to co-culture. The experiments were performed on two separate occasions, n = 8,
(for thermal and shaking stress aggregates from mAb1) and n = 5 for mAb2. Comparisons of the means (± SEM) between groups are made. Statistical significance
of the results obtained was calculated using one way ANOVA. *p < 0.05,**p < 0.01,***p < 0.001. coupling to superparamagnetic iron oxide nanoparticles
(SPIONs) to form HSP70-SPION conjugates. HSP70-
SPIONs have shown effective delivery of immunogenic peptides from tumor lysates to DCs, stimulating a tumor-spe-
cific, CD8+ cytotoxic T cell response in experimental glioma
models [48]. It is also known that HSP–peptide complexes can coupling to superparamagnetic iron oxide nanoparticles
(SPIONs) to form HSP70-SPION conjugates. HSP70-
SPIONs have shown effective delivery of immunogenic 51
Page 12 of 14 Pharm Res (2019) 36: 51 51 REFERENCES 1. Li J, Zhu Z. Research and development of next generation of
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Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which per-
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MedImmune to carry out this research project. J. Derrick
and the University of Manchester have received research
grants from MedImmune. The other authors declare that they
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2327–43.
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Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop Pemrograman Secara Daring
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Abstrak Pelatihan dalam bentuk pembelajaran melalui daring kini semakin sering dilakukan, terutama di bidang pemrograman yang
merupakan dasar utama di rumpun ilmu komputer. Salah satu hal yang mendorong pelaksanaan pembelajaran daring ini adalah
situasi global yang mengharuskan pembatasan jarak akibat adanya pandemi Covid-19. Kemajuan teknologi berupa penyebaran
jaringan internet dan ketersediaan berbagai aplikasi yang menjadi penunjang pembelajaran daring ini. Hal ini juga dibantu
dengan tersedianya berbagai aplikasi lain yang bisa membantu untuk pengajaran dalam bentuk presentasi maupun untuk
penilaian ujian yang siap digunakan untuk pembelajaran daring. Kemajuan teknologi ini tentunya merupakan hal yang baik,
namun ternyata masih banyak kendala yang dihadapi bagi para peserta pembelajaran daring untuk mengadopsi teknologi
tersebut. Berdasarkan permasalahan tersebut dirumuskan sebuah rumusan masalah untuk penelitian ini yaitu bagaimana
melakukan identifikasi faktor-faktor yang mempengaruhi kesuksesan proses pelatihan pemrograman yang dilakukan secara
daring? Dan Faktor-faktor apa yang berdampak pada pemahaman materi bagi peserta pelatihan? Proses identifikasi faktor-faktor
yang dapat mendorong kesuksesan pelatihan berbasis online merupakan fokus dari penelitian ini. Langkah awal adalah dengan
melakukan proses pengumpulan data menggunakan survei dan kuesioner yang sudah disusun berdasarkan pada metode
Technology Acceptance Model (TAM), kemudian data dianalisis berdasarkan statistika deskriptif. Penelitian ini berhasil
melakukan identifikasi bagaimana seharusnya pelatihan berbasis daring dilakukan dengan efektif berdasarkan faktor-faktor
penting yang sudah teridentifikasi. Kata Kunci: Adopsi, Teknologi, Pelatihan, Online, Pemrograman. Kata Kunci: Adopsi, Teknologi, Pelatihan, Online, Pemrograman. Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau
Workshop Pemrograman Secara Daring Laura Mahendratta Tjahjono1, Adi Suryaputra Paramita2* 1Program Studi Informatika, Universitas Ciputra Surabaya, Surabaya, Jawa Timur
2Program Studi Sistem Informasi, Universitas Ciputra Surabaya, Surabaya, Jawa Timur
Email: 1laura@ciputra.ac.id, 2*adi.suryaputra@ciputra.ac.id (Naskah masuk: 26 Jul 2021, direvisi: 20 Okt 2021, diterima: 27 Okt 2021) (Naskah masuk: 26 Jul 2021, direvisi: 20 Okt 2021, diterima: 27 Okt 2021) 176 176 Tjahjono, M.L., et.al.: Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring Tjahjono, M.L., et.al.: Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring I. PENDAHULUAN yang mendukung teori ini antara lain penelitian Hodjat
Hamidi, Amir Chavoshi yang dilakukan pada tahun 2017, yang
didalamnya diungkapkan faktor-faktor yang berperan penting
terhadap suksesnya adopsi teknologi untuk pembelajaran
daring. Faktor-faktor tersebut antara lain adalah kemudahan
penggunaan teknologi (ease of use), tingkat kepercayaan
terhadap teknologi, karakter dan kepribadian pengguna,
konteks, manfaat dari penggunaan teknologi terkait (perceived
usefulness of using), kesungguhan niat peserta, dan latar
belakang budaya [2]. Penelitian menarik lainnya oleh
Marcelino Arrosagaray pada 2018 menyebutkan bahwa
terdapat kaitan erat antara jenis kelamin, usia dan latar
belakang pekerjaan dari peserta dengan tingkat adopsi mereka
akan
teknologi,
yang
pada
akhirnya
mempengaruhi
peningkatan kepercayaan diri dalam melakukan pembelajaran
daring. Peserta laki-laki dengan usia yang relatif muda dan
yang kegiatan utamanya adalah belajar atau bekerja
menunjukkan kepercayaan diri yang lebih besar akan
kemampuan mereka dalam menggunakan teknologi dalam
pembelajaran daring [3]. Hal ini senada dengan yang
diungkapkan pada penelitian yang diterbitkan tahun 2019 yang
menyatakan bahwa perbedaan jenis kelamin menjadi hal yang
menarik untuk diteliti dalam adopsi teknologi [4]. Berdasarkan
hasil yang telah disajikan pada penelitian-penelitian terdahulu
ini, ada beberapa hal yang bisa dilanjutkan pada penelitian ini. Pada penelitian ini akan dilakukan analisis lebih mendalam
mengenai hubungan antara kemudahan peserta dalam
menerima
pelatihan
dalam
bentuk
daring
dengan
memperhatikan faktor-faktor berikut: Pertemuan rutin dan seminar merupakan kegiatan yang
sering dilakukan oleh berbagai komunitas sosial. Kegiatan ini
dilakukan baik secara rutin maupun insidentil dan biasanya
merupakan inti dari terbentuknya komunitas tersebut. Selain
pertemuan dan seminar ini, komunitas sosial juga ada kalanya
menyelenggarakan pelatihan-pelatihan bagi anggotanya. Pelatihan ini merupakan salah satu kegiatan penting guna
meningkatkan pengetahuan dan kemampuan anggotanya. Sayangnya sejak akhir tahun 2019 ini, segala jenis kegiatan
yang melibatkan banyak orang berkumpul di satu tempat harus
dihindari karena adanya virus Covid-19. Salah satu aspek yang
terdampak tentunya adalah aspek sosial, dimana ada aturan
ketat terkait pembatasan jarak fisik untuk mencegah
penyebaran virus ini. Dengan adanya pembatasan jarak ini,
maka kegiatan atau acara yang memerlukan temu muka
langsung tidak lagi dapat diselenggarakan seperti biasa. Sebagai solusi untuk pembatasan jarak di masa pandemi
Covid-19 ini, internet dan aplikasi jenis ini menjadi kebutuhan
dasar untuk terselenggaranya kegiatan mereka, seperti
pertemuan, seminar dan pelatihan-pelatihan. Pada pelaksanaannya, kegiatan pembelajaran dengan
menggunakan media daring ini ternyata masih sulit dan
menemui banyak permasalahan, terutama pada kegiatan
berupa pelatihan. Pada pelatihan, selain memahami materi
yang berupa teori, biasanya peserta juga harus melakukan
praktek langsung dengan bimbingan pengajar. Tjahjono, M.L., et.al.: Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring Tjahjono, M.L., et.al.: Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring 177 I. PENDAHULUAN Hal yang
dulunya mudah diajarkan secara langsung pada saat pelatihan
kini menjadi tantangan tersendiri. Inilah yang menjadi
perhatian untuk dilakukannya penelitian mengenai faktor-
faktor apa saja yang sebenarnya bisa mendukung adopsi
pembelajaran dan pelatihan lewat daring. Tujuan penelitian ini
adalah melakukan identifikasi faktor-faktor yang mendukung
pembelajaran
daring
yang
berupa
pelatihan
untuk
menghasilkan sebuah panduan bagi instruktur pemrograman
pada pelatihan berbasis daring. Panduan yang dihasilkan
tersebut bertujuan untuk memberikan gambaran solusi untuk
pelatihan atau Workshop pemrograman secara daring di masa
pandemi virus Covid-19 ini diperkirakan masih akan berlanjut
sehingga perlu diberlakukan kebisaan normal yang baru, yang
dikenal dengan the new normal. Pada era new normal ini dapat
diperkirakan pelatihan berbasis daring akan semakin banyak
dan
perlu
segera
ditemukan
model
dari
pelatihan
pemrograman yang efektif. p
1. Demografi peserta seperti usia, pendidikan dan kegiatan
rutinitas. 2. Kondisi ekonomi yang tentunya mempengaruhi kualitas
koneksi internet dan jenis perangkat keras yang mereka
miliki. 3. Latar belakang pengetahuan peserta mengenai TIK
(Teknologi Informasi dan Komunikasi). Important Factors in The Delivery of Online Programming
Training Abstract Training in the form of online learning is now increasingly being carried out, especially in programming, which is the main
foundation in the computer science family. One thing that encourages the implementation of online learning is the global
situation that requires distance restrictions due to the Covid-19 pandemic. Technological advancement, the spread of the internet
network, and various applications support this online learning. This is also helped by the availability of various other
applications that can help teach in the form of presentations and exam assessments ready for online learning. This technological
advancement is undoubtedly a good thing, but it turns out that there are still many obstacles faced for online learning
participants to adopt the technology. Based on these problems, a problem formulation for this research is formulated: how to
identify the factors that influence the success of the online programming training process? And what factors have an impact on
the understanding of the material for the trainees? The process of identifying factors that can drive the success of online-based
training is the focus of this research. The first step is to carry out the data collection process using surveys and questionnaires
prepared based on the Technology Acceptance Model (TAM) method, and then the data is analyzed based on descriptive
statistics. This study succeeded in identifying how online-based training should be carried out effectively based on the essential
factors that have been identified. Keywords: Adoption, Technology, Training, Online, Programming. DOI: 10.34148/teknika.v10i3.392 TEKNIKA, Volume 10(3), November 2021, pp. 176-180
ISSN 2549-8037, EISSN 2549-8045 II. METODOLOGI PENELITIAN Tahapan penelitian diawali dengan menyusun instrumen
penelitian berdasarkan variabel yang ada di framework
Technology Acceptance Model (TAM). Framework pada
model TAM sudah banyak digunakan untuk mengukur faktor-
faktor yang berpengaruh dalam adopsi suatu teknologi. Setelah
instrumen selesai, proses pengumpulan data dilakukan dengan
survei dan
kuesioner
yang
sudah disusun. Melalui
pengumpulan data tersebut akan didapatkan data primer yang
nantinya akan siap diolah. Hasil analisis data yang didapatkan
akan disajikan dalam bentuk statistika deskriptif sesuai dengan
kaidah dalam ilmu statistika. Langkah selanjutnya adalah
dengan membuat ringkasan berdasarkan analisis statistika
deskriptif untuk kemudian disusun menjadi pedoman
pelaksanaan pelatihan pemprograman online. Proses dan
tahapan penelitian terlihat pada Gambar 1 di bawah. Berbagai penelitian terdahulu sudah banyak dilakukan
untuk melihat tingkat efektifitas dan meningkatkan kualitas
dari pembelajaran via daring. Agar pembelajaran via daring ini
terlaksana dengan baik serta hasil yang diperoleh dapat
maksimal, maka perlu diperhatikan berbagai faktor yang
mempengaruhinya. Teori adopsi teknologi yang sering
digunakan dan dikenal adalah Technology Acceptance Model
(TAM), yang memiliki empat variabel utama yang diteliti yaitu
perceived usefulness (PU), perceived ease of use (PEU), usage
behavior (UB), dan intention to use (IU). TAM bisa menjadi
model untuk validasi hasil penelitian di bidang adopsi
teknologi dan mengalami beberapa perkembangan model
penelitian di penelitian-penelitian terkini [1]. Penelitian sejenis DOI: 10.34148/teknika.v10i3.392 TEKNIKA, Volume 10(3), November 2021, pp. 176-180
ISSN 2549-8037, EISSN 2549-8045 178
Tjahjono, M.L., et.al.: Faktor-Faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring
Gambar 3. Jenis Kelamin Responden
Start
Pengumpulan
Data Primer
Penyusunan
Instrumen
Penelitian Tjahjono, M.L., et.al.: Faktor-Faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring Tjahjono, M.L., et.al.: Faktor-Faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring 178 Gambar 1. Tahapan Penelitian
Start
Stop
Pengumpulan
Data Primer
Analisa Data
Menggunakan Regresi
Linier
Hasil Pembahasan
Kesimpulan
Penyusunan
Instrumen
Penelitian Gambar 1. Tahapan Penelitian
Start
Stop
Pengumpulan
Data Primer
Analisa Data
Menggunakan Regresi
Linier
Hasil Pembahasan
Kesimpulan
Penyusunan
Instrumen
Penelitian Gambar 3. Jenis Kelamin Responden Gambar 3. Jenis Kelamin Responden Pada Gambar 2 terlihat bahwa responden adalah kelahiran
tahun 2000 ke atas dimana secara usia tergolong muda dan
seharusnya tidak ada kendala dalam menggunakan teknologi
untuk
mengikuti
kegiatan
pelatihan
berbasis
daring. Berdasarkan penelitian terdahulu dikatakan bahwa internet
yang lancar merupakan hal mutlak yang harus tersedia demi
terjadinya pembelajaran daring. Tanpa adanya koneksi
internet, maka pembelajaran daring tidak mungkin bisa
dilakukan [6]. II. METODOLOGI PENELITIAN Kendala internet ini bisa menjadi salah satu
faktor,
mengapa
pembelajaran
daring
lambat
perkembangannya di negara-negara yang sedang berkembang. Pada Gambar 4 terlihat bahwa belum 100% responden
memiliki internet di rumah dan masih mengandalkan kuota
data yang sangat bergantung pada sinyal seluler. Pada Gambar
5 dapat terlihat tingkat kelancaran koneksi internet pada saat
mengikuti pelatihan. Gambar 4. Fasilitas yang Dimiliki Responden
Gambar 5. Tingkat Kelancaran Koneksi Internet Gambar 4. Fasilitas yang Dimiliki Responden Gambar 1. Tahapan Penelitian Data pada penelitian ini diambil dari para peserta pelatihan
atau workshop pemrograman yang dilakukan secara daring,
data yang yang sudah terkumpul melalui kuesioner akan diolah
menggunakan statistika deskriptif dimana analisis kuantitatif
statistika deskriptif adalah suatu analisis statistik yang
digunakan untuk menganalisis data dengan cara
mendeskripsikan atau menggambarkan data yang telah
terkumpul [5]. Pada penelitian ini akan dihasilkan sebuah
model pelatihan pemrograman berbasis daring. Gambar 4. Fasilitas yang Dimiliki Responden Gambar 5. Tingkat Kelancaran Koneksi Internet III. HASIL Pada
Gambar 9 dapat diidentifikasi faktor paling penting dari
pelaksanaan pelatihan berbasis daring sesi diskusi dengan
pengajar dan disusul dengan kecepatan bicara pengajar, faktor
penting lainnya adalah diskusi kelompok dengan peserta lain
serta tersedianya materi yang bisa dibaca terlebih dahulu oleh
peserta. Pada penelitian ini juga digali apakah responden lebih
memilih pelatihan berbasis daring atau berbasis tatap muka. Hasil penggalian tersebut dapat dilihat dari Gambar 10 di
bawah ini. Gambar 10. Preferensi Pelatihan Oleh Responden
Pada Gambar 10 terlihat bahwa peserta lebih banyak yang
memilih offline workshop atau berbasis tatap muka, tetapi
terlihat juga 31,9% yang memilih sama saja dan 23,1%
memilih online workshop. Melalui hasil ini dapat terlihat
bahwa metode pelatihan daring bisa menjadi alternatif di masa
pandemi covid-19. Model pelatihan berbasis daring yang
disarankan berdasarkan hasil penelitian ini dapat dilihat pada
Gambar 11 di bawah ini. 179 Gambar 8, dimana sebagian besar peserta akan memutar
kembali apabila rekaman materi pelatihan telah ada. Pada
Gambar 9 dapat diidentifikasi faktor paling penting dari
pelaksanaan pelatihan berbasis daring sesi diskusi dengan
pengajar dan disusul dengan kecepatan bicara pengajar, faktor
penting lainnya adalah diskusi kelompok dengan peserta lain
serta tersedianya materi yang bisa dibaca terlebih dahulu oleh
peserta. Gambar 8, dimana sebagian besar peserta akan memutar
kembali apabila rekaman materi pelatihan telah ada. Pada
Gambar 9 dapat diidentifikasi faktor paling penting dari
pelaksanaan pelatihan berbasis daring sesi diskusi dengan
pengajar dan disusul dengan kecepatan bicara pengajar, faktor
penting lainnya adalah diskusi kelompok dengan peserta lain
serta tersedianya materi yang bisa dibaca terlebih dahulu oleh
peserta. Gambar 6. Lama Durasi Pelatihan yang Diharapkan p
Pada penelitian ini juga digali apakah responden lebih
memilih pelatihan berbasis daring atau berbasis tatap muka. Hasil penggalian tersebut dapat dilihat dari Gambar 10 di
bawah ini. Gambar 6. Lama Durasi Pelatihan yang Diharapkan Gambar 7. Kebutuhan Rekaman Materi Pelatihan Gambar 10. Preferensi Pelatihan Oleh Responden Gambar 7. Kebutuhan Rekaman Materi Pelatihan Gambar 7. Kebutuhan Rekaman Materi Pelatihan Gambar 10. Preferensi Pelatihan Oleh Responden Gambar 8. Kemungkinan Responden Memutar Kembali
Rekaman Materi
Gambar 9. Faktor-Faktor Penting Dalam Terselenggaranya
Pelatihan Berbasis Daring Gambar 8. Kemungkinan Responden Memutar Kembali
Rekaman Materi Pada Gambar 10 terlihat bahwa peserta lebih banyak yang
memilih offline workshop atau berbasis tatap muka, tetapi
terlihat juga 31,9% yang memilih sama saja dan 23,1%
memilih online workshop. Melalui hasil ini dapat terlihat
bahwa metode pelatihan daring bisa menjadi alternatif di masa
pandemi covid-19. III. HASIL Pada penelitian ini didapatkan responden sebanyak 91
orang, yang terdiri dari 76,9% pria dan 23,1% wanita. Profil
dari responden dapat terlihat di Gambar 2 dan Gambar 3 di
bawah ini. Gambar 5. Tingkat Kelancaran Koneksi Internet Pada Gambar 5 terlihat bahwa masih ada peserta yang
koneksi internetnya tidak selalu lancar pada setiap pelaksanaan
pelatihan. Hasil
temuan
pada
penelitian
selanjutnya
menggambarkan berapa durasi waktu pelatihan yang dirasa
ideal oleh responden dan komponen dari suatu pelatihan yang
dirasa perlu ada ketika pelatihan dilaksanakan. Hal ini
digambarkan di Gambar 6 hingga Gambar 9. TEKNIKA, Volume 10(3), November 2021, pp. 176-180
ISSN 2549-8037, EISSN 2549-8045
DOI: 10.34148/teknika.v10i3.392
Gambar 2. Tahun Kelahiran Responden
Pada Gambar 5 terlihat bahwa masih ada peserta yang
koneksi internetnya tidak selalu lancar pada setiap pelaksanaan
pelatihan. Hasil
temuan
pada
penelitian
selanjutnya
menggambarkan berapa durasi waktu pelatihan yang dirasa
ideal oleh responden dan komponen dari suatu pelatihan yang
dirasa perlu ada ketika pelatihan dilaksanakan. Hal ini
digambarkan di Gambar 6 hingga Gambar 9. Gambar 2. Tahun Kelahiran Responden Gambar 2. Tahun Kelahiran Responden DOI: 10.34148/teknika.v10i3.392 TEKNIKA, Volume 10(3), November 2021, pp. 176-180
ISSN 2549-8037, EISSN 2549-8045 TEKNIKA, Volume 10(3), November 2021, pp. 176-180
ISSN 2549-8037, EISSN 2549-8045 179
Tjahjono, M.L., et.al.: Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring
Gambar 6. Lama Durasi Pelatihan yang Diharapkan
Gambar 7. Kebutuhan Rekaman Materi Pelatihan
Gambar 8, dimana sebagian besar peserta akan memutar
kembali apabila rekaman materi pelatihan telah ada. Pada
Gambar 9 dapat diidentifikasi faktor paling penting dari
pelaksanaan pelatihan berbasis daring sesi diskusi dengan
pengajar dan disusul dengan kecepatan bicara pengajar, faktor
penting lainnya adalah diskusi kelompok dengan peserta lain
serta tersedianya materi yang bisa dibaca terlebih dahulu oleh
peserta. Pada penelitian ini juga digali apakah responden lebih
memilih pelatihan berbasis daring atau berbasis tatap muka. Hasil penggalian tersebut dapat dilihat dari Gambar 10 di
bawah ini. Gambar 10. Preferensi Pelatihan Oleh Responden Tjahjono, M.L., et.al.: Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring Tjahjono, M.L., et.al.: Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring 179
Tjahjono, M.L., et.al.: Faktor-faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring
Gambar 6. Lama Durasi Pelatihan yang Diharapkan
Gambar 7. Kebutuhan Rekaman Materi Pelatihan
Gambar 8. Kemungkinan Responden Memutar Kembali
Rekaman Materi
Gambar 8, dimana sebagian besar peserta akan memutar
kembali apabila rekaman materi pelatihan telah ada. REFERENSI [1] Gallego, D., Bueno, S.M., Racero, F.J. & Noyes, J. (2015). Open Source Software: The Effects of Training
on Acceptance. Computers in Human Behavior, Vol. 49,
pp. 390-399. Pada penelitian teridentifikasi faktor penting dalam adopsi
pelatihan dalam daring yaitu tersedianya sesi diskusi dengan
pengajar serta bagaimana cara pengajar menyampaikan materi. Hal ini melengkapi hasil penelitian di tahun 2019 oleh Hodjat
Hamidi dan Amir Chavoshi yang menjabarkan bahwa orang
yang berpengaruh di sekitar pengguna seperti dosen, teman
dan keluarga serta kondisi lingkungan akan mempengaruhi
proses adopsi teknologi oleh peserta [8][9]. pp
[2] Hamidi, H. & Chavoshi, A. (2018). Analysis of the
Essential Factors for the Adoption of Mobile Learning in
Higher Education: a Case Study of Students of the
University of Technology. Telematics and Informatics,
Vol. 35(4), pp. 1053–1070. ( ) pp
[3] Arrosagaray, M., González-Peiteado, M., Pino-Juste, M. & Rodríguez-López, B. (2019). A Comparative Study of
Spanish Adult Students’ Attitudes to ICT in Classroom,
Blended and Distance Language Learning Modes. Computers & Education, Vol. 134, pp. 31–40. i
i
d
i
l Sebagian besar responden pada penelitian berusia diantara
19 tahun ke atas dan didominasi oleh pria. Hal ini menjawab
lebih detail dari temuan pada penelitian yang dilakukan
Marcelino Arrosagaray menyebutkan bahwa peserta laki-laki
dengan usia yang relatif muda dan yang kegiatan utamanya
adalah belajar atau bekerja menunjukkan kepercayaan diri
yang
lebih
besar
akan
kemampuan
mereka
dalam
menggunakan
teknologi
dalam
pembelajaran
daring. Penelitian ini kemudian diterbitkan tahun 2019 yang
menyatakan bahwa perbedaan jenis kelamin menjadi hal yang
menarik untuk diteliti dalam adopsi teknologi [4]. p
pp
[4] Maican, C.I., Cazan, A.M., Lixandroiu, R.C. & Dovleac,
L. (2019). A Study on Academic Staff Personality and
Technology Acceptance: The Case of Communication
and
Collaboration
Applications. Computers
&
Education, Vol. 128, pp. 113–131. pp
[5] Lubis, R.K. (2020). Pengaruh Kompensasi Dan
Lingkungan Kerja Terhadap Turnover Intention Di Pt
Axa Mandiri Cabang Imam Bonjol Medan. SAINTEK,
Vol. 1(2), pp. 32-39. pp
[6] Acharya, B. & Lee, J. (2018). Users’ Perspective on the
Adoption of E-learning in Developing Countries: The
Case of Nepal with a Conjoint-based Discrete Choice
Approach. Telematics and Informatics, Vol. 35(6), pp. 1733–1743. Pada penelitian berikutnya hal menarik yang untuk
dilakukan penelitian lebih lanjut adalah membangun model
berdasarkan korelasi analisis atau path analysis. Apabila hal
ini akan dilakukan tentu diperlukan sebuah instrumen
penelitian yang lebih solid dan detail dalam menggali data
untuk membangun model tersebut [7] Saade, R. G. & Kira, D. REFERENSI (2004). Effectiveness of an
Interactive Application to Assist Learning: A Test Case. Journal of Information Systems Education, Vol. 15(4). [8] F
h
T (2012) A
l i
h T
h
l
A f
f
y
[8] Farahat, T. (2012). Applying the Technology Acceptance
Model to Online Learning in the Egyptian Universities. Procedia - Social and Behavioral Sciences, Vol. 64, pp. 95–104. [9] Chavoshi, A. & Hamidi, H. (2019). Social, Individual,
Technological and Pedagogical Factors Influencing
Mobile Learning Acceptance in Higher Education: a Case
From Iran. Telematics and Informatics, Vol. 38, pp. 133–
165. III. HASIL Model pelatihan berbasis daring yang
disarankan berdasarkan hasil penelitian ini dapat dilihat pada
Gambar 11 di bawah ini. Gambar 8. Kemungkinan Responden Memutar Kembali
Rekaman Materi Gambar 8. Kemungkinan Responden Memutar Kembali
Rekaman Materi Gambar 11. Panduan Pelatihan Daring Berdasarkan Hasil
Penelitian. Gambar 9. Faktor-Faktor Penting Dalam Terselenggaranya
Pelatihan Berbasis Daring Pada Gambar 6 terlihat bahwa durasi pelatihan yang
diharapkan tiap sesi tidak lebih dari 2 jam agar bisa terserap
dan dipahami terlebih dahulu sebelum nantinya dilanjutkan
aktifitas pelatihan selanjutnya. Sedangkan pada Gambar 7
terlihat bahwa rekaman materi pelatihan diharapkan ada untuk
dapat diputar kembali ketika peserta masih belum paham
100% terhadap materi pelatihan. Hal ini terlihat juga di Gambar 11. Panduan Pelatihan Daring Berdasarkan Hasil
Penelitian. Pada Gambar 11 dapat dilihat halaman depan website yang
memuat model pelatihan berbasis daring yang disarankan
berdasarkan hasil penelitian yang dilakukan. Model tersebut DOI: 10.34148/teknika.v10i3.392 TEKNIKA, Volume 10(3), November 2021, pp. 176-180
ISSN 2549-8037, EISSN 2549-8045 Tjahjono, M.L., et.al.: Faktor-Faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring Tjahjono, M.L., et.al.: Faktor-Faktor Penting Dalam Penyampaian Pelatihan Atau Workshop
Pemrograman Secara Daring 180 bisa diakses melalui alamat https://sites.google.com/view/
laurazone/panduan?authuser=0 bisa diakses melalui alamat https://sites.google.com/view/
laurazone/panduan?authuser=0 baik serta menyediakan waktu untuk berdiskusi dengan
peserta, selain itu keterlibatan asisten pengajar/tutor juga akan
membantu proses pelatihan berbasis daring. Hal yang bersifat
umum tetapi wajib tersedia dengan baik adalah fasilitas yang
dimiliki peserta terutama koneksi internet yang lancar dan
tidak mudah terputus. Penelitian ini juga menghasilkan sebuah
model pelatihan pemrograman yang berbasis daring beserta
panduannya. Penelitian ini masih bisa dikembangkan dengan
meneliti lebih lanjut faktor-faktor yang berkorelasi dalam
sebuah pelatihan berbasis daring menggunakan metode
analisis jalur atau Technology Acceptance Model (TAM) versi
terbaru. IV. PEMBAHASAN Pada penelitian ini ditemukan sebuah model pelatihan
untuk pemrograman berbasis daring. Ada beberapa hal yang
selaras dengan temuan pada penelitian terdahulu seperti
penelitian di tahun 2004 yang mengatakan kemudahan dalam
adopsi teknologi ini sangat dipengaruhi kemampuan
penyerapan kognitif oleh setiap individu yang ikut sebagai
peserta dalam pembelajaran daring. Hal ini menentukan
seberapa cepat seorang peserta dapat memahami manfaat dan
cara penggunaan teknologi. Semakin tinggi kemampuan
kognitif, maka semakin cepat proses adopsi teknologi ini
terjadi [7] V. KESIMPULAN Pada penelitian ini ditemukan bahwa faktor penting dalam
sebuah pelatihan pemrograman berbasis daring adalah
bagaimana instruktur/pengajar mempersiapkan materi dengan DOI: 10.34148/teknika.v10i3.392 TEKNIKA, Volume 10(3), November 2021, pp. 176-180
ISSN 2549-8037, EISSN 2549-8045
|
https://openalex.org/W2891971398
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https://europepmc.org/articles/pmc6125871?pdf=render
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English
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Investigating factors associated with success of breastfeeding in first-time mothers undergoing epidural analgesia: a prospective cohort study
|
International breastfeeding journal - Electronic Edition -
| 2,018
|
cc-by
| 6,595
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Investigating factors associated with
success of breastfeeding in first-time
mothers undergoing epidural analgesia: a
prospective cohort study Daryl Jian An Tan1, John Paul Lew2, Maria Binte Jumhasan3, Cynthia Pang3, Rehena Sultana4
and Ban Leong Sng1,5* Tan et al. International Breastfeeding Journal (2018) 13:42
https://doi.org/10.1186/s13006-018-0184-7 Tan et al. International Breastfeeding Journal (2018) 13:42
https://doi.org/10.1186/s13006-018-0184-7 Open Access Background nulliparous, adult parturients at ≥36 weeks gestation,
carrying a singleton fetus, presenting in early labour
(cervical dilation ≤5 cm), requesting labour epidural
analgesia and who were suitable for a combined spinal-
epidural technique. Parturients with multiple pregnan-
cies, a non-cephalic fetal presentation, a history of
obstetric or medical complications, contraindications to
neuraxial blockade, or who had received parenteral
opioids within the last 2 h or had an unintentional dural
puncture at the initiation of the epidural were excluded. The participants, attending midwives and medical team
were all blinded to the participant assignment and treat-
ment allocation. g
The 2011 National Breastfeeding Survey for Singapore,
which included 1962 new mothers from nine hospitals
in Singapore, found that while 96% of the new mothers
were breastfeeding on the day of discharge, only 80%
and 42% of them were still breastfeeding at 2 months
and 6 months respectively [1]. This is despite the fact
that most Singaporean mothers know that “breastfeeding
is the best form of feeding for a newborn” (93%), “breast
milk is the best for baby” (79%) and that “breast milk
protects baby from a wide range of disease” (54%) [1]. The most commonly cited reasons for stopping breast-
feeding included being “not able to supply enough milk”
(61%) and the “need to return to work” (24%) [1]. Post-
partum anxiety and depression have been shown to have
negative associations to continued breastfeeding out-
comes [2–4]. Women with symptoms of postpartum
anxiety are less likely to initiate breastfeeding and, if
breastfeeding were to be commenced, are more likely to
terminate breastfeeding earlier and supplement with
infant formula [2]. Postpartum depression is also nega-
tively associated with decreased breastfeeding duration,
increased breastfeeding difficulties and decreased levels
of breastfeeding self-efficacy [3, 4]. Self-reported questionnaires on data of anxiety (State--
Trait Anxiety Inventory (STAI)), depression (Edinburgh
Postnatal Depression Scale (EPDS)), stress (Perceived
Stress Scale (PSS)), pain susceptibility (Pain Catastrophiz-
ing Scale (PCS)) and pain perception (Short Form McGill
Pain Questionnaire (SFMPQ)) were collected from the
participant at baseline after they had just received
labour epidural analgesia. We selected to use these
questionnaires because they were not only convenient
for the participants, but also the reliability and validity of
the questionnaires used in our study have been well-estab-
lished [5–10]. Participant demographics, obstetric and an-
aesthetic data were also collected in this study. Abstract International Breastfeeding Journal (2018) 13:42 Page 2 of 9 Page 2 of 9 Background While there have been various studies that demon-
strate associations between postpartum anxiety and
depression and the likelihood of breastfeeding, the rela-
tionship between peripartum anxiety and depression and
its effects on breastfeeding has not been well researched. We hypothesize that the psychological vulnerabilities the
mother experiences during delivery, particularly peripar-
tum anxiety and depression, can have negative impacts
on the mother’s inclination to breastfeed. The primary
aim of this study is to investigate the associations
between psychological vulnerabilities, specifically peri-
partum anxiety and depression, and the likelihood of
breastfeeding at 5 to 9 weeks postpartum. The secondary
aim of this study is to investigate demographic, obstetric
and pain characteristics that may also influence breast-
feeding at 5 to 9 weeks postpartum. A phone interview would be done at 5 to 9 weeks
postpartum. This time period was selected to assess the
short term breastfeeding status of participants, as well as
to screen for postnatal depression. The participant
would be asked to complete another series of question-
naires including the EPDS, STAI, and the Breastfeeding
Questionnaire via a phone interview conducted by a
blinded research study team member. The Breastfeeding
Questionnaire has been included in this manuscript (see
Additional file 1). All data collected was entered into an
electronic REDCap database, by two research team
members and subsequently crosschecked after entry, to
ensure the accuracy of the data recording. Any discrep-
ancies and inconsistencies in the data entry were
reviewed and de-conflicted by a third investigator. Par-
ticipants who responded to this 5 to 9 weeks postpartum
phone interview were included into the analysis. Abstract Background: We investigated the possible risk factors that could influence the likelihood of breastfeeding at 5 to
9 weeks postpartum with our primary aim being to analyse the associations between psychological vulnerabilities,
such as peripartum depression and anxiety, and continued breastfeeding. Our secondary aim was to investigate
other non-psychological factors’ influence on continued breastfeeding. Methods: A prospective cohort study was conducted in KK Women’s and Children’s Hospital in Singapore. Healthy
nulliparous parturients at ≥36 weeks gestation with a singleton fetus who received epidural analgesia were
recruited. Demographic and anaesthetic data were obtained. Self-reported psychological and pain determinants
such as anxiety (State-Trait Anxiety Inventory), depression (Edinburgh Postnatal Depression Scale), stress (Perceived
Stress Scale), pain susceptibility (Pain Catastrophizing Scale) and pain perception (McGill Pain Questionnaire) were
also recorded at baseline. A phone interview was then performed at 5 to 9 weeks postpartum to obtain
information on breastfeeding status. Results: 329 participants were included into this study, of which 263 (79.9%) of them were still breastfeeding at
5 weeks postpartum. Multivariate logistic regression analysis showed that a higher State-Trait Anxiety Inventory
score (Adjusted Odds Ratio [AOR] 0.97; 95% Confidence Interval [CI] 0.94, 1.00) at baseline, higher intrapartum blood
loss (AOR 0.76; 95% CI 0.61, 0.93), and occurrence of fetal anomalies (AOR 0.15; 95% CI 0.03, 0.72) were associated
with reduced likelihood of breastfeeding at 5 to 9 weeks postpartum. Indians (AOR 0.56; 95% CI 0.20, 1.53), Malays
(AOR 0.30; 95% CI 0.14, 0.62) and other ethnicities (AOR 0.36; 95% CI 0.16, 0.83) were less likely to continue
breastfeeding compared to Chinese participants. On the other hand, receiving any support services on
breastfeeding during the participants’ hospital stay was 3.3 times more likely (AOR 3.30; 95% CI 1.21, 9.02) to
increase the likelihood of breastfeeding at 5 to 9 weeks postpartum. Conclusion: We identified 5 independent association factors that could have significant influences on
breastfeeding at 5 to 9 weeks postpartum. Healthcare providers could utilize this risk stratification to identify
parturients likely to have poorer breastfeeding outcomes and undertake interventions that may help safeguard
optimization of breastfeeding outcomes and parturient care. Trial registration: Clinicaltrials.gov NCT02278601. Registered 26 October 2014. Keywords: Breastfeeding, Risk factors, Predictors, Cohort study, Singapore * Correspondence: sng.ban.leong@singhealth.com.sg
1Duke-NUS Medical School, Singapore, Singapore
5Department of Women’s Anaesthesia, KK Women’s and Children’s Hospital,
Singapore, Singapore
Full list of author information is available at the end of the article Tan et al. Methods KK Women’s and Children’s Hospital (KKH) is a tertiary
level care hospital in Singapore that provides specialized
medical
care
to
the
obstetric,
gynaecological
and
pediatric populations. This cohort study uses data col-
lected in an ongoing trial: The Collaborative Outcomes
with Labour Epidural Use Study (COLEUS). The COLEUS
study is registered on clinicaltrials.gov (NCT02278601). The main study is a double blinded, randomized con-
trolled trial, with the primary aim being to investigate the
efficacy of 3 different epidural maintenance regimens. Sample size calculation and statistical analysis Sample size calculation and statistical analysis
The planned sample size for the study was 320 partici-
pants, which is based on the following assumption: 80%
of mothers will continue breastfeeding till 2–4 months
after delivery based on the National Breastfeeding Sur-
vey conducted in Singapore [1], with a 95% confidence
interval (CI) (precision) of 79.96% to 80.04% i.e. a width
of confidence interval of 0.086%, using the Wilson score
interval method for CI calculation [11, 12]. Our primary
objective was to investigate for possible determinants The study population included healthy (American
Society of Anesthesiologists physical statuses 1 and 2), Tan et al. International Breastfeeding Journal (2018) 13:42 Page 3 of 9 Page 3 of 9 that may influence the mother’s likelihood of breastfeed-
ing at 5 to 9 weeks postpartum. Peduzzi et al., Concato
et al. and Vittinghoff et al. recommended that multivari-
able logistic regression models should be used with at
least 10 events per predictor variable [13–15]. From our
data, the prevalence of participants who breastfed at 5 to
9 weeks postpartum was 82.4% (263/319). Based on the
recommendations, we could adjust for a maximum of
263/10 ≈26 variables in the multivariate model. Our study
was adequately powered (> 80%) with 320 patients based
on following assumptions: proportion of participants who
“breastfed 5 to 9 weeks postpartum” as 80%, odds ratio
(OR) of 3.5 (or 0.29) and alpha or type I error rate as 5%. cardiac anomalies, 2 newborns had orthopedic anomalies
and 1 newborn had neurological anomalies. When grad-
ing their birth experience, 91.8% of participants were
above satisfied. Results from self-reported questionnaires are shown in
Table 2. From the Breastfeeding Questionnaire, 94.2% of
participants had some form of breastfeeding support, of
which help from the nurses in the maternity ward
(82.1%) and from a lactation consultant (37.4%) were the
most common. The mean infant age for cessation of
breastfeeding was 34 days. The most common reasons
for cessation reported by participants were having insuf-
ficient breast milk (75.8%) and choosing to stop breast-
feeding (33.3%). (
)
(
)
p
yp
The primary outcome “status of continued breastfeed-
ing” was treated as binary data with categories of “yes” or
“no”. Demographic, clinical, self-reported questionnaires,
obstetric and anesthetic data were summarized as mean
with standard deviation (SD) for continuous variables, and
frequency with corresponding proportion for categorical
variables. Discussion In our study, we have found that ethnic differences are
independently associated with the likelihood to breast-
feed for at least 5 to 9 weeks postpartum. Participants
with no fetal anomalies, lesser intrapartum blood loss,
lower state anxiety score and who received support or
guidance on breastfeeding are more likely to be breast-
feeding at 5 to 9 weeks postpartum. We did not find any
