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219487736 | pes2o/s2orc | v3-fos-license | Improving the ability of entrepreneurs to use alternative learning models in the automotive field
Entrepreneurial activity is believed to be a tool to encourage economic growth and to solve other economic problems such as unemployment. Entrepreneurship has been widely applied in schools, especially in vocational schools that aim to produce graduates so they can work and be entrepreneurs. There are many learning models that exist in vocational schools at this time, but the learning model does not have an impact on students to have a soul or desire to become entrepreneurs. Therefore, we have conducted a literature review to explain an alternative learning model, which is a self-designed project learning model that is expected to trigger students to become entrepreneurs. A systematic mapping study is used as a basic method for conducting literature reviews. Various relevant journals, which are learning models that can enhance one’s entrepreneurship from various circles, were selected by researchers as preliminary data for a literature review. The various learning models that currently exist do not have enough impact to foster a sense of entrepreneurship in students, whereas learning in SMK should every productive learning must make students master and can make students have a sense of entrepreneurship when learning has ended. In this study describes a self-designed project learning model that will be applied in the automotive field in light vehicle engine tune-up subjects. This alternative learning model has been used by other studies, the researcher applies this alternative learning model in the field of machining that produces an impact on students, i.e. students can increase or make it have the desire to become entrepreneurs.
Introduction
Vocational High Schools (SMK) have a strategic role to organize education and training tailored to the needs of employment and entrepreneurship. Aiming to be a workforce, must have the knowledge, skills, and attitudes in accordance with the qualifications of the workforce [1]. To be able to work and compete in industry and entrepreneurship, vocational schools must have competencies, as well as the abilities needed to complete certain jobs in the world of work and some are related to these abilities. Competencies that are suitable for work in the industrial world are grouped in the Indonesian National Work Qualifications (KKNI) while recognition of these competencies can be carried out by the National Professional Certification Agency (BNSP) through its extension made by the Professional Certification Agency (LSP). Not only in Vocational Education, but will become the term "entrepreneurship" is now a daily keyword, because everyone is talking about it, both in a macro context or even an individual context. In the macro context, he opposed it as a driver of economic growth and other economic [2][3][4][5]. Educational institutions are under greater pressure to instill entrepreneurship in their various curriculums, accessible to all students, regardless of their discipline, including higher education institutions [6][7][8][9]. In the world of Vocational Education in Indonesia, entrepreneurship subjects so that students can actualize themselves in entrepreneurial relationships will teach entrepreneurship learning in Vocational Schools currently conducted using lecture, recitation, and reading textbook methods. Entrepreneurship education in vocational schools should be in accordance with the needs of industry, there is research that questions, entrepreneurial universities commercialize their knowledge and complete it in the process of transferring and utilizing technology. With so meaning that issued here is not just the school, but must be questioned by the school and the government [10].
Because entrepreneurship is believed to be the solution to solving the unemployment problem of SMK graduates, we must, therefore, find a way to create new entrepreneurs, right after or even before they graduate. In other words, we must produce more graduate entrepreneurs and to do that, higher education in general and entrepreneurship education, in particular, can help promote entrepreneurial activities among students [11,12]. The existence of entrepreneurship learning in schools is good to start introducing an entrepreneurial spirit early on even though the entrepreneurial learning model is still an Oriented text-book. The model is the implementation of learning by using the lecture method that is varied with the discussion method has not emphasized the students' thinking processes independently. Because in general the discussion was conducted in large classes that were still dominated by teachers, the material discussed was not in accordance with the context and moral issues that were developing in society, especially those related to entrepreneurship. There is a tendency for students to be merely listeners to the teacher's explanation or merely to complete the Student Worksheet (LKS). His condition became increasingly serious because educators lacked developing their learning material according to students' needs. Even though paying attention to students' interests, a teacher will be able to teach effectively. The impact of this should be thought about increasing entrepreneurial skills for vocational graduates through alternative learning models. There are so many learning models in schools today, but the management of the learning process in schools is still dominated by uniformity models, which pay less attention to students' cultural backgrounds so that boredom and boredom arise from students to learn them because they are only directed to memorizing them. This happens because all this time the material learned has not touched their needs. Therefore the concept of the self-design project learning model was initiated for vocational students so that they have the ability to work in relevant fields so that it is expected to overcome the problem of unemployment in Indonesia because through a study entitled "The Impact of Entrepreneurship Education on Entrepreneurial Outcomes." reported that entrepreneurship education had a positive impact on entrepreneurial production, where many graduates began to open their own businesses, which eventually grew into strong companies [11]. Studies also show that individuals with high entrepreneurial qualities will be active, flexible, able to adapt to the learning environment and able to see change as an opportunity [11,13].
Method
This research uses a systematic mapping study, which aims to help the authors carry out a systematic review. Systematic reviews are valuable data, where the authors summarize and analyze literature reviews that are relevant to the topic then compare them so that different researchers can use them as reference material for further research [14,15].
Strategy
This stage the authors choose articles related to learning models that can improve abilities or lead to a desire for entrepreneurship both within students and the general public. The search is done through a journal publisher database or proceeding, then some from internet blogs and books or theses, and dissertations. The search was conducted using the keywords "entrepreneurship", "learning model", "automotive", "learning model self-designed project". The search for this article was carried out randomly as needed, in 2004, and was vulnerable from 2007 to 2018. The next stage of the article was chosen according to the topic.
Inclusion and exclusion criteria
The inclusion and exclusion criteria will be explained in table 1. According to the exclusion criteria, articles that have fulfilled the requirements are selected, except those using languages other than English and Indonesian. Then a time less than 2004 was deleted. Seeing the appropriate article is done by looking at the appropriate title and abstract based on the relevance of the article related to learning that enhances entrepreneurship or activities that generate interest in entrepreneurship. In this stage, a total of 32 articles, books, the internet, and other sources in accordance with the inclusion criteria and relevant to this literature research. Table 1. Inclusion and exclusion criteria.
Data extraction and analysis
The final step, 42 full-text data which will contribute to this literature review for an overall review. A thorough data check to extract and summarize the information needed in order to answer the objectives of this research literature. Based on the information needed, we consider several classifications and criteria that fit the objectives. Based on the analysis of data extraction, we can achieve the best results and recommendations. Criteria are the author, year of publication, type of article, journal or conference, sample size, context, type of data, enhancement or growing sense of entrepreneurship. After reviewing and summarizing the articles collected.
Results and discussion
Many ways or methods are used to make students or the community have an entrepreneurial spirit, one method that gives good results to influence or add intentions to become entrepreneurs is higher, the selfefficacy method [16]. In schools, although entrepreneurship education does not teach students to pursue entrepreneurial careers, but to apply what they learn to their future work whether they are entrepreneurial or working in government or industrial agencies [17]. However, it is expected that by having the provision of entrepreneurship learning that has been carried out at school, especially on productive subjects tune up engines, students are expected to have an initial intention to entrepreneurship, at least it will reduce unemployment in Indonesia. In order to achieve this, learning in Vocational High Schools must use learning models that will trigger students to foster a sense of entrepreneurship, one of which is by using this Self Designed Project learning model because this learning model is related to entrepreneurship, as similar research reveals that entrepreneurship education is important for raises students' intention to start a business [12].
Self designed project learning model
Learning is the process of student interaction with educators and learning resources in a learning environment. Another opinion says that learning is a process carried out by individuals to obtain a new behavior change as a whole, as a result of the experience of the individual itself in interaction with his environment [18,19]. By doing so, it means that a student is expected to have a provision