significant associations between EPDS scores and the
likelihood of breastfeeding at 5 to 9 weeks postpartum. Sample size calculation and statistical analysis Univariate and multivariate logistic regression
models were used to identify possible risk factors of
breastfeeding at 5 to 9 weeks postpartum. Associations
drawn from the logistic regression models were character-
ized using ORs with corresponding 95% CI. Variables with
p-values < 0.20 in the univariate analysis and clinically
important variables were selected for the multivariate lo-
gistic regression model. The union of the variables from
forward, backward and stepwise method were used to
finalize the list of variables in the multivariate model with
entry and stay criteria as 0.2 and 0.05 respectively. Then
we used likelihood ratio test followed by area under the
curve (AUC) to determine the final multivariate model. The variables identified in the multivariate analysis were
further analysed for the strength of their associations to
the likelihood of breastfeeding at 5 to 9 weeks postpartum
through a Receiver Operating Characteristic (ROC) curve. AUC from ROC was also reported. The significance level
was set at 0.05 and all tests were two-tailed. Data were
analysed using SAS version 9.3 software (SAS Institute
Inc.; Cary, NC, USA). The univariate and multivariate regression analyses are
shown in Table 3. Covariates that are independently
associated with breastfeeding at 5 to 9 weeks postpar-
tum are: receiving any support services on breastfeed-
ing (p = 0.02), achieving a lower state anxiety score at
baseline
(p = 0.03),
not
having
any
form
of
fetal
anomalies (p = 0.02) and achieving lesser intrapartum
blood loss (p = 0.01). Ethnic differences are also independ-
ently associated with the likelihood to breastfeed at 5 to
9 weeks postpartum (p = 0.01). Indians (Adjusted OR
[AOR] 0.56; 95% CI 0.20, 1.53), Malays (AOR 0.30; 95%
CI 0.14, 0.62) and other ethnicities (AOR 0.36; 95% CI
0.16, 0.83) were less likely to breastfeed at 5 to 9 weeks
postpartum as compared to Chinese participants. The
AUC based on multivariate analysis was 0.7065, demon-
strating that the association factors utilised in this study
and the identified covariates are moderately associated
with breastfeeding at 5 to 9 weeks postpartum (see
Additional file 2). Results A total of 464 participants were selected for this study,
of which 135 participants were lost to follow up and
hence were not included into the study. The participant
demographic characteristics are shown in Table 1. We
found that 263 (79.9%) of the participants were still
breastfeeding at 5 weeks postpartum. The proportions of
those who breastfed at 5–9 weeks postpartum in each
ethnic group were 84.6% (176/208) Chinese, 80.0% (24/30)
Indian, 66.0% (33/50) Malay and 73.2% (30/41) of other
ethnicities. Figure 1 illustrates the flowchart of the study. The 2011 National Breastfeeding Survey for Singapore
showed that the prevalence of breastfeeding differed
across the various ethnic groups, with higher prevalence
found in the Chinese and Indians as compared to Malays
and other ethnicities [1]. Similarly, Pang et al. conducted
a cohort study involving 1030 Singaporean women in
early pregnancy and found that the prevalence of
breastfeeding at 6 months postpartum varies among
the different ethnic groups even after adjusting for
maternal education [16]. In our study, we found We also found that 7 (2.1%) participants had newborns
with congenital disorders, of which 4 newborns had Tan et al. International Breastfeeding Journal (2018) 13:42 Page 4 of 9 Table 1 Participants’ demographic and obstetric characteristics (n = 329)
Characteristics
Participant still breastfeeding 5–9 weeks postpartum? Results Total
(n = 329)
Yes (n = 263)
No (n = 66)
State-Trait Anxiety Inventory
At baseline
State anxiety score
36.0 ± 8.93
38.2 ± 12.55
36.4 ± 9.77
Trait anxiety score
37.5 ± 6.41
38.8 ± 8.56
37.7 ± 6.90
Total score
73.5 ± 14.19
77.0 ± 19.87
74.2 ± 15.51
At 5–9 weeks postpartum
State anxiety score
30.0 ± 8.88
30.7 ± 9.48
30.2 ± 8.99
Trait anxiety score
35.0 ± 7.14
36.0 ± 8.00
35.2 ± 7.32
Total score
65.1 ± 15.21
66.7 ± 16.72
65.4 ± 15.51
Edinburgh Postnatal Depression Scale
At baseline
7.0 ± 3.76
7.7 ± 5.33
7.2 ± 4.13
At 5–9 weeks postpartum
2.7 ± 3.79
3.3 ± 4.35
2.8 ± 3.91
Short-form McGill Pain Questionnaire (at baseline)
42.4 ± 34.30
48.5 ± 41.74
43.7 ± 35.99
Perceived Stress Scale (at baseline)
18.9 ± 4.60
19.3 ± 5.21
19.0 ± 4.72
Pain Catastrophizing Scale (at baseline)
Rumination score
8.2 ± 4.34
8.7 ± 4.67
8.3 ± 4.40
Magnification score
4.3 ± 2.86
5.1 ± 3.15
4.4 ± 2.93
Helplessness score
9.4 ± 6.17
10.1 ± 6.06
9.6 ± 6.15
Total score
22.6 ± 12.59
24.5 ± 12.67
23.0 ± 12.61
Use of support services on breastfeeding (at 5–9 weeks postpartum)
Help from lactation consultant during hospital stay
105 (39.9)
18 (27.3)
123 (37.4)
Help from ward nurse during hospital stay
218 (82.9)
52 (78.8)
270 (82.1)
Attended lactation clinic
12 (4.6)
1 (1.5)
13 (4.0)
Contacted hospital helpline
5 (1.9)
2 (3.0)
7 (2.1)
Not at all
11 (4.2)
8 (12.1)
19 (5.8)
Reported reasons for stopping breastfeeding (at 5–9 weeks postpartum)
Insufficient milk supply
50 (75.8)
Participant’s volition
22 (33.3)
Difficulty with latching onto breast
8 (12.1)
Medical reasons (either baby or mother)
6 (9.1)
Fatigue/Tiredness
5 (7.6)
Need to return to work after maternity leave
6 (9.1)
Sore nipples
1 (1.5)
Lack of breastfeeding facilities at workplace
1 (1.5)
Values are represented either as mean ± SD or frequency (proportion) Table 2 Results from self-reported questionnaires collected from participants at baseline after they had just received labour epidural
analgesia and at 5–9 weeks postpartum Lack of breastfeeding facilities at workplace lack of social and cultural acceptance and support,
language barriers, and lifestyle choices [17]. similar prevalence (79.1% vs 79.9%) of breastfeeding
at 5 to 9 weeks postpartum as compared to the Na-
tional Breastfeeding Survey in 2011 [1]. Results Total
(n = 329)
Yes (n = 263)
No (n = 66)
Age (years)
30.2 ± 4.15
29.5 ± 5.58
30.1 ± 4.47
Ethnicity
Chinese
176 (66.9)
32 (48.5)
208 (63.2)
Indian
24 (9.1)
6 (9.1)
30 (9.1)
Malay
33 (12.5)
17 (25.8)
50 (15.2)
Others
30 (11.4)
11 (16.7)
41 (12.5)
Mode of delivery
Normal vaginal delivery
153 (58.2)
34 (51.5)
187 (56.8)
Instrument-assisted delivery
46 (17.5)
9 (13.6)
55 (16.7)
Emergency Caesarean Section
64 (24.3)
23 (34.8)
87 (26.4)
Satisfaction with birth experience
Extremely satisfied
42 (16.0)
11 (16.7)
53 (16.1)
Very satisfied
74 (28.1)
17 (25.8)
91 (27.7)
Satisfied
126 (47.9)
32 (48.5)
158 (48.0)
Unsatisfied
19 (7.2)
3 (4.5)
22 (6.7)
Extremely unsatisfied
2 (0.8)
3 (4.5)
5 (1.5)
Complications experienced intrapartum
Premature rupture of membranes
39 (14.8)
15 (22.7)
54 (16.4)
Maternal pyrexia
72 (27.4)
21 (31.8)
93 (28.3)
Non-reassuring fetal status
27 (10.3)
5 (7.6)
32 (9.7)
Failure to progress
32 (12.2)
13 (19.7)
45 (13.7)
Intrapartum blood loss (per 100 ml)
2.5 ± 1.04
3.0 ± 1.76
2.6 ± 1.23
Occurrence of fetal anomalies
3 (1.1)
4 (6.1)
7 (2.1)
Values are represented either as mean ± SD or number (proportion) Table 1 Participants’ demographic and obstetric characteristics (n = 329) Fig. 1 Flow chart of study Tan et al. International Breastfeeding Journal (2018) 13:42 Page 5 of 9 l
l
(
%
%)
f b
f
d
l
k
f
l
d
l
l
d
Table 2 Results from self-reported questionnaires collected from participants at baseline after they had just received labour epidural
analgesia and at 5–9 weeks postpartum
Characteristics
Participant still breastfeeding 5–9 weeks postpartum? Results The ethnic
differences
and
effects
on
breastfeeding
at
5
to
9 weeks postpartum in our study also mirror those
found in the National Breastfeeding Survey and Pang
et al.’s study. Some barriers to breastfeeding which
might account for such ethnic differences include the The Baby Friendly Hospital Initiative was jointly
launched by the World Health Organization and the
United Nations Children’s Fund in 1991 as a global
effort to implement practices that promote, protect and
support breastfeeding [18]. This includes
improved
support of breastfeeding in hospitals, actions to protect Tan et al. Results This is however confounded by the fact that those
participants who are more likely to want to breastfeed
also tend to seek out such support services. Nonetheless,
published literature have supported the efficacy of sup-
port services on the inclination to breastfeed and its
continued duration postpartum [19, 20]. in a high dependency unit or in an intensive care unit. A
delayed initiation of breastfeeding has been shown to
increase the risk of breastfeeding cessation. Thompson
et al. conducted a study of 206 mothers looking at asso-
ciations between postpartum hemorrhage and breast-
feeding experiences, and showed that women with
greater blood loss are more likely to have delayed initi-
ation of breastfeeding and shortened duration of breast-
feeding [24]. Similarly, Brown and Jordan found, in their
cross-sectional survey of 284 mothers, that postpartum
hemorrhage was significantly associated with shorter
breastfeeding duration [25]. The presence of fetal anomalies having a negative
impact on breastfeeding at 5 to 9 weeks postpartum
could be explained by the increased psychosocial burden
placed on the families. Mothers of infants with fetal
anomalies may find it more difficult to feed their babies
and have increased distress over the overall care of their
newborns [26–28]. This burden could be intensified if
the newborns had been admitted into the intensive care
unit [29]. That being said, it is still possible to improve
breastfeeding outcomes in newborns with congenital
anomalies so long as the primary care team adopts the
necessary measures [30]. Torowicz et al.’s prospective
cohort study of 62 mothers has shown that the attitudes
of the institution and the advocacy for breastfeeding are
key for the initiation and maintenance of breastfeeding
in mothers who have infants with complex congenital
heart diseases [31]. Our study showed an inverse association between
STAI scores at baseline and breastfeeding at 5 to 9 weeks
postpartum. Participants with more postpartum anxiety
were more likely to stop breastfeeding by 5 to 9 weeks. In a systematic review by Fallon et al. on postpartum
anxiety and infant-feeding outcomes, the authors found
that women with postpartum anxiety are less likely to
breastfeed exclusively and more likely to stop breastfeed-
ing earlier [2]. In those who do breastfeed, postpartum
anxiety reduces self-efficacy, increases breastfeeding dif-
ficulties, and may negatively affect breastfeeding behav-
iors and even breast milk composition. Heterogeneous
outcomes and methodological limitations however limit
the ability to compare across the studies in the review. Results (Ref: No)
3.160 (1.217, 8.207)
0.0181
3.304 (1.210, 9.020)
0.0197
Satisfaction with birth experience (Ref: Satisfied)
0.3410+
Extremely satisfied
0.970 (0.449, 2.092)
0.5452
Very satisfied
1.106 (0.575, 2.127)
0.2763
Unsatisfied
1.608 (0.448, 5.773)
0.1800
Very unsatisfied
0.169 (0.027, 1.057)
0.0406 nd multivariate logistic regression analysis of covariates of breastfeeding 5 to 9 weeks postpartum Page 7 of 9 Page 7 of 9 Tan et al. International Breastfeeding Journal (2018) 13:42 Page 7 of 9 breastfeeding by national policy implementation, and
public promotion campaigns. KKH was accredited as a
baby-friendly hospital under the Initiative from 2014. We have also developed multiple support services to
encourage breastfeeding, including training for obstetric
ward nurses, arranging a visit to the breastfeeding dyad
from a lactation consultant while in hospital and estab-
lishing a pre- and postnatal lactation clinic. Our partici-
pants also had access to a lactation telephone helpline
and to the lactation consultant via phone after discharge. Other support services include the nationwide Breast-
feeding Mothers’ Support Group Helpline and the Joyful
Parenting and Breastfeeding Helpline. We found that
participants who received support services on breast-
feeding were 3.3 times more likely to be breastfeeding at
5 to 9 weeks postpartum compared to those who did
not. This is however confounded by the fact that those
participants who are more likely to want to breastfeed
also tend to seek out such support services. Nonetheless,
published literature have supported the efficacy of sup-
port services on the inclination to breastfeed and its
continued duration postpartum [19, 20]. breastfeeding by national policy implementation, and
public promotion campaigns. KKH was accredited as a
baby-friendly hospital under the Initiative from 2014. We have also developed multiple support services to
encourage breastfeeding, including training for obstetric
ward nurses, arranging a visit to the breastfeeding dyad
from a lactation consultant while in hospital and estab-
lishing a pre- and postnatal lactation clinic. Our partici-
pants also had access to a lactation telephone helpline
and to the lactation consultant via phone after discharge. Other support services include the nationwide Breast-
feeding Mothers’ Support Group Helpline and the Joyful
Parenting and Breastfeeding Helpline. We found that
participants who received support services on breast-
feeding were 3.3 times more likely to be breastfeeding at
5 to 9 weeks postpartum compared to those who did
not. Results International Breastfeeding Journal (2018) 13:42 Page 6 of 9 Table 3 Univariate and multivariate logistic regression analysis of covariates of breastfeeding 5 to 9 weeks postpartum
Characteristics
Unadjusted
OR (95% CI)
P – value
Adjusted OR
(95% CI)
P – value
Age (years)
1.036 (0.975, 1.102)
0.2545
Mode of delivery (Ref: Normal vaginal delivery)
0.2180+
Instrument-assisted delivery
0.618 (0.338, 1.132)
0.0872
Emergency Caesarean Section
1.136 (0.508, 2.541)
0.3528
Race (Ref: Chinese)
0.0217+
0.0052+
Indian
0.727 (0.276, 1.920)
0.557 (0.203, 1.528)
0.7416
Malay
0.353 (0.176, 0.708)
0.294 (0.140, 0.618)
0.0668
Others
0.496 (0.226, 1.089)
0.362 (0.157, 0.833)
0.3129
State-Trait Anxiety Inventory
At baseline
State anxiety score
0.977 (0.951, 1.004)
0.1002
0.968 (0.940, 0.997)
0.0304
Trait anxiety score
0.973 (0.935, 1.011)
0.1628
Total score
0.985 (0.969, 1.003)
0.0977
At 5–9 weeks postpartum
State anxiety score
0.993 (0.964, 1.022)
0.6244
Trait anxiety score
0.982 (0.948, 1.018)
0.3180
Total score
0.994 (0.977, 1.010)
0.4501
Edinburgh Postnatal Depression Scale
At baseline
0.961 (0.902, 1.025)
0.2306
At 5–9 weeks postpartum
0.966 (0.905, 1.031)
0.3002
Short-form McGill Pain Questionnaire
0.996 (0.988, 1.003)
0.2472
Perceived Stress Scale
0.985 (0.929, 1.044)
0.6133
Pain Catastrophizing Scale
Rumination score
0.977 (0.919, 1.039)
0.4576
Magnification score
0.907 (0.830, 0.992)
0.0332
Helplessness score
0.982 (0.940, 1.025)
0.4010
Total score
0.989 (0.968, 1.010)
0.2896
Complications experienced intrapartum (Ref: No)
Premature rupture of membranes
0.592 (0.303, 1.155)
0.1243
Maternal pyrexia
0.808 (0.450, 1.449)
0.4742
Non-reassuring fetal status
1.395 (0.516, 3.774)
0.5115
Failure to progress
0.565 (0.278, 1.149)
0.1150
Intrapartum blood loss per 100 ml
0.789 (0.646, 0.962)
0.0191
0.756 (0.614, 0.930)
0.0081
Occurrence of fetal anomalies (Ref: No)
0.179 (0.039, 0.820)
0.0267
0.148 (0.030, 0.722)
0.0182
Use of support services on breastfeeding
Help from lactation consultant during hospital stay (Ref: No)
1.772 (0.977, 3.214)
0.0596
Help from ward nurse during hospital stay (Ref: No)
1.304 (0.666, 2.553)
0.4382
Attended lactation clinic (Ref: No)
3.105 (0.397, 24.306)
0.2804
Contacted hospital helpline (Ref: No)
0.620 (0.118, 3.266)
0.5725
Did you receive any of these support services on
breastfeeding or giving breast milk to your baby? Results Our findings are consistent with those found in similar
studies such as a study in Pennsylvania which showed
that the positive STAI score of 192 parturients were
associated with poorer breastfeeding outcomes in the
first 6 months postpartum [21]. Our study did not show any significant associations
between EPDS scores and breastfeeding at 5 to 9 weeks
postpartum. This finding differs from those seen in other
studies, which show that higher EPDS scores were asso-
ciated with cessation of breastfeeding [32–34]. Likewise,
a systematic review of existing literature on breastfeed-
ing and maternal depression has shown that both preg-
nancy
and
postpartum
depression
predict
shorter
breastfeeding durations [35]. We hypothesize that the
lack of an association in our study could be the short
follow-up period (5 to 9 weeks postpartum) as opposed
to the time point assessments adopted in other studies. We found that intrapartum blood loss is negatively as-
sociated with breastfeeding at 5 to 9 weeks postpartum. We hypothesize that the amount of blood lost intrapar-
tum is an indicator of the difficulty and complexity of
the delivery process. To the participants, this can trans-
late into birth traumas that can become a source of psy-
chological distress. It has been shown in other studies
that traumatic stressors can influence and truncate the
duration of breastfeeding in mothers [22, 23]. Addition-
ally, it can be argued that substantial hemorrhages can
lead to delayed initiation of breastfeeding. This could be
due to the mothers feeling fatigued from anemia, or that
the mothers had to receive specialized care postpartum This study is limited by confounders present in our
study such as participants with preconceived intentions
to breastfeed and participants with background prenatal
anxiety and depression. Other considerations such as
smoking status and body mass index that may serve as
potential confounders were also not taken into account
in our study. Another limitation is our broader inclusion
criteria of participants with 59 who breastfed exclusively
and 97 who breastfed non-exclusively, rather than spe-
cifically analysing participants who breastfed exclusively. Furthermore, we only looked at participants who have
received labour epidural analgesia. In addition, our study
conducted follow ups at 5 to 9 weeks postpartum, and Tan et al. International Breastfeeding Journal (2018) 13:42 Page 8 of 9 Page 8 of 9 Page 8 of 9 did not follow up at 6 months postpartum. Competing interests
Th
h
d
l
h Breastfeeding is substantially beneficial to both mother
and child. Given the importance of breastfeeding, it is
essential to identify risk factors that can influence
breastfeeding outcomes. In our study, we have ascer-
tained several determinants that have significant impact
on breastfeeding at 5 to 9 weeks postpartum. Health-
care providers could utilize this risk stratification to
identify parturients likely to have poorer breastfeeding
outcomes and undertake interventions that may help
safeguard optimization of breastfeeding outcomes and
parturient care. Results While breast-
feeding is recommended for at least the first 6 months,
we recognise that psychological vulnerabilities such as
anxiety and depression are prevalent in the weeks
following labour, thus our selection of follow up time
points. We only analysed participants who completed all
stages of this study, and we did not analyse the differ-
ences between these participants and those with incom-
plete stages. Finally, we found that insufficient milk
supply was a common reason for cessation of breast-
feeding according to responses. However, we did not ex-
plore the milk insufficiency in these women. version to be submitted. JPL: Data collection, data analysis, drafted the
manuscript, revised the article critically for important intellectual content and
final approval for the version to be submitted. MBJ: data analysis, revised the
article and final approval of the version to be submitted. CP: data analysis,
revised the article and final approval of the version to be submitted. RS: data
analysis, revised the article and final approval of the version to be submitted. BLS: study design, data analysis, revised the article and final approval of the
version to be submitted. version to be submitted. JPL: Data collection, data analysis, drafted the
manuscript, revised the article critically for important intellectual content and
final approval for the version to be submitted. MBJ: data analysis, revised the
article and final approval of the version to be submitted. CP: data analysis,
revised the article and final approval of the version to be submitted. RS: data
analysis, revised the article and final approval of the version to be submitted. BLS: study design, data analysis, revised the article and final approval of the
version to be submitted. Author details
1 1Duke-NUS Medical School, Singapore, Singapore. 2Department of
Anaesthesiology, Singapore General Hospital, Singapore, Singapore. 3Division
of Nursing, KK Women’s and Children’s Hospital, Singapore, Singapore. 4Centre for Quantitative Medicine, Duke-NUS Medical School, Singapore,
Singapore. 5Department of Women’s Anaesthesia, KK Women’s and
Children’s Hospital, Singapore, Singapore. Received: 6 February 2018 Accepted: 27 August 2018 Received: 6 February 2018 Accepted: 27 August 2018 Availability of data and materials The data that support the findings of this study are available from the
corresponding author upon reasonable request. 10. Chen H, Bautista D, Ch'ng YC, Li W, Chan E, Rush AJ. Screening for postnatal
depression in Chinese-speaking women using the Hong Kong translated
version of the Edinburgh postnatal depression scale. Asia Pac Psychiatry. 2013;5:E64–72. Ethics approval and consent to participate This study was approved by the Singhealth Centralised Institutional Review
Board (Singhealth CIRB reference number: 2014/670/D) and the trial is
registered on clinicaltrials.gov (NCT02278601). All participants provided their
written informed consent before enrolment. Consent for publication
Not applicable. Consent for publication
Not applicable. Abbreviations 4. Henderson JJ, Evans SF, Straton JA, Priest SR, Hagan R. Impact of postnatal
depression on breastfeeding duration. Birth. 2003;30:175–80. 4. Henderson JJ, Evans SF, Straton JA, Priest SR, Hagan R. Impact of postnatal
depression on breastfeeding duration. Birth. 2003;30:175–80. AUC: Area under the ROC curve; CI: Confidence interval; CIRB: Centralised
Institutional Review Board; COLEUS: Collaborative Outcomes with Labour
Epidural Use Study; EPDS: Edinburgh postnatal depression scale; KKH: KK
Women’s and Children’s Hospital; OR: Odds ratio; PCS: Pain catastrophizing
scale; PSS: Perceived stress scale; ROC: Receiver operating characteristic;
SAS: Statistical analysis software; SD: Standard deviation; SFMPQ: Short form
McGill pain questionnaire; STAI: State-Trait anxiety inventory 5. Gunning MD, Denison FC, Stockley CJ, Ho SP, Sandhu HK, Reynolds RM. Assessing maternal anxiety in pregnancy with the state-trait anxiety
inventory (STAI): issues of validity, location and participation. J Reprod Infant
Psychol. 2010;28:266–73. 5. Gunning MD, Denison FC, Stockley CJ, Ho SP, Sandhu HK, Reynolds RM. Assessing maternal anxiety in pregnancy with the state-trait anxiety
inventory (STAI): issues of validity, location and participation. J Reprod Infant
Psychol. 2010;28:266–73. 6. Grafton KV, Foster NE, Wright CC. Test-retest reliability of the short-form
McGill pain questionnaire: assessment of intraclass correlation coefficients
and limits of agreement in patients with osteoarthritis. Clin J Pain. 2005;
21:73–82. 6. Grafton KV, Foster NE, Wright CC. Test-retest reliability of the short-form
McGill pain questionnaire: assessment of intraclass correlation coefficients
and limits of agreement in patients with osteoarthritis. Clin J Pain. 2005;
21:73–82. Acknowledgements This study used data collected from the COLEUS study. We would like to
thank Ms. Agnes Teo (Clinical Research Coordinator) for her administrative
support. 7. Gibson J, McKenzie-McHarg K, Shakespeare J, Price J, Gray R. A systematic
review of studies validating the Edinburgh postnatal depression scale
in antepartum and postpartum women. Acta Psychiatr Scand. 2009;
119:350–64. References Additional file 1: Breastfeeding Questionnaire. This is a copy of the
Breastfeeding Questionnaire which was conducted via a telephone
interview to the participant at 5 to 9 weeks postpartum. (PDF 42 kb)
Additional file 2: Receiver Operating Characteristic (ROC) curve. The
Receiver Operating Characteristic (ROC) curve for the independent
covariates for breastfeeding 5 to 9 weeks postpartum. The area under the
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Congenital multiple colonic atresias with intestinal malrotation: a case report
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Surgical case reports
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© The Author(s). 2020 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/. Background congenital multiple colonic atresias (TypeI) with intes-
tinal malrotation, along with a literature review. Congenital intestinal atresia develops in 1 in 1500 to 20,
000 births [1, 2]. Colonic atresia, which accounts for
1.8–15% of intestinal atresia cases [3, 4], is accompanied
by other gastrointestinal atresias such as small intestinal
atresia, gastroschisis, imperforate anus, and intestinal
malformation in 47–80% [5]. Although a report shows
that patients with multiple colonic atresias are 8.9% of
those with colonic atresia [6], there have been three re-
ports of multiple colonic atresias with malrotation ac-
cording to our most extensive search. However all three
reports were TypeIII atresias. We report a case of Ishii et al. Surgical Case Reports (2020) 6:60
https://doi.org/10.1186/s40792-020-00822-z Ishii et al. Surgical Case Reports (2020) 6:60
https://doi.org/10.1186/s40792-020-00822-z Open Access Abstract Background: Congenital intestinal atresia develops in 1 in 1500 to 20,000 births. Colonic atresia, which accounts for
1.8–15% of intestinal atresia cases, is accompanied by other gastrointestinal atresias such as small intestinal atresia,
gastroschisis, imperforate anus, and intestinal malformation in 47–80%. Although a report shows that patients with
multiple colonic atresias are 8.9% of those with colonic atresia. Case presentation: A male infant did not have the first bowel movement within 36 h of birth and had abdominal
distention/vomiting. Radiography showed significant dilation of the intestinal tract. A contrast enema examination
at 3 days of age showed a microcolon and disruption in the descending colon. We performed an emergency
decompressive loop enterostomy in the distended segment. At the age of 7 months, imaging from the stoma
showed disruption of the contrast medium in the intestinal tract at the right lower abdomen, and the continuity of
the intestinal tract was not clarified. Intestinal malrotation was found during the second surgery, and the
enterostomy was located in the ileum proximal to Bauhin’s valve. Continuity of the intestinal serosal surface was
maintained. However, multiple membranous obstructions (three atresias and one stenosis) were observed in the
distal segment of the bowel, which was penetrated by intraluminal advancement of a urethral catheter. Therefore,
he was diagnosed with multiple colonic atresias. The intestinal tract was longitudinally incised, and
membranectomy and mucosal/lateral suture were performed. Conclusions: It is important for neonates with intestinal atresia to evaluate and prepare for distal patency of the
colon before radical anastomosis. In addition, anomalies associated with colon atresia should also be assessed. Keywords: Colonic atresia, Multiple, Intestinal malrotation, Microcolon Daisuke Ishii*, Hisayuki Miyagi, Masatoshi Hirasawa and Kazutoshi Miyamoto Daisuke Ishii*, Hisayuki Miyagi, Masatoshi Hirasawa and Kazutoshi Miyamoto * Correspondence: d-ishii@asahikawa-med.ac.jp
Department of Pediatric Surgery, Asahikawa Medical University, 2-1-1,
Midorigaoka-higashi Asahikawashi, Hokkaido 078-8510, Japan Case presentation The course of pregnancy was normal. The infant was
born at a gestational age of 38 weeks and 6 days by nor-
mal vaginal delivery, and birth weight was 2790 g at the
previous hospital. The infant did not have the first bowel
movement within 36 h of birth and had abdominal dis-
tention
and
vomiting. Plain
abdominal radiography
showed significant dilation of the intestinal tract and no
gas in the pelvic cavity (Fig. 1), which did not respond to
catheterization with a Nelaton’s catheter or enema. A
contrast enema examination at 3 days of age (Fig. 2)
showed a microcolon and disruption in the descending * Correspondence: d-ishii@asahikawa-med.ac.jp
Department of Pediatric Surgery, Asahikawa Medical University, 2-1-1,
Midorigaoka-higashi Asahikawashi, Hokkaido 078-8510, Japan Page 2 of 5 Page 2 of 5 Ishii et al. Surgical Case Reports (2020) 6:60 (2020) 6:60 Ishii et al. Surgical Case Reports observed. After puncturing the dilated intestinal tract
and depressurizing, we performed only emergency enter-
ostomy with double orifices for the dilated intestinal
tract. During the operation, we wanted to confirm distal
patency and associated anomalies and make a definitive
diagnosis, but we could not do it due to prominent in-
testinal dilatation and his unsteady breathing condition. Fig. 1 Plain abdominal radiography (36 h after birth). Plain
abdominal radiography showed a significant dilation of the intestinal
tract and no gas in the pelvic cavity A biopsy of the intestinal tract at the site of enterostomy
showed ganglion cells in the Meissner’s and Auerbach’s
plexus. And a biopsy of the rectal mucosa showed ganglion
cells in the Meissner’s plexus. Therefore, Hirschsprung’s
disease was ruled out. As associated anomalies, the infant
had Ebstein’s anomaly (mild) and mild pyelectasis on the
left side (I–II). His family history was noncontributory. He was admitted to our department to undergo radical
operation at 7 months of age. The height was 65.1 cm
and weight was 6.53 kg. Preoperative contrast enema
from the anus showed a microcolon and disruption of
the contrast medium in the descending colon. Imaging
from the stoma showed a disruption of the contrast
medium in the intestinal tract at the right lower abdo-
men, and the continuity of the intestinal tract on both
sides was not clear (Fig. 3). Fig. 1 Plain abdominal radiography (36 h after birth). Plain
abdominal radiography showed a significant dilation of the intestinal
tract and no gas in the pelvic cavity g
Laparotomy, with transection in the lower abdomen,
showed a complicated non-rotation type of intestinal
malrotation, and the enterostomy was located in the
ileum proximal to Bauhin’s valve. We removed the ileos-
tomy once to confirm distal patency. The continuity of
the intestinal serosal surface was maintained, whereas in
the lumen, there were 3 membranous atretic sites and 1
stenotic site; (1) a membrane-like atresia in the Bauhin’s
valve, (2) a membrane-like atresia in the 2-cm anal side
of Bauhin’s valve, (3) a membrane-like atresia in the
transverse colon, and (4) a stricture in the 2-cm anal
side of the atresia (Fig. 4). For the three membrane-like
atresias (TypeI), the intestinal tract was longitudinally
incised, and membranectomy and mucosal/lateral suture
were performed. Discussion Colonic atresia, a type of congenital intestinal atresia
that develops in the colon, was first reported by Binnin-
ger et al. in 1873, and a lifesaving case was reported by
Gaub et al. in 1922 [7]. Congenital intestinal atresia de-
velops in 1 in 1500 to 20,000 births [1, 2], and colonic
atresia is shown to account for 1.8–15% of them [3, 4]. We had 75 patients with congenital intestinal atresia be-
tween 1989 and 2017 in our department, of whom, five
patients (6.7%) had colonic atresia. Although a report
shows that patients with multiple colonic atresias are
8.9% of those with colonic atresia [6], there have been
three reports of multiple colonic atresias with malrota-
tion according to our most extensive search (Table 1) [8,
9] (excluding patients with familial intestinal polyatresia
(FIPA) syndrome stated below). All three reports were
TypeIII atresias with our case being the first reported
case of TypeI atresias. At 9 months of age, he was admitted for the ileostomy
closure. Simulated stool had been injected into the intes-
tinal tract in the anal side of the stoma 7 days before
surgery. A contrast enema examination confirmed the
improvement in disuse atrophy of the intestinal tract
and its passage. But at operation a stricture was observed
in the region where the membrane-like atresia in the
transverse colon was removed. We performed a balloon Fig. 4 Operative findings. The continuity of the intestinal serosal
surface was maintained, while, as for the lumen, there were
membranous atresias in the Bauhin’s valve and anus, 2 cm from the
Bauhin’s valve (circle), and a membranous atresia in the transverse
colon and stricture in the anus, 2 cm from the atresia (arrow) The causes of congenital intestinal atresia are ex-
plained by the theory of developmental anomaly due to
failure of recanalization by Tandler et al. [10] and the
theory of vascular insufficiency by Louw et al. [11]. In
the present case, atresia was categorized as type I in
Louw’s classification; there was no finding that suggests
vascular insufficiency, including no abnormality in the
mesentery; and the pathological diagnosis showed find-
ings in which developmental anomaly was suspected, so Fig. 4 Operative findings. Imaging from the
stoma showed a disruption of the contrast medium inside the
intestinal tract in the right lower abdomen (circle), and the
continuity of the cecum and intestinal tract on both sides was
not clear Fig. 5 Pathological diagnosis. The wall structure in membranous
atresias was maintained, and mucosal epithelial regenerative
changes were observed. In the resected membranes of the atresias
in the Bauhin’s valve and ascending colon, the muscle layer
structure was unclear in the entire specimens, and the muscle layer
was occupied with hyperplastic connective tissue dilation and the ileostomy closure. The postoperative
course was favorable, and the baby was discharged on
day 11 after surgery. Currently, he has outpatient follow-
up visits, and the control of defecation is favorable. Fig. 3 Preoperative contrast enema examination (7 months of age). Imaging from the anus showed a microcolon and disruption of the
contrast medium in the descending colon (arrow). Imaging from the
stoma showed a disruption of the contrast medium inside the
intestinal tract in the right lower abdomen (circle), and the
continuity of the cecum and intestinal tract on both sides was
not clear We only performed longitudinal inci-
sion/lateral suture for the constricted intestinal tract. Fi-
nally, the ileostomy was reconstructed at the same ileal
site. The operative time was 2 h 59 min, and blood loss
was 55 g. The postoperative course was favorable, and he
was discharged on day 14 postoperatively. colon. Taking intestinal atresia, Hirschsprung’s disease,
and hypoplastic left hemicolon as a preoperative diagno-
sis into consideration, he was transferred to our hospital. Because of his unsteady breathing condition, we per-
formed emergency enterostomy at 3 days after birth. Laparotomy, with 3 cm transection in the upper right
abdomen, observed serous ascites, but no adhesion was colon. Taking intestinal atresia, Hirschsprung’s disease,
and hypoplastic left hemicolon as a preoperative diagno-
sis into consideration, he was transferred to our hospital. Because of his unsteady breathing condition, we per-
formed emergency enterostomy at 3 days after birth. Laparotomy, with 3 cm transection in the upper right
abdomen, observed serous ascites, but no adhesion was Fig. 2 Contrast enema examination (3 days of age). The arrow
showed a microcolon and disruption in the descending colon Pathologically, the wall structure of the membrane-like
atresias was maintained, and mucosal epithelial regen-
erative changes were observed (Fig. 5). Ganglion cells
were present in the Meissner’s and Auerbach’s plexus. In the resected membranes of the atresias in the Bau-
hin’s valve and ascending colon, the muscle layer struc-
ture was unclear in the entire specimens, and the muscle
layer was occupied by hyperplastic connective tissue,
mainly composed of collagen fibers, in which defect and
developmental abnormalities of the muscle layer were
suspected as the causes of atresia. Fig. 2 Contrast enema examination (3 days of age). The arrow
showed a microcolon and disruption in the descending colon Page 3 of 5 Ishii et al. Surgical Case Reports (2020) 6:60 (2020) 6:60 Ishii et al. Surgical Case Reports Fig. 5 Pathological diagnosis. The wall structure in membranous
atresias was maintained, and mucosal epithelial regenerative
changes were observed. In the resected membranes of the atresias
in the Bauhin’s valve and ascending colon, the muscle layer
structure was unclear in the entire specimens, and the muscle layer
was occupied with hyperplastic connective tissue Fig. 3 Preoperative contrast enema examination (7 months of age). Imaging from the anus showed a microcolon and disruption of the
contrast medium in the descending colon (arrow). Abbreviations Abbreviations
FIPA: Familial intestinal polyatresia FIPA: Familial intestinal polyatresia Discussion The continuity of the intestinal serosal
surface was maintained, while, as for the lumen, there were
membranous atresias in the Bauhin’s valve and anus, 2 cm from the
Bauhin’s valve (circle), and a membranous atresia in the transverse
colon and stricture in the anus, 2 cm from the atresia (arrow) Ishii et al. Surgical Case Reports (2020) 6:60 Page 4 of 5 Table 1 Reports of multiple colonic atresias with malrotation
Birth weight
Gastational age
Time of first surgery
Atresias
Type
First surgery
Anomalies
Ref
1
nm
40 weeks
52 h
A, T, D
III
Ostomy
Malrotation
8
2
nm
40 weeks
48 h
T, Sp
III
Ostomy
Malrotation
8
3
nm
34 weeks
12 h
A, H, T
III
Ostomy
Gastroschisis, malrotation
9
4
2790 g
38 weeks
72 h
A, T
I
Ostomy
Ebstein’s anomaly, pyelectasis, malrotation
Our case
nm not mentioned, A ascending colon, T transverse colon, Sp sigmoid colon, D descending colon Table 1 Reports of multiple colonic atresias with malrotation failure of recanalization was considered to be the cause
of the disease. But we cannot explain the cause of mul-
tiple atresias because of only failure of recanalization. As
a cause of multiple atresias, vascular insufficiency due to
gastroschisis and FIPA syndrome [12, 13] are common
causes to be discriminated. The present case had no
findings to suggest them. So, multiple atresias can be
caused by various factors. Further case accumulation
and analysis are required. performed emergency enterostomy. In a case of mild
dilation of the intestinal tract, one-stage surgery can be
performed. However, in a case of potential multiple atre-
sias, as in our patient, and a case of colonic atresia, com-
bined malformation is common, and we consider that by
performing multiple-stage surgery, radical operation,
after a preoperative sufficient examination of the intes-
tinal tract, is safe. Combined malformation is shown to occur in 47–80% of
patients with colonic atresia [5, 6], and atresia of the other
gastrointestinal tracts, such as small intestinal atresia, gas-
troschisis, imperforate anus, intestinal malrotation, cardiac
malformation, omphalocele, and Hirschsprung’s disease,
have been reported [6]. The most common of the com-
bined malformation is gastroschisis. Intestinal malrotation
is less common as the combined malformation [14, 15]. Ethics approval and consent to participate
Not applicable. Ethics approval and consent to participate
Not applicable. Funding None. Authors’ contributions These authors saw this case in the department of surgery, and equally
contributed for preparation of this manuscript. DI, HM, MH, and KM
performed the surgical procedure. DI contributed to the drafting of the
manuscript and literature review. DI obtained consent from the patient. KM
is the director of the department. DI, HM, and MH managed to preoperative
and postoperative course. HM, MH, and KM instructed of the overall
description of this manuscript. All the authors critically appraised the
manuscript and contributed to make necessary changes in the article. All
authors read and approved the final manuscript. Consent for publication Written informed consent was obtained from the patient for publication of
this case report and any accompanying images. Conclusions It is important for neonates with intestinal atresia to
evaluate and prepare for distal patency of the colon be-
fore radical anastomosis. In addition, anomalies associ-
ated with colon atresia should also be assessed. In a case
of potential multiple atresias, we consider that by per-
forming multiple-stage surgery, radical operation, after a
preoperative sufficient examination of the intestinal
tract, is safe. [
,
]
Since in colonic atresia, due to the presence of the
Bauhin’s valve, which is different from intestinal atresia
in other regions, closed loop obstruction occurs, the on-
set of abdominal distention and vomiting is delayed, and
symptoms commonly occur after 24 h of birth. There-
fore, necrosis and perforation easily occur, and early
diagnosis is necessary. A report showed that, in patients
who underwent surgery over 72 h after birth, the mortal-
ity is significantly higher than those who had surgery
within 72 h after birth, and the cause of death is perfor-
ation due to dilation of the occluded colon in the rostral
portion [8]. For the atresia in the right colon, one-stage
surgery, in which anastomosis is performed at the first
surgery, and for the atresia in the left colon, multiple-
stage surgery, in which anastomosis is performed after
colostomy, have been recommended [16]. However, one-
stage surgery is advantageous in that it requires fewer
surgeries, but the risks of ileocecal resection, anasto-
motic leak, and sepsis are shown to be high [6]. Multiple-stage surgery allows safe decompression of an
anastomotic site as well as a dilated intestinal tract, and
this is advantageous in the conservation of the ileocecal
valve. However, it is disadvantageous in that it requires
more number of surgeries. Because our patient had a
significant dilation of the intestinal tract at the first sur-
gery, we could not confirm distal patency and make a
definitive diagnosis and we judged that the risk of
complication would be high in one-stage surgery, and Availability of data and materials
Not applicable. Availability of data and materials
Not applicable. Acknowledgements
h
k d Acknowledgements
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Fruhen Embryonalstadien. Gegenbaurs Morphol Jahrb. 1902;29:187–215. 12. Mishalany HG, Der Kaloustian VM. Familial multiple-level intestinal atresias:
report of two siblings. J Pediatr. 1971;79:124–5. 13. Cole C, Freitas A, Clifton MS, et al. Hereditary multiple intestinal atresias: 2
new cases and review of the literature. J Pediatr Surg. 2010;45:21–5. 14. El-Asmar KM, Abdel-Latif M, Abdel-Hamid A, El-Kassaby, et al. Colonic atresia
association with other anomalies. J Neonat Surg. 2016;5:47. 15. Karnak I, Ciftci AO. Mehmet Emin Senocak, et al. Colonic atresia: surgical
management and outcome. Pediatr Surg Int. 2001;17:631–5. 16. Benson CD, Lofti MW, Brough AJ. Congenital atresia and stenosis of the
colon. J Pediatr Surg. 1968;3:253–7. Competing interests
Th
h
h The authors have no competing interests to declare. The authors have no competing interests to declare. Page 5 of 5 Page 5 of 5 Ishii et al. Surgical Case Reports (2020) 6:60 Ishii et al. Surgical Case Reports Ishii et al. Surgical Case Reports (2020) 6:60 Received: 23 November 2019 Accepted: 19 March 2020 References
1. Evans CH. Atresias of the gastrointestinal tract. Int Abstr Surg. 1951;92:1–8. 2. Webb CH, Wangsteen OH. Congenital intestinal atresia. Am J Dis Child. 1931;41:262–84. 3. Pohlson EC, Hatch EI Jr, Glick PL, et al. Individualized management of
colonic atresia. Am J Surg. 1988;155:690–2. 4. Dalla Vecchia LK, Grosfeld JL, West KW, et al. Intestinal atresia and stenosis: a
25-year experience with 277 cases. Arch Surg. 1998;133:490–7. 5. Wilson BJ, Nelson A, Harshbarger M. Congenital atresia of the colon. Surg
Gynecol Obstet. 1954;99:34–41. 6. Takenouchi A, Yoshida H, Matsunaga T, et al. A case of congenital colonic
atresia with ileal stenosis. J Jpn Soc Pediatr Surg. 2004;40:884–9. 7. Fuller JW, Scarano VR. Congenital atresia of the colon. South Med J. 1977;70:
987–90. 8. Etensel B, Temir G, Karkiner A, et al. Atresia of the colon. J Pediatr Surg. 2005;40:1258–68. 9. Barsoom MJ, Prabulos A, Rodis JF, et al. Vanishing gastroschisis and short-
bowel syndrome. Obstet Gynecol. 2000;96:818–9. 10. Tandler I. Zur Entwicklungsgeschichte des Menschlichen Duodenum in
Fruhen Embryonalstadien. Gegenbaurs Morphol Jahrb. 1902;29:187–215. 11. Louw JH, Barnard CN. Congenital intestinal atresia; observations on its
origin. Lancet. 1955;269:1065–7. 12. Mishalany HG, Der Kaloustian VM. Familial multiple-level intestinal atresias:
report of two siblings. J Pediatr. 1971;79:124–5. 13. Cole C, Freitas A, Clifton MS, et al. Hereditary multiple intestinal atresias: 2
new cases and review of the literature. J Pediatr Surg. 2010;45:21–5. 14. El-Asmar KM, Abdel-Latif M, Abdel-Hamid A, El-Kassaby, et al. Colonic atresia:
association with other anomalies. J Neonat Surg. 2016;5:47. 15. Karnak I, Ciftci AO. Mehmet Emin Senocak, et al. Colonic atresia: surgical
management and outcome. Pediatr Surg Int. 2001;17:631–5. 16. Benson CD, Lofti MW, Brough AJ. Congenital atresia and stenosis of the
colon. J Pediatr Surg. 1968;3:253–7. Publisher’s Note
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A feasibility study of perioperative vitamin D supplementation in
patients undergoing colorectal cancer resection Edinburgh Research Explorer Citation for published version:
Vaughan-shaw, P, Buijs, LF, Blackmur, J, Ewing, A, Theodoratou, E, Ooi, LY, Din, FVN, Farrington, SM &
Dunlop, MG 2023, 'A feasibility study of perioperative vitamin D supplementation in patients undergoing
colorectal cancer resection', Frontiers in Nutrition, vol. 10, 1106431, pp. 1106431. https://doi.org/10.3389/fnut.2023.1106431 Citation for published version:
Vaughan-shaw, P, Buijs, LF, Blackmur, J, Ewing, A, Theodoratou, E, Ooi, LY, Din, FVN, Farrington, SM &
Dunlop, MG 2023, 'A feasibility study of perioperative vitamin D supplementation in patients undergoing
colorectal cancer resection', Frontiers in Nutrition, vol. 10, 1106431, pp. 1106431. https://doi.org/10.3389/fnut.2023.1106431 OPEN ACCESS OPEN ACCESS
EDITED BY
William K. K. Wu,
The Chinese University of Hong Kong, China
REVIEWED BY
Ronan Lordan,
University of Pennsylvania, United States
Vera Wesselink,
Wageningen University and Research,
Netherlands
*CORRESPONDENCE
M. G. Dunlop
malcolm.dunlop@ed.ac.uk
SPECIALTY SECTION
This article was submitted to
Clinical Nutrition,
a section of the journal
Frontiers in Nutrition
RECEIVED 23 November 2022
ACCEPTED 09 March 2023
PUBLISHED 31 March 2023
CITATION
Vaughan-Shaw PG, Buijs LF, Blackmur JP,
Ewing A, Becher H, Theodoratou E, Ooi LY,
Din FVN, Farrington SM and Dunlop MG (2023)
A feasibility study of perioperative vitamin D
supplementation in patients undergoing
colorectal cancer resection. Front. Nutr. 10:1106431. doi: 10.3389/fnut.2023.1106431 OPEN ACCESS
EDITED BY
William K. K. Wu,
The Chinese University of Hong Kong, China
REVIEWED BY
Ronan Lordan,
University of Pennsylvania, United States
Vera Wesselink,
Wageningen University and Research,
Netherlands
*CORRESPONDENCE
M. G. Dunlop
malcolm.dunlop@ed.ac.uk
SPECIALTY SECTION
This article was submitted to
Clinical Nutrition,
a section of the journal
Frontiers in Nutrition
RECEIVED 23 November 2022
ACCEPTED 09 March 2023
PUBLISHED 31 March 2023
CITATION
Vaughan-Shaw PG, Buijs LF, Blackmur JP,
Ewing A, Becher H, Theodoratou E, Ooi LY,
Din FVN, Farrington SM and Dunlop MG (2023)
A feasibility study of perioperative vitamin D
supplementation in patients undergoing
colorectal cancer resection. Front. Nutr. 10:1106431. doi: 10.3389/fnut.2023.1106431 P. G. Vaughan-Shaw1,2, L. F. Buijs1,2, J. P. Blackmur1,2, A. Ewing1,2,
H. Becher1, E. Theodoratou2,3, L. Y. Ooi1,2,4, F. V. N. Din2,
S. M. Farrington2 and M. G. Dunlop
1,2* 1MRC Human Genetics Unit, Institute of Genetics and Cancer, The University of Edinburgh, Edinburgh,
United Kingdom, 2Cancer Research UK Edinburgh Centre, Institute of Genetics and Cancer,
The University of Edinburgh, Edinburgh, United Kingdom, 3Centre for Global Health Research, Usher
Institute for Population Health Sciences and Informatics, The University of Edinburgh, Edinburgh,
United Kingdom, 4Department of Pathology, National University Hospital, Singapore, Singapore Background: Vitamin D supplementation improves colorectal cancer (CRC)
survival outcomes in randomized trials. The aim of this study was to test the
feasibility, safety and efficacy of vitamin D supplementation in the pre- and
perioperative period in patients undergoing CRC surgery. Methods:
Patients
were
given
3200IU oral
cholecalciferol
(D3)
per
day
perioperatively. Serial serum 25-hydroxyvitamin (25OHD) was measured by liquid
chromatography tandem mass spectrometry and compared to untreated CRC
controls. 25OHD and C-reactive protein (CRP) levels were compared using
adjusted generalized linear mixed-effects models. © 2023 Vaughan-Shaw, Buijs, Blackmur, Ewing,
Becher, Theodoratou, Ooi, Din, Farrington and
Dunlop. 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. colorectal < cancer type, vitamin D, nutraceuticals, supplementation, surgery KEYWORDS Frontiers in Nutrition Take down policy Take down policy
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PUBLISHED 31 March 2023
DOI 10.3389/fnut.2023.1106431 TYPE Original Research
PUBLISHED 31 March 2023
DOI 10.3389/fnut.2023.1106431 Introduction to undergo high dose pre- and perioperative oral vitamin
D supplementation (3200IU/day cholecalciferol, Fultium-D3,
Internis Pharmaceuticals Ltd, Huddersfield, UK), alongside a
contemporaneous and historical control group (2). Any patients
receiving neo-adjuvant therapy were sampled and provided
supplementation after completion of pre-operative oncological
therapy. The chosen dose was the maximum available within
the hospital formulary and with reference to doses supplied in
relevant CRC vitamin D trials (9). All patients over 16 years of
age and eligible for supplementation but not already established
on vitamin D or multivitamin supplementation were considered
for inclusion. Patients were excluded if contra-indicated to
high-dose vitamin D supplementation (severe kidney disease,
hypercalcemia, hyperparathyroidism, atherosclerosis, sarcoidosis,
histoplasmosis, lymphoma, female and child bearing age, or
taking thiazide diuretics, digoxin or other cardiac glycosides). Whether the patient was recruited by our research nurse,
or
surgical
research
fellows
(PVS/LB)
determined
whether
they were allocated to the control or supplementation group,
respectively, itself determined by timetabling and clinic availability
of respective recruiter. Patients in the supplementation arm
took 3200IU cholecalciferol (D3) daily preoperatively, including
on the morning of surgery, and in the early post-operative
period until discharge except where post-operative ileus occurred. As this was a pragmatic feasibility study we were unable to
dictate operative scheduling, and so patients took preoperative
supplementation from the pre-operative surgical clinic until
their surgery, which in our unit is ∼4 weeks. Supplementation
was given in the early post-operative period and discontinued
at the point of discharge from hospital. The study protocol
allowed a maximum of 12 weeks’ supplementation to be given. Recruitment was paused during the first wave of the COVID-19
pandemic, significantly impacting on total study recruitment, while
restrictions on hospital appointments and non-clinical sampling
limited the number of post-discharge samples that could be
taken. Management and reporting of adverse events and serious
adverse events was as per ACCORD (Academic and clinical
office for research support, University of Edinburgh) standard
operating procedures. Despite substantial improvement in mortality from colorectal
cancer (CRC) over recent years, ∼16,600 people die from CRC
each year in the UK (1). Indeed, the overall global cancer
burden is increasing year on year, with ∼10 million deaths per
year (1). Whilst surgery remains the mainstay of therapy for
the majority of solid cancers, new therapeutic approaches are
required. Abbreviations: 25OHD,
25-hydroxyvitamin
D;
AJCC,
American
Joint
Committee on Cancer stage; BMI, body mass index; CI, confidence interval;
CRC, colorectal cancer; CRP, C-reactive protein; SIR, systemic inflammatory
response; SCOVIDS, Scottish vitamin D study; SOCCS, Study of Colorectal
Cancer in Scotland. Introduction Compelling evidence from observational studies (2)
demonstrate a link between low vitamin D and poor outcomes
following a diagnosis of colorectal cancer (3–8), while, crucially,
a recent meta-analysis of trial data demonstrates a 30% reduction
in adverse CRC outcomes with supplementation (9). Plasma
25-hydroxyvitamin D (25OHD) levels in CRC patients are
consistently lower than in population controls (10), yet a key
unanswered question is whether supplementation could provide
benefit from the time of diagnosis. Abdominal surgery is a major
physiological insult and 25OHD (the best marker and storage
form of vitamin D) falls dramatically following CRC surgery
(2). Meanwhile, 25OHD is also known to decrease following
orthopedic, cardiac and gynecological surgery (11–16). Suggested
explanations include circulatory fluid changes, i.e., hemodilution
(14, 17) and/or systemic inflammatory responses to surgery. It
follows that because 25OHD depletion is associated with adverse
survival outcomes and supplementation can improve survival,
studies to define when to initiate supplementation are required. While no study to date has assessed the impact of vitamin D
supplements in patients undergoing colorectal cancer resection,
cholecalciferol (vitamin D3) supplements have been shown to
significantly improve 25OHD levels in patients with a historical
diagnosis of CRC (18) and in patients undergoing chemotherapy
for CRC (19). Here, we have investigated the perioperative temporal variation
in plasma 25OHD and CRP by serially sampling patients
undergoing CRC resectional surgery and aimed to demonstrate
that vitamin D supplementation is feasible, safe and effective in
the perioperative period. These data will be essential in informing
the design of future randomized trials of vitamin D and survival
outcomes in patients with colorectal cancer. OPEN ACCESS doi: 10.3389/fnut.2023.1106431 © 2023 Vaughan-Shaw, Buijs, Blackmur, Ewing,
Becher, Theodoratou, Ooi, Din, Farrington and
Dunlop. 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. Results: A total of 122 patients underwent serial perioperative sampling, including
41 patients given high-dose perioperative supplementation. Supplementation
was well-tolerated with no adverse or serious adverse events related to
supplementation reported. Pre-operative supplementation increased 25OHD
levels on the day of surgery (103.9 vs. 42.5 nmol/l, P = 8.2E–12). Supplementation
increased 25OHD levels at all post-operative timepoints (P < 0.001) and
attenuated the post-operative drop in 25OHD (46 vs. 24% drop, P = 3.0E–4). Rate of vitamin D peri-operative insufficiency was significantly less in those on
supplementation (e.g., day 3–5, 14 vs. 84%, P = 1.41E–08), with multivariate
modeling across all timepoints indicating a ∼59 nmol/l higher 25OHD compared
to control patients (P = 3.7E–21). Post-operative CRP was lower in patients taking
supplementation (e.g., day 3–5 timepoint; 129 vs. 81 mg/l, P = 0.04). Conclusion: High dose pre-operative vitamin D supplementation is associated
with higher perioperative 25OHD levels, lower rates of vitamin D insufficiency and
reduced early post-operative CRP. Alongside published evidence for a beneficial
effect of vitamin D on CRC survival outcomes, these novel findings provide strong
rationale for early initiation of vitamin D supplementation after a diagnosis of CRC. 01 01 Frontiers in Nutrition frontiersin.org 10.3389/fnut.2023.1106431 10.3389/fnut.2023.1106431 Vaughan-Shaw et al. frontiersin.org Materials and methods Samples were taken at preoperative assessment clinic and/or
the day of surgery, and post-operatively (on the ward at 1–2, 3–
5, 6–9 days) and at the first post-operative clinic appointment
(30–120 days, Figure 1). Venepuncture was performed from a
peripheral arm vein using a 4 ml S-Monovette
R⃝Serum Gel
tube (Sarstedt AG & Co., Nümbrecht, Germany), with serum
extracted using recommended centrifugation settings of 2,500 × g
at 20◦C for 10 min. Samples were submitted for 25OHD
analysis in batches, with samples from an individual patient
analyzed in the same batch to reduce intra-patient variation. Furthermore, to minimize any potential analytical variation in
plasma 25OHD measurement, all samples were assayed in a
single United Kingdom Accreditation Service (UKAS) accredited
laboratory using a method traceable to National Standards
of Science and Technology standard reference material (20). All participants provided informed written consent, and
research was approved by local research ethics committees
(SOCCS 11/SS/0109 and 01/0/05; SCOVIDS 13/SS/0248), National
Health Service management (SOCCS 2013/0014, 2003/W/GEN/05;
SCOVIDS 2014/0058) and registered with www.clinicaltrials.gov
(NCT05506696). Clinical and sampling variables were collected
from patient case notes and pathology records, entered into
a prospective study database and extracted for analysis. We
recruited patients undergoing curative colorectal cancer resection 02 frontiersin.org Frontiers in Nutrition Vaughan-Shaw et al. 10.3389/fnut.2023.1106431 FIGURE 1
May-adjusted peri-operative 25-hydroxy vitamin D (25OHD) in supplemented and control groups Median 25OHD levels charted in nmol/l. Number
of patients in each group and P-values given. False discovery rate (FDR) P-value given adjusted in mixed-effects model for gender, age, body mass
index (BMI), cancer site, operative approach, and American Joint Committee on Cancer stage (AJCC). May-adjusted peri-operative 25-hydroxy vitamin D (25OHD) in supplemented and control groups Median 25OHD levels charted in nmol/l. Number
of patients in each group and P-values given. False discovery rate (FDR) P-value given adjusted in mixed-effects model for gender, age, body mass
index (BMI), cancer site, operative approach, and American Joint Committee on Cancer stage (AJCC). Patient, tumor and treatment-related
variables Total 25OHD (25OHD2 and 25OHD3) was measured by liquid
chromatography tandem mass spectrometry with low coefficient
of variation (<10%) in previously published data (20) and
analyzed using the Waters
R⃝Xevo
R⃝TQ-S system (Waters Limited,
Wilmslow, UK). We considered and adjusted for patient-related factors
previously established to influence 25OHD levels [age, sex, body
mass index (BMI), neo-adjuvant therapy and adjuvant therapy
use, operative approach and cancer site] (22–24). Tumor and
treatment-related variables were collected to investigate putative
effect on 25OHD, including American Joint Committee on Cancer
(AJCC) stage. Information on tumor site, multiplicity and clinico-
pathologic staging were obtained from clinical records, along with
preoperative imaging. By using collated pathology, imaging, and
clinical data; tumor stage was mapped onto the AJCC staging
system (AJCC stage I to IV). Sample size considerations Data from our previously published control group (2)
was used to inform a power calculation, with the current
study design powered to identify a 50% suppression of the
day 1–2 post-operative drop in 25OHD with supplementation
[α = 0.05 (one-tailed); β = 0.10, calculated sample size in each
arm = 54]. Plasma CRP assay To assess for potential relevance of the systemic inflammatory
response, samples were assayed for CRP in an NHS Biochemistry
Laboratory serving our hospital. CRP was measured using
the
Abbott
Architect
C
series
clinical
chemistry
analyzer
to standard sensitivity protocol, with the range of output
values
0.2–480
mg/l. CRP
internal
quality
control
was
performed daily, with three control samples assayed twice
per
day. Target
mean
of
the
three
control
samples
was
3.2, 8.3, and 27.5 mg/l with actual observed mean over
6 months of 3.2, 8.4, and 27.8 mg/l, respectively, from 5,151
completed assays. Coefficients of variation were 3.42, 2.34, and
2.05%, respectively. Statistical analysis All statistical analysis was undertaken in R (version 4.1.0) (25). To account for seasonal differences in vitamin D status, reported
25OHD level was May-standardized by adjusting for sampling
month using differences in age- and sex-adjusted monthly averages
generated from SOCCS control data. Baseline data in the control
and treatment groups were compared using Mann-Whitney, Chi-
squared or Fisher’s exact test, with significant difference considered
as P < 0.05. Baseline 25OHD was compared between groups
using a multivariable linear regression model adjusting for gender,
AGE, BMI, AJCC, cancer site and operative approach. We also
tested for baseline differences between the two control groups
in the same way. Next, we used the “lme4” package (26) to
generate a linear mixed-effects model of the serially sampled
data to examine the association between perioperative changes in
25OHD (log-transformed) and treatment group. This multivariable
model adjusted for relevant clinico-demographic factors (gender,
age, BMI, AJCC, and operative approach). Additional multivariable
linear mixed-effects analyses assessing the intervention against
each control group separately and including neo-adjuvant/adjuvant
therapy use were performed and included in Supplementary
material. The package “emmeans” (27) was used to compute
contrasts between estimated marginal means to evaluate adjusted
differences in 25OHD between treatment groups at each timepoint. The post-operative drop in 25OHD was calculated as a fold-
change between pre- and post-operative values and compared
between treatment groups using Mann-Whitney test. Finally, a
binomial generalized linear mixed-effects model was applied to
test for differences in rate of vitamin D insufficiency between Frontiers in Nutrition frontiersin.org Results Supplemented patients had higher 25OHD levels on the day
of surgery (103.9 vs. 42.5 nmol/l, P = 8.2E–12, Supplementary
Table 2 and Figure 2) and in the early post-operative period (e.g.,
1–2 days 80.9 vs. 26.7 nmol/l, P = 3.6E–15). Multivariate mixed-
effects modeling confirmed a significant and clinically relevant
impact of supplementation on perioperative 25OHD levels across
all timepoints, after adjusting for age, gender, BMI, cancer site,
operative approach and AJCC stage (25OHD estimate 59.4, 95%
CI 49.5–70.9 nmol/l, P = 3.7E–21, Supplementary Table 3). Supplementation also reduced the prevalence of perioperative
vitamin D insufficiency (25OHD<50 nmol/l), with just 14% of
supplemented patients having early post-operative insufficiency
compared to 84% of control patients (day 3–5 timepoint, P = 1.41E–
08, Table 2). Finally, although 25OHD levels dropped in all
treatment groups following surgery, supplementation attenuated We recruited a total of 122 patients, including 41 patients
who received high-dose perioperative vitamin D supplementation
(Table 1). No significant differences in baseline clinicopathological
variables between the groups were observed, while no differences
between the previously published control group and newly
recruited control group were observed, including in baseline
25OHD (46.3 vs. 47.6 nmol/l, P = 0.75; Supplementary Table 1). Patient and public involvement This
project
is
relevant
to
biomarker
prediction
and
risk
stratification
and
was
assigned
high
priority
in
the
seminal
Association
of
Coloproctology
of
Great
Britain
and
Ireland
(ACPGBI)
Delphi
process
(21). After
draft
protocol
design,
we
developed
our
lay
summary
and
PPI questionnaire in collaboration with the University of
Edinburgh
Patient
Advisory
Group
and
Patient
Liaison
Group
of
the
ACPGBI. Thereafter
we
surveyed
eight
patients
with
colorectal
cancer
to
assess
the
acceptability
of the proposed serial sampling study and help inform our
final study design. 03 frontiersin.org Vaughan-Shaw et al. 10.3389/fnut.2023.1106431 10.3389/fnut.2023.1106431 supplementation, which was well-tolerated in study participants. No
episodes
of
hypercalcemia
or
renal
impairment
were
reported, and no statistically significant changes in calcium
or estimated glomerular filtration rate (eGFR) were noted
during a previous study of 12 weeks’ supplementation at
the
same
dose
(28). Supplementation
induced
a
median
1.9-fold-increase in 25OHD level in the lead up to surgery
(48.2,
95%
CI
20.7–75.6
nmol/l;
P
=
0.01,
Figure
2). Overall,
patients
took
preoperative
supplementation
for
median 25 days (range 9–84). Dose diaries (N = 8 subjects),
indicated
high
compliance
with
daily
supplementation
(316
out
of
319
days
perioperative
supplementation
completed). treatment groups at each peri-operative timepoint, adjusting for
all relevant clinic-demographic factors. “emmeans” was evaluate
adjusted differences in rate of insufficiency between treatment
groups at each timepoint. To investigate effect on CRP level, a linear mixed-effects model
was applied to the serially sampled data to examine the association
between perioperative changes in CRP (log-transformed) and
treatment group adjusting for clinico-demographic factors. As
above the package “emmeans” (27) was used to evaluate adjusted
differences in CRP between treatment groups at each timepoint. *Three additional patients who underwent neo-adjuvant therapy classified as ypT0 on histological analysis. †Includes laparoscopic and robotic-assisted surgery. ‡Four patients chemo-
radiotherapy and three patients short-course radiotherapy in each group. py
g
p Frontiers in Nutrition Efficacy and safety of perioperative
supplementation No
adverse
or
serious
adverse
events
related
to
supplementation
occurred
in
those
taking
perioperative TABLE 1 Clinicopathological demographics of included patients. p
g
g
p
p
Control
VitD
P
N
81
41
–
Sampling years
2012–2020
2020
–
Age (years, SD)
66.4 (13.0)
64.7 (14.1)
0.72
Gender (F)
37 (46%)
17 (41%)
0.70
BMI (kg/m2, SD)
27.0 (4.4)
28.5 (5.7)
0.11
Baseline 25OHD (median, IQR)
42.5 (31.9)
50.49 (27.79)
0.35
Cancer site
0.06
Colon
49 (60%)
17 (41%)
–
Rectum
32 (40%)
24 (59%)
–
Surgical approach
–
Open
36 (44%)
13 (32%)
0.18
Minimally invasive†
45 (56%)
28 (68%)
–
Oncological treatment
–
Neo-adjuvant‡
7 (9%)
7 (17%)
0.23
Adjuvant
18 (22%)
16 (39%)
0.05
Cancer stage
0.74
AJCC 1
26 (32%)
8* (21%)
–
AJCC 2
27 (33%)
12 (32%)
–
AJCC 3
19 (23%)
17 (45%)
–
AJCC 4
9 (11%)
1 (3%)
−
*Three additional patients who underwent neo-adjuvant therapy classified as ypT0 on histological analysis. †Includes laparoscopic and robotic-assisted surgery. ‡Four patients chemo-
di
h
d h
i
h
di
h
i
h 04 frontiersin.org 10.3389/fnut.2023.1106431 Vaughan-Shaw et al. FIGURE 2
Study flow diagram* indicates sample point. Median sampling days from surgery were comparable between control and supplemented subjects at
timepoints 1–2, and 3–5 (P > 0.05). However, it was noted that fewer supplemented subjects were sampled at timepoint 6–9 and 30–120 days, with
sampling occurring earlier in the supplemented patients (6 vs. 7 days, P = 0.04; 39 vs. 67 days, P = 0.0001). FIGURE 2 FIGURE 2
Study flow diagram* indicates sample point. Median sampling days from surgery were comparable between control and supplemented subjects at
timepoints 1–2, and 3–5 (P > 0.05). However, it was noted that fewer supplemented subjects were sampled at timepoint 6–9 and 30–120 days, with
sampling occurring earlier in the supplemented patients (6 vs. 7 days, P = 0.04; 39 vs. 67 days, P = 0.0001). TABLE 2 25-hydroxyvitamin D (25OHD) insufficiency at each peri-operative timepoint in control and supplemented patients. Efficacy and safety of perioperative
supplementation Timepoint
Control
High-dose vit D
N (%)
N (%)
P†
OR (95% CI)*
P*
Pre–op
55 (68%)
3 (8%)
3.06E–09
5.59 (3.40–7.77)
5.37E–07
1–2 days
69 (86%)
11 (28%)
1.37E–10
5.20 (3.23–7.17)
2.18E–07
3–5 days
64 (84%)
5 (14%)
7.45E–14
6.52 (4.27–8.78)
1.41E–08
6–9 days
52 (87%)
4 (20%)
1.04E–07
5.66 (3.3–8.01)
2.52E–06
30–120 days
51 (84%)
1 (7%)
2.29E–09
7.77 (4.36–11.17)
7.92E–06
Vitamin D insufficiency <50 nmol/l. †Univariate P-value. *Odds ratio (OR) and false discovery rate (FDR) P-value given adjusted in mixed effects model for gender, age, body mass index
(BMI), American Joint Committee on Cancer stage (AJCC), cancer site and operative approach. lower in patients on supplementation (15.2, 95% CI 13.8–
15.6 mg/l). the early post-operative drop (e.g., 46 drop vs. 24% drop
at days 1–2, P = 3.0E–04) (Figure 2 and Supplementary
Table 4). lower in patients on supplementation (15.2, 95% CI 13.8–
15.6 mg/l). Frontiers in Nutrition frontiersin.org Impact of supplementation on
post-operative CRP levels However, we demonstrate that despite the
insult of resectional cancer surgery, and inherent changes in gut
motility and absorption, perioperative supplementation induces a
marked increase in preoperative 25OHD levels and attenuates the
drop in 25OHD following surgery. Given the tight physiological
autoregulation that exists, it is unlikely that mechanisms that
confer the observed beneficial impact of vitamin D on CRC
survival occur in a linear manner, but rather that a threshold
effect exists. Given that our previous work suggests that a
25OHD threshold of ∼45–50 nmol/l appears to most strongly
associate with survival, it is relevant that in the current study
supplementation significantly reduced post-operative vitamin D
insufficiency (25OHD<50 nmol/l). Effects on inflammatory processes are consistent with the lower
post-operative CRP levels in supplemented patients in the
current study, supporting the notion that vitamin D might
be causally implicated in the SIR response following surgery. This provides further potential mechanism to improved survival
outcomes with supplementation given that CRP, an established
marker of inflammation, is strongly correlated with CRC survival
(35, 36). The
current
study
has
a
number
of
limitations. We
acknowledge that supplemented patients recruited in 2020 were
compared against a combined control group including patients
recruited in 2012. As such, unmeasured historical differences in
demographics, lifestyle (e.g., physical activity, dietary vitamin D
intake or UV-B exposure), genetic background [e.g., vitamin D
receptor or pathway SNPs (37)], exact sampling times, or clinical
factors (e.g., neo-adjuvant therapy prior to supplementation
and surgery, post-operative recovery, diet and discharge) may
confound the observed effect of supplementation. However, we
observed no difference in baseline or peri-operative 25OHD
between the two control groups. Furthermore, mixed-modeling
identified a significant association between supplementation
and increased 25OHD level when comparing supplemented
patients with the contemporaneous control cohort alone. Despite
this, we cannot fully exclude the possibility that differences
in perioperative 25OHD levels are due to factors other than
supplementation because this was not a randomized study. Next,
given the pragmatic methodology, no alteration to the normal
patient pathway occurred and there was marked variation in
duration of preoperative supplementation between patients, in
part due to delays and disruption from the COVID-19 pandemic
which also impacted total study recruitment and sampling. Impact of supplementation on
post-operative CRP levels High-dose perioperative vitamin D supplementation is safe
and well-tolerated in patients undergoing colorectal cancer
surgery. Compared to control patients, supplementation induces
a significant increase in perioperative 25OHD levels, a smaller
relative drop in 25OHD following surgery and lower rates of post-
operative vitamin D insufficiency. Meanwhile, early post-operative
CRP levels may be lower in patients on supplementation supporting
the role of vitamin D in regulation of inflammatory processes. As expected, CRP increased post-operatively in all groups,
yet
the
increase
appeared
less
in
those
taking
high-dose
supplementation, with lower early post-operative CRP levels seen
in these patients (e.g., 3–5 days 80.5 vs. 129 mg/l, P = 0.04,
Table 3 and Supplementary Figure 1). On multivariate mixed-
effects modeling supplementation had a non-significant impact
on peri-operative CRP levels across all timepoints (P = 0.07;
Supplementary Table 5), with post-operative CRP levels ∼15 mg/l This study provides evidence for a beneficial effect of vitamin
D supplementation on perioperative vitamin D status while a 05 frontiersin.org 10.3389/fnut.2023.1106431 Vaughan-Shaw et al. TABLE 3 Perioperative C-reactive protein (CRP) levels in control and supplemented patients. TABLE 3 Perioperative C-reactive protein (CRP) levels in control and supplemented patients. TABLE 3 Perioperative C-reactive protein (CRP) levels in control and supplemented patients. Control
VitD
Timepoint
N
CRP (mg/l, iqr)
N
CRP (mg/l, iqr)
P†
P*
Pre-op
59
5.0 (11)
38
3.0 (9)
0.16
0.04
1–2 days
58
97.5 (62.25)
39
78.5 (48)
0.049
0.38
3–5 days
57
129.0 (121)
39
80.5 (72.6)
0.017
0.04
6–9 days
46
61.0 (58)
18
54.0 (75)
0.95
0.85
Median C-reactive protein (CRP) levels given in mg/l with inter-quartile range (IQR). †Univariate P-value. *False discovery rate (FDR) P-value given from multivariable mixed-effects model. CRP) levels given in mg/l with inter-quartile range (IQR). †Univariate P-value. *False discovery rate (FDR) P-value given from multivariable mixed-effects model. recent meta-analysis of RCT data reported a significant reduction
in CRC mortality with vitamin D supplementation (9, 29). Taken
together, these data support early initiation of supplementation
at the point of CRC diagnosis. While vitamin D repletion
has already been shown to be feasible in patients undergoing
chemotherapy (3, 4, 19), no study to date has explored its use
in CRC patients in the perioperative period. Vitamin D levels
are known to drop following surgery (2), which is confirmed
here in all groups. Frontiers in Nutrition frontiersin.org Funding surrounding which assay provides the best marker of vitamin D
status (39, 40). Given the marked changes in circulating 25OHD
observed with supplementation, we would not expect any material
changes in the observed differences in our results or conclusions
after adjustment for albumin or DBP level. We acknowledge that
efforts to assay 1,25OHD in target tissue (i.e., colon or rectum)
would be of value in future mechanistic studies. Also, we have
reported CRP as an easily assayed and recognized marker of the
systemic inflammatory to surgery but acknowledge that this is a
non-specific marker. Other markers of inflammation, including
pro-calcitonin and interleukins, may provide fuller understanding
of specific inflammatory responses to supplementation, surgery
or CRC itself. Finally, while our data supports the early initiation
of supplementation in patients undergoing CRC surgery given
its effects on 25OHD level and extrapolating from previous trial
evidence of survival benefit, we do not provide direct evidence of
survival benefit from early supplementation, nor do we consider
other clinical outcomes (e.g., post-operative morbidity), or patient
reported outcomes (e.g., quality of life). Future trials must consider
such outcomes in the context of clinically relevant patient benefit. surrounding which assay provides the best marker of vitamin D
status (39, 40). Given the marked changes in circulating 25OHD
observed with supplementation, we would not expect any material
changes in the observed differences in our results or conclusions
after adjustment for albumin or DBP level. We acknowledge that
efforts to assay 1,25OHD in target tissue (i.e., colon or rectum)
would be of value in future mechanistic studies. Also, we have
reported CRP as an easily assayed and recognized marker of the
systemic inflammatory to surgery but acknowledge that this is a
non-specific marker. Other markers of inflammation, including
pro-calcitonin and interleukins, may provide fuller understanding
of specific inflammatory responses to supplementation, surgery
or CRC itself. Finally, while our data supports the early initiation
of supplementation in patients undergoing CRC surgery given
its effects on 25OHD level and extrapolating from previous trial
evidence of survival benefit, we do not provide direct evidence of
survival benefit from early supplementation, nor do we consider
other clinical outcomes (e.g., post-operative morbidity), or patient
reported outcomes (e.g., quality of life). Future trials must consider
such outcomes in the context of clinically relevant patient benefit. This study received a grant from Bowel Disease Research
Foundation
(now
Bowel
Research
UK). 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. Acknowledgments We acknowledge the excellent technical support from Marion
Walker and Stuart Reid. We are grateful to Donna Markie,
and all those who continue to contribute to recruitment, data
collection, and data curation for the Study of Colorectal Cancer
in Scotland studies. We acknowledge the nursing and study
facilities provided by the Edinburgh Wellcome Trust Clinical
Research Facility. Finally, we acknowledge that these studies
would not be possible without the patients and surgeons
who
take
part. The
manuscript
is
available
on
medRxiv,
MEDRXIV/2022/278022. Data availability statement The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. 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. Ethics statement The studies involving human participants were reviewed and
approved by South East Scotland Regional Ethics Committee 01,
(SOCCS 11/SS/0109 and 01/0/05; SCOVIDS 13/SS/0248). The
patients/participants provided their written informed consent to
participate in this study. Impact of supplementation on
post-operative CRP levels Heterogeneity in exact sampling day within the respective
timepoints between the treated and control subjects, driven by
differences between the contemporary and historical cohorts is
also acknowledged, yet we do not believe that these factors would
impact our conclusions given the magnitude and significance of
differences in 25OHD levels between supplemented and control
patients. Furthermore, given the immediate post-operative drop
in 25OHD that is observed across all patients, early sampling in
the supplemented patients would be expected to deflate, rather
than inflate observed differences with the control cohort. Next, we
did not record compliance with post-operative supplementation
in regards to return to oral intake or complications precluding
oral supplementation. We did not collect albumin or vitamin
D binding protein (DBP) levels in this study. We acknowledge
such levels may impact levels of available 1,25-dihydroxyvitamin
D3 (38), the active form of 25OHD. Meanwhile, there is debate To date, no trial has investigated the optimum time to
initiate supplementation. Indeed, few of the supplementation
trials in CRC patients include patients undergoing curative
resection, with one such trial (AMATERASU trial), recruiting
patients at the first outpatient visit after surgery (5). It is
relevant
that
in
population
trials
where
recruitment
and
supplementation occur before the diagnosis of incident cases
of CRC, a beneficial effect of vitamin D supplementation on
survival is still seen. Furthermore, 25OHD levels sampled
preoperatively
and
at
the
earliest
post-operative
timepoint
(<6 months) are already associated with survival in observational
data
(2). Therefore,
given
that
a
cheap
supplement
has
now
been
shown
to
be
safe
and
well-tolerated
in
the
perioperative period, there is compelling rationale to start at
the point of diagnosis. Previous pre-clinical and human intervention studies provide
clues to the mechanisms that may underlie the beneficial effect
of supplementation on CRC survival and provide no contra-
indication to earlier supplementation. 1,25-dihydroxyvitamin D3
modulates immune and inflammatory pathway genes in large
bowel epithelium (30) and CRC cell lines (31) while oral
supplementation induces transcriptomic changes in rectal mucosa
that are linked to anti-tumor effects (32). Vitamin D also
regulates multiple inflammatory processes both in vitro and
in vivo, including those involved in CRC such as oxidative
stress and the cyclooxygenase and NF-kB pathways (33, 34). 06 frontiersin.org Vaughan-Shaw et al. 10.3389/fnut.2023.1106431 Author contributions PV-S, LB, JB, LO, FD, SF, and MD: study concepts. PV-S,
LB, LO, JB, ET, SF, and MD: study design. PV-S, LO, LB, and
JB: data acquisition. PV-S and AE: quality control of data and
algorithms. PV-S, AE, and HB: data analysis and interpretation and
statistical analysis. PV-S, LB, JB, AE, HB, SF, and MD: manuscript
preparation and editing. PV-S, ET, AE, FD, SF, and MD: manuscript
review. PV-S, SF, and MD: funding acquisition. MD: project
administration. MD and SF: supervision. All authors contributed
to the article and approved the submitted version. Funding This
work
was
supported by funding for the infrastructure and staffing of
the Edinburgh CRUK Cancer Research Centre; CRUK program
grant C348/A18927 (MD and SF) and CRUK Career Development
Fellowship (C31250/A22804, ET). PV-S was supported by a MRC
Clinical Research Training Fellowship (MR/M004007/1). LO was
supported by a Cancer Research UK Research Training Fellowship
(C10195/A12996). This work was also funded by a grant to MD as
Project Leader with the MRC Human Genetics Unit Centre Grants
(U127527202 and U127527198 from 1/4/18). JB was supported
by an Edinburgh Clinical Academic Track (ECAT) linked Cancer
Research UK Clinical Research Fellowship (C157/A23218). FD was
supported by a senior fellowship from the Chief Scientist Office,
Scotland (SCAF/16/01) and previously from Cancer Research UK
(C26031/A11378). MD was supported by a Cancer Research UK
programme grant (DRCPGM/100012). In conclusion, we report for the first time on the feasibility
and safety of perioperative vitamin D supplementation in patients
undergoing colorectal cancer surgery. We identified a positive effect
of supplementation on perioperative 25OHD levels with lower
rates of post-operative vitamin D insufficiency and reduced early
post-operative CRP. Our findings provide compelling rationale for
early initiation of vitamin D supplementation after a diagnosis of
CRC. Future randomized trials of supplementation with a defined
endpoint of a beneficial effect on survival outcomes should consider
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application. Ann Epidemiol. (2009) 19:73–8. doi: 10.1016/j.annepidem.2007.12.001 40. Holick M. Vitamin D status: measurement, interpretation, and clinical
application. Ann Epidemiol. (2009) 19:73–8. doi: 10.1016/j.annepidem.2007.12.001 08 Frontiers in Nutrition frontiersin.org
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A Psicologia na Atenção Básica e a Saúde Coletiva
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Abstract The article aims to describe the psychologist’s challenges in primary care health. We conducted
a qualitative, exploratory, and descriptive research. We analyzed ten scientific articles, published
between 2001 and 2017 at the Portal de Periódicos da Capes. These articles subject were
psychologist’s narratives of work at primary care health. They were analyzed from the perspective of
Laville and Dionne’s analysis of content. Results point to the necessity of a board perspective of health
by the psychologists and the development of a collective health approach to mental health. Thus, we
were capable to enable preventive, humanized attention to health in all dimensions at primary care. Furthermore, there is a need for necessary changes in psychologist’s formation and objectives policies
guidelines to mental health at the primary care.i Keywords: Psychology, primary care, mental health, collective health Resumen El artículo objetiva describir los desafíos del profesional en psicología en la atención básica à la salud. Se ha hecho una investigación cualitativa, exploratoria y descriptiva. Fueron analizados diez artículos
publicados entre 2001 y 2017 en lo Portal de Periódicos da Capes. Estos artículos presentaban
narrativas de actuación de profesionales de la psicología en la atención básica à la salud y fueran
analizados en la perspectiva del análisis de contenido de Laville e Dionne. Los resultados apuntan la
necesidad de una visión ampliada de salud en la perspectiva de la salud colectiva por los psicólogos. Con esto, es posible crear una asistencia integral à la salud con una actuación humanizada, preventiva
y ampliada en la atención básica. Y más, se necesita transformar la formación de los profesionales de
psicología y elaborar directrices más claras y objetivas para la salud mental en la atención básica. Palabras clave: Psicología atención básica salud mental salud colectiva Resumoi O objetivo do artigo é descrever os desafios do profissional da psicologia na atenção básica à saúde. Trata-se de uma pesquisa de delineamento qualitativo, exploratório e descritivo. Foram analisados
10 artigos publicados entre 2001 e 2017 em revistas científicas pertencentes às bases de dados do
Portal de Periódicos da Capes. Os artigos apresentavam relatos de experiência de profissionais da
psicologia que atuam na atenção básica à saúde e foram analisados na perspectiva da análise de
conteúdo proposta por Laville e Dionne. Os resultados apontam a necessidade de o profissional da
psicologia adotar uma visão ampliada de saúde na perspectiva da saúde coletiva. Deste modo, pode
se desenvolver um atendimento integral à saúde por meio de atuação humanizada, preventiva e
ampliada na atenção básica. Mais ainda, busca-se a transformação na formação dos profissionais da
psicologia e a elaboração de diretrizes norteadoras mais claras e objetivas para a saúde mental na
atenção básica. Palavras-chave: Psicologia, atenção básica à saúde, saúde mental, saúde coletiva A Psicologia na Atenção Básica e a Saúde Coletiva
Psychology at Primary Care and Public Health
La Psicología en la Atención Básica y la Salud Colectiva Vanessa Santos Lemos1
Cristina Lhullier
Universidade de Caxias do Sul (UCS) 1 Endereço de contato: Rua das Palmeiras, 878, Cruzeiro, Caxias do Sul, RS. CEP 95074-310. E-mail:
neh.lemos@gmail.com Revista Psicologia e Saúde. Revista Psicologia e Saúde. doi: http://dx.doi.org/10.20435/pssa.vi.1076 A Psicologia na Atenção Básica e a Saúde Coletiva
Psychology at Primary Care and Public Health
La Psicología en la Atención Básica y la Salud Colectiva Introdução A atenção básica constitui-se de um conjunto de ações em saúde localizadas nos terri
tórios, voltadas à promoção e à prevenção de saúde. Ela é o principal meio de acesso aos
serviços de saúde do Sistema Único de Saúde (SUS) e conta com projetos que visam à abran
gência e ao acesso da população a esses serviços (https://atencaobasica.saude.rs.gov.br/ Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 ISSN: 2177-093X Revista Psicologia e Saúde. 178 programas-e-acoes). Um dos programas que contribuem para essas finalidades, além das
ações dos agentes comunitários, é a Estratégia de Saúde da Família (ESF). Esta objetiva o
atendimento interdisciplinar da população, sendo um dos projetos da Política Nacional de
Atenção Básica (Ministério da Saúde, 2013). A saúde coletiva caracteriza-se como uma prática sanitária, que se utiliza de distintos sa
beres com o intuito de ampliar as bases epidemiológicas e sociais (Campos, 2000). Atua com
base na integralidade, questionando o paradigma biomédico e promovendo a autonomia do
usuário em relação à sua saúde. Essa prática deve ser proposta desde a formação profissio
nal, para que torne parte da atuação comprometida com o social (Bernandes & Guareschi,
2010; Campos, 2000). Diante do compromisso da saúde pública com a saúde coletiva, buscando integralidade e
interdisciplinaridade, houve a regulamentação da Estratégia Saúde da Família, pela Portaria
n. 648/GM (Ministério da Saúde, 2006). Nesta, a saúde mental não se encontra considerada. Contudo a assistência à saúde mental deve ser trabalhada no cotidiano da atenção básica,
sendo o sofrimento psíquico algo que vai além dos diagnósticos, considerando-se a subje
tividade, as diferenças culturais e o atendimento humanizado (Ministério da Saúde, 2013). i Desse modo, há a exigência da mudança do modelo hegemônico para um cuidado multi
disciplinar compartilhado, ressaltando a necessidade de uma formação profissional adequa
da a esse princípio de intervenção (Ministério da Saúde, 2009). Com o apoio matricial, a atenção básica à saúde mental passou a ter mais espaço na rede
(Ministério da Saúde, 2013). O matriciamento atua no suporte técnico e pedagógico dos
serviços de saúde, ampliando as relações entre funcionários e usuários. Os investimentos
financeiros para esse fim começaram a partir da criação dos Núcleos de Apoio à Saúde da
Família (Nasfs), possibilitando a formação de uma equipe com profissionais de diferentes
áreas apoiando tecnicamente as Unidades Básicas de Saúde (UBSs) que atuam com a ESF
diretamente nos territórios (Ministério da Saúde, 2008). Revista Psicologia e Saúde. 179 Com o psicólogo consolidando seu espaço na atenção básica, surgem desafios a serem
superados. Inicialmente, há os estigmas diante da profissão psicólogo. Com base no estu
do dirigido pelo Conselho Federal de Psicologia (CFP, 2011), pode-se perceber que tanto
usuários quanto profissionais da saúde ainda não compreendem a prática psicológica como
fundamental nas intervenções à saúde. A pesquisa aponta que o estigma do setting terapêu
tico dificulta o entendimento das formas de atuação e das intervenções psíquicas dentro da
saúde pública (CFP, 2011). Outro desafio encontrado foi aliar a saúde mental e a atenção básica à saúde, atuando de
forma integral, como precede o “princípio do SUS de integralidade, com objetivo de organi
zar o sistema e trabalhar com ações preventivas” (Spink & Matta, 2010, p. 45), além de mo
dificar a cultura que rotula o doente mental, construindo um olhar para esse sujeito como
pessoa que sofre (CFP, 2011; Spink & Matta, 2010). i Há ainda que se considerar a falta de políticas públicas que estabeleçam diretrizes para
o fazer psi no cuidado da atenção primária, auxiliando e norteando o trabalho (CFP, 2011). Dessa forma, constata-se a urgência na criação de políticas públicas que acolham as deman
das sociais da atualidade, além do reconhecimento da importância e do papel do profissio
nal psicólogo na atenção básica à saúde como profissional que está na saúde pública como
agente de saúde coletiva, lutando pela cidadania diante das propostas governamentais em
saúde (Bernardes & Guareschi, 2010). A partir do exposto, o artigo propõe-se a descrever os desafios do profissional da psicolo
gia na atenção básica à saúde, tomando como ponto de partida a perspectiva desse nível de
atenção como prática coletiva e integradora. Introdução O principal desafio para a atenção básica é transformar o paradigma de atendimento indi
vidual em uma reforma que possibilite “cuidados primários, como um conjunto de valores e
princípios para orientar o desenvolvimento dos sistemas de saúde” (Organização Mundial da
Saúde [OMS], 2008, p. 9), promova inovações que possibilitem cobertura universal, promova
emancipação do usuário, necessidade de reforma nas políticas públicas e uma liderança do
Estado mais inclusiva, “baseada em negociação e participação, mais adequada à complexi
dade dos sistemas de saúde contemporâneos” (OMS, 2008, p. 9). A qualidade de vida e o estresse estão relacionados ao surgimento de doenças mentais. Mesmo considerando a complexidade do termo qualidade de vida, vale pensar sobre o im
pacto desta na saúde. Segundo estudo realizado com 1.466 pacientes que frequentam a
assistência básica de saúde no estado de São Paulo e do Rio de Janeiro, foi possível mostrar
a relação da qualidade de vida e o adoecimento psíquico na população de baixa renda, con
siderando variáveis como estresse, estrutura familiar, relações sociais, problemas de saúde,
mudanças e rompimentos abruptos, violência, entre outros (Silva & Santana, 2012). De acor
do com pesquisa publicada pelo Instituto Brasileiro de Geografia e Estatística (IBGE, 2014),
em 2013, a região Sul e a Sudeste apresentaram maiores índices de depressão no país. Cerca
de 12,6% da população do Sul do país tem diagnóstico de depressão, fato que acaba se ma
nifestando na rede de atenção básica em saúde. Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 ISSN: 2177-093X Revista Psicologia e Saúde. Método A pesquisa realizada teve delineamento qualitativo, de caráter exploratório e descritivo. Considera-se a investigação exploratória, pois se buscou construir gradativamente uma com
preensão dos dados, de modo a esclarecer o objetivo proposto (Gil, 2010; Laville & Dionne,
1999). O aspecto descritivo da pesquisa constituiu-se na elaboração de um panorama das
publicações sobre a temática escolhida em um dado período de tempo (Gil, 2010).iiíi Foram utilizados 10 artigos escritos em língua portuguesa publicados em revistas científi
cas que fazem parte do Portal de Periódicos da Capes. Foram utilizados os descritores: saúde
pública, saúde mental na atenção básica e psicologia na atenção básica.i Foram utilizados 10 artigos escritos em língua portuguesa publicados em revistas científi
cas que fazem parte do Portal de Periódicos da Capes. Foram utilizados os descritores: saúde
pública, saúde mental na atenção básica e psicologia na atenção básica.i Os critérios de seleção dos artigos foram os seguintes: 1) relatos de experiência de psicólogos que atuam na atenção básica em UBS e nas equi
pes de Nasf, nas regiões do Sul e Sudeste do Brasil. Optou-se por focar nessas duas
regiões do país, visto que essas apresentam os maiores índices de transtornos mentais
leves diagnosticados na atenção básica; 2) artigos publicados entre 2001 e 2017. O marco de 2001 corresponde à data de promul
gação da lei da Reforma Psiquiátrica brasileira, que reestrutura os serviços de atenção
à saúde mental no país.i Os artigos selecionados apresentam-se na Tabela 1. Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 ISSN: 2177-093X Revista Psicologia e Saúde. Desafios para o Psicólogo na Atenção Básica à Saúde Nos 10 artigos analisados, destaca-se a importância do compromisso social do profissio
nal da psicologia na atenção básica à saúde. Esse compromisso social encontra-se atrelado
à perspectiva de saúde coletiva (Archanjo & Schraiber, 2012; Cintra & Bernardo, 2017; Costa
& Olivo, 2009; Freire & Pichelli, 2013; Hori & Nascimento, 2014; Jimenez, 2011; Klein &
Oliveira, 2017; Ferreira Neto, 2008; Parise & De Antoni, 2014; Sundfeld, 2010). Segundo os
artigos, o compromisso social não se constitui como uma área de atuação específica, mas
sim como um posicionamento ético e político, causador de transformações a partir da mo
bilização do saber profissional (Archanjo & Schraiber, 2012; Cintra & Bernardo, 2017; Costa
& Olivo, 2009; Freire & Pichelli, 2013; Hori & Nascimento, 2014; Jimenez, 2011; Klein &
Oliveira, 2017; Ferreira Neto, 2008; Parise & Antoni, 2014; Sundfeld, 2010). i Klein e Oliveira (2017), no Artigo 9, após acompanhar o trabalho de uma equipe que atua
no Nasf em São Paulo, destacam a perspectiva territorial como base primordial nas inter
venções, chamando a atenção para uma realidade de atuação profissional que diverge do
ideal proposto pelo SUS. Na entrevista com três psicólogos que atuam em UBS de Campinas,
Cintra e Bernardo (2017), no Artigo 10, analisam se as práticas da psicologia estão em conso
nância com a atuação em saúde coletiva como esperado pelo SUS, mostrando a possibilida
de de efetivar e ampliar o modelo clínico, com intervenções contextualizadas na comunida
de. O compromisso social também aparece no Artigo 5 (Archanjo & Schraiber, 2012), no qual
as autoras entrevistaram 17 psicólogos da atenção básica com o objetivo de compreender
as mudanças e as necessidades diante da atuação desses profissionais na saúde pública,
trazendo a necessidade de serem incluídas na atenção básica práticas clínico-sanitárias e
melhor articulação e regulamentação político-institucional.i Sobre o papel social do psicólogo, o Artigo 4, escrito por Jimenez (2011), traz a experiência
de grupos para discussão de casos entre equipe de uma UBS da grande São Paulo, de forma
a refletir sobre a importância do psicólogo nesse contexto, potencializando o entendimento
sociocultural atrelado à saúde, por meio da atuação nos Nasfs. Nesse sentido, o Artigo 3, de
Sundfeld (2010), contribui com a necessidade de o psicólogo ser inserido na atenção básica
com o intuito de efetivação da clínica ampliada por meio de matriciamento, promovendo a
atuação em saúde coletiva. Método 177-188 ISSN: 2177-093X Revista Psicologia e Saúde. Resultados Método Artigos Selecionados
Título
Ano
Autoria
Periódico
1
Práticas transversalizadas da clínica em
saúde mental
2008
Ferreira Neto
Psicologia: Reflexão
e Crítica
2
Novos sentidos para a atuação do
psicólogo no Programa Saúde da Família
2009
Costa & Olivo
Ciência & Saúde
Coletiva
3
Clínica ampliada na atenção básica e
processos de subjetivação: relato de
uma experiência
2010
Sundfeld
Physis: Revista de
Saúde Coletiva
4
Psicologia na atenção básica à saúde:
demanda, território e integralidade
2011
Jimenez
Psicologia &
Sociedade
5
A atuação dos psicólogos em Unidades
Básicas de Saúde na cidade de São Paulo
2012
Archanjo &
Schraiber
Saúde & Sociedade
6
O psicólogo apoiador matricial:
percepções e práticas na atenção básica
2013
Freire & Pichelli
Psicologia: Ciência e
Profissão
7
A psicologia na atenção primária
à saúde: práticas psicossociais,
interdisciplinaridade e intersetorialidade
2014
Parise & Antoni
Ciência & Cultura
8
Projeto Terapêutico Singular e as práticas
de saúde mental no Núcleo de Apoio à
Saúde da Família (NASF) em Guarulhos
(SP), Brasil
2014
Hora & Nascimento
Ciência & Saúde
Coletiva
9
O “cabo de força” da assistência:
concepção e prática de psicólogos
sobre o Apoio Matricial no Núcleo de
Apoio à Saúde da Família
2017
Klein, Pires &
d’Oliveira
Cadernos de Saúde
Pública
10 Atuação do psicólogo na atenção básica
do SUS e a Psicologia Social
2017
Cintra & Bernardo
Psicologia: Ciência e
Profissão Após a seleção dos artigos, foi feita a codificação deles, permitindo a sistematização e a
estruturação das ideias relevantes para a análise (Gibbs, 2009). Foi elaborado um primeiro grupo de categorias, assim nomeadas: 1) transformação na
atuação do psicólogo; 2) formação insuficiente dos psicólogos para a atuação na saúde pú
blica; 3) políticas públicas que direcionam o fazer psi; 4) integralidade no quesito saúde; 5)
mais investimentos à saúde; 6) modelo biomédico ultrapassado; 7) tratar a saúde mental no
senso comum. No decorrer do processo de análise dos dados, foi realizada uma revisão das
categorias, reagrupando-as em duas categorias nomeadas de Desafios para o Psicólogo na
Atenção Básica e Visão Ampliada de Saúde. A análise do conteúdo dos artigos foi realizada com base nos passos propostos por Laville
e Dionne (1999). Utilizou-se do sistema de categorização no modelo misto e buscou-se ela
borar a compreensão dos dados por meio da construção interativa de uma explicação (Laville
& Dionne, 1999). Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. Desafios para o Psicólogo na Atenção Básica à Saúde No Artigo 6, Freire e Pichelli (2013) entrevistaram 10 psicólogos que trabalham como
apoiadores matriciais em João Pessoa, PB. Os entrevistados defendem essa postura técnica
do psicólogo, apostando na transformação social ao estimular o usuário na busca pelo seu
próprio bem-estar, por meio de uma função ativa no seu plano de intervenções em saúde. i Essa ação deve ser pensada como um direito e um dever do sujeito, como refere o Artigo
7 (Parise & De Antoni, 2014). Nesse, foram entrevistados seis psicólogos da atenção básica
com o objetivo de compreender o entendimento de cada profissional sobre suas práticas. Os
autores destacam que uma atuação voltada à saúde coletiva, atenta às necessidades e aos
recursos do próprio território, permite o surgimento de novas formas de saber e de fazer,
fortalecendo o vínculo dos usuários à saúde, de forma a construir um “espaço de resistência
às formas de disciplinarização” (Parise & De Antoni, 2014, p. 1094), apostando na emancipa
ção do coletivo, construindo uma “clínica viva” (p. 1095), movimentando o desejo do sujeito, Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 ISSN: 2177-093X Revista Psicologia e Saúde. 182 atrelado à qualidade de vida, abrindo-se para o cuidado e promovendo o cuidar (Parise & De
Antoni, 2014). No Artigo 4, Jimenez (2011) afirma que, quando as práticas são descontextualizadas, ten
demos à “patologização dos indivíduos colocando o trabalho psicológico a serviço da ma
nutenção da desigualdade, da injustiça e da reprodução da violência” (p. 136). Sobretudo,
a análise dos artigos mostra que esse fazer pautado no social exige um posicionamento que
necessita ser construído desde a formação dos profissionais, pois esse olhar só é possível
diante de uma desconstrução do saber. O psicólogo deve estar disponível para aprender e
entender diferentes realidades, buscando intervenções adequadas e corresponsabilizando
os demais envolvidos. As autoras Freire e Pichelli (2013), no Artigo 6, concluem que os psicólogos que atuam
no eixo público ainda se distanciam desse compromisso social, estando atrelados a um fazer
clínico e individual. Observa-se tal distanciamento nas falas destacadas do Artigo 2 (Costa &
Olivo, 2009): “Bom, eu acho que a minha prática, no momento, como é iniciante, está mais
voltada para o atendimento clínico. Então, eu atendo crianças, adolescentes, adultos (psicó
loga 3)” (p. 1387); ou ainda: “Quando eu iniciei, a gente começou pelos grupos, me inseri em
todos os grupos que tinham. ISSN: 2177-093X Desafios para o Psicólogo na Atenção Básica à Saúde Depois disso, parti para o atendimento individual. Estavam me
pedindo nos grupos e a demanda estava grande para atendimento clínico, mas esse não era
nosso objetivo inicial (psicóloga 2)” (p. 1387). Há também o entrave entre o compromisso curativo e preventivo das UBSs, gerando
nos funcionários uma confusão diante das demandas exacerbadas. No Artigo 5 (Archanjo
& Schraiber, 2012), alguns entrevistados apontam que a demanda de ações voltadas para a
“doença é muito maior do que de saúde” (p. 358), não tendo muito espaço para interven
ções preventivas. A “exigência é para que se atenda o maior número de pessoas” (Archanjo
& Schraiber, 2012, p. 358), apontando esse fato como uma exigência política e territorial. Revista Psicologia e Saúde. Revista Psicologia e Saúde. 183 Estratégia a gente faz pouca coisa, são poucos os trabalhos que a gente faz [juntos] (E4)”;
ou então: “Antes, quando eu entrei, eu achei que seria uma coisa muito mais assistencia
lista, hoje eu acho que o meu maior papel é de ser matriciadora, sabe, de explicar, mesmo
que seja explicar 200 mil vezes a mesma informação, mas acho que é isso de ensinar essas
pessoas a trabalharem de uma forma, enfim [...] (E6)” (Klein & Oliveira, 2017, p. 5). Diante
dessas falas, pode-se pensar na importância da desconstrução do saber, distanciando-se da
fantasia de “o poder do psicólogo”, com uma postura de inserção, aberta ao conhecimento e
ao valor das diversidades sociais, assumindo a profissão como um agente de transformação
e construção social, apreciando a cidadania e o respeito mútuo. No Artigo 1, Ferreira Neto (2008) percebe a promoção de saúde como geradora de trans
formação social e ainda ressalta a necessidade de um atendimento interdisciplinar, desta
cando esse aspecto como o bem maior da atenção básica. Contudo o autor aponta que esse
reconhecimento e prática ainda não estão internalizados nas equipes técnicas de forma ge
ral. Ainda há muita coisa para se refletir e construir, e uma mudança crucial é a integralidade
da saúde e o compromisso social do servidor público, internalizando uma prática em saúde
coletiva (Ferreira Neto, 2008). Visão Ampliada de Saúde A busca pela integralidade na saúde ainda carece de investimentos e de profissionais ca
pacitados, conforme aponta o Artigo 10 (Cintra & Bernardo, 2017). Outro aspecto descrito
no artigo é a fragmentação do cuidado em saúde como uma das principais deficiências da
atenção básica em saúde (Cintra & Bernardo, 2017). Os Artigos 8 (Hori & Nascimento, 2014) e 9 (Klein & Oliveira, 2017) destacam a falta de
harmonização dos gestores com a integralidade e a sobrecarga de trabalho como dificultado
ra de uma atuação coletiva em saúde. Já os Artigos 5 (Archanjo & Schraiber, 2012) e 7 (Parise
& De Antoni, 2014) indicam a necessidade de transformação da formação acadêmica do
profissional como principal meio para exercer a integralidade em saúde. E Freire e Pichellli
(2013), no Artigo 6, apontam o modelo biomédico, ainda enraizado na atenção básica, como
um fator que dificulta o processo de intervenções coletivas em saúde.ii Os Artigos 3 (Sundfeld, 2010) e 4 (Jimenez, 2011) utilizam-se do conceito de complexi
dade para refletir sobre a atuação integral em saúde, chamando a atenção para práticas em
saúde coletiva como principal estratégia na transformação do paradigma individualizante. Da mesma forma, os Artigos 1 (Ferreira Neto, 2008) e 2 (Costa & Olivo, 2009) destacam essa
mudança de paradigma, começando com a formação acadêmica do profissional. O levantamento feito a partir das reuniões de equipe observadas no Artigo 8 (Hori &
Nascimento, 2014) também dá a dimensão da falta de preparo para integralidade na aten
ção básica. Como fatores que justificam esse despreparo, está a formação insuficiente para
atuação na área pública dos profissionais ou falta de capacitações e suporte técnico efetivo
para atuar nesse nível de atenção. “A presença de diversos profissionais de diferentes áreas
foi insuficiente para qualificar as discussões; em alguns momentos, foi visível clima de cons
trangimento, desconfiança e distanciamento entre as equipes (Hori & Nascimento, 2014, p. 3566)”, fragilizando o olhar ampliado para saúde, de forma a desmotivar os profissionais
para práticas coletivas. O levantamento feito a partir das reuniões de equipe observadas no Artigo 8 (Hori &
Nascimento, 2014) também dá a dimensão da falta de preparo para integralidade na aten
ção básica. Como fatores que justificam esse despreparo, está a formação insuficiente para
atuação na área pública dos profissionais ou falta de capacitações e suporte técnico efetivo
para atuar nesse nível de atenção. a, UCDB - Campo Grande, MS Dessa forma, em meio a essa desconstituição de papéis e compromissos, a função do
psicólogo não é bem compreendida na atenção básica. O profissional não tem clareza nem
há diretrizes suficientes que respaldem sua função nessa esfera. Assim, os funcionários e os
usuários acabam esperando que o psicólogo atue em formato individual, ou ainda como um
profissional “quebra-galho”, que está ali para intervir em todas as demandas emergentes,
como aparece na fala de uma entrevistada no Artigo 5: “[...] um pouco como bombeiro, tem
alguém surtando, corre chamar o psicólogo, é interessante que nessa hora eles não chamam
o médico, que é o detentor do poder” (Archanjo & Schraiber, 2012. p. 359). i Diante disso, pode-se pensar na importância de novas referências para a profissão do
psicólogo, principalmente no que tange à prática em saúde coletiva dentro do setor público. No Artigo 9, Klein e Oliveira (2017), após entrevistarem psicólogos que atuam em equipes
Nasf no município de São Paulo, chamam a atenção para o “excesso de liberdade (p. 3)”,
referindo-se à falta de diretrizes que viabilizem o trabalho e a construção de práticas con
textualizadas de projetos importantes como o Apoio Matricial e a equipe Nasf, por exemplo,
sem o intuito de burocratizar os serviços. Os entrevistados na pesquisa do Artigo 9 mostram entendimentos divergentes em rela
ção a esses projetos. Muitos ainda não têm a compreensão da sua função, como pode-se
perceber nas falas extraídas do artigo: “Compartilhado com a Estratégia, engraçado isso... né? A gente é apoio para a Estratégia e trabalha mais com o Nasf, é isso mesmo. Com a Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 ISSN: 2177-093X Revista Psicologia e Saúde. 184 A integralidade na saúde corresponde à prática de voltar o fazer profissional para atender
às necessidades subjetivas do coletivo, considerando a saúde um estado integral de bem-es
tar e qualidade de vida, conforme indicam Costa e Olivo (2009) no Artigo 2. “Eu acho que a
gente deveria pensar numa saúde mental que pudesse incluir mais as pessoas na comuni
dade, no seu bem-estar familiar, e não uma coisa separada, excluída, que vem consultar, e é
o que está sendo feito na realidade em saúde primária no PSF (psicóloga 4)” (Costa & Olivo,
2009, p. 1391). No Artigo 4, Jimenez (2011) chama a atenção para a importância de um cui
dado integral, sem separar saúde mental de saúde. Também é importante ressaltar a importância de uma compreensão ampla no processo
de saúde, considerando o sofrimento um sintoma social, pois “as pessoas sofrem por maze
las que são produzidas socialmente [...] dilemas sociais chegam fantasiados de sofrimento
psíquico e é por isso que é fundamental ter a dimensão do social no cuidado em saúde”
(Cintra & Bernardo, 2017, p. 890); ou ainda, no Artigo 5, (Archanjo & Schraiber, 2012): “as
demandas de saúde estão cada vez mais associadas a demandas sociais e [...] o psicólogo é
o ‘termômetro mais sensível da demanda social’” (p. 359). Nas citações acima, percebe-se a necessidade de desconstrução da hegemonia do saber,
para atuar de forma humanizada e contextualizada. Esse movimento de saúde coletiva é um
fator complexo, que exige uma reformulação da formação dos profissionais, assim como um
interesse pela humanização da equipe técnica na atenção básica. Archanjo e Schraiber (2012), no Artigo 5, notam que o interesse pela atuação na saúde
pública não estava relacionado a um posicionamento de interesse social, e sim à estabilidade
profissional e financeira: “[...] acho que tem duas, duas coisas né, uma é salário garantido,
né, porque o, o consultório dependia de ter clientes, de ter indicação, [...] tem acho que ou
tra coisa que, um pouco, é, tem um [sic] tradição” (p. 357). Para obter efetividade no quesito saúde, é preciso sair do paradigma de atendimentos
individualizados e com foco na cura de sintomas. Ampliar o entendimento sobre saúde de
forma integral e investir na atuação pautada em saúde coletiva sem “distinção entre saúde e
saúde mental [...] (E4)” (Parise & De Antoni, 2014. p. 78). Visão Ampliada de Saúde “A presença de diversos profissionais de diferentes áreas
foi insuficiente para qualificar as discussões; em alguns momentos, foi visível clima de cons
trangimento, desconfiança e distanciamento entre as equipes (Hori & Nascimento, 2014, p. 3566)”, fragilizando o olhar ampliado para saúde, de forma a desmotivar os profissionais
para práticas coletivas. Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 ISSN: 2177-093X Revista Psicologia e Saúde. Discussão Com base na análise das fontes selecionadas, apresentada nos Resultados, percebe-se
a necessidade de qualificar o processo de formação dos psicólogos que atuam na atenção
básica, visando a intervenções coletivas e integradoras, principalmente no que se refere ao
compromisso social desses profissionais diante de suas intervenções. O fato de o profissional
da psicologia trabalhar com sujeitos que pensam, agem, transformam a cultura e a socieda
de evidencia a complexidade e a diversidade de sua atuação. Assim, há o dever, enquanto
profissionais de saúde, em assumir uma postura ética que preconize a responsabilidade so
cial (Codo & Lane, 1989; Dimenstein, 2001). Também foi possível perceber que o papel do psicólogo na saúde pública não está claro,
nem para a legislação vigente nem para a classe profissional. Isso fragiliza a atuação desse
profissional, desqualificando seu potencial principalmente no que se refere à atenção básica
de saúde. Boing e Crepaldi (2010) apontam, em seu estudo documental, que as políticas
públicas vigentes não incluem de forma clara e efetiva o psicólogo. Quando há o profissional
psicólogo inserido na atenção básica, suas intervenções acabam em atendimentos individu
alizados, justificados pela resistência da equipe diante de outras propostas de intervenção
(Dimenstein, 2000). Essa transformação do modelo biomédico para uma perspectiva coletiva de se produzir
saúde, principalmente na atenção básica, corresponde à principal premissa da saúde coleti
va (Campos, 2013), apostando em uma prática horizontal e integrada. Na pesquisa realizada
para este artigo, integralidade é um conceito-chave na construção de uma atenção básica
plural e efetiva. Fica evidente que a fragmentação da saúde impede a ampliação do conheci
mento ante o ser humano, desconsiderando os atravessamentos históricos e culturais desse
saber (Codo & Lane, 1989; Dimenstein, 2000). Segundo Codo e Lane (1989), as fronteiras
entre as áreas do conhecimento devem ser permeáveis, permitindo a integralidade e a in
tersetorialidade para uma compreensão qualificada da complexidade que são as interações
do indivíduo com o social. De acordo com o Conselho Federal de Psicologia (CFP, 2011), uma das dificuldades da atu
ação do psicólogo na atenção básica à saúde é a concepção de profissionais e gestores sobre
o entendimento do processo saúde e doença. O conceito de saúde vai além da ausência de
doença, trata-se de um bem-estar integral, considerando aspectos físicos, sociais e mentais
(https://www.paho.org/bra/index.php?option=com_content&view=article&id=5263:opas
-oms-apoia-governos-no-objetivo-de-fortalecer-e-promover-a-saude-mental-da-popula
cao&Itemid=839). Revista Psicologia e Saúde. Pensando nessas mudanças de atuação e envolvimento com a saúde, Sundfeld (2010)
coloca, no Artigo 3, que “a reforma da assistência pressupõe a reforma do pensamento de
seus atores: profissionais e comunidade e, sobretudo, um sim às incertezas e inventividade
do cotidiano” (p. 1094), além de entender essas mudanças em saúde pública, com foco
em saúde coletiva, como “uma prática à espera de teoria” (Ferreira Neto, 2008, p. 112),
diferenciando-se do modelo clínico, individualizado, considerando saúde mental como algo
indissociável à saúde. Outro apontamento importante nos artigos é a questão da medicalização como conduta
de tratamento corriqueiro na UBS (Jimenez, 2011). “O foco das ações em saúde é centrado
no uso exagerado de medicamentos […] sem possibilitar um espaço de escuta para esse
sofrimento como forma de evitar o desenfreado sistema de medicamentação” (Freire &
Pichelli, 2013, p. 168). Com a Reforma Psiquiátrica, deixa-se de ter um olhar para patologização, apostando no
funcionamento adaptativo do sujeito por meio de serviços substitutivos que estimulem
essa visão de saúde com olhar humanizado. “O objeto a ser abordado deixa de ser a doen
ça, abarcando os fatores sociais, culturais, políticos e econômicos como determinantes do Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 ISSN: 2177-093X Revista Psicologia e Saúde. 185 processo de adoecimento. Consequentemente, os meios de trabalho migram das práticas
predominantemente medicamentosas para o sujeito como agente fundamental do trata
mento” (Jimenez, 2011, p. 131). Conclusões Entende-se que a adoção do paradigma da saúde coletiva na atenção à saúde é conside
rada como a possibilidade mais eficaz para que se torne efetivo o cuidado integral preconi
zado pelo SUS. Isto porque esse paradigma favorece o desenvolvimento saudável da popula
ção, ao incluir a saúde mental na atenção, bem como transforma a assistência por meio das
ações humanizadas. Evidenciou-se que o objetivo do psicólogo que atua na atenção básica de saúde é a pro
posição de intervenções coletivas e integradas na busca de uma comunidade mais autônoma
e consciente, evitando uma assistência curativa em saúde fundamentada na medicalização. Com o profissional de psicologia inserido na atenção básica, também são afetados os modos
de atuação dos servidores, ampliando o entendimento sobre saúde, diminuindo estigmas e
mediando as relações com o usuário e entre as equipes. i Para se alcançar os objetivos mencionados, é necessária uma revisão das diretrizes que
fundamentam as práticas do psicólogo na atenção básica. Tendo a noção do prolongado pe
ríodo para esse processo de reformulação da legislação, sugere-se que essa assistência seja
desenvolvida nos Nasfs, que estão constituídos nas regulamentações do SUS, viabilizando o
movimento em prol da saúde coletiva. Com o psicólogo atuando nas equipes Nasf de forma direcionada ao apoio técnico no
território, é possível, com uso de diálogos, capacitações e apoio ao profissional de saúde em
intervenções complexas, dar suporte e promover ações em saúde coletiva, além de auxiliar
na humanização do atendimento, com a promoção de uma escuta qualificada e criação de
vínculo profissional, facilitando a adesão do usuário aos tratamentos. Dessa forma, direciona-se para ações em saúde coletiva, trabalhando a partir da clínica
ampliada, como preveem as normativas do SUS, de forma a gerar menos gastos com a saú
de pública, levando em consideração a diminuição de medicalização e menores índices de
adoecimento. Pode-se inferir, com isso, a ampliação dos recursos financeiros para práticas
em saúde coletiva. Outra possibilidade de promover maior assistência e organização do sistema são as par
cerias entre instituições de ensino e rede pública, com estágios acadêmicos que possibilitem
experiências em saúde coletiva, somadas ao apoio dos estagiários de diferentes áreas, inte
grando ensino e aprendizagem à formação − uma forma de promover compromisso social do
profissional com práticas que permitam conhecer a diversidade e a realidade das demandas
encontrada nos territórios. Revista Psicologia e Saúde. 186 A formação acadêmica é significativa nessa transformação do modelo biomédico e
Dimenstein (2000) sugere ampliar a argumentação a respeito da formação do profissional
na graduação com o uso de referências mais atualizadas. Aponta que a graduação não prepa
ra para uma “intervenção adequada aos espaços territoriais, locais que demandam um alto
grau de potência de resposta/ação, de articulação intersetorial, de mobilização de parcerias
e de estratégias específicas” (Dimenstein, 2000, p. 62). Discussão Sendo assim, a atenção à saúde mental deve ser considerada uma prática
de significância dentro dos núcleos de atenção básica, não sendo tratada como um aspecto
paralelo à saúde. O profissional psicólogo necessita buscar e concretizar seu espaço neste
âmbito primário, em virtude do seu conhecimento diante do desenvolvimento humano e
social, além de compreender e viabilizar relações pessoais de qualidade e promover a saúde
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dx.doi.org/10.1590/S0103-73312010000400002 Recebido em: 30/07/2019
Última revisão: 23/10/2019
Aceite final: 21/01/2020 Sobre as autoras: Sobre as autoras: Vanessa Santos Lemos: Psicóloga. Docente no curso de Psicologia da Universidade de Caxias do Sul
(UCS). E-mail: at.psi.zoi@gmail.com, Orcid: http://orcid.org/0000-0002-7078-6059 Cristina Lhullier: Doutora em Ciências na área de Psicologia. Professora da Graduação em
Psicologia da Universidade de Caxias do Sul (UCS). E-mail: cris.lhullier@yahoo.com.br, Orcid:
http://orcid.org/0000-0002-5440-2916 Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 Revista Psicologia e Saúde, v. 12, n. 3, jul./set. 2020, p. 177-188 ISSN: 2177-093X
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https://openalex.org/W3103370225
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https://zenodo.org/record/3599255/files/CHEP2019_320.pdf
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English
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Likelihood preservation and statistical reproduction of searches for new physics
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EPJ web of conferences
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cc-by
| 1,627
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Matthew Feickert, Lukas Heinrich, Giordon Stark
on behalf of the ATLAS collaboration
matthew.feickert@cern.ch
CHEP 2019
November 7th, 2019 matthew.feickert@cern.ch
CHEP 2019
November 7th, 2019 1 / 22 High information-density summary of
analysis
Almost everything we do in the analysis
ultimately affects the likelihood and is
encapsulated in it
Trigger
Detector
Systematic Uncertainties
Event Selection
Unique representation of the analysis to
preserve
Why is the likelihood important? High information-density summary of
analysis
Almost everything we do in the analysis
ultimately affects the likelihood and is
encapsulated in it
Trigger
Detector
Systematic Uncertainties
Event Selection
Unique representation of the analysis to
preserve
Why is the likelihood important? High information-density summary of
analysis Almost everything we do in the analysis
ultimately affects the likelihood and is
encapsulated in it Trigger Detector Systematic Uncertainties Event Selection Unique representation of the analysis to
preserve 1 / 22 Likelihood serialization... ...making good on 19 year old agreement to publish likelihood This hadn't been done in HEP until now In an "open world" of statistics this is a difØcult problem to solve In an "open world" of statistics this is a difØcult problem to solve What to preserve and how? All of ROOT? Idea: Focus on a single more tractable binned model Ørst 2 / 22 Enter HistFactory A Ùexible p.d.f. template to build statistical models from binned distributions and data
Developed by Cranmer, Lewis, Moneta, Shibata, and Verkerke (CERN-OPEN-2012-016)
Widely used by the HEP community for standard model measurements and BSM searches A Ùexible p.d.f. template to build statistical models from binned distributions and data Widely used by the HEP community for standard model measurements and BSM searches Widely used by the HEP community for standard model measurements and BSM searches 3 / 22 tFactory Template
ltiple disjoint channels (or regions) of binned distributions with multiple samples contributing to each with
al (possibly shared) systematics between sample estimates
f
,
,
=
Pois n
ν
,
c
a
χ
(n⃗a⃗∣η⃗χ⃗)
c∈channels
∏
b∈bins
c
∏
(
cb∣cb (η⃗χ⃗))
χ ∈
χ⃗
∏
χ ( χ∣)
ν
(
,
) =
(ν
(
,
) +
)
cb η⃗χ⃗
s ∈samples
∑
multiplicative
κ
(
,
)
(
κ ∈κ⃗
∑
scb η⃗χ⃗)
scb
0
η⃗χ⃗
additive
Δ
(
,
)
Δ∈Δ⃗
∑
scb η⃗χ⃗ HistFactory Template multiplicative Use: Multiple disjoint channels (or regions) of binned distributions with multiple samples contributing to each with
additional (possibly shared) systematics between sample estimates Main pieces: Main Poisson p.d.f. for simultaneous measurement of multiple channels Main Poisson p.d.f. for simultaneous measurement of multiple channels Event rates
from nominal rate
and rate modiØers and
ν
cb
ν
scb
0
κ
Δ Event rates
from nominal rate
and rate modiØers and
ν
cb
ν
scb
0
κ
Δ Constraint p.d.f. (+ data) for "auxiliary measurements" Constraint p.d.f. (+ data) for "auxiliary measurements" encoding systematic uncertainties (normalization, shape, etc) encoding systematic uncertainties (normalization, shape, etc) : events, : auxiliary data, : unconstrained pars, : constrained pars
n⃗
a⃗
η⃗
χ⃗ : events, : auxiliary data, : unconstrained pars, : constrained pars
n⃗
a⃗
η⃗
χ⃗ 4 / 22 This is a mathematical representation! Nowhere is any software Preservation: Likelihood stored in the binary ROOT format
Challenge for long-term preservation (i.e. HEPData)
Why is a histogram needed for an array of numbers? Why is a histogram needed for an array of numbers? To start using HistFactory p.d.f.s Ørst have to learn ROOT, RooFit, RooStats
Problem for our theory colleagues (generally don't want to) DifØcult to use for reinterpretation 5 / 22 Has a JSON spec that fully describes the HistFactory model Open source tool for all of HEP Originated from a DIANA/HEP project fellowship and now an IRIS-HEP supporte Used for reinterpretation in phenomenology paper (DOI: 10.1007/JHEP04(2019)14 Used internally in ATLAS for pMSSM SUSY large scale reinterpretation 6 / 22 Example pyhf JSON spec JSON deØning a single channel, two bin counting experiment with systematics 7 / 22 Live demo time! Live demo time! Just click the button! Just click the button! launch
launch binder
binder 8 / 22 Example using pyhf CLI
CL
s 9 / 22 JSON Patch for new signal models Original model
New Signal (JSON Patch Øle)
Reinterpretation
10 / 22 Original model
New Signal (JSON Patch Øle) Original model
New Original model New Signal (JSON Patch Øle) New Signal (JSON Patch Øle) R i t
t ti Reinterpretation 10 / 22 JSON Patch for new signal models s (model A)
Recast analysis (model B)
or new signal models
11 / 22 Original analysis (model A)
Recast analysis (model B)
11 / 22 Original analysis (model A) Original analysis (model A) Recast analysis (model B) Recast analysis (model B) 11 / 22 Likelihoods preserved on HEPData Background-only model JSON stored Signal models stored as JSON Patch Øles Together are able to fully preserve the full model (with own DOI! D O I
D O I 10.17182/hepdata.89408.v1/r2
10.17182/hepdata.89408.v1/r2 ) Together are able to fully preserve the full model (with own DOI! D O I
D O I 10.17182/hepdata.89408.v1/r2
10.17182/hepdata.89408.v1/r2 ) 12 / 22 ...can be streamed from HEPData Background-only model JSON stored Signal models stored as JSON Patch Øles to fully preserve the full model (with own DOI! D O I
D O I 10.17182/hepdata.89408.v1/r2
10.17182/hepdata.89408.v1/r2 )
13 / 22 Together are able to fully preserve the full model (with own DOI! D O I
D O I 10.17182/hepdata.89408.v1/r2
10.17182/hepdata.89408.v1/r2 ) Together are able to fully preserve the full model (with own DO Together are able to fully preserve the full model (with own DOI! D O I
D O I 10.17182/hepdata.89408.v1/r2
10.17182/hepdata.89408.v1/r2 ) 13 / 22 Likelihood serialization and reproduction ATLAS PUB note on the JSON schema for serialization and reproduction of results (ATL-PHYS-PUB-2019-
029) Contours: █ original ROOT+XML, █ pyhf JSON, █ JSON converted back to ROOT+XML Contours: █ original ROOT+XML, █ pyhf JSON, █ JSON converted back to ROOT+XML 14 / 22 Independent pure-Python implementation of HistFactory + hypo Publication for the Ørst time of the full likelihood of a search fo Likelihood serialization and reproduction ATLAS PUB note on the JSON schema for serialization and reproduction of results (ATL-PHYS-PUB-2019-
029) Contours: █ original ROOT+XML, █ pyhf JSON, █ JSON converted back to ROOT+XML Contours: █ original ROOT+XML, █ pyhf JSON, █ JSON converte
Overlay of contours nice visualization of near perfect agreement █
g
█py
█
Overlay of contours nice visualization of near perfect agreement Overlay of contours nice visualization of near perfect agreement Overlay of contours nice visualization of near perfect agreement Serialized likelihood and reproduced results of ATLAS Run-2 search for sbottom quarks (CERN-EP-2019-142) an Serialized likelihood and reproduced results of ATLAS Run-2 search for sbottom quarks (CERN-EP-2019-142) and published to HEPData Serialized likelihood and reproduced results of ATLAS Run-2 search for sbottom qua Shown to reproduce results but faster! ROOT: 10+ hours pyhf: < 30 minutes 15 / 22 Bidirectional translation of likelihood speciØcations
ROOT workspaces ↔ JSON Bidirectional translation of likelihood speciØcations
ROOT workspaces ↔ JSON (1st Workshop on ConØdence Limits, CERN, 2000)
(ATLAS 2019)
Summary
Through pyhf are able to provide:
JSON speciØcation of likelihoods
human/machine readable, versionable, HEPData friendly, orders of magnitude smaller
Bidirectional translation of likelihood speciØcations
ROOT workspaces ↔ JSON
Independent pure-Python implementation of HistFactory + hypothesis testing
Publication for the Ørst time of the full likelihood of a search for new physics Through pyhf are able to provide: JSON speciØcation of likelihoods
human/machine readable, versionable, HEPData friendly, orders of magnitude s (1st Workshop on ConØdence Limits, CERN, 2000) 16 / 22 17 / 22 17 / 22 ROOT + XML to JSON and back 18 / 22 Best-Øt parameter values 19 / 22 JSON Patch Øles for new signal models $ pyhf cls example.json | jq .CLs_obs
0.053994246621274014 $ cat new_signal.json $ pyhf cls example.json --patch new_signal.json | jq .CLs_obs
0.3536906623262466 $ pyhf cls example.json --patch new_signal.json | jq .CLs_obs
0.3536906623262466 20 / 22 20 / 22 elihoods can be streamed from HEPData
signal model
l -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
-O -xzv RegionA/BkgOnly.json | \
f cls --patch <(curl -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
ar -O -xzv RegionA/patch.sbottom_1300_205_60.json) | \
CLs_obs
4363799054463
ifferent signal model
l -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
-O -xzv RegionA/BkgOnly.json | \
f cls --patch <(curl -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
ar -O -xzv RegionA/patch.sbottom_1300_230_100.json) | \
CLs_obs # One signal model g
$ curl -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
tar -O -xzv RegionA/BkgOnly.json | \
pyhf cls --patch <(curl -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
tar -O -xzv RegionA/patch.sbottom_1300_205_60.json) | \ g
L https://doi.org/10.17182/hepdata.89408.v1/r2 | jq .CLs_obs 0.2444363799054463 0.2444363799054463 0.2444363799054463 0.2444363799054463 # A different signal model # A different signal model # A different signal model
$ curl -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
tar -O -xzv RegionA/BkgOnly.json | \
pyhf cls --patch <(curl -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
tar -O -xzv RegionA/patch.sbottom_1300_230_100.json) | \ $ curl -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
tar -O -xzv RegionA/BkgOnly.json | \
pyhf cls --patch <(curl -sL https://doi.org/10.17182/hepdata.89408.v1/r2 | \
tar -O -xzv RegionA/patch.sbottom_1300_230_100.json) | \ jq .CLs_obs
0.040766026035752724 21 / 22 21 / 22 References 2. ROOT collaboration, K. Cranmer, G. Lewis, L. Moneta, A. Shibata and W. Verkerke, HistFactory: A tool for
creating statistical models for use with RooFit and RooStats, 2012. 3. L. Heinrich, H. Schulz, J. Turner and Y. Zhou, Constraining
Leptonic Flavour Model Parameters at Colliders
and Beyond, 2018. A
4 4. ATLAS collaboration, Search for bottom-squark pair production with the ATLAS detector in Ønal states containing
Higgs bosons, b-jets and missing transverse momentum, 2019 5. ATLAS collaboration, Reproducing searches for new physics with the ATLAS experiment through publication of
full statistical likelihoods, 2019 6. ATLAS collaboration, Search for bottom-squark pair production with the ATLAS detector in Ønal states containing
Higgs bosons, b-jets and missing transverse momentum: HEPData entry, 2019 22 / 22 22 / 22 22 / 22
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https://openalex.org/W2887598267
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https://philpapers.org/archive/ELLLEI.pdf
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English
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Logical Entropy: Introduction to Classical and Quantum Logical Information Theory
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Entropy
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cc-by
| 16,611
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Article David Ellerman ID Department of Philosophy, University of California Riverside, Riverside, CA 92521, USA; david@ellerman Received: 10 August 2018; Accepted: 4 September 2018; Published: 6 September 2018 Abstract: Logical information theory is the quantitative version of the logic of partitions just as logical
probability theory is the quantitative version of the dual Boolean logic of subsets. The resulting notion
of information is about distinctions, differences and distinguishability and is formalized using the
distinctions (“dits”) of a partition (a pair of points distinguished by the partition). All the definitions of
simple, joint, conditional and mutual entropy of Shannon information theory are derived by a uniform
transformation from the corresponding definitions at the logical level. The purpose of this paper is to
give the direct generalization to quantum logical information theory that similarly focuses on the
pairs of eigenstates distinguished by an observable, i.e., qudits of an observable. The fundamental
theorem for quantum logical entropy and measurement establishes a direct quantitative connection
between the increase in quantum logical entropy due to a projective measurement and the eigenstates
(cohered together in the pure superposition state being measured) that are distinguished by the
measurement (decohered in the post-measurement mixed state). Both the classical and quantum
versions of logical entropy have simple interpretations as “two-draw” probabilities for distinctions. The conclusion is that quantum logical entropy is the simple and natural notion of information for
quantum information theory focusing on the distinguishing of quantum states. Keywords: logical entropy; partition logic; qudits of an observable 1. Introduction The formula for “classical” logical entropy goes back to the early Twentieth Century [1]. It is the
derivation of the formula from basic logic that is new and accounts for the name. The ordinary Boolean
logic of subsets has a dual logic of partitions [2] since partitions (=equivalence relations = quotient sets)
are category-theoretically dual to subsets. Just as the quantitative version of subset logic is the notion
of logical finite probability, so the quantitative version of partition logic is logical information theory
using the notion of logical entropy [3]. This paper generalizes that “classical” (i.e., non-quantum)
logical information theory to the quantum version. The classical logical information theory is briefly
developed before turning to the quantum version. Applications of logical entropy have already been
developed in several special mathematical settings; see [4] and the references cited therein. entropy entropy Entropy 2018, 20, 679; doi:10.3390/e20090679 2. Duality of Subsets and Partitions The foundations for classical and quantum logical information theory are built on the logic of
partitions ([2,5]), which is dual (in the category-theoretic sense) to the usual Boolean logic of subsets. This duality can be most simply illustrated using a set function f : X →Y. The image f (X) is a subset
of the codomain Y and the inverse-image or coimage f −1 (Y) is a partition on the domain X, where a
partition π = {B1, . . . , BI} on a set U is a set of subsets or blocks Bi that are mutually disjoint and jointly
exhaustive (∪iBi = U) In category theory, the duality between subobject-type constructions (e.g., limits)
and quotient-object-type constructions (e.g., colimits) is often indicated by adding the prefix “co-” to Entropy 2018, 20, 679; doi:10.3390/e20090679 www.mdpi.com/journal/entropy 2 of 22 Entropy 2018, 20, 679 the latter. Hence, the usual Boolean logic of “images” has the dual logic of “coimages”. However,
the duality runs deeper than between subsets and partitions. The dual to the notion of an “element”
(an “it”) of a subset is the notion of a “distinction” (a “dit”) of a partition, where (u, u′) ∈U × U is
a distinction or dit of π if the two elements are in different blocks. Let dit (π) ⊆U × U be the set of
distinctions or ditset of π. Similarly an indistinction or indit of π is a pair (u, u′) ∈U × U in the same
block of π. Let indit (π) ⊆U × U be the set of indistinctions or inditset of π. Then, indit (π) is the
equivalence relation associated with π, and dit (π) = U × U −indit (π) is the complementary binary
relation that might be called apartition relation or an a partness relation. The notions of a distinction and
indistinction of a partition are illustrated in Figure 1. Figure 1. Distinctions and indistinctions of a partition. Figure 1. Distinctions and indistinctions of a partition. The relationships between Boolean subset logic and partition logic are summarized in Figure 2,
which illustrates the dual relationship between the elements (“its”) of a subset and the distinctions
(“dits”) of a partition. Figure 2. Dual logics: Boolean subset logic of subsets and partition logic. Figure 2. Dual logics: Boolean subset logic of subsets and partition logic. 3. From the Logic of Partitions to Logical Information Theory In Gian-Carlo Rota’s Fubini Lectures [6] (and in his lectures at MIT), he remarked in view of duality
between partitions and subsets that, quantitatively, the “lattice of partitions plays for information the
role that the Boolean algebra of subsets plays for size or probability” ([7] p. 30) or symbolically: Logical Probability Theory
Boolean Logic of Subsets = Logical Information Theory
Logic of Partitions
. Logical Probability Theory
Boolean Logic of Subsets = Logical Information Theory
Logic of Partitions
. as suggested that information theory should start with sets, not probabilities. Andrei Kolmogorov has suggested that information theory should start with sets, not probabilities Entropy 2018 20 679
3 of 2 3 of 22 Entropy 2018, 20, 679 Information theory must precede probability theory, and not be based on it. By the very
essence of this discipline, the foundations of information theory have a finite combinatorial
character. ([8] p. 39) The notion of information-as-distinctions does start with the set of distinctions, the information set,
of a partition π = {B1, . . . , BI} on a finite set U where that set of distinctions (dits) is: dit (π) = {(u, u′) : ∃Bi, Bi′ ∈π, Bi ̸= Bi′, u ∈Bi, u′ ∈Bi′}. The normalized size of a subset is the logical probability of the event, and the normalized size of
the ditset of a partition is, in the sense of measure theory, the measure of the amount of information in
a partition. Thus, we define the logical entropy of a partition π = {B1, . . . , BI}, denoted h (π), as the
size of the ditset dit (π) ⊆U × U normalized by the size of U × U: h (π) = |dit(π)|
|U×U| = |U×U|−∑I
i=1|Bi×Bi|
|U×U|
= 1 −∑I
i=1
|Bi|
|U|
2
= 1 −∑I
i=1 Pr (Bi)2. In two independent draws from U, the probability of getting a distinction of π is the probability
of not getting an indistinction. In two independent draws from U, the probability of getting a distinction of π is the probability
of not getting an indistinction. Given any probability measure p : U →[0, 1] on U = {u1, . . . , un}, which defines pi = p (ui) for
i = 1, . . . 3. From the Logic of Partitions to Logical Information Theory , n, the product measure p × p : U × U →[0, 1] has for any binary relation R ⊆U × U the
value of: p × p (R) = ∑(ui,uj)∈R p (ui) p
uj
= ∑(ui,uj)∈R pipj. The logical entropy of π in general is the product-probability measure of its ditset dit (π) ⊆U × U,
where Pr (B) = ∑u∈B p (u): h (π) = p × p (dit (π)) = ∑(ui,uj)∈dit(π) pipj = 1 −∑B∈π Pr (B)2. The standard interpretation of h (π) is the two-draw probability of getting a distinction of the
partition π, just as Pr (S) is the one-draw probability of getting an element of the subset-event S. The standard interpretation of h (π) is the two-draw probability of getting a distinction of the
partition π, just as Pr (S) is the one-draw probability of getting an element of the subset-event S. 4. Compound Logical Entropies , pn} on U, the joint logical entropy is the product probability measure
on the union of ditsets: 4 of 22 Entropy 2018, 20, 679 Entropy 2018, 20, 679 h (π, σ) = h (π ∨σ) = p × p (dit (π) ∪dit (σ)) = 1 −∑i,j Pr
Bi ∩Cj
2. h (π, σ) = h (π ∨σ) = p × p (dit (π) ∪dit (σ)) = 1 −∑i,j Pr
Bi ∩Cj
2. The information set for the conditional logical entropy h (π|σ) is the difference of ditsets, and thus,
that logical entropy is: h (π|σ) = p × p (dit (π) −dit (σ)) = h (π, σ) −h (σ). The information set for the logical mutual information m (π, σ) is the intersection of ditsets, so that
logical entropy is: m (π, σ) = p × p (dit (π) ∩dit (σ)) = h (π, σ) −h (π|σ) −h (σ|π) = h (π) + h (σ) −h (π, σ). m (π, σ) = p × p (dit (π) ∩dit (σ)) = h (π, σ) −h (π|σ) −h (σ|π) = h (π) + h (σ) −h (π, σ). Since all the logical entropies are the values of a measure p × p : U × U →[0, 1] on subsets of
U × U, they automatically satisfy the usual Venn diagram relationships as in Figure 3. Figure 3. Venn diagram for logical entropies as values of a probability measure p × p on U × U. Figure 3. Venn diagram for logical entropies as values of a probability measure p × p on U × U. At the level of information sets (w/o probabilities), we have the information algebra I (π, σ),
which is the Boolean subalgebra of ℘(U × U) generated by ditsets and their complements. 4. Compound Logical Entropies The compound notions of logical entropy are also developed in two stages, first as sets and then,
given a probability distribution, as two-draw probabilities. After observing the similarity between the
formulas holding for the compound Shannon entropies and the Venn diagram formulas that hold for
any measure (in the sense of measure theory), the information theorist, Lorne L. Campbell, remarked in
1965 that the similarity: suggests the possibility that H (α) and H (β) are measures of sets, that H (α, β) is the
measure of their union, that I (α, β) is the measure of their intersection, and that H (α|β)
is the measure of their difference. The possibility that I (α, β) is the entropy of the
“intersection” of two partitions is particularly interesting. This “intersection,” if it existed,
would presumably contain the information common to the partitions α and β. ([9] p. 113) suggests the possibility that H (α) and H (β) are measures of sets, that H (α, β) is the
measure of their union, that I (α, β) is the measure of their intersection, and that H (α|β)
is the measure of their difference. The possibility that I (α, β) is the entropy of the
“intersection” of two partitions is particularly interesting. This “intersection,” if it existed,
would presumably contain the information common to the partitions α and β. ([9] p. 113) Yet, there is no such interpretation of the Shannon entropies as measures of sets, but the logical
entropies precisely fulfill Campbell’s suggestion (with the “intersection” of two partitions being the
intersection of their ditsets). Moreover, there is a uniform requantifying transformation (see the next
section) that obtains all the Shannon definitions from the logical definitions and explains how the
Shannon entropies can satisfy the Venn diagram formulas (e.g., as a mnemonic) while not being defined
by a measure on sets. Given partitions π = {B1, . . . , BI} and σ =
C1, . . . , CJ
on U, the joint information set is the
union of the ditsets, which is also the ditset for their join: dit (π) ∪dit (σ) = dit (π ∨σ) ⊆U × U. Given probabilities p = {p1, . . . 5. Deriving the Shannon Entropies from the Logical Entropies , B2n} with Pr (Bi) =
1
2n ,
i.e., that it takes to uniquely encode each distinct element, is n, so the Shannon–Hartley entropy [13] is
the canonical bit-count: n = log2 (2n) = log2
1
1/2n
= log2
1
Pr(Bi)
. The general case of Shannon entropy is the average of these canonical bit-counts log2
1
Pr(Bi)
: The general case of Shannon entropy is the average of these canonical bit-counts log2
1
Pr(Bi)
: H (π) = ∑i Pr (Bi) log2
1
Pr(Bi)
. H (π) = ∑i Pr (Bi) log2
1
Pr(Bi)
. The dit-bit transform essentially replaces the canonical dit-counts by the canonical bit-counts. First, express any logical entropy concept (simple, joint, conditional or mutual) as an average of
canonical dit-counts 1 −Pr (Bi), and then, substitute the canonical bit-count log
1
Pr(Bi)
to obtain the
corresponding formula as defined by Shannon. Figure 4 gives examples of the dit-bit transform. Figure 4. Summary of the dit-bit transform. Figure 4. Summary of the dit-bit transform. For instance, h (π|σ) = h (π, σ) −h (σ) = ∑i,j Pr
Bi ∩Cj
1 −Pr
Bi ∩Cj
−∑j Pr
Cj
1 −Pr
Cj
is the expression for h (π|σ) as an average over 1 −Pr
Bi ∩Cj
and 1 −Pr
Cj
, so applying the dit-bit
transform gives: h (π|σ) = h (π, σ) −h (σ) = ∑i,j Pr
Bi ∩Cj
1 −Pr
Bi ∩Cj
−∑j Pr
Cj
1 −Pr
Cj
is the expression for h (π|σ) as an average over 1 −Pr
Bi ∩Cj
and 1 −Pr
Cj
, so applying the dit-bit
transform gives: i,j Pr
Bi ∩Cj
log
1/ Pr
Bi ∩Cj
−∑j Pr
Cj
log
1/ Pr
Cj
= H (π, σ) −H (σ) = H (π|σ). ∑i,j Pr
Bi ∩Cj
log
1/ Pr
Bi ∩Cj
−∑j Pr
Cj
log
1/ Pr
Cj
= H (π, σ) −H (σ) = H (π|σ). 5. Deriving the Shannon Entropies from the Logical Entropies Instead of being defined as the values of a measure, the usual notions of simple and compound
entropy ‘burst forth fully formed from the forehead’ of Claude Shannon [10] already satisfying the
standard Venn diagram relationships (one author surmised that “Shannon carefully contrived for
this ‘accident’ to occur” ([11] p. 153)). Since the Shannon entropies are not the values of a measure,
many authors have pointed out that these Venn diagram relations for the Shannon entropies can
only be taken as “analogies” or “mnemonics” ([9,12]). Logical information theory explains this
situation since all the Shannon definitions of simple, joint, conditional and mutual information can
be obtained by a uniform requantifying transformation from the corresponding logical definitions,
and the transformation preserves the Venn diagram relationships. This transformation is possible since the logical and Shannon notions of entropy can be seen as
two different ways to quantify distinctions; and thus, both theories are based on the foundational idea
of information-as-distinctions. f
Consider the canonical case of n equiprobable elements, pi =
1
n. The logical entropy of
1 = {B1, . . . , Bn} where Bi = {ui} with p =
n
1
n, . . . , 1
n
o
is: Consider the canonical case of n equiprobable elements, pi =
1
n. The logical entropy of
1 = {B1, . . . , Bn} where Bi = {ui} with p =
n
1
n, . . . , 1
n
o
is: |U×U−∆|
|U×U|
= n2−n
n2
= 1 −1
n = 1 −Pr (Bi). The normalized number of distinctions or ‘dit-count’ of the discrete partition 1 is 1 −1
n =
1 −Pr (Bi). The general case of logical entropy for any π = {B1, . . . , BI} is the average of the dit-counts
1 −Pr (Bi) for the canonical cases: h (π) = ∑i Pr (Bi) (1 −Pr (Bi)). 5 of 22 Entropy 2018, 20, 679 Entropy 2018, 20, 679 In the canonical case of 2n equiprobable elements, the minimum number of binary partitions
(“yes-or-no questions” or “bits”) whose join is the discrete partition 1 = {B1, . . . 5. Deriving the Shannon Entropies from the Logical Entropies The dit-bit transform is linear in the sense of preserving plus and minus, so the Venn diagram
formulas, e.g., h (π, σ) = h (σ) + h (π|σ), which are automatically satisfied by logical entropy since it
is a measure, carry over to Shannon entropy, e.g., H (π, σ) = H (σ) + H (π|σ) as in Figure 5, in spite
of it not being a measure (in the sense of measure theory): Figure 5. Venn diagram mnemonic for Shannon entropies. Figure 5. Venn diagram mnemonic for Shannon entropies. 6 of 22 Entropy 2018, 20, 679 6. Logical Entropy via Density Matrices The transition to quantum logical entropy is facilitated by reformulating the classical logical theory
in terms of density matrices. Let U = {u1, . . . , un} be the sample space with the point probabilities
p = (p1, . . . , pn). An event S ⊆U has the probability Pr (S) = ∑uj∈S pj. For any event S with Pr (S) > 0, let: p
(p
p )
For any event S with Pr (S) > 0, let: |S⟩=
1
√
Pr(S)(χS (u1) √p1, . . . , χS (un) √pn)t (the superscript t indicates transpose) which is a normalized column vector in Rn where χS : U →{0, 1}
is the characteristic function for S, and let ⟨S| be the corresponding row vector. Since |S⟩is normalized,
⟨S|S⟩= 1. Then, the density matrix representing the event S is the n × n symmetric real matrix: (the superscript t indicates transpose) which is a normalized column vector in Rn where χS : U →{0, 1}
is the characteristic function for S, and let ⟨S| be the corresponding row vector. Since |S⟩is normalized,
⟨S|S⟩= 1. Then, the density matrix representing the event S is the n × n symmetric real matrix: ρ (S) = |S⟩⟨S| so that (ρ(S))j,k =
(
1
Pr(S)
√pjpk for uj, uk ∈S
0 otherwise
. Then, ρ (S)2 = |S⟩⟨S|S⟩⟨S| = ρ (S), so borrowing language from quantum mechanics, ρ (S) is
said to be a pure state density matrix. hen, ρ (S)2 = |S⟩⟨S|S⟩⟨S| = ρ (S), so borrowing language from quantum mechanics, ρ (S) is
be a pure state density matrix. y
Given any partition π = {B1, . . . , BI} on U, its density matrix is the average of the block
density matrices: ρ (π) = ∑i Pr (Bi) ρ (Bi). ρ (π) = ∑i Pr (Bi) ρ (Bi). ρ (π) = ∑i Pr (Bi) ρ (Bi). Then, ρ (π) represents the mixed state, experiment or lottery where the event Bi occurs with
probability Pr (Bi). A little calculation connects the logical entropy h (π) of a partition with the density
matrix treatment: h (π) = 1 −∑I
i=1 Pr (Bi)2 = 1 −tr
h
ρ (π)2i
= h (ρ (π)) where ρ (π)2 is substituted for Pr (Bi)2 and the trace is substituted for the summation. 6. Logical Entropy via Density Matrices For the throw of a fair die, U = {u1, u3, u5, u2, u4, u6} (note the odd faces ordered before the even
faces in the matrix rows and columns) where uj represents the number j coming up, the density matrix
ρ (0) is the “pure state” 6 × 6 matrix with each entry being 1
6. ρ (0) =
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
1/6
u1
u3
u5
u2
u4
u6
. The nonzero off-diagonal entries represent indistinctions or indits of the partition 0 or, in quantum
terms, “coherences” where all 6 “eigenstates” cohere together in a pure “superposition” state. All pure
states have a logical entropy of zero, i.e., h (0) = 0 (i.e., no dits) since tr [ρ] = 1 for any density matrix,
so if ρ (0)2 = ρ (0), then tr
h
ρ (0)2i
= tr [ρ (0)] = 1 and h (0) = 1 −tr
h
ρ (0)2i
= 0. h
i
h
i
The logical operation of classifying undistinguished entities (like the six faces of the die before a
throw to determine a face up) by a numerical attribute makes distinctions between the entities with
different numerical values of the attribute. Classification (also called sorting, fibering or partitioning
([14] Section 6.1)) is the classical operation corresponding to the quantum operation of “measurement”
of a superposition state by an observable to obtain a mixed state. Now classify or “measure” the die-faces by the parity-of-the-face-up (odd or even) partition
(observable) π = {Bodd, Beven} = {{u1, u3, u5} , {u2, u4, u6}}. Mathematically, this is done by the
Lüders mixture operation ([15] p. 6. Logical Entropy via Density Matrices 279), i.e., pre- and post-multiplying the density matrix ρ (0) by Podd
and by Peven, the projection matrices to the odd or even components, and summing the results: 7 of 22 Entropy 2018, 20, 679 Poddρ (0) Podd + Pevenρ (0) Peven
=
1/6
1/6
1/6
0
0
0
1/6
1/6
1/6
0
0
0
1/6
1/6
1/6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1/6
1/6
1/6
0
0
0
1/6
1/6
1/6
0
0
0
1/6
1/6
1/6
=
1/6
1/6
1/6
0
0
0
1/6
1/6
1/6
0
0
0
1/6
1/6
1/6
0
0
0
0
0
0
1/6
1/6
1/6
0
0
0
1/6
1/6
1/6
0
0
0
1/6
1/6
1/6
= 1
2ρ (Bodd) + 1
2ρ (Beven) = ρ (π). Theorem 1 ( Fundamental(classical)). The increase in logical entropy, h (ρ (π)) −h (ρ (0)), due to a Lüders
mixture operation is the sum of amplitudes squared of the non-zero off-diagonal entries of the beginning density
matrix that are zeroed in the final density matrix. Proof. Since for any density matrix ρ, tr
ρ2 = ∑i,j
ρij
2 ([16] p. 77), we have: h (ρ (π)) −h (ρ (0)) =
1 −tr
h
ρ (π)2i
−
1 −tr
h
ρ (0)2i
=
tr
h
ρ (0)2i
−tr
h
ρ (π)2i
=
∑i,j
ρij (0)
2 −
ρij (π)
2
,
if (ui, ui′) ∈dit (π), then and only then are the off-diagonal terms corresponding to ui and ui′ zeroed
by the Lüders operation. Proof. Since for any density matrix ρ, tr
ρ2 = ∑i,j
ρij
2 ([16] p. 77), we have: h (ρ (π)) −h (ρ (0)) =
1 −tr
h
ρ (π)2i
−
1 −tr
h
ρ (0)2i
=
tr
h
ρ (0)2i
−tr
h
ρ (π)2i
=
∑i,j
ρij (0)
2 −
ρij (π)
2
,
if (ui, ui′) ∈dit (π), then and only then are the off-diagonal terms corresponding to ui and ui′ zeroed
by the Lüders operation. (
)
( )
by the Lüders operation. 6. Logical Entropy via Density Matrices The classical fundamental theorem connects the concept of information-as-distinctions to the
process of “measurement” or classification, which uses some attribute (like parity in the example) or
“observable” to make distinctions. In the comparison with the matrix ρ (0) of all entries 1
6, the entries that got zeroed in the Lüders
operation ρ (0) −→ρ (π) correspond to the distinctions created in the transition 0 = {{u1, . . . , u6}} −→
π
=
{{u1, u3, u5} , {u2, u4, u6}}, i.e., the odd-numbered faces were distinguished from the
even-numbered faces by the parity attribute. The increase in logical entropy = sum of the squares
of the off-diagonal elements that were zeroed = h (π) −h (0) = 2 × 9 ×
1
6
2
= 18
36 = 1
2. The usual
calculations of the two logical entropies are: h (π) = 2 ×
1
2
2
= 1
2 and h (0) = 1 −1 = 0. e: h (π) = 2 ×
1
2
2
= 1
2 and h (0) = 1 −1 = 0.
Since, in quantum mechanics, a projective measurement’s effect on a density matrix is the Lüders
mixture operation, that means that the effects of the measurement are the above-described “making
distinctions” by decohering or zeroing certain coherence terms in the density matrix, and the sum of
the absolute squares of the coherences that were decohered is the increase in the logical entropy. 7. Quantum Logical Information Theory: Commuting Observables The idea of information-as-distinctions carries over to quantum mechanics. [Information] is the notion of distinguishability abstracted away from what we are
distinguishing, or from the carrier of information. . . . And we ought to develop a theory of
information which generalizes the theory of distinguishability to include these quantum
properties... ([17] p. 155) Let F : V →V be a self-adjoint operator (observable) on a n-dimensional Hilbert space V with
the real eigenvalues φ1, . . . , φI, and let U = {u1, . . . , un} be an orthonormal (ON) basis of eigenvectors
of F. The quantum version of a dit, a qudit, is a pair of states definitely distinguishable by some
observable. Any nondegenerate self-adjoint operator such as ∑n
k=1 kP[uk], where P[uk] is the projection 8 of 22 Entropy 2018, 20, 679 to the one-dimensional subspace generated by uk, will distinguish all the vectors in the orthonormal
basis U, which is analogous classically to a pair (u, u′) of distinct elements of U that are distinguishable
by some partition (i.e., 1). In general, a qudit is relativized to an observable, just as classically a distinction
is a distinction of a partition. Then, there is a set partition π = {Bi}i=1,...,I on the ON basis U so that
Bi is a basis for the eigenspace of the eigenvalue φi and |Bi| is the “multiplicity” (dimension of the
eigenspace) of the eigenvalue φi for i = 1, . . . , I. Note that the real-valued function f : U →R takes
each eigenvector in uj ∈Bi ⊆U to its eigenvalue φi so that f −1 (φi) = Bi contains all the information
in the self-adjoint operator F : V →V since F can be reconstructed by defining it on the basis U as
Fuj = f
uj
uj. j
j
j
The generalization of “classical” logical entropy to quantum logical entropy is straightforward
using the usual ways that set-concepts generalize to vector-space concepts: subsets →subspaces,
set partitions →direct-sum decompositions of subspaces (hence the “classical” logic of partitions on
a set will generalize to the quantum logic of direct-sum decompositions [18] that is the dual to the
usual quantum logic of subspaces ), Cartesian products of sets →tensor products of vector spaces
and ordered pairs (uk, uk′) ∈U × U →basis elements uk ⊗uk′ ∈V ⊗V. 7. Quantum Logical Information Theory: Commuting Observables That is, there are always two eigenvectors uk and uk′ that have
different eigenvalues both by F and by G. g
y
y
In a measurement, the observables do not provide the point probabilities; they come from the
pure (normalized) state ψ being measured. Let |ψ⟩= ∑n
j=1
uj|ψ
uj
= ∑n
j=1 αj
uj
be the resolution
of |ψ⟩in terms of the orthonormal basis U = {u1, . . . , un} of simultaneous eigenvectors for F and G. Then, pj = αjα∗
j (α∗
j is the complex conjugate of αj) for j = 1, . . . , n are the point probabilities on U,
and the pure state density matrix ρ (ψ) = |ψ⟩⟨ψ| (where ⟨ψ| = |ψ⟩† is the conjugate-transpose) has the
entries: ρjk (ψ) = αjα∗
k, so the diagonal entries ρjj (ψ) = αjα∗
j = pj are the point probabilities. Figure 7
gives the remaining parallel development with the probabilities provided by the pure state ψ where
we write ρ (ψ)† ρ (ψ) as ρ (ψ)2. Figure 7. The development of classical and quantum logical entropies for commuting F and G. Figure 7. The development of classical and quantum logical entropies for commuting F and G. The formula h (ρ) = 1 −tr
ρ2
is hardly new. Indeed, tr
ρ2
is usually called the purity of the
density matrix since a state ρ is pure if and only if tr
ρ2 = 1, so h (ρ) = 0, and otherwise, tr
ρ2 < 1,
so h (ρ) > 0; and the state is said to be mixed. Hence, the complement 1 −tr
ρ2
has been called the
“mixedness” ([20] p. 5) or “impurity” of the state ρ. The seminal paper of Manfredi and Feix [21]
approaches the same formula 1 −tr
ρ2
(which they denote as S2) from the viewpoint of Wigner
functions, and they present strong arguments for this notion of quantum entropy (thanks to a referee
for this important reference to the Manfredi and Feix paper). This notion of quantum entropy is also
called by the misnomer “linear entropy” even though it is quadratic in ρ, so we will not continue
that usage. See [22] or [23] for references to that literature. 7. Quantum Logical Information Theory: Commuting Observables The eigenvalue function
f : U →R determines a partition
f −1 (φi)
i∈I on U, and the blocks in that partition generate the
eigenspaces of F, which form a direct-sum decomposition of V. g
p
p
Classically, a dit of the partition
f −1 (φi)
i∈I on U is a pair (uk, uk′) of points in distinct blocks
of the partition, i.e., f (uk) ̸= f (uk′). Hence, a qudit of F is a pair (uk, uk′) (interpreted as uk ⊗uk′ in
the context of V ⊗V) of vectors in the eigenbasis definitely distinguishable by F, i.e., f (uk) ̸= f (uk′),
distinct F-eigenvalues. Let G : V →V be another self-adjoint operator on V, which commutes with F
so that we may then assume that U is an orthonormal basis of simultaneous eigenvectors of F and
G. Let
γj
j∈J be the set of eigenvalues of G, and let g : U →R be the eigenvalue function so a pair
(uk, uk′) is a qudit of G if g (uk) ̸= g (uk′), i.e., if the two eigenvectors have distinct eigenvalues of G. As in classical logical information theory, information is represented by certain subsets (or, in the
quantum case, subspaces) prior to the introduction of any probabilities. Since the transition from
classical to quantum logical information theory is straightforward, it will be presented in table form
in Figure 6 (which does not involve any probabilities), where the qudits (uk, uk′) are interpreted as
uk ⊗uk′. Figure 6. The parallel development of classical and quantum logical information prior to probabilities Figure 6. The parallel development of classical and quantum logical information prior to probabiliti Figure 6. The parallel development of classical and quantum logical information prior to probabilities. The information subspace associated with F is the subspace [qudit (F)] ⊆V ⊗V generated by
the qudits uk ⊗uk′ of F. If F = λI is a scalar multiple of the identity I, then it has no qudits, so 9 of 22 Entropy 2018, 20, 679 its information space [qudit (λI)] is the zero subspace. It is an easy implication of the common dits
theorem of classical logical information theory (([19] (Proposition 1)) or ([5] (Theorem 1.4))) that any
two nonzero information spaces [qudit (F)] and [qudit (G)] have a nonzero intersection, i.e., have a
nonzero mutual information space. 8. Two Theorems about Quantum Logical Entropy The proof shows that they are both equal to classical
logical entropy of the partition π (F : ψ) defined on the ON basis U = {u1, . . . , un} of F-eigenvectors
by the F-eigenvalues with the point probabilities pj = α∗
j αj. That is, the inverse images Bi = φ−1 (φi)
of the eigenvalue function φ : U →R define the eigenvalue partition π (F : ψ) = {B1, . . . , BI} on
the ON basis U = {u1, . . . , un} with the point probabilities pj = α∗
j αj provided by the state |ψ⟩for
j = 1, . . . , n. The classical logical entropy of that partition is: h (π (F : ψ)) = 1 −∑I
i=1 p (Bi)2 where
p (Bi) = ∑u ∈B pj j
j = 1, . . . , n. The classical logical entropy of that partition is: h (π (F : ψ)) = 1 −∑I
i=1 p (Bi)2 where
p (Bi) = ∑uj∈Bi pj. j
j = 1, . . . , n. The classical logical entropy of that partition is: h (π (F : ψ)) = 1 −∑I
i=1 p (Bi)2 where
p (Bi) = ∑uj∈Bi pj. h
i We first show that h (F : ψ) = tr
h
P[qudit(F)]ρ (ψ) ⊗ρ (ψ)
i
= h (π (F : ψ)). Now, qudit (F) =
uj ⊗uk : φ
uj
̸= φ (uk)
, and [qudit (F)] is the subspace of V ⊗V generated by it. The n × n
pure state density matrix ρ (ψ) has the entries ρjk (ψ) = αjα∗
k, and ρ (ψ) ⊗ρ (ψ) is an n2 × n2
matrix. The projection matrix P[qudit(F)] is an n2 × n2 diagonal matrix with the diagonal entries,
indexed by j, k = 1, . . . , n:
h
P[qudit(F)]
i
jjkk = 1 if φ
uj
̸= φ (uk) and zero otherwise. Thus, in the
product P[qudit(F)]ρ (ψ) ⊗ρ (ψ), the nonzero diagonal elements are the pjpk where φ
uj
̸= φ (uk),
and so, the trace is ∑n
j.k=1
pjpk : φ
uj
̸= φ (uk)
, which by definition, is h (F : ψ). 8. Two Theorems about Quantum Logical Entropy Classically, a pair of elements
uj, uk
either “cohere” together in the same block of a partition
on U, i.e., are an indistinction of the partition, or they do not, i.e., they are a distinction of the
partition. In the quantum case, the nonzero off-diagonal entries αjα∗
k in the pure state density matrix
ρ (ψ) = |ψ⟩⟨ψ| are called quantum “coherences” ([27] p. 303; [15] p. 177) because they give the
amplitude of the eigenstates
uj
and |uk⟩“cohering” together in the coherent superposition state
vector |ψ⟩= ∑n
j=1
uj|ψ
uj
= ∑j αj
uj
. The coherences are classically modeled by the nonzero
off-diagonal entries √pjpk for the indistinctions
uj, uk
∈Bi × Bi, i.e., coherences ≈indistinctions. √
j
j
For an observable F, let φ : U →R be for F-eigenvalue function assigning the eigenvalue
φ (ui) = φi for each ui in the ON basis U = {u1, . . . , un} of F-eigenvectors. The range of φ is the
set of F-eigenvalues {φ1, . . . , φI}. Let Pφi : V →V be the projection matrix in the U-basis to the
eigenspace of φi. The projective F-measurement of the state ψ transforms the pure state density matrix
ρ (ψ) (represented in the ON basis U of F-eigenvectors) to yield the Lüders mixture density matrix
ρ′ (ψ) = ∑I
i=1 Pφiρ (ψ) Pφi ([15] p. 279). The off-diagonal elements of ρ (ψ) that are zeroed in ρ′ (ψ) are
the coherences (quantum indistinctions or quindits) that are turned into “decoherences” (quantum
distinctions or qudits of the observable being measured). For any observable F and a pure state ψ, a quantum logical entropy was defined as h (F : ψ) =
tr
h
P[qudit(F)]ρ (ψ) ⊗ρ (ψ)
i
. That definition was the quantum generalization of the “classical” logical
entropy defined as h (π) = p × p (dit (π)). When a projective F-measurement is performed on ψ,
the pure state density matrix ρ (ψ) is transformed into the mixed state density matrix by the quantum
Lüders mixture operation, which then defines the quantum logical entropy h (ρ′ (ψ)) = 1 −tr
h
ρ′ (ψ)2i
. The first test of how the quantum logical entropy notions fit together is showing that these two
entropies are the same: h (F : ψ) = h (ρ′ (ψ)). 7. Quantum Logical Information Theory: Commuting Observables The logical entropy is also the quadratic
special case of the Tsallis–Havrda–Charvat entropy ([24,25]) and the logical distance special case [19]
of C. R. Rao’s quadratic entropy [26]. What is new here is not the formula, but the whole back story of partition logic outlined above,
which gives the logical notion of entropy arising out of partition logic as the normalized counting
measure on ditsets of partitions; just as logical probability arises out of Boolean subset logic as the
normalized counting measure on subsets. The basic idea of information is differences, distinguishability
and distinctions ([3,19]), so the logical notion of entropy is the measure of the distinctions or dits of a
partition, and the corresponding quantum version is the measure of the qudits of an observable. Entropy 2018, 20, 679 10 of 22 10 of 22 8. Two Theorems about Quantum Logical Entropy h (ρ′ (ψ)) −h (ρ (ψ)) =
1 −tr
h
ρ′ (ψ)2i
−
1 −tr
h
ρ (ψ)2i
= ∑jk
ρjk (ψ)
2
−
ρ′
jk (ψ)
2
. If uj and uk are a qudit of F, then and only then are the corresponding off-diagonal terms zeroed by
the Lüders mixture operation ∑I
i=1 Pφiρ (ψ) Pφi to obtain ρ′ (ψ) from ρ (ψ). Proof. h (ρ′ (ψ)) −h (ρ (ψ)) =
1 −tr
h
ρ′ (ψ)2i
−
1 −tr
h
ρ (ψ)2i
= ∑jk
ρjk (ψ)
2
−
ρ′
jk (ψ)
2
. If uj and uk are a qudit of F, then and only then are the corresponding off-diagonal terms zeroed by
the Lüders mixture operation ∑I
i=1 Pφiρ (ψ) Pφi to obtain ρ′ (ψ) from ρ (ψ). Density matrices have long been a standard part of the machinery of quantum mechanics. The fundamental theorem for logical entropy and measurement shows there is a simple, direct and
quantitative connection between density matrices and logical entropy. The theorem directly connects
the changes in the density matrix due to a measurement (sum of absolute squares of zeroed
off-diagonal terms) with the increase in logical entropy due to the F-measurement h (F : ψ) =
tr
h
P[qudit(F)]ρ (ψ) ⊗ρ (ψ)
i
= h (ρ′ (ψ)) (where h (ρ (ψ)) = 0 for the pure state ψ). h
[q
( )]
i
This direct quantitative connection between state discrimination and quantum logical entropy
reinforces the judgment of Boaz Tamir and Eliahu Cohen ([28,29]) that quantum logical entropy is a
natural and informative entropy concept for quantum mechanics. We find this framework of partitions and distinction most suitable (at least conceptually)
for describing the problems of quantum state discrimination, quantum cryptography and
in general, for discussing quantum channel capacity. In these problems, we are basically
interested in a distance measure between such sets of states, and this is exactly the kind of
knowledge provided by logical entropy [Reference to [19]]. ([28] p. 1) Moreover, the quantum logical entropy has a simple “two-draw probability” interpretation,
i.e., h (F : ψ) = h (ρ′ (ψ)) is the probability that two independent F-measurements of ψ will yield
distinct F-eigenvalues, i.e., will yield a qudit of F. 8. Two Theorems about Quantum Logical Entropy Then, grouping the j-th diagonal elements for uj ∈Bi gives ∑uj∈Bi pjp (Bi) = p (
Hence, the whole trace is: tr
h
ρ′ (ψ)2i
= ∑I
i=1 p (Bi)2, and thus: ∑n
k=1
n
α∗
j αkαjα∗
k : φ
uj
= φ (uk)
o
= ∑n
k=1
pjpk : φ
uj
= φ (uk)
= pjp (Bi) ∑n
k=1
n
α∗
j αkαjα∗
k : φ
uj
= φ (uk)
o
= ∑n
k=1
pjpk : φ
uj
= φ (uk)
= pjp (Bi)
where uj ∈Bi. Then, grouping the j-th diagonal elements for uj ∈Bi gives ∑uj∈Bi pjp (Bi) = p (Bi)2. Hence, the whole trace is: tr
h
ρ′ (ψ)2i
= ∑I
i=1 p (Bi)2, and thus: uj ∈Bi. Then, grouping the j-th diagonal elements for uj ∈Bi gives ∑uj∈Bi pjp (Bi) = p (Bi)2. j
j
Hence, the whole trace is: tr
h
ρ′ (ψ)2i
= ∑I
i=1 p (Bi)2, and thus: Hence, the whole trace is: tr
h
ρ′ (ψ)2i
= ∑I
i=1 p (Bi)2, and thus: h (ρ′ (ψ)) = 1 −tr
h
ρ′ (ψ)2i
= 1 −∑I
i=1 p (Bi)2 = h (F : ψ). This completes the proof of the following theorem. Theorem 2. h (F : ψ) = h (π (F : ψ)) = h (ρ′ (ψ)). Measurement creates distinctions, i.e., turns coherences into “decoherences”, which, classically,
is the operation of distinguishing elements by classifying them according to some attribute like
classifying the faces of a die by their parity. The fundamental theorem about quantum logical entropy
and projective measurement, in the density matrix version, shows how the quantum logical entropy
created (starting with h (ρ (ψ)) = 0 for the pure state ψ) by the measurement can be computed directly
from the coherences of ρ (ψ) that are decohered in ρ′ (ψ). Theorem 3 (Fundamental (quantum)). The increase in quantum logical entropy, h (F : ψ) = h (ρ′ (ψ)),
due to the F-measurement of the pure state ψ, is the sum of the absolute squares of the nonzero off-diagonal
terms (coherences) in ρ (ψ) (represented in an ON basis of F-eigenvectors) that are zeroed (‘decohered’) in the
post-measurement Lüders mixture density matrix ρ′ (ψ) = ∑I
i=1 Pφiρ (ψ) Pφi. Proof. 8. Two Theorems about Quantum Logical Entropy Since ∑n
j=1 pj =
∑I
i=1 p (Bi) = 1,
∑I
i=1 p (Bi)
2
= 1 = ∑I
i=1 p (Bi)2 + ∑i̸=i′ p (Bi) p (Bi′). By grouping the pjpk in the
trace according to the blocks of π (F : ψ), we have: h (F : ψ) = tr
h
P[qudit(F)]ρ (ψ) ⊗ρ (ψ)
i
= ∑n
j.k=1
pjpk : φ
uj
̸= φ (uk)
= ∑i̸=i′ ∑
pjpk : uj ∈Bi, uk ∈Bi′
= ∑i̸=i′ p (Bi) p (Bi′)
= 1 −∑I
i=1 p (Bi)2 = h (π (F : ψ)).
pjpk
j
k
i
i̸
i p (
= 1 −∑I
i=1 p (Bi)2 = h (π (F : ψ)). To show that h (ρ′ (ψ)) = 1 −tr
h
ρ′ (ψ)2i
= h (π (F : ψ)) for ρ′ (ψ) = ∑I
i=1 Pφiρ (ψ) Pφi, we need
to compute tr
h
ρ′ (ψ)2i
. An off-diagonal element in ρjk (ψ) = αjα∗
k of ρ (ψ) survives (i.e., is not zeroed
and has the same value) the Lüders operation if and only if φ
uj
= φ (uk). Hence, the j-th diagonal
element of ρ′ (ψ)2 is: To show that h (ρ′ (ψ)) = 1 −tr
h
ρ′ (ψ)2i
= h (π (F : ψ)) for ρ′ (ψ) = ∑I
i=1 Pφiρ (ψ) Pφi, we need
to compute tr
h
ρ′ (ψ)2i
. An off-diagonal element in ρjk (ψ) = αjα∗
k of ρ (ψ) survives (i.e., is not zeroed
and has the same value) the Lüders operation if and only if φ
uj
= φ (uk). Hence, the j-th diagonal
element of ρ′ (ψ)2 is: Entropy 2018, 20, 679 11 of 22 ∑n
k=1
n
α∗
j αkαjα∗
k : φ
uj
= φ (uk)
o
= ∑n
k=1
pjpk : φ
uj
= φ (uk)
= pjp (Bi)
where uj ∈Bi. 9. Quantum Logical Entropies of Density Operators The, n the qudit subspaces are the subspaces of (V ⊗V)2 generated
by the qudit sets of generators: •
[qudit (1X)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′i
;
•
[qudit (1Y)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: j ̸= j′i
;
•
[qudit (1X, 1Y)] = [qudit (1X) ∪qudit (1Y)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′ or j ̸= j′i
;
•
[qudit (1X|1Y)] = [qudit (1X) −qudit (1Y)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′ and j = j′i
;
•
[qudit (1Y|1X)] = [qudit (1Y) −qudit (1X)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i = i′ and j ̸= j′i
; and
•
[qudit (1Y&1X)] = [qudit (1Y) ∩qudit (1X)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′ and j ̸= j′i
. •
[qudit (1X)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′i
;
•
[qudit (1Y)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: j ̸= j′i
;
•
[qudit (1X, 1Y)] = [qudit (1X) ∪qudit (1Y)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′ or j ̸= j′i
;
•
[qudit (1X|1Y)] = [qudit (1X) −qudit (1Y)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′ and j = j′i
;
•
[qudit (1Y|1X)] = [qudit (1Y) −qudit (1X)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i = i′ and j ̸= j′i
; and
•
[qudit (1Y&1X)] = [qudit (1Y) ∩qudit (1X)] =
h ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′ and j ̸= j′i
. Then, as qudit sets: qudit (1X, 1Y) = qudit (1X|1Y) ⊎qudit (1Y|1X) ⊎qudit (1Y&1X), and the
corresponding qudit subspaces stand in the same relation where the disjoint union is replaced by the
disjoint sum. Then, as qudit sets: qudit (1X, 1Y) = qudit (1X|1Y) ⊎qudit (1Y|1X) ⊎qudit (1Y&1X), and the
corresponding qudit subspaces stand in the same relation where the disjoint union is replaced by the
disjoint sum. The density operator ρ is represented by the diagonal density matrix ρX in its own ON basis X with
(ρX)ii = λi and similarly for the diagonal density matrix τY with (τY)jj = µj. 8. Two Theorems about Quantum Logical Entropy In contrast, the von Neumann entropy has no
such simple interpretation, and there seems to be no such intuitive connection between pre- and
post-measurement density matrices and von Neumann entropy, although von Neumann entropy also
increases in a projective measurement ([30] Theorem 11.9, p. 515). p
j
p
The development of the quantum logical concepts for two non-commuting operators
(see Appendix B) is the straightforward vector space version of the classical logical entropy treatment
of partitions on two set X and Y (see Appendix A). Entropy 2018, 20, 679 12 of 22 9. Quantum Logical Entropies of Density Operators The extension of the classical logical entropy h (p) = 1 −∑n
i=1 p2
i of a probability distribution
p = (p1, . . . , pn) to the quantum case is h (ρ) = 1 −tr
ρ2
where a density matrix ρ replaces the
probability distribution p and the trace replaces the summation. An arbitrary density operator ρ, representing a pure or mixed state on V, is also a self-adjoint
operator on V, so quantum logical entropies can be defined where density operators play the double
role of providing the measurement basis (as self-adjoint operators), as well as the state being measured. Let ρ and τ be two non-commuting density operators on V. Let X = {ui}i=1,...,n be an orthonormal
(ON) basis of ρ eigenvectors, and let {λi}i=1,...,n be the corresponding eigenvalues, which must be
non-negative and sum to one, so they can be interpreted as probabilities. Let Y =
vj
j=1,...,n be an
ON basis of eigenvectors for τ, and let
µj
j=1,...,n be the corresponding eigenvalues, which are also
non-negative and sum to one. Each density operator plays a double role. For instance, ρ acts as the observable to supply the
measurement basis of {ui}i and the eigenvalues {λi}i, as well as being the state to be measured
supplying the probabilities {λi}i for the measurement outcomes. In this section, we define quantum
logical entropies using the discrete partition 1X on the set of “index” states X = {ui}i and similarly for
the discrete partition 1Y on Y =
vj
j, the ON basis of eigenvectors for τ. j
The qudit sets of (V ⊗V) ⊗(V ⊗V) are then defined according to the identity and
difference on the index sets and independent of the eigenvalue-probabilities, e.g., qudit (1X) =
n ui ⊗vj
⊗
ui′ ⊗vj′
: i ̸= i′o
. 9. Quantum Logical Entropies of Density Operators The density operators
ρ, τ on V define a density operator ρ ⊗τ on V ⊗V with the ON basis of eigenvectors
ui ⊗vj
i,j and the
eigenvalue-probabilities of
λiµj
i,j. The operator ρ ⊗τ is represented in its ON basis by the diagonal
density matrix ρX ⊗τY with diagonal entries λiµj where 1 = (λ1 + · · · + λn) (µ1 + · · · + µn) =
∑n
i,j=1 λiµj. The probability measure p
ui ⊗vj
= λiµj on V ⊗V defines the product measure p × p
on (V ⊗V)2 where it can be applied to the qudit subspaces to define the quantum logical entropies
as usual. In the first instance, we have: h (1X : ρ ⊗τ) = p × p ([qudit (1X)]) = ∑
n
λiµjλi′µj′ : i ̸= i′o
= ∑i̸=i′ λiλi′ ∑j,j′ µjµj′ = ∑i̸=i′ λiλi′ = 1 −∑i λ2
i = 1 −tr
ρ2 = h (ρ) and similarly h (1Y : ρ ⊗τ) = h (τ). Since all the data are supplied by the two density operators, we
can use simplified notation to define the corresponding joint, conditional and mutual entropies: Entropy 2018, 20, 679
13 of 22 13 of 22 ntropy 2018, 20, 679
13 Entropy 2018, 20, 679 •
h (ρ, τ) = h (1X, 1Y : ρ ⊗τ) = p × p ([qudit (1X) ∪qudit (1Y)]);
•
h (ρ|τ) = h (1X|1Y : ρ ⊗τ) = p × p ([qudit (1X) −qudit (1Y)]);
•
h (τ|ρ) = h (1Y|1X : ρ ⊗τ) = p × p ([qudit (1Y) −qudit (1X)]); and
•
m (ρ, τ) = h (1Y&1X : ρ ⊗τ) = p × p ([qudit (1Y) ∩qudit (1X)]). Then, the usual Venn diagram relationships hold for the probability measure p × p on
(V ⊗V)2, e.g., Then, the usual Venn diagram relationships hold for the probability measure p × p on
(V ⊗V)2, e.g., h (ρ, τ) = h (ρ|τ) + h (τ|ρ) + m (ρ, τ), and probability interpretations are readily available. There are two probability distributions λ = {λi}i
and µ =
µj
j on the sample space {1, . . . , n}. Two pairs (i, j) and (i′, j′) are drawn with replacement;
the first entry in each pair is drawn according to λ and the second entry according to µ. 9. Quantum Logical Entropies of Density Operators Then, h (ρ, τ)
is the probability that i ̸= i′ or j ̸= j′ (or both); h (ρ|τ) is the probability that i ̸= i′ and j = j′, and so
forth. Note that this interpretation assumes no special significance to a λi and µi having the same
index since we are drawing a pair of pairs. and probability interpretations are readily available. There are two probability distributions λ = {λi}i
and µ =
µj
j on the sample space {1, . . . , n}. Two pairs (i, j) and (i′, j′) are drawn with replacement;
the first entry in each pair is drawn according to λ and the second entry according to µ. Then, h (ρ, τ)
is the probability that i ̸= i′ or j ̸= j′ (or both); h (ρ|τ) is the probability that i ̸= i′ and j = j′, and so
forth. Note that this interpretation assumes no special significance to a λi and µi having the same
index since we are drawing a pair of pairs. In the classical case of two probability distributions p = (p1, . . . , pn) and q = (q1, . . . , qn) on
the same index set, the logical cross-entropy is defined as: h (p||q) = 1 −∑i piqi and interpreted as
the probability of getting different indices in drawing a single pair, one from p and the other from q. However, this cross-entropy assumes some special significance to pi and qi having the same index. However, in our current quantum setting, there is no correlation between the two sets of “index”
states {ui}i=1,...,n and
vj
j=1,...,n. However, when the two density operators commute, τρ = ρτ,
then we can take {ui}i=1,...,n as an ON basis of simultaneous eigenvectors for the two operators with
respective eigenvalues λi and µi for ui with i = 1, . . . , n. In that special case, we can meaningfully
define the quantum logical cross-entropy as h (ρ||τ) = 1 −∑n
i=1 λiµi, but the general case awaits
further analysis below. 10. The Logical Hamming Distance between Two Partitions The development of logical quantum information theory in terms of some given commuting
or non-commuting observables gives an analysis of the distinguishability of quantum states using
those observables. Without any given observables, there is still a natural logical analysis of the
distance between quantum states that generalizes the “classical” logical distance h (π|σ) + h (σ|π)
between partitions on a set. In the classical case, we have the logical entropy h (π) of a partition
where the partition plays the role of the direct-sum decomposition of eigenspaces of an observable in
the quantum case. However, we also have just the logical entropy h (p) of a probability distribution
p = (p1, . . . , pn) and the related compound notions of logical entropy given another probability
distribution q = (q1, . . . , qn) indexed by the same set. First, we review that classical treatment to motivate the quantum version of the logical Hamming
distance between states. A binary relation R ⊆U × U on U = {u1, . . . , un} can be represented by an
n × n incidence matrix I(R) where: I (R)ij =
(
1 if
ui, uj
∈R
0 if
ui, uj
/∈R. Taking R as the equivalence relation indit (π) associated with a partition π = {B1, . . . , BI},
the density matrix ρ (π) of the partition π (with equiprobable points) is just the incidence matrix
I (indit (π)) rescaled to be of trace one (i.e., the sum of diagonal entries is one): Taking R as the equivalence relation indit (π) associated with a partition π = {B1, . . . , BI},
the density matrix ρ (π) of the partition π (with equiprobable points) is just the incidence matrix
I (indit (π)) rescaled to be of trace one (i.e., the sum of diagonal entries is one): ρ (π) =
1
|U| I (indit (π)). ρ (π) =
1
|U| I (indit (π)). From coding theory ([31] p. 66), we have the notion of the Hamming distance between two
0, 1 vectors or matrices (of the same dimensions), which is the number of places where they differ. Since logical information theory is about distinctions and differences, it is important to have a 14 of 22 Entropy 2018, 20, 679 classical and quantum logical notion of Hamming distance. 10. The Logical Hamming Distance between Two Partitions The powerset ℘(U × U) can be viewed
as a vector space over Z2 where the sum of two binary relations R, R′ ⊆U × U is the symmetric
difference, symbolized as R∆R′ = (R −R′) ∪(R′ −R) = R ∪R′ −R ∩R′, which is the set of
elements (i.e., ordered pairs
ui, uj
∈U × U) that are in one set or the other, but not both. Thus,
the Hamming distance DH (I (R) , I (R′)) between the incidence matrices of two binary relations is
just the cardinality of their symmetric difference: DH (I (R) , I (R′)) = |R∆R′|. Moreover, the size of
the symmetric difference does not change if the binary relations are replaced by their complements:
|R∆R′| =
U2 −R
∆
U2 −R′ .
Hence, given two partitions π = {B1, . . . , BI} and σ =
C1, . . . , CJ
on U, the unnormalized
Hamming distance between the two partitions is naturally defined as (this is investigated in Rossi [32]) D (π, σ) = DH (I (indit (π)) , I (indit (σ))) = |indit (π) ∆indit (σ)| = |dit (π) ∆dit (σ)|,
and the Hamming distance between π and σ is defined as the normalized D (π, σ): D (π, σ) = DH (I (indit (π)) , I (indit (σ))) = |indit (π) ∆indit (σ)| = |dit (π) ∆dit (σ)|, D (π, σ) = DH (I (indit (π)) , I (indit (σ))) = |indit (π) ∆indit (σ)| = |dit (π) ∆dit (σ)|,
and the Hamming distance between π and σ is defined as the normalized D (π, σ): (
)
( (
( ))
(
( )))
|
( )
( )|
|
(
and the Hamming distance between π and σ is defined as the normalized D (π, σ): the Hamming distance between π and σ is defined as the normalized D (π, σ): d (π, σ) = D(π,σ)
|U×U| = |dit(π)∆dit(σ)|
|U×U|
= |dit(π)−dit(σ)|
|U×U|
+ |dit(σ)−dit(π)|
|U×U|
= h (π|σ) + h (σ|π). This motivates the general case of point probabilities p = (p1, . . . , pn) where we define the
Hamming distance between the two partitions as the sum of the two logical conditional entropies: d (π, σ) = h (π|σ) + h (σ|π) = 2h(π ∨σ) −h (π) −h (σ). 10. The Logical Hamming Distance between Two Partitions To motivate the bridge to the quantum version of the Hamming distance, we need to calculate it
using the density matrices ρ (π) and ρ (σ) of the two partitions. To compute the trace tr [ρ (π) ρ (σ)],
we compute the diagonal elements in the product ρ (π) ρ (σ) and add them up: [ρ (π) ρ (σ)]kk =
∑l ρ (π)kl ρ (σ)lk = ∑l
√pkpl
√plpk where the only nonzero terms are where uk, ul ∈B ∩C for some
B ∈π and C ∈σ. Thus, if uk ∈B ∩C, then [ρ (π) ρ (σ)]kk = ∑ul∈B∩C pkpl. Therefore, the diagonal
element for uk is the sum of the pkpl for ul in the same intersection B ∩C as uk, so it is pk Pr (B ∩C). Then, when we sum over the diagonal elements, then for all the uk ∈B ∩C for any given B, C,
we just sum ∑uk∈B∩C pk Pr (B ∩C) = Pr (B ∩C)2 so that tr [ρ (π) ρ (σ)] = ∑B∈π,C∈σ Pr (B ∩C)2 =
1 −h (π ∨σ). Hence, if we define the logical cross-entropy of π and σ as: h(π||σ) = 1 −tr [ρ (π) ρ (σ)], then for partitions on U with the point probabilities p = (p1, . . . , pn), the logical cross-entropy h (π||σ)
of two partitions is the same as the logical joint entropy, which is also the logical entropy of the join: then for partitions on U with the point probabilities p = (p1, . . . , pn), the logical cross-entropy h (π||σ)
of two partitions is the same as the logical joint entropy, which is also the logical entropy of the join: h (π||σ) = h (π, σ) = h (π ∨σ). Thus, we can also express the logical Hamming distance between two partitions entirely in terms
of density matrices: d (π, σ) = 2h (π||σ) −h (π) −h (σ) = tr
h
ρ (π)2i
+ tr
h
ρ (σ)2i
−2 tr [ρ (π) ρ (σ)]. 11. The Quantum Logical Hamming Distance The quantum logical entropy h (ρ) = 1 −tr
ρ2
of a density matrix ρ generalizes the classical
h (p) = 1 −∑i p2
i for a probability distribution p = (p1, . . . , pn). As a self-adjoint operator, a density
matrix has a spectral decomposition ρ = ∑n
i=1 λi |ui⟩⟨ui| where {|ui⟩}i=1,...,n is an orthonormal basis
for V and where all the eigenvalues λi are real, non-negative and ∑n
i=1 λi = 1. Then, h (ρ) = 1 −∑i λ2
i
so h (ρ) can be interpreted as the probability of getting distinct indices i ̸= i′ in two independent
measurements of the state ρ with {|ui⟩} as the measurement basis. Classically, it is the two-draw
probability of getting distinct indices in two independent samples of the probability distribution
λ = (λ1, . . . , λn), just as h (p) is the probability of getting distinct indices in two independent draws Entropy 2018, 20, 679
15 of 22 Entropy 2018, 20, 679 15 of 22 on p. For a pure state ρ, there is one λi = 1 with the others zero, and h (ρ) = 0 is the probability of
getting distinct indices in two independent draws on λ = (0, . . . , 0, 1, 0, . . . , 0). In the classical case of the logical entropies, we worked with the ditsets or sets of distinctions
of partitions. However, everything could also be expressed in terms of the complementary sets of
indits or indistinctions of partitions (ordered pairs of elements in the same block of the partition) since:
dit (π) ⊎indit (π) = U × U. When we switch to the density matrix treatment of “classical” partitions,
then the focus shifts to the indistinctions. For a partition π = {B1, . . . , BI}, the logical entropy is the
sum of the distinction probabilities: h (π) = ∑(uk,ul)∈dit(π) pkpl, which in terms of indistinctions is: h (π) = 1 −∑(uk,ul)∈indit(π) pkpl = 1 −∑I
i=1 Pr (Bi)2. When expressed in the density matrix formulation, then tr
h
ρ (π)2i
is the sum of the
indistinction probabilities: When expressed in the density matrix formulation, then tr
h
ρ (π)2i
is the sum of the
indistinction probabilities: tr
h
ρ (π)2i
= ∑(uk,ul)∈indit(π) pkpl = ∑I
i=1 Pr (Bi)2. 11. The Quantum Logical Hamming Distance The nonzero entries in ρ (π) have the form √pkpl for (uk, ul) ∈indit (π); their squares are the
indistinction probabilities. That provides the interpretive bridge to the quantum case. 2 The quantum analogue of an indistinction probability is the absolute square |ρkl|2 of a nonzero
entry ρkl in a density matrix ρ, and tr
ρ2 = ∑k,l |ρkl|2 is the sum of those “indistinction” probabilities. The nonzero entries in the density matrix ρ might be called “coherences” so that ρkl may be interpreted
as the amplitudes for the states uk and ul to cohere together in the state ρ, so tr
ρ2
is the sum
of the coherence probabilities, just as tr
h
ρ (π)2i
= ∑(uk,ul)∈indit(π) pkpl is the sum of the indistinction
probabilities. The quantum logical entropy h (ρ) = 1 −tr
ρ2
may then be interpreted as the sum
of the decoherence probabilities, just as h (ρ (π)) = h (π) = 1 −∑(uk,ul)∈indit(π) pkpl is the sum of the
distinction probabilities. This motivates the general quantum definition of the joint entropy h (π, σ) = h (π ∨σ) = h (π||σ),
which is the: h (ρ||τ) = 1 −tr
ρ†τ
quantum logical cross-entropy. h (ρ||τ) = 1 −tr
ρ†τ
quantum logical cross-entropy. To work out its interpretation, we again take ON eigenvector bases {|ui⟩}n
i=1 for ρ and
vj
n
j=1
for τ with λi and µj as the respective eigenvalues and compute the operation of τ†ρ : V →V. Now, |ui⟩= ∑j
vj|ui
vj
so ρ |ui⟩= λi |ui⟩= ∑j λi
vj|ui
vj
, and then, for τ† = ∑j
vj
vj
µj,
so τ†ρ |ui⟩= ∑j λiµj
vj|ui
vj
. Thus, τ†ρ in the {ui}i basis would have the diagonal entries
ui|τ†ρ|ui
= ∑j λiµj
vj|ui
ui|vj
, so the trace is: tr
τ†ρ
= ∑i
ui|τ†ρ|ui
= ∑i,j λiµj
vj|ui
ui|vj
= tr
ρ†τ
which is symmetrical. The other information we have is the ∑i λi = 1 = ∑j µj, and they are
non-negative. The classical logical cross-entropy of two probability distributions is h (p||q) =
1 −∑i,j piqjδij, where two indices i and j are either identical or totally distinct. However, in the
quantum case, the “index” states |ui⟩and
vj
have an “overlap” measured by the inner product
ui|vj
. The trace tr
ρ†τ
is real since
vj|ui
=
ui|vj
∗and
vj|ui
ui|vj
=
ui|vj
2 =
vj|ui
2
is the probability of getting λi when measuring vj in the ui basis and the probability of getting µj when
measuring ui in the vj basis. The twofold nature of density matrices as states and as observables then
allows tr
ρ†τ
to be interpreted as the average value of the observable ρ when measuring the state τ
or vice versa. We may call
vj|ui
ui|vj
the proportion or extent of overlap for those two index states. Thus, tr
ρ†τ
is the sum of all the probability combinations λiµj weighted by the overlaps
vj|ui
ui|vj
for the index states |ui⟩and
vj
. The quantum logical cross-entropy can be written in a
number of ways: Entropy 2018, 20, 679 Entropy 2018, 20, 679 16 of 22 16 of 22 h (ρ||τ) = 1 −tr
ρ†τ
= 1 −∑i,j λiµj
vj|ui
ui|vj
= tr
τ† (I −ρ)
= ∑i,j (1 −λi) µj
vj|ui
ui|vj
= tr
ρ† (I −τ)
= ∑i,j λi
1 −µj
vj|ui
ui|vj
. Classically, the “index state” {i} completely overlaps with {j} when i = j and has no overlap
with any other {i′} from the indices {1, . Theorem 4. Hilbert–Schmidt norm = quantum logical Hamming distance. h (ρ||τ) = 1 −tr
ρ†τ
quantum logical cross-entropy. . . , n}, so the “overlaps” are, as it were, ⟨j|i⟩⟨i|j⟩= δij,
the Kronecker delta. Hence, the classical analogue formulas are: Classically, the “index state” {i} completely overlaps with {j} when i = j and has no overlap
with any other {i′} from the indices {1, . . . , n}, so the “overlaps” are, as it were, ⟨j|i⟩⟨i|j⟩= δij,
the Kronecker delta. Hence, the classical analogue formulas are: h (p||q) = 1 −∑i,j piqjδij = ∑i,j (1 −pi) qjδij = ∑i,j pi
1 −qj
δij. The quantum logical cross-entropy h (ρ||τ) can be interpreted by considering two measurements,
one of ρ with the {|ui⟩}i measurement basis and the other of τ with the
vj
j measurement
basis. If the outcome of the ρ measurement was ui with probability λi, then the outcome of the
τ measurement is different than vj with probability 1 −µj; however, that distinction probability
λi
1 −µj
is only relevant to the extent that ui and vj are the “same state” or overlap, and that extent
is
vj|ui
ui|vj
. Hence, the quantum logical cross-entropy is the sum of those two-measurement
distinction probabilities weighted by the extent that the states overlap. The interpretation of h (ρ) and
h (τ||ρ), as well as the later development of the quantum logical conditional entropy h (ρ|τ) and the
quantum Hamming distance d (ρ, τ) are all based on using the eigenvectors and eigenvalues of density
matrices, which Michael Nielsen and Issac Chuang seem to prematurely dismiss as having little or no
“special significance” ([30] p. 103). When the two density matrices commute, ρτ = τρ, then (as noted above) we have the essentially
classical situation of one set of index states {|ui⟩}i which is an orthonormal basis set of simultaneous
eigenvectors for both ρ and τ with the respective eigenvalues {λi}i and
µj
j. Then,
uj|ui
ui|uj
=
δij, so h (ρ||τ) = ∑i,j λi
1 −µj
δij is the probability of getting two distinct index states ui and uj for
i ̸= j in two independent measurements, one of ρ and one of τ in the same measurement basis of
{|ui⟩}i. This interpretation includes the special case when τ = ρ and h (ρ||ρ) = h (ρ). h (ρ||τ) = 1 −tr
ρ†τ
quantum logical cross-entropy. {| i⟩}i
p
p
ρ
(ρ||ρ)
(ρ)
We saw that classically, the logical Hamming distance between two partitions could be p
p
ρ
(ρ||ρ)
(ρ)
hat classically, the logical Hamming distance between two partitions could be defined as: d (π, σ) = 2h (π||σ) −h (π) −h (σ) = tr
h
ρ (π)2i
+ tr
h
ρ (σ)2i
−2 tr [ρ (π) ρ (σ)] so this motivates the quantum definition. Nielsen and Chuang suggest the idea of a Hamming distance
between quantum states, only to then dismiss it. “Unfortunately, the Hamming distance between two
objects is simply a matter of labeling, and a priori there are not any labels in the Hilbert space arena of
quantum mechanics!” ([30] p. 399). They are right that there is no correlation, say, between the vectors
in the two ON bases {ui}i and
vj
j for V, but the cross-entropy h (ρ||τ) uses all possible combinations
in the terms λi
1 −µj
vj|ui
ui|vj
; thus, the definition of the Hamming distance given below does
not use any arbitrary labeling or correlations. d (ρ, τ) = 2h (ρ||τ) −h (ρ) −h (τ) = tr
ρ2 + tr
τ2 −2 tr
ρ†τ
This is the definition of the quantum logical Hamming distance between two quantum states. This is the definition of the quantum logical Hamming distance between two quantum st There is another distance measure between quantum states, namely the Hilbert–Schmidt norm,
which has been recently investigated in [29] (with an added 1
2 factor). It is the square of the Euclidean
distance between the quantum states, and ignoring the 1
2 factor, it is the square of the “trace distance”
([30] Chapter 9) between the states. tr
h
(ρ −τ)2i
Hilbert–Schmidt norm, where we write A2 for A†A. Then, the naturalness of this norm as a “distance” is enhanced by the fact
that it is the same as the quantum Hamming distance: where we write A2 for A†A. Then, the naturalness of this norm as a “distance” is enhanced by the fact
that it is the same as the quantum Hamming distance: Theorem 4. Hilbert–Schmidt norm = quantum logical Hamming distance. Theorem 4. Hilbert–Schmidt norm = quantum logical Hamming distance. 17 of 22 Entropy 2018, 20, 679 Proof. Theorem 5 (Fundamental (quantum)). tr
h
(ρ −ˆρ)2i
= h ( ˆρ) −h (ρ). Proof. tr
h
(ρ −ˆρ)2i
=
tr[
ρ† −ˆρ† (ρ −ˆρ)]
=
tr
ρ2 + tr
ˆρ2 −tr [ρˆρ] −tr [ˆρρ] where ρ†
=
ρ,
ˆρ†
=
ˆρ, tr [ρˆρ]
=
tr [ˆρρ] and tr [ρˆρ]
=
tr
h
ρ ∑I
i=1 PφiρPφi
i
=
tr
∑ρPφiρPφi
. Also tr
ˆρ2
=
tr
h ∑i PφiρPφi
∑j PφjρPφj
i
= tr
h
∑i PφiρP2
φiρPφi
i
using the orthogonality of the distinct projection
operators. Then, using the idempotency of the projections and the cyclicity of the trace, tr
ˆρ2 =
tr
∑i ρPφiρPφi
so tr [ρˆρ] = tr [ˆρρ] = tr
ˆρ2
, and hence, tr
h
(ρ −ˆρ)2i
= tr
ρ2−tr
ˆρ2 = h (ˆρ)−h (ρ). Proof. tr
h
(ρ −ˆρ)2i
=
tr[
ρ† −ˆρ† (ρ −ˆρ)]
=
tr
ρ2 + tr
ˆρ2 −tr [ρˆρ] −tr [ˆρρ] where ρ†
=
ρ,
ˆρ†
=
ˆρ, tr [ρˆρ]
=
tr [ˆρρ] and tr [ρˆρ]
=
tr
h
ρ ∑I
i=1 PφiρPφi
i
=
tr
∑ρPφiρPφi
. Also tr
ˆρ2
=
tr
h ∑i PφiρPφi
∑j PφjρPφj
i
= tr
h
∑i PφiρP2
φiρPφi
i
using the orthogonality of the distinct projection
operators. Then, using the idempotency of the projections and the cyclicity of the trace, tr
ˆρ2 =
tr
∑i ρPφiρPφi
so tr [ρˆρ] = tr [ˆρρ] = tr
ˆρ2
, and hence, tr
h
(ρ −ˆρ)2i
= tr
ρ2−tr
ˆρ2 = h (ˆρ)−h (ρ). 12. Results Logical information theory arises as the quantitative version of the logic of partitions just as
logical probability theory arises as the quantitative version of the dual Boolean logic of subsets. Philosophically, logical information is based on the idea of information-as-distinctions. The Shannon
definitions of entropy arise naturally out of the logical definitions by replacing the counting of
distinctions by the counting of the minimum number of binary partitions (bits) that are required,
on average, to make all the same distinctions, i.e., to encode the distinguished elements uniquely,
which is why the Shannon theory is so well adapted for the theory of coding and communication. y
y
p
y
g
This “classical” logical information theory may be developed with the data of two partitions on a
set with point probabilities. Section 7 gives the generalization to the quantum case where the partitions
are provided by two commuting observables (the point set is an ON basis of simultaneous eigenvectors),
and the point probabilities are provided by the state to be measured. In Section 8, the fundamental
theorem for quantum logical entropy and measurement established a direct quantitative connection
between the increase in quantum logical entropy due to a projective measurement and the eigenstates
(cohered together in the pure superposition state being measured) that are distinguished by the
measurement (decohered in the post-measurement mixed state). This theorem establishes quantum
logical entropy as a natural notion for a quantum information theory focusing on distinguishing states. The classical theory might also start with partitions on two different sets and a probability
distribution on the product of the sets (see Appendix A). Appendix B gives the quantum generalization
of that case with the two sets being two ON bases for two non-commuting observables, and the
probabilities are provided by a state to be measured. The classical theory may also be developed just
using two probability distributions indexed by the same set, and this is generalized to the quantum case
where we are just given two density matrices representing two states in a Hilbert space. Sections 10
and 11 carry over the Hamming distance measure from the classical to the quantum case where it is
equal to the Hilbert–Schmidt distance (square of the trace distance). h (ρ||τ) = 1 −tr
ρ†τ
quantum logical cross-entropy. tr
h
(ρ −τ)2i
= tr
ρ2 + tr
τ2 −2 tr
ρ†τ
= 2h (ρ||τ) −h (ρ) −h (τ) = d (ρ, τ) . Hence, the information inequality holds trivially for the quantum logical Hamming distance: d (ρ, τ) ≥0 with equality iff ρ = τ. d (ρ, τ) ≥0 with equality iff ρ = τ. The fundamental theorem can be usefully restated in this broader context of density operators
instead of in terms of the density matrix represented in the ON basis for F-eigenvectors. Let ρ be
any state to be measured by an observable F, and let ˆρ = ∑I
i=1 PφiρPφi be the result of applying the
Lüders mixture operation (where the Pφi are the projection operators to the eigenspaces of F). Then,
a natural question to ask is the Hilbert–Schmidt norm or quantum logical Hamming distance between
the pre- and post-measurement states. It might be noted that the Hilbert–Schmidt norm and the Lüders
mixture operation are defined independently of any considerations of logical entropy. Theorem 5 (Fundamental (quantum)). tr
h
(ρ −ˆρ)2i
= h ( ˆρ) −h (ρ). Theorem 5 (Fundamental (quantum)). tr
h
(ρ −ˆρ)2i
= h ( ˆρ) −h (ρ). 13. Discussion The overall argument is that quantum logical entropy is the simple and natural notion of
information-as-distinctions for quantum information theory focusing on the distinguishing of quantum
states. These results add to the arguments already presented by Manfredi and Feix [21] and many
others (see [23]) for this notion of quantum entropy. There are two related classical theories of information, classical logical information theory
(focusing on information-as-distinctions and analyzing classification) and the Shannon theory (focusing
on coding and communications theory). Generalizing to the quantum case, there are also two related
quantum theories of information, the logical theory (using quantum logical entropy to focus on
distinguishing quantum states and analyzing measurement as the quantum version of classification)
and the conventional quantum information theory (using von Neumann entropy to develop a quantum
version of the Shannon treatment of coding and communications). Funding: This research received no external funding. Funding: This research received no external funding. Conflicts of Interest: The author declares no conflicts of interest. Conflicts of Interest: The author declares no conflicts of interest. 12. Results The general fundamental theorem
relating measurement and logical entropy is that the Hilbert–Schmidt distance (=quantum logical
Hamming distance) between any pre-measurement state ρ and the state ˆρ resulting from a projective
measurement of the state is the difference in their logical entropies, h ( ˆρ) −h (ρ). This “classical” logical information theory may be developed with the data of two partitions on a
set with point probabilities. Section 7 gives the generalization to the quantum case where the partitions
are provided by two commuting observables (the point set is an ON basis of simultaneous eigenvectors),
and the point probabilities are provided by the state to be measured. In Section 8, the fundamental
theorem for quantum logical entropy and measurement established a direct quantitative connection
between the increase in quantum logical entropy due to a projective measurement and the eigenstates
(cohered together in the pure superposition state being measured) that are distinguished by the
measurement (decohered in the post-measurement mixed state). This theorem establishes quantum
logical entropy as a natural notion for a quantum information theory focusing on distinguishing states. The classical theory might also start with partitions on two different sets and a probability
distribution on the product of the sets (see Appendix A). Appendix B gives the quantum generalization
of that case with the two sets being two ON bases for two non-commuting observables, and the
probabilities are provided by a state to be measured. The classical theory may also be developed just
using two probability distributions indexed by the same set, and this is generalized to the quantum case
where we are just given two density matrices representing two states in a Hilbert space. Sections 10
and 11 carry over the Hamming distance measure from the classical to the quantum case where it is
equal to the Hilbert–Schmidt distance (square of the trace distance). The general fundamental theorem
relating measurement and logical entropy is that the Hilbert–Schmidt distance (=quantum logical
Hamming distance) between any pre-measurement state ρ and the state ˆρ resulting from a projective
measurement of the state is the difference in their logical entropies, h ( ˆρ) −h (ρ). Entropy 2018, 20, 679 18 of 22 18 of 22 Appendix A. Classical Logical Information Theory with Two Sets X and Y The usual (“classical”) logical information theory for a probability distribution {p (x, y)} on X × Y
(finite) in effect uses the discrete partition on X and Y [3]. For the general case of quantum logical
entropy for not-necessarily commuting observables, we need to first briefly develop the classical case
with general partitions on X and Y. Given two finite sets X and Y and real-valued functions f : X →R with values {φi}I
i=1 and
g : Y →R with values
γj
J
j=1, each function induces a partition on its domain: π =
f −1 (φi)
i∈I = {B1, . . . , BI} on X, and σ =
g−1 γj
j∈J =
C1, . . . , CJ
on Y. We need to define logical entropies on X × Y, but first, we need to define the ditsets or
information sets. A partition π = {B1, . . . , BI} on X and a partition σ =
C1, . . . , CJ
on Y define a product partition
π × σ on X × Y whose blocks are
Bi × Cj
i,j. Then, π induces π × 0Y on X × Y (where 0Y is the
indiscrete partition on Y), and σ induces 0X × σ on X × Y. The corresponding ditsets or information
sets are: •
dit (π × 0Y) = {((x, y) , (x′, y′)) : f (x) ̸= f (x′)} ⊆(X × Y)2;
2 •
dit (π × 0Y) = {((x, y) , (x′, y′)) : f (x) ̸= f (x′)} ⊆(X × Y)2;
(
)
{((
) ( ′
′))
( ) ̸
( ′)}
(
)2 •
dit (π × 0Y) = {((x, y) , (x′, y′)) : f (x) ̸= f (x′)} ⊆(X × Y)2;
•
dit (0X × σ) = {((x, y) , (x′, y′)) : g (y) ̸= g (y′)} ⊆(X × Y)2; dit (π × 0Y)
{((x, y) , (x , y )) : f (x) ̸
f (x )} ⊆(X × Y) ;
•
dit (0X × σ) = {((x, y) , (x′, y′)) : g (y) ̸= g (y′)} ⊆(X × Y)2;
di (
)
di (
)
di (
)
d
f
h •
dit (π × σ) = dit (π × 0Y) ∪dit (0X × σ); and so forth. Appendix A. Classical Logical Information Theory with Two Sets X and Y •
dit (π × σ) = dit (π × 0Y) ∪dit (0X × σ); and so forth. Given a joint probability distribution p : X × Y →[0, 1], the product probability distribution is
p × p : (X × Y)2 →[0, 1]. All the logical entropies are just the product probabilities of the ditsets and their union, differences
and intersection: •
h (π × 0Y) = p × p (dit (π × 0Y));
•
h (0X × σ) = p × p (dit (0X × σ));
•
h (π × σ) = p × p (dit (π × σ)) = p × p (dit (π × 0Y) ∪dit (0X × σ));
•
h (π × 0Y|0X × σ) = p × p (dit (π × 0Y) −dit (0X × σ));
•
h (0X × σ|π × 0Y) = p × p (dit (0X × σ) −dit (π × 0Y));
•
m (π × 0Y, 0X × σ) = p × p (dit (π × 0Y) ∩dit (0X × σ)). •
h (π × σ) = p × p (dit (π × σ)) = p × p (dit (π × 0Y) ∪dit (0X × σ
•
h (π × 0Y|0X × σ) = p × p (dit (π × 0Y) −dit (0X × σ));
•
h (0X × σ|π × 0Y) = p × p (dit (0X × σ) −dit (π × 0Y));
•
m (π × 0Y, 0X × σ) = p × p (dit (π × 0Y) ∩dit (0X × σ)). All the logical entropies have the usual two-draw probability interpretation where the two
independent draws from X × Y are (x, y) and (x′, y′) and can be interpreted in terms of the f-values
and g-values: 19 of 22 Entropy 2018, 20, 679 •
h (π × 0Y) = probability of getting distinct f-values;
•
h (0X × σ) = probability of getting distinct g-values;
•
h (π × σ) = probability of getting distinct f- or g-values;
•
h (π × 0Y|0X × σ) = probability of getting distinct f-values, but the same g-values;
•
h (0X × σ|π × 0Y) = probability of getting distinct g-values, but the same f-values;
•
m (π × 0Y, 0X × σ) = probability of getting distinct f- and distinct g-values. We have defined all the logical entropies by the general method of the product probabilities on
the ditsets. Appendix A. Classical Logical Information Theory with Two Sets X and Y In the first three cases, h (π × 0Y), h (0X × σ) and h (π × σ), they were the logical entropies
of partitions on X × Y, so they could equivalently be defined using density matrices. The case of
h (π × σ) illustrates the general case. If ρ (π) is the density matrix defined for π on X and ρ (σ) the
density matrix for σ on Y, then ρ (π × σ) = ρ (π) ⊗ρ (σ) is the density matrix for π × σ defined on
X × Y, and: h (π × σ) = 1 −tr
h
ρ (π × σ)2i
. The marginal distributions are: pX (x) = ∑y p (x, y) and pY (y) = ∑x p (x, y). Since π is a partition
on X, there is also the usual logical entropy h (π) = pX × pX (dit (π)) = 1 −tr
h
ρ (π)2i
= h (π × 0Y)
where dit (π) ⊆X × X and similarly for pY. The marginal distributions are: pX (x) = ∑y p (x, y) and pY (y) = ∑x p (x, y). Since π is a partition
on X, there is also the usual logical entropy h (π) = pX × pX (dit (π)) = 1 −tr
h
ρ (π)2i
= h (π × 0Y)
where dit (π) ⊆X × X and similarly for pY. Since the context should be clear, we may henceforth adopt the old notation from the case where
π and σ were partitions on the same set U, i.e., h (π) = h (π × 0Y), h (σ) = h (0X × σ), h (π, σ) =
h (π × σ), etc. (
)
Since the logical entropies are the values of a probability measure, all the usual identities hold
where the underlying set is now (X × Y)2 instead of U2, as illustrated in Figure A1. Figure A1. Venn diagram for logical entropies as values of a probability measure p × p on (X × Y)2. Figure A1. Venn diagram for logical entropies as values of a probability measure p × p on (X × Y)2. The previous treatment of h (X), h (Y), h (X, Y), h (X|Y), h (Y|X) and m (X, Y) in [3] was just the
special cases where π = 1X and σ = 1Y. Appendix B. Quantum Logical Entropies with Non-Commuting Observables Tt is again an easy implication of the
aforementioned common dits theorem that any two nonzero information spaces [qudit (π)] and
[qudit (σ)] have a nonzero intersection, so the mutual information space [qudit (π) ∩qudit (σ)] is
not the zero space. A normalized state |ψ⟩on V ⊗V defines a pure state density matrix ρ (ψ) = |ψ⟩⟨ψ|. Let αx,y =
⟨x ⊗y|ψ⟩, so if P[x⊗y] is the projection to the subspace (ray) generated by x ⊗y in V ⊗V, then a
probability distribution on X × Y is defined by: p (x, y) = αx,yα∗
x,y = tr
h
P[x⊗y]ρ (ψ)
i
, p (x, y) = αx,yα∗
x,y = tr
h
P[x⊗y]ρ (ψ)
i
, p (x, y) = αx,yα∗
x,y = tr
h
P[x⊗y]ρ (ψ)
i
, or more generally, for a subspace T ⊆V ⊗V, a probability distribution is defined on the subspaces by:
Pr (T) = tr [PTρ (ψ)]. Then, the product probability distribution p × p on the subspaces of (V ⊗V)2 defines the quantum
logical entropies when applied to the information subspaces: •
h (F : ψ) = p × p ([qudit (π)]) = tr
h
P[qudit(π)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
h (G : ψ) = p × p ([qudit (σ)]) = tr
h
P[qudit(σ)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
h (F, G : ψ) = p × p ([qudit (π) ∪qudit (σ)]) = tr
h
P[qudit(π)∪qudit(σ)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
h (F|G : ψ) = p × p ([qudit (π) −qudit (σ)]) = tr
h
P[qudit(π)−qudit(σ)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
h (G|F : ψ) = p × p ([qudit (σ) −qudit (π)]) = tr
h
P[qudit(σ)−qudit(π)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
m (F, G : ψ) = p × p ([qudit (π) ∩qudit (σ)]) = tr
h
P[qudit(π)∩qudit(σ)] (ρ (ψ) ⊗ρ (ψ))
i
. Appendix B. Quantum Logical Entropies with Non-Commuting Observables As before in the case of commuting observables, the quantum case can be developed in close
analogy with the previous classical case. Given a finite-dimensional Hilbert space V and not necessarily
commuting observables F, G : V →V, let X be an orthonormal basis of V of F-eigenvectors, and let Y
be an orthonormal basis for V of G-eigenvectors (so |X| = |Y|). I Let f : X →R be the eigenvalue function for F with values {φi}I
i=1, and let g : Y →R be the
eigenvalue function for G with values
γj
J
j=1. j
1
Each eigenvalue function induces a partition on its domain: π =
f −1 (φi)
= {B1, . . . , BI} on X, and σ =
g−1 γj
=
C1, . . . , CJ
on Y. We associated with the ordered pair (x, y), the basis element x ⊗y in the basis {x ⊗y}x∈X,y∈Y for
V ⊗V. Then, each pair of pairs ((x, y) , (x′, y′)) is associated with the basis element (x ⊗y) ⊗(x′ ⊗y′)
in (V ⊗V) ⊗(V ⊗V) = (V ⊗V)2. 20 of 22 Entropy 2018, 20, 679 Instead of ditsets or information sets, we now have qudit subspaces or information subspaces. For R ⊆(V ⊗V)2, let [R] be the subspace generated by R. We simplify the notation of qudit (π × 0Y) =
qudit (π) = {(x ⊗y) ⊗(x′ ⊗y′) : f (x) ̸= f (x′)}, etc. •
[qudit (π)] = [{(x ⊗y) ⊗(x′ ⊗y′) : f (x) ̸= f (x′)}]; •
[qudit (π)] = [{(x ⊗y) ⊗(x′ ⊗y′) : f (x) ̸= f (x′)}]; •
[qudit (σ)] = [{(x ⊗y) ⊗(x′ ⊗y′) : g (y) ̸= g (y′)}]; •
[qudit (σ)] = [{(x ⊗y) ⊗(x′ ⊗y′) : g (y) ̸= g (y′)}]; •
[qudit (π, σ)] = [qudit (π) ∪qudit (σ)]; and so forth. Tt is again an easy implication of the
aforementioned common dits theorem that any two nonzero information spaces [qudit (π)] and
[qudit (σ)] have a nonzero intersection, so the mutual information space [qudit (π) ∩qudit (σ)] is
not the zero space. •
[qudit (π, σ)] = [qudit (π) ∪qudit (σ)]; and so forth. Appendix B. Quantum Logical Entropies with Non-Commuting Observables •
h (F : ψ) = p × p ([qudit (π)]) = tr
h
P[qudit(π)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
h (G : ψ) = p × p ([qudit (σ)]) = tr
h
P[qudit(σ)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
h (F, G : ψ) = p × p ([qudit (π) ∪qudit (σ)]) = tr
h
P[qudit(π)∪qudit(σ)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
h (F|G : ψ) = p × p ([qudit (π) −qudit (σ)]) = tr
h
P[qudit(π)−qudit(σ)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
h (G|F : ψ) = p × p ([qudit (σ) −qudit (π)]) = tr
h
P[qudit(σ)−qudit(π)] (ρ (ψ) ⊗ρ (ψ))
i
;
•
m (F, G : ψ) = p × p ([qudit (π) ∩qudit (σ)]) = tr
h
P[qudit(π)∩qudit(σ)] (ρ (ψ) ⊗ρ (ψ))
i
. The observable F : V →V defines an observable F ⊗I : V ⊗V →V ⊗V with the eigenvectors
x ⊗v for any nonzero v ∈V and with the same eigenvalues φ1, . . . , φI (the context should suffice to
distinguish the identity operator I : V →V from the index set I for the F-eigenvalues). Then, in two
independent measurements of ψ by the observable F ⊗I, we have: h (F : ψ) = probability of getting distinct eigenvalues φi and φi′, i.e., of getting a qudit of F. In a similar manner, G : V →V defines the observable I ⊗G : V ⊗V →V ⊗V with the
eigenvectors v ⊗y and with the same eigenvalues γ1, . . . , γJ. Then, in two independent measurements
of ψ by the observable I ⊗G, we have: h (G : ψ) = probability of getting distinct eigenvalues γj and γj′. The two observables F, G : V →V define an observable F ⊗G : V ⊗V →V ⊗V with the
eigenvectors x ⊗y for (x, y) ∈X × Y and eigenvalues f (x) g (y) = φiγj. To interpret the compound
logical entropies cleanly, we assume there is no accidental degeneracy, so there are no φiγj = φi′γj′ for
i ̸= i′ and j ̸= j′. Appendix B. Quantum Logical Entropies with Non-Commuting Observables Then, for two independent measurements of ψ by F ⊗G, the compound quantum
logical entropies can be interpreted as the following “two-measurement” probabilities: •
h (F, G : ψ) = probability of getting distinct eigenvalues φiγj ̸= φi′γj′ where i ̸= i′ or j ̸= j′;
•
h (F|G : ψ) = probability of getting distinct eigenvalues φiγj ̸= φi′γj where i ̸= i′; •
h (F, G : ψ) = probability of getting distinct eigenvalues φiγj ̸= φi′γj′ where i ̸= i′ or j ̸= j •
h (F, G : ψ) = probability of getting distinct eigenvalues φiγj ̸= φi′γj′ where i ̸= i′ or j ̸= j′; h (F|G : ψ) = probability of getting distinct eigenvalues φiγj ̸= φi′γj where i ̸= i′; 21 of 22 Entropy 2018, 20, 679 •
h (G|F : ψ) = probability of getting distinct eigenvalues φiγj ̸= φiγj′ where j ̸= j′; j
j
•
m (F, G : ψ) = probability of getting distinct eigenvalues φiγj ̸= φi′γj′ where i ̸= i′ and j All the quantum logical entropies have been defined by the general method using the information
subspaces, but in the first three cases h (F : ψ), h (G : ψ) and h (F, G : ψ), the density matrix method of
defining logical entropies could also be used. Then, the fundamental theorem could be applied relating
the quantum logical entropies to the zeroed entities in the density matrices indicating the eigenstates
distinguished by the measurements. The previous set identities for disjoint unions now become subspace identities for direct sums
such as: udit (π) ∪qudit (σ)] = [qudit (π) −qudit (σ)] ⊕[qudit (π) ∩qudit (σ)] ⊕[qudit (σ) −qudit (π)]. [qudit (π) ∪qudit (σ)] = [qudit (π) −qudit (σ)] ⊕[qudit (π) ∩qudit (σ)] ⊕[qudit (σ) −qudit (π)]. Hence, the probabilities are additive on those subspaces as shown in Figure A2: h (F, G : ψ) = h (F|G : ψ) + m (F, G : ψ) + h (G|F : ψ). Figure A2. Venn diagram for quantum logical entropies as probabilities on (V ⊗V)2. Figure A2. Venn diagram for quantum logical entropies as probabilities on (V ⊗V)2. References 1. Gini, C. Variabilità e Mutabilità; Tipografia di Paolo Cuppini: Bologna, Italy, 1912. (In Italian) 2. Ellerman, D. An Introduction of Partition Logic. Logic J. IGPL 2014, 22, 94–125. [CrossRef] 3. Ellerman, D. Logical Information Theory: New Foundations for Information Theory. Logic J. IGPL 2017, 25,
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Information in Product MV-Algebras. Entropy 2018, 20, 129. [CrossRef] 5. Ellerman, D. The Logic of Partitions: Introduction to the Dual of the Logic of Subsets. Rev. Symb. Logic 2010,
3, 287–350. [CrossRef] 6. Rota, G.-C. Twelve Problems in Probability No One Likes to Bring up. In Algebraic Combinatorics and Computer
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USA, 2009. 8. Kolmogorov, A. Combinatorial Foundations of Information Theory and the Calculus of Probabilities. Russ. Math. Surv. 1983, 38, 29–40. [CrossRef] 9. Campbell, L. Lorne. Entropy as a Measure. IEEE Trans. Inf. Theory 1965, 11, 112–114. [CrossRef] 10. Shannon, C.E. A Mathematical Theory of Communication. Bell Syst. Tech. J. 1948, 27, 379–423, 623–656. [CrossRef] 11. Rozeboom, W.W. The Theory of Abstract Partials: An Introduction. Psychometrika 1968, 33, 133–167. [CrossRef] [PubMed] 12. Abramson, N. Information Theory Coding; McGraw-Hill: New York, NY, USA, 1963. 3. Hartley, R.V. Transmission of information. Bell Syst. Tech. J. 1928, 7, 553–563. [CrossRef] 14. Lawvere, F. William and Stephen Schanuel. In Conceptual Mathematics: A First Introduction to Categories;
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15. Auletta, G.; Mauro, F.; Giorgio, P. Quantum Mechanics; Cambridge University Press: Cambridge, UK, 2009. 22 of 22 Entropy 2018, 20, 679 16. Fano, U. Description of States in Quantum Mechanics by Density Matrix and Operator Techniques. Rev. Mod. Phys. 1957, 29, 74–93. [CrossRef] 17. Bennett, C.H. Quantum Information: Qubits and Quantum Error Correction. Int. J. Theor. Phys. 2003, 42,
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Subspaces. Logic J. IGPL 2018, 26, 1–13. [CrossRef] 19. Ellerman, D. Counting Distinctions: On the Conceptual Foundations of Shannon’s Information Theory. Synthese 2009, 168, 119–149. [CrossRef] 20. Jaeger, G. Quantum Information: An Overview; Springer: New York, NY, USA, 2007. 21. Manfredi, G.; Feix, M.R. Entropy and Wigner Functions. Phys. Rev. E 2000, 62, 4665. arXiv:quant-ph/0203102. [CrossRef] 22. References Buscemi, F.; Bordone, P.; Bertoni, A. Linear Entropy as an Entanglement Measure in Two-Fermion Systems. Phys. Rev. 2007, 75, 032301, arXiv:quant-ph/0611223v2. [CrossRef] 23. Woldarz, J.J. Entropy and Wigner Distribution Functions Revisited. Int. J. Theor. Phys. 2003, 42, 1075–1084. [CrossRef] 24. Havrda, J.; Charvát, F. Quantification Methods of Classification Processes:
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Alpha-Entropy. Kybernetika 1967, 3, 30–35. 25. Tsallis, C. Possible Generalization for Boltzmann-Gibbs Statistics. J. Stat. Phys. 1988, 52, 479–487. [Cr 25. Tsallis, C. Possible Generalization for Boltzmann-Gibbs Statistics. J. Stat. Phys. 1988, 52, 479–487. [CrossRef]
26. Rao, C.R. Diversity and Dissimilarity Coefficients: A Unified Approach. Theor. Popul. Biol. 1982, 21, 24–43. [CrossRef] y
26. Rao, C.R. Diversity and Dissimilarity Coefficients: A Unified Approach. Theor. Popul. Biol. 1982, 21, 24–43. [CrossRef] 27. Cohen-Tannoudji, C.; Laloe, F.; Diu, B. Quantum Mechanics; John Wiley and Sons: New York, NY, USA, 2005;
Volume 1. 28. Tamir, B.; Cohen, E. Logical Entropy for Quantum States. arXiv 2014, arXiv:1412.0616v2. 29. Tamir, B.; Cohen, E. A Holevo-Type Bound for a Hilbert Schmidt Distance Measure. J. Quantum Inf. Sci. 2015,
5, 127–133. [CrossRef] 30. Nielsen, M.; Chuang, I. Quantum Computation and Quantum Information; Cambridge University Press:
Cambridge, UK, 2000. 31. McEliece, R.J. The Theory of Information and Coding: A Mathematical Framework for Communication. In Encyclopedia of Mathematics and Its Applications; Addison-Wesley: Reading, MA, USA, 1977; Volume 3. 32. Rossi, G. Partition Distances. arXiv 2011, arXiv:1106.4579v1. c⃝2018 by the author. 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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Trajectories of Loneliness During Adolescence Predict Subsequent Symptoms of Depression and Positive Wellbeing
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Abstract There is a need to identify the outcomes of changes in loneliness during adolescence, and to consider this within a
multidimensional framework of loneliness. This study considered the effects of different trajectories of change in Isolation
Loneliness and in Friendship Loneliness upon both positive wellbeing and symptoms of depression. To achieve this, 1782
(43% female; 12.92 years old at the start of the study, SD = 1.60) young people took part in a longitudinal study with four
data points across 2 years. Four Isolation Loneliness trajectories and five Friendship Loneliness trajectories were identified. Youth who experienced low levels of Isolation Loneliness that subsequently increased appear to be at particular risk for poor
outcomes. Similarly, initially high levels of Friendship Loneliness that decreased rapidly, or which began at a low level and
only increased marginally, seem to also be a risk. Loneliness is a multi-dimensional construct and its development during
adolescence impacts upon young people’s depressive symptomatology and positive mental wellbeing. Keywords:
Loneliness
●Trajectories
●Depression
●Positive Mental Wellbeing Simon C. Hunter
1,2
●Rebecca Seth3
●Stephen Houghton2,4
●David Lawrence3
●Corinne Zadow2
●
Michael Rosenberg2
●Lisa Wood5
●Pamela Qualter6
●Trevor Shilton3 Received: 19 September 2023 / Accepted: 30 November 2023 / Published online: 21 December 2023
© The Author(s) 2023 6
University of Manchester, Oxford Road, Manchester M13 9PL
England, UK * Simon C. Hunter
simon.hunter@gcu.ac.uk
1
Glasgow Caledonian University, Cowcaddens Road, Glasgow G4
0BA Scotland, UK
2
University of Western Australia, 35 Stirling Highway,
Crawley 6009 WA, Australia
3
Curtin University, Kent Street, Perth, WA 6102, Australia
4
University of Strathclyde, 16 Richmond Street, Glasgow G1 1XQ
Scotland, UK
5
University of Notre Dame, 23 High Street, Fremantle, WA,
Australia
6
University of Manchester, Oxford Road, Manchester M13 9PL
England, UK Journal of Youth and Adolescence (2024) 53:1078–1090
https://doi.org/10.1007/s10964-023-01925-0 Journal of Youth and Adolescence (2024) 53:1078–1090
https://doi.org/10.1007/s10964-023-01925-0 EMPIRICAL RESEARCH Introduction Moreover, loneliness is associated with later
reports of mental health problems, physical health risk beha-
viours, poorer employment prospects (Leigh-Hunt et al., 2017;
Matthews et al., 2018), poorer sleep quality (Matthews et al.,
2017), withdrawal from social activities and relationships
(Böger & Huxhold, 2018), suicidal behaviours (Heinrich &
Gullone, 2006), cardiovascular disease (Leigh-Hunt et al.,
2017) and both morbidity and mortality (see Hawkley &
Cacioppo, 2010). Given that loneliness experienced in ado-
lescence can have both immediate and lasting implications for
outcomes in later years, studying loneliness in young people is
important. policy involving children and young people in the UK
(Garrat et al., 2022; Scottish Government, 2020). Cross-
sectional work with adolescents has supported that a
negative association exists between positive mental well-
being and loneliness (Houghton et al., 2016; Lyyra et al.,
2021). Cross-lagged panel analyses using longitudinal data
collected across the COVID-19 pandemic, in contrast,
indicates that levels of loneliness may not be associated
with positive mental wellbeing (Houghton et al., 2022). While positive mental wellbeing is negatively correlated
with indices of psychological maladjustment such as
depressive symptomatology (Clarke et al., 2011) it is also
the case that depressive symptomatology is more strongly
associated with subsequent positive mental wellbeing than
vice-versa among adolescents (Zadow et al., 2017). Indeed,
in adolescence, low levels of positive mental wellbeing can
occur in the presence of low levels of depressive sympto-
matology and high levels of positive mental wellbeing can
occur even in the presence of mental illness (Patalay &
Fitzsimons, 2018), and there is significant change between
groups over time (Petersen et al., 2022). Thus, negative and
positive mental wellbeing are distinctive concepts and the
degree to which tackling loneliness can contribute to
improving either health outcome is not clear. The degree to which different trajectories of loneliness are
associated with psycho-social adjustment has often been the
focus of published work (see Table 1). Confidently naming
trajectories relating to loneliness is problematic since there is
no accepted definition of low, medium, or high loneliness. However, across studies, youth in stable-low trajectory groups
tend to report better psychological and social adjustment than
those in other groups, including lower depressive symptoma-
tology (Jobe-Shields et al., 2011; Ladd & Ettekal, 2013;
Qualter et al., 2013; Schinka et al., 2013; Vanhalst et al.,
2013), higher social skills (Schinka et al., 2013), better self-
esteem (Vanhalst et al., 2013), and higher academic outcomes
(Benner, 2011). Introduction Young people with chronically high levels of
loneliness tend to be most at risk for depressive symptoma-
tology (Ladd & Ettekal, 2013; Qualter et al., 2013; Schinka
et al., 2013; Vanhalst et al., 2013), alcohol misuse (Qualter
et al., 2013), suicidal ideation (Schinka et al., 2013), and
poorer general health (Harris et al., 2013; Qualter et al., 2013). Among those with trajectories of loneliness that display
change over time, there is evidence that increasing levels of
loneliness are a marker for later maladjustment (Benner, 2011;
Jobe-Shields et al., 2011; Qualter et al., 2013) and there is
some support for a scar hypothesis (Rohde et al., 1990)
whereby higher starting levels that subsequently reduce are
still associated with poor outcomes (Harris et al., 2013). As well as focussing only on negative indices of
adjustment as outcomes of loneliness, the current literature
examining trajectories of loneliness has not addressed the
multi-dimensional nature of the construct. Contemporary
theory and measurement of loneliness considers it to have
between two and four dimensions (Goossens et al., 2009;
Houghton et al., 2014; Majorano et al., 2015). Parent (or
family)-related and peer-related loneliness sub-scales have
been reported in two separate studies (Goossens et al., 2009;
Marjorano et al., 2015), and to these have been added two
attitudinal factors reflecting positive and negative attitudes
toward solitude (Goossens et al., 2009). While there exists
support for the existence of positive and negative attitudinal
factors, they may reflect attitudes toward “aloneness” (e.g.,
“I have discovered the benefits of being alone”) rather than
loneliness per se (Houghton et al., 2014). Furthermore,
parent (or family)-related and peer-related factors risk
confusing the construct with the situations in which it may
be expressed and thus it has been proposed that factors
relating to friendship loneliness (e.g., “I feel part of a group
of friends”, where higher scores reflect a positive outcome)
and isolation loneliness (e.g., “I have nobody to talk to”,
where higher scores reflect a negative outcome) are more
theoretically distinct constructs (Houghton et al., 2014). There is clear evidence that feelings of isolation and quality
of friendships are highly, inversely correlated among ado-
lescents (Houghton et al., 2020). As is clear from Table 1, symptoms of depression have
frequently been the focus of research seeking to document
the outcomes of different loneliness trajectories. Introduction loneliness) and considered the effects of these on both positive
mental wellbeing and symptoms of depression. Loneliness is associated with numerous indices of maladaptive
adjustment and there is an attendant need to identify the out-
comes of changes in loneliness during adolescence. Empirical
study has begun to investigate these relationships but has
neither considered positive mental wellbeing as an outcome
nor addressed the multidimensional nature of loneliness. This
study identified trajectories of change in relation to two
dimensions of loneliness (Isolation loneliness and Friendship Loneliness is the sense that one’s social relationships are
not commensurate with one’s desired social relationships
(Cacioppo et al., 2015a). Considering loneliness across the
life-span, it is evident that adolescence and early adulthood
are times when loneliness peaks (Heinrich & Gullone, 2006;
Xerxa et al., 2023). According to the Office for National
Statistics (2018) nearly half of 10–12-year-olds report
feeling lonely at least some of the time, with this rising to
almost 60% in 16–24-year-olds. Brain development during
adolescence in regions involved in social processing, a
desire for increased peer interaction and friendships (Orben
et al., 2020), but increased sensitivity to social rejection
(Blakemore & Mills, 2014), and developmental shifts and
transitions in social networks (e.g., moving from primary to
secondary school, or leaving home) are all known risk
factors for increased loneliness (Siva, 2020; Sundqvist &
Hemberg, 2021). * Simon C. Hunter
simon.hunter@gcu.ac.uk Adolescence is a particularly vulnerable period for the
development of mental health problems, with half of all mental
health problems emerging before the age of 14 (Kessler et al.,
2007) and the peak age of incidence coinciding with the
transition from “childhood/adolescence” to “adult” life (Tha-
par & Riglin, 2020). Loneliness in adolescence is a known risk 5
University of Notre Dame, 23 High Street, Fremantle, WA,
Australia 1079 Journal of Youth and Adolescence (2024) 53:1078–1090 factor for anxiety (Maes et al., 2019), depression (Fontaine
et al., 2009; Lasgaard et al., 2011), suicidal ideation (Gallagher
et al., 2014) and diminished positive mental health (Lyyra
et al., 2021). Introduction However,
notably absent from this literature examining trajectories of
change in loneliness is information concerning associations
with positive mental wellbeing. Positive mental wellbeing is
neither the polar opposite, nor absence, of psychological
maladjustment (World Health Organisation WHO (2004))
and includes both hedonic (i.e., happiness, subjective well-
being) and eudemonic (i.e., positive functioning) aspects of
wellbeing (Clarke et al., 2011). Promoting positive mental
wellbeing among adolescents is a national priority in many
countries and has been placed at the centre of Government Studying both isolation and friendships during ado-
lescence is important because it is a period where Journal of Youth and Adolescence (2024) 53:1078–1090 1080 Table 1 Studies estimating trajectory classes of loneliness among children and young people
Study
Sample
Design
Trajectory classesa
Results
Harris et al. (2013)
N = 209. Aged 8
years at T1. Data collected three times, at 18-
month intervals. Two-class solution: Relatively High, Reducing
(48%), and Low Stable (52%). At 11 years, the Relatively High, Reducing group had
higher levels of depressive symptoms, poorer general
health, and poorer sleep. Benner (2011)
N = 640. Grade 9 at
T1. Data collected twice in Grades 9
and 10. Three-class solution: Consistently Low (78%),
High (11%), and Low but Increasing (11%). High and Low Increasing classes had poorer academic
outcomes than the Low class. Jobe-Shields et al. (2011)
N = 170. Aged 9
years at T1. Data collected annually at ages 9,
10 and 11 years. Three-class solution: Stable Low (65%), Increasers
(23%), and Decreasers (12%). Stable Low had generally positive peer functioning;
Increasers at risk group for developing later internalising
symptomatology; and Decreasers had mixed pattern of
peer functioning at age 9 years, but were
indistinguishable from the Stable Low group
subsequently. Verboon et al. (2022)
N-130. 9 years. Data collected at ages 9, 13, 16, and
21. Three-class solution: Stable-Low (51–61%), Low-
Increasing (21–32%), and High-Declining
(7–22%). NB. Ranges are reported here as three
separate clustering techniques were employed. Qualter et al. (2013) N = 586. Age 7 years
at T1. Data collected biannually for ten
years. Four-class solution: Low Stable (37%), Moderate
Decliners (23%), Moderate Increasers (18%),
Relatively High Stable (22%). Both the High Stable and Moderate Increasing
trajectories were associated with depressive symptoms at
age 17 years. Ladd & Ettekal
(2013)
N = 478. Age 12
years at T1. Data collected annually for six
years. Participants At T1 (see Procedure for details of data collection points),
there were 1544 youth (45% female), at T2 there were 1703
(45% female), at T3 there were 1782 (43% female), and at
T4 there were 1591 (44% female). Average age at T1 was
12.92 years old (SD = 1.60; range = 10 to 16) and across
the four waves of data collection 77–80% of young people
reported living in an urban environment. Numbers of par-
ticipants varied somewhat across waves because young
people were invited to participate if they joined classes that
were already involved in the study. One thousand two
hundred and twelve participants took part in all four waves,
while 524 took part in three, 101 took part in two, and 83
took part in a single session Participants attended 38 separate schools (N = 34 State
Government; N = 4 non-Government) which themselves
reflected a range of socio-economic status (SES) areas as
indexed by their Socio-Economic Index for Areas (Aus-
tralian Bureau of Statistics, 2011). All eligible students were
invited to participate and both information sheets and con-
sent forms were sent to parents of students explaining that
involvement would consist of data gathering over approxi-
mately three school years. Five primary schools were in low
SES areas, three in mid SES areas, and five were in high
SES areas. Among the high schools, three in low SES areas,
nine in mid SES areas and 13 in high SES areas. Introduction Five-class solution: Stable Non-Lonely (19%),
Stable Low (20%), Stable High (Chronic) (14%),
Moderate Decliners (42%), and Steep Decliners
(6%). Stable High had the highest level of symptoms of
depression. Schinka et al. (2013) N = 832. Age 9 years
at T1. Loneliness assessed at ages 9, 11,
and 15 years. Five-class solution: Stable Low (49%), Moderate
Increasing (32%), High Increasing (5%),
Decreasing (11%), and Chronic (4%). Depression assessed at 7 years old predicted greater
likelihood of being in: High Increasing/Chronic groups
than Stable Low. All groups reported higher levels of
depression at age 15 when compared to the Stable. Vanhalst et al. (2013)
N = 389. Mage at
T1 = 15.22 years. Cohort-sequential (two-groups,
ages 15 and 16 at T1). Data
collected annually for 5 years. Five-class solution: Chronically High (3%), High
Decreasing (6%), Moderate Decreasing (8%), Low
Increasing (17%), and Stable Low (65%). Those who never experienced loneliness were best
adjusted (lowest stress, least depressive symptoms,
highest self-esteem), whereas the Chronically High
group had the most problems (highest stress, most
depressive symptoms, lowest self-esteem). Vanhalst et al. (2015, 2018)
N = 730. Mage at
T1 = 15.43 years. Data collected annually for 4 years. Five-class solution: Chronic (3%), Low Stable
(47%), Moderate-Stable (27%), Moderate-
Increasing (14%), High Decreasing (9%). Cognitive and emotional responses of the Chronic group
seem to perpetuate, rather than reduce, loneliness (e.g.,
hypersensitivity to social exclusion and hyposensitivity
to social inclusion). aTrajectory labels used here are those used by the original authors Journal of Youth and Adolescence (2024) 53:1078–1090 1081 Present Study There is a need to identify the outcomes of changes in
loneliness during adolescence, and to consider this within a
multidimensional framework of loneliness. The present
study considers the effects of different trajectories of change
in two forms of loneliness (isolation loneliness and friend-
ship loneliness) upon both positive wellbeing and symp-
toms of depression. Based on existing research utilising
single-factor loneliness scales, it is expected that four or five
trajectories of loneliness will be evidenced for both isolation
loneliness and friendship loneliness. The largest trajectory
group in both cases is expected to be a consistently
unproblematic loneliness trajectory (i.e., low isolation
loneliness, high friendship related loneliness). A chronically
problematic trajectory in each case (i.e., high isolation
loneliness,
low
friendship
related
loneliness)
is
also
expected. Based on the two existing studies that most clo-
sely resemble the present study in terms of participant age,
it is expected that there will be a third trajectory reflecting
loneliness which is problematic but subsequently becomes
less so (isolation loneliness decreasing, friendship lone-
liness increasing). After controlling for earlier levels of both
positive mental wellbeing and symptoms of depression, it is
expected that youth in the chronically problematic lone-
liness trajectory groups will report significantly higher
symptoms of depression, and significantly lower levels of
positive mental wellbeing, than youth in the other trajectory
groups. Methods belonging to a peer group is a major concern among
young people (Rubin et al., 2008) and peer interactions
and relationships become increasingly more important
(Qualter et al., 2015; Rubin et al., 2009). For some
young people, insufficient connections to others can lead
to profound and long-standing negative consequences,
while having quality friendships can provide numerous
social and emotional benefits (Houghton et al., 2014). The turbulence of significant life transitions during
adolescence means some young people drift in or out of
loneliness while others experience loneliness persistently
(Matthews et al., 2023). Cross-sectional research with
adolescents shows that positive mental wellbeing is
positively associated with “friendship” loneliness (where
higher scores represent lower loneliness) and is nega-
tively associated with “isolation” loneliness (where
higher scores represent higher loneliness) (Houghton
et al., 2016). This speaks to the importance of con-
sidering loneliness as a multi-dimensional construct,
which no published work on loneliness trajectories and
adjustment has yet considered. belonging to a peer group is a major concern among
young people (Rubin et al., 2008) and peer interactions
and relationships become increasingly more important
(Qualter et al., 2015; Rubin et al., 2009). For some
young people, insufficient connections to others can lead
to profound and long-standing negative consequences,
while having quality friendships can provide numerous
social and emotional benefits (Houghton et al., 2014). The turbulence of significant life transitions during
adolescence means some young people drift in or out of
loneliness while others experience loneliness persistently
(Matthews et al., 2023). Cross-sectional research with
adolescents shows that positive mental wellbeing is
positively associated with “friendship” loneliness (where
higher scores represent lower loneliness) and is nega-
tively associated with “isolation” loneliness (where
higher scores represent higher loneliness) (Houghton
et al., 2016). This speaks to the importance of con-
sidering loneliness as a multi-dimensional construct,
which no published work on loneliness trajectories and
adjustment has yet considered. Loneliness The self-report Perth A-Loneliness scale (PALs: Houghton
et al., 2014) which includes both a friendship related
loneliness (e.g., “My friends will stand by me in almost any
difficulty”, “I feel part of a group of friends”), and isolation
loneliness (e.g., “I feel like I do not have a friend in the
world”, “I am not close to anyone”) scale was utilised. Both
scales comprised six items and responses are recorded using
a six-point Likert scale (1 = “never”, 2 = “rarely”, 3 =
“sometimes”, 4 = “often”, 5 = “very often”, 6 = “always”). Factor scores were obtained by summing the relevant items
so that scores could range from 6 to 36, and Cronbach’s
alpha
was
good
for
both
scales
(αT1friendship = 0.91,
αT2friendship = 0.91;
αT3friendship = 0.92;
αT4friendship = 0.92;
αT1isolation = 0.87,
αT2isolation = 0.91,
αT3isolation = 0.91,
αT4isolation = 0.92). Higher scores indicated higher levels of
the relevant construct (i.e. “worse” loneliness with regards
to Isolation loneliness but “better” loneliness with respect to
Friendship loneliness). Journal of Youth and Adolescence (2024) 53:1078–1090 1082 Statistical Analyses MPlus Version 8.7 was used to perform a latent trajectory
analysis for both friendship-related loneliness and isolation-
related loneliness measures over four time points. As only
four time points were available, trajectories were fitted as
linear across the four waves. Latent trajectory analysis
identifies distinct subgroups within the cohort who have
similar
trajectories
of
friendship-related
loneliness
or
isolation-related loneliness across waves. The appropriate
number of classes for both loneliness measures was deter-
mined by starting with an unconditional model with no
latent trajectory classes, and conducting subsequent ana-
lyses adding one trajectory class at a time and comparing fit. Intercept and slope were permitted to vary across trajectory
classes. The most appropriate number of classes was chosen
using a combination of (i) smaller Bayesian information
criterion (BIC), (ii) higher entropy (>0.8) (Asparouhov &
Muthén
(2014)). MPlus
implements
Full
Information
Maximum Likelihood (FIML) to address missing data. Symptoms of depression Depression, and Positive Mental Wellbeing all assessed). In addition to the aforementioned measures, young
people’s screen use was also assessed and those data are
reported elsewhere (see Houghton et al., 2015; Rosen-
berg et al., 2018). The Children’s Depression Inventory 2 (CDI 2: Kovacs,
2004) is a 12-item self-report assessment of cognitive,
affective, and behavioural symptoms of depression in chil-
dren and adolescents aged 7–17 years. Each item has three
separate sentence response options which describe partici-
pants’ feelings and experiences over the past 2 weeks (e.g.,
0 = “I am sad once in a while”, 1 = “I am sad many times”,
or 2 = “I am sad all the time”). One item on this scale was
not used when calculating scale scores for participants since
it related to feelings of loneliness: “I do not feel alone, I feel
alone many times, I feel alone all the time”. Scores could
range from 0 to 22, with higher scores reflecting higher
depressive symptomatology, and the scale evidenced good
internal reliability (αT1 = 0.81, αT4 = 0.85). One teacher in each school was responsible for liais-
ing with the research team and for administering the
survey. These teachers were provided with written
instructions to ensure standardisation of administration. The electronic survey remained open for ~4 weeks across
each of the test administrations. Close monitoring of the
survey administrations resulted in attrition rates being
maintained below 6% per data collection period. Positive mental wellbeing The Short Warwick-Edinburgh Mental Wellbeing Scale
(SWEMWBS: Stewart-Brown et al., 2009) is a 7-item self-
report scale related to positive feelings, answered on a five-
point Likert scale (1 = “none of the time”, 2 = “rarely”, 3 =
“some of the time”, 4 = “often”, 5 = “all of the time”). Example item are “I have been feeling useful” and “I’ve been
feeling relaxed”. The total score is a mean of the item
responses, though the scale was modified by excluding the
item “I have been feeling close to other people” since it was
related to feelings of loneliness (hence, the current scale was 6,
not 7, items). Scores could range from 1–5, with higher scores
reflecting better positive mental wellbeing, and the scale evi-
denced good internal reliability (αT1 = 0.86; αT4 = 0.91). Results other category combinations included less than 3% of the total
sample. Sensitivity analyses confirmed the same optimal
number of classes among young people who were present at all
four data points and that these were comparable (see Online
Supplement 1). Trajectories of Loneliness For Isolation Loneliness, four trajectories were identified (see
Table 2, and Fig. 1): Low Stable (74.7%), Elevated Stable
(17.4%), Low Increasing (5.1%), and High Decreasing (2.8%). For Friendship Loneliness, there were five groups: High Stable
(54.5%), Average Stable (25.9%), High Decreasing (12.5%),
Average Increasing (3.8%), and Low Increasing (3.3%) (see
Table 2, and Fig. 2). When class membership relating to both
forms of loneliness is examined (see Table 3), the most com-
mon category combinations are High Stable Friendship + Low
Stable Isolation (48.2% of all participants), Average Stable
Friendship + Low Stable Isolation (14.7%), Average Stable
Friendship + Elevated Stable Isolation (10.0%), and High
Decreasing Friendship + Low Stable Isolation (8.7%). All Procedure To investigate the relationship between latent trajec-
tories of loneliness and measures of wellbeing, general-
ised
linear
modelling
was
undertaken
using
the
GENMOD procedure in SAS Version 9.4. Four separate
models were fitted, two with CDI depression score
(without loneliness item) at Wave 4 as outcome variable,
and two with WEMWBS score (without loneliness item)
at Wave 4 as outcome variable. In each case the model
adjusted for age, gender, and CDI or WEMWBS score at
Wave 1. Because of the high degree of correlation
between
friendship
related
loneliness
and
isolation
loneliness, separate models were fitted with friendship
related loneliness trajectory class or isolation loneliness
trajectory class as independent variable. The Human Research Ethics Committees of The Uni-
versity of Western Australia and the Western Australian
Department of Education granted permission to conduct
this research. Informed consent was obtained from all
individual
participants,
and
their
parents/guardians,
included in the study. All participants were provided
with a unique identification code, which allowed them to
log on to an electronic survey at each administration. This unique code also ensured that all information pro-
vided was confidential and that data could be linked
across waves via these codes for the purposes of data
analysis. Data were collected on six occasions, four of
which are relevant to the current study: T1 (November/
December, 2013: Loneliness, Symptoms of Depression,
and
Positive
Mental
Wellbeing
all
assessed),
T2
(August/September,
2014;
Loneliness
assessed),
T3
(March/April,
2015;
Loneliness
assessed),
and
T4
(August/September, 2015: Loneliness, Symptoms of Sensitivity analyses were also conducted by restrict-
ing the aforementioned analyses to those adolescents
who only participated in all four waves of data collec-
tion. This involved a smaller sample of 1212 young
people. Journal of Youth and Adolescence (2024) 53:1078–1090 1083 Results
Trajectories of Loneliness
For Isolation Loneliness, four trajectories were identified (see
Table 2, and Fig. 1): Low Stable (74.7%), Elevated Stable
(17.4%), Low Increasing (5.1%), and High Decreasing (2.8%). For Friendship Loneliness, there were five groups: High Stable
(54.5%), Average Stable (25.9%), High Decreasing (12.5%),
Average Increasing (3.8%), and Low Increasing (3.3%) (see
Table 2, and Fig. 2). When class membership relating to both
other category combinations included less than 3% of the total
sample. Sensitivity analyses confirmed the same optimal
number of classes among young people who were present at all
four data points and that these were comparable (see Online
Supplement 1). Procedure Descriptive statistics
In Table 4, descriptive statistics for the measures of positive
mental wellbeing and depressive symptomatology at the start
and end of the study are shown by trajectory class. For
Table 2 Entropy and Bayesian
Information Criterion (BIC)
relating to number of loneliness
trajectories for both Friendship
Loneliness and Isolation
Loneliness subscales
Number of trajectories
Friendship Loneliness
Isolation Loneliness
Entropy
BIC
Change in BIC
Entropy
BIC
Change in BIC
p1
p2
p1
p2
2
0.608
39538
0.0000
0.0000
0.823
37226
0.0000
0.0000
3
0.637
39447
0.0000
0.0000
0.877
36950
0.0028
0.0035
4
0.640
39398
0.0045
0.0054
0.883
36654
0.0000
0.0000
5
0.689
39371
0.0028
0.0035
0.813
36562
0.0903
0.0917
6
0.668
39359
0.2462
0.2628
-
-
-
-
Change in BIC was assessed using the Vuong-Lo_Mendell-Rubin Likelihood Ratio Test (p1 above) and the
Lo-Mendell-Rubin Adjusted Likelihood Ratio Test (p2 above)
Fig. 1 Isolation loneliness
trajectories Table 2 Entropy and Bayesian
Information Criterion (BIC)
relating to number of loneliness
trajectories for both Friendship
Loneliness and Isolation
Loneliness subscales
Number of trajectories
Friendship Loneliness
Isolation Loneliness
Entropy
BIC
Change in BIC
Entropy
BIC
Change in BIC
p1
p2
p1
p2
2
0.608
39538
0.0000
0.0000
0.823
37226
0.0000
0.0000
3
0.637
39447
0.0000
0.0000
0.877
36950
0.0028
0.0035
4
0.640
39398
0.0045
0.0054
0.883
36654
0.0000
0.0000
5
0.689
39371
0.0028
0.0035
0.813
36562
0.0903
0.0917
6
0.668
39359
0.2462
0.2628
-
-
-
-
Change in BIC was assessed using the Vuong-Lo_Mendell-Rubin Likelihood Ratio Test (p1 above) and the
Lo-Mendell-Rubin Adjusted Likelihood Ratio Test (p2 above) oneliness Table 4 Descriptive statistics for
positive mental wellbeing and
depressive symptomatology at
start and end of the study, by
trajectory class Descriptive statistics In Table 4, descriptive statistics for the measures of positive
mental wellbeing and depressive symptomatology at the start
and end of the study are shown by trajectory class. For
symptoms of depression, the largest changes in mean scores
were evident among the Average Increasing and High
Decreasing Friendship related loneliness trajectory groups,
though these changes occurred within the context of substantial
variation in scores at each time point. For the Isolation lone-
liness trajectory groups, the High Decreasing and Low Journal of Youth and Adolescence (2024) 53:1078–1090 1084 Fig. 2 Friendship loneliness
trajectories
Table 3 Number of participants
in each loneliness trajectories for
both Friendship Loneliness and
Isolation Loneliness subscales
Isolation Loneliness
Row total
(Column %)
High Decreasing Low Increasing Low Stable Elevated Stable
Friendship Loneliness
Average Increasing
4
5
36
26
71 (3.8%)
High Decreasing
14
22
164
35
235 (12.5%)
Low Increasing
7
7
21
27
62 (3.3%)
Average Stable
9
14
276
189
488 (25.9%)
High Stable
19
48
908
51
1026 (54.5%)
Column total (Row %) 53 (2.8%)
96 (5.1%)
1405
(74.7%)
328 (17.4%)
Table 4 Descriptive statistics for
positive mental wellbeing and
depressive symptomatology at
start and end of the study, by
trajectory class
Loneliness
Trajectory Group
Depression
Positive Mental Wellbeing
T1
T4
T1
T4
Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. Friendship Subscale
Average Increasing
5.27
4.43
3.81
3.99
2.21
0.71
2.82
0.48
High Decreasing
3.74
3.53
6.50
5.16
2.63
0.55
2.15
0.72
Low Increasing
8.80
5.04
9.03
4.54
1.82
0.80
1.95
0.61
Average Stable
5.61
3.60
5.60
4.30
2.28
0.46
2.35
0.53
High Stable
2.69
2.78
2.83
3.12
2.74
0.46
2.83
0.48
Isolation Subscale
High Decreasing
7.43
6.53
5.22
5.25
2.59
0.78
2.46
0.76
Low Increasing
4.40
4.40
8.86
6.75
2.54
0.62
2.47
0.91
Elevated Stable
6.25
3.77
6.30
4.43
2.22
0.53
2.30
0.61
Low Stable
3.11
2.97
3.40
3.43
2.64
0.52
2.67
0.56 Fig. Descriptive statistics 2 Friendship loneliness
trajectories
Table 3 Number of participants
in each loneliness trajectories for
both Friendship Loneliness and
Isolation Loneliness subscales
Isolation Loneliness
Row total
(Column %)
High Decreasing Low Increasing Low Stable Elevated Stable
Friendship Loneliness
Average Increasing
4
5
36
26
71 (3.8%)
High Decreasing
14
22
164
35
235 (12.5%)
Low Increasing
7
7
21
27
62 (3.3%)
Average Stable
9
14
276
189
488 (25.9%)
High Stable
19
48
908
51
1026 (54.5%)
Column total (Row %) 53 (2.8%)
96 (5.1%)
1405
(74.7%)
328 (17.4%)
Table 4 Descriptive statistics for
positive mental wellbeing and
depressive symptomatology at
start and end of the study, by
trajectory class
Loneliness
Trajectory Group
Depression
Positive Mental Wellbeing
T1
T4
T1
T4
Mean
S.D. Mean
S.D. Mean
S.D. Mean
S.D. Friendship Subscale
Average Increasing
5.27
4.43
3.81
3.99
2.21
0.71
2.82
0.48
High Decreasing
3.74
3.53
6.50
5.16
2.63
0.55
2.15
0.72
Low Increasing
8.80
5.04
9.03
4.54
1.82
0.80
1.95
0.61
Average Stable
5.61
3.60
5.60
4.30
2.28
0.46
2.35
0.53
High Stable
2.69
2.78
2.83
3.12
2.74
0.46
2.83
0.48
Isolation Subscale
High Decreasing
7.43
6.53
5.22
5.25
2.59
0.78
2.46
0.76
Low Increasing
4.40
4.40
8.86
6.75
2.54
0.62
2.47
0.91
Elevated Stable
6.25
3.77
6.30
4.43
2.22
0.53
2.30
0.61
Low Stable
3.11
2.97
3.40
3.43
2.64
0.52
2.67
0.56 Fig. Isolation loneliness For positive wellbeing, the same two Friendship related
loneliness trajectory groups again showed the largest
changes and these changes were of almost a full standard
deviation. Finally, for Isolation Loneliness, there were only
relatively small changes observed. Compared to the reference group, all other groups reported
significantly higher levels of depressive symptomatology. The biggest effect was observed when comparing the Low
Increasing trajectory group (i.e. Isolation Loneliness wor-
sening) where the model estimates a difference between
these groups of more than 5 points, a large effect given that
the scale only ranged from 0–20. As shown in Fig. 1, this
group finished with the highest loneliness scores. The other
two groups (High Decreasing and Elevated Stable) were
approximately 1.5 points higher than the Low Stable group. As before, sensitivity analyses confirmed that the classes
had very similar characteristics to the previously derived
classes (see Online Supplement 2). Descriptive statistics 2 Friendship loneliness
trajectories Table 3 Number of participants
in each loneliness trajectories for
both Friendship Loneliness and
Isolation Loneliness subscales Journal of Youth and Adolescence (2024) 53:1078–1090 1085 Table 5 Results of generalised
linear modelling analyses
investigating the relationship
between latent trajectories of
loneliness and measures of
wellbeinga Loneliness Trajectory
Group
/Covariate
Depression
Positive Mental Wellbeing
Estimateb
Sig
Estimateb
Sig
Friendship Subscale
Average Increasing
−0.16 (−1.25, 0.93)
0.771
0.13 (−0.02, 0.28)
0.095
High Decreasing
3.35 (2.69, 4.01)
<0.001
−0.64 (−0.73, −0.55)
<0.001
Low Increasing
4.46 (2.97, 5.95)
<0.001
−0.71 (−0.91, −0.52)
<0.001
Average Stable
1.84 (1.26, 2.43)
<0.001
−0.37 (−0.45, −0.29)
<0.001
High Stable
Reference category
Gender
Males
−0.86 (−1.31, −0.41)
<0.001
0.15 (0.09, 0.21)
<0.001
Females
Reference category
Age (years) at wave 1
0.13 (−0.01, 0.27)
0.078
−0.02 (−0.04, 0.01)
0.061
Depression at wave 1
0.33 (0.26, 0.40)
<0.001
Positive Mental Wellbeing at Wave 1
0.28 (0.22, 0.34)
<0.001
Isolation Subscale
High Decreasing
1.68 (0.29, 3.07)
0.018
−0.24 (−0.50, −0.00)
0.012
Low Increasing
5.33 (4.32, 6.35)
<0.001
−0.19 (−0.34, −0.04)
0.015
Elevated Stable
1.48 (0.85, 2.10)
<0.001
−0.18 (−0.27, −0.08)
<0.001
Low Stable
Reference category
Gender
Males
−0.84 (−1.29, −0.38)
<0.001
0.14 (0.07, 0.21)
0.035
Females
Reference category
Age (years) at wave 1
0.09 (−0.04, 0.24)
0.183
−0.03 (−0.05, −0.01)
0.015
Depression at wave 1
0.37 (0.30, 0.44)
<0.001
Positive Mental Wellbeing at Wave 1
0.35 (0.28, 0.41)
<0.001
aAge, Gender, and Baseline score on the relevant dependent variable were all controlled for as covariates
bEstimates are unstandardised and include confidence intervals aAge, Gender, and Baseline score on the relevant dependent variable were all controlled for as covariates
bEstimates are unstandardised and include confidence intervals symptomatology, but girls reported significantly more
symptoms of depression than boys (see Table 5 for results). Increasing groups showed most change and, even in the con-
text of high variation in scores, the Low Increasing group
increased by a full standard deviation. Associations with Depressive Symptomatology at T4
Controlling for T1 Depressive Symptomatology The Low Stable Isolation Loneliness group was used as the
reference group when estimating possible effects relating to
Isolation Loneliness, as this was the most frequently
occurring group (74.7% of youth). Similarly, when esti-
mating effects for Friendship Isolation, the High Stable
Friendship group was used as the reference group (54.5% of
youth). Across both analyses, age did not predict depressive Table 5 Results of generalised
linear modelling analyses
investigating the relationship
between latent trajectories of
loneliness and measures of
wellbeinga Friendship related loneliness The reference group reported significantly fewer symptoms
of depression that the High Decreasing Friendship Lone-
liness group, Low Increasing Friendship Loneliness group,
and Average Stable Friendship Loneliness group. However, Journal of Youth and Adolescence (2024) 53:1078–1090 1086 loneliness (Isolation loneliness and Friendship loneliness)
and considered the effects of these on both positive mental
wellbeing
and
symptoms of
depression. Expectations
relating to the number of trajectories (four or five), and the
form that trajectories would take, were largely confirmed. Identification of different numbers of trajectory groups for
each of the two different forms of loneliness is consistent
with a multidimensional conceptualisation of loneliness. Additionally, youth in different trajectory groups evidenced
clear differences in their mental health outcomes, support-
ing the argument that it is important to consider change and
development in loneliness. the reference group did not differ from the Average
Increasing Friendship Loneliness group. The biggest effect
was evident when comparing the Low Increasing Friend-
ship Loneliness group to the reference group, with an
estimated difference on final depressive symptomatology of
4.46 points. The High Decreasing Friendship Loneliness
group was also significantly higher than the High Stable
group by 3.35 points. Friendship related loneliness The High Decreasing, Low Increasing, and Average Stable
Friendship Loneliness groups all reported significantly lower
positive mental wellbeing than the reference group. These
reductions ranged from 0.37 to 0.71. However, the Average
Increasing group did not differ from the reference group. Since this is the first report of trajectories for two separate
loneliness factors, the ways in which these may co-occur was
documented. Four combinations were most prevalent: Low
Stable Isolation Loneliness with either High Stable Friendship
Loneliness (48%), High Decreasing Friendship Loneliness
(9%), or Average Stable Friendship Loneliness (15%), and
Elevated Stable Isolation Loneliness with Average Stable
Friendship Loneliness (10%). All other trajectory group com-
binations occur at a prevalence of under 3%, though there were
at least some young people in all 20 possible combination of
trajectories. These combinations speak to the need for theory to
consider the ways in which dimensions of loneliness may
interact to produce outcomes. Of particular interest here is the
possible disparity between desired and experienced levels of
loneliness (Cacioppo et al., 2015b; Qualter et al., 2013; Rook,
1984) as the existence of these combinations suggests that
youth may hold different beliefs about such a mismatch across
different factors of loneliness. Alternatively, it may be that one
or other dimension is associated with shorter, or fewer, fluc-
tuations over time, and experience sampling assessments (e.g.,
van Roekel et al., 2014) offer appropriate ways to assess such a Sensitivity analyses again provided results with aligned with
the main results reported above (see Online Supplement 2). Only one parameter was more than trivially different, and this
was still non-significantly different. Low Increasing friendship
subscale as a predictor or depression, co-efficient changed from
4.46 (2.97–5.95) to 2.52 (1.03–4.00). Associations with Positive Mental Wellbeing at T4
Controlling for T1 Positive Mental Wellbeing The same reference groups were again employed in ana-
lyses relating to positive wellbeing (see section 3.2). Across
both analyses, boys reported significantly higher levels of
positive wellbeing than girls, though only in the Isolation
Loneliness model did age negatively predict positive well-
being (see Table 3 for results). It was hypothesised that there would exist four or five
trajectories of loneliness. Four trajectories were found for
Isolation loneliness and five trajectories for Friendship related
loneliness. The largest trajectory group in both cases, and as
expected, is a consistently unproblematic one: 75% of youth
were in the Low Stable Isolation Loneliness trajectory groups
and 55% were in the High Stable Friendship Loneliness tra-
jectory (with a further 26% reporting Average Stable
Friendship Loneliness). However, contrary to expectation
(Ladd & Ettekal, 2013; Qualter et al., 2013; Schinka et al.,
2013; Vanhalst et al., 2013, 2015, 2018), there was not a
chronically high trajectory for Isolation loneliness, though in
line with expectations there was a group of adolescents who
reported consistently low levels of Friendship related lone-
liness. Given the relationships between trajectory membership
and adjustment, discussed below, these prevalence rates are
very encouraging because they indicate that most youth were
not experiencing problematic levels of loneliness and were
not therefore at risk for the problematic outcomes associated
with such membership. Isolation loneliness All three comparison groups reported significantly lower
levels of positive mental wellbeing at the end of the study as
compared to the reference group. Estimated reductions
ranged from 0.18 to 0.24 (on a scale of 1–5). Discussion There is a need to identify the outcomes of changes in
loneliness during adolescence, and to consider this within a
multidimensional framework of loneliness. When experi-
enced in adolescence, loneliness can impose long-term
distress and significant adverse outcomes in later years,
regardless of whether it recurs or persists over time (Mat-
thews et al., 2023). Therefore, the importance of interven-
tions to break the cycle of loneliness during the early years
is critical. To achieve this, the present study identified tra-
jectories of change in relation to two dimensions of Journal of Youth and Adolescence (2024) 53:1078–1090 1087 proposition in future. However, it is encouraging to see that the
most prevalent group is that which combines both of the least
problematic trajectories of the two forms of loneliness. wellbeing. This is the first research to establish a link between
trajectories of loneliness during adolescence and low levels of
positive mental health and extends results reported in correla-
tional studies (Houghton et al., 2016; Lyyra et al., 2021). While
the present results contrast with Houghton et al.’s (2022), the
unique context of the COVID-19 pandemic that took place
during that study may have had an impact on their results. Thus, interventions that can successfully tackle loneliness
(Eccles & Qualter (2021)) may usefully be used alongside, or
integrated into, existing interventions to enhance positive
mental wellbeing for young people (see Cilar et al., 2020). These results speak to the multidimensionality of lone-
liness (Goossens et al., 2009; Houghton et al., 2014;
Majorano et al., 2015). Different numbers of trajectory
classes for each type of loneliness is evidence in support of
a multidimensional conceptualisation. In addition, youth did
not always report “equivalent” trajectories on each measure
of loneliness as might be expected if the two scales were
simply mirror-images of one another. For example, only
23% of young people in the Low Increasing Isolation
Loneliness trajectory were also in the High Decreasing
Friendship Loneliness trajectory. Similarly, only 13% of
young people in the High Decreasing Isolation Loneliness
trajectory were also in the Low-Increasing Friendship
Loneliness trajectory. These results strengthen the case for
considering Friendship loneliness and Isolation loneliness
as distinctive dimensions, reflecting the importance of
considering both the quality of friendships and the degree to
which youth feel isolated from others. The results reported here may not generalise to adoles-
cents outside of Western Australia. Discussion In addition, a reliance on
self-reports may mean the results are subject to limitations
relating to mono-method approaches such as shared-method
variance. However, accurate and reliable reports of sub-
jective dispositions such as those which were the subject of
the current study can be difficult to obtain from third parties
such as teachers or parents (see Frick et al., 2009). The
number of trajectories for each of the two forms of lone-
liness that were investigated here may be sample-specific,
though one of the strengths of the study was the recruitment
of a large demographically representative sample, a long-
itudinal design with low levels of attrition, and the use of
state-of-the-art statistical techniques. A final limitation
relates to the collection of both loneliness and outcome data
at the same final time point, which may restrict causal
interpretations of these data. The benefits of having stable, low trajectories of loneliness
with respect to symptoms of depression have been reported in
previous research (e.g., Harris et al., 2013; Qualter et al., 2013;
Schinka et al., 2013; Vanhalst et al., 2013). This was echoed on
the current findings, with the Low Stable Isolation Loneliness
trajectory group reporting fewer symptoms of depression than
all other youth in this study. The difference was particularly
notable concerning other youth in the Low Increasing Isolation
loneliness trajectory group, whose levels of depressive symp-
tomatology increased by more than a standard deviation from
the start to the end of the study. The reasons underpinning why
young people experiencing increasing levels of loneliness
across adolescence are at particular risk of experiencing
symptoms of depression is unknown, but the disconnection
between desired and perceived relationships which is the core
of loneliness (e.g. Cacioppo et al., 2015b) may offer some
insight. Specifically, an increasing awareness of how far one’s
desires are from what one actually has, especially in the context
of having had it previously, may differentiate this group from
those who experience decreasing, or consistently high, levels of
Isolation Loneliness. Such a pattern of results could provide
support for a scar hypothesis (Rohde et al., 1990) pertaining to
loneliness. Future research should seek to interrogate this
possibility in more detail. Conclusion Loneliness can be best described as multidimensional dur-
ing adolescence, yet there is little understanding of the ways
in which different dimensions of loneliness develop during
this period. The results of this study imply that change and
stability in these dimensions are differentially associated
with fluctuations in symptoms of depression and positive
mental health across a 2-year period. Young people who
experienced low levels of Isolation Loneliness that subse-
quently increased, and those for whom their quality of
friendships
were
consistently
low
(despite
increases),
appear to be at particular risk for poor outcomes. Taken
together, it appears that being cognisant of the ways in
which loneliness is experienced over time is an important
additional consideration when seeking to influence young
people’s mental health. Novel findings reported here also indicated that specific
trajectories of loneliness during adolescence can negatively
impact positive mental wellbeing even after controlling for
earlier levels of that construct. Outcomes for positive mental
wellbeing were very similar to those reported above for
symptoms of depression, although the risk associated with the
Low Increasing Isolation loneliness trajectory group for
symptoms of depression was not apparent for positive mental Supplementary information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s10964-023-01925-0. Acknowledgements We are indebted to the Department of Education
(Western Australia) for providing access to schools. We acknowledge
Karen Martin of the University of Western Australia who contributed Journal of Youth and Adolescence (2024) 53:1078–1090 1088 Equation
Modeling:
A
Multidisciplinary
Journal,
21(3),
329–341. https://doi.org/10.1080/10705511.2014.915181. to the development of the study. Preregistration: This research project
was not preregistered. to the development of the study. Preregistration: This research project
was not preregistered. to the development of the study. Preregistration: This research project
was not preregistered. Australian Bureau of Statistics. Census of population and housing: socio-
economic index for areas (SEIFA), Australia, State Suburb, Indexes,
SEIFA 2011. http://www.abs.gov.au Accessed 15 Feb 2018. Authors’ Contributions S.H. conceptualised the study, developed the
instruments, coordinated and supervised data collection and drafted the
initial manuscript; D.L. conceptualised and conducted the data ana-
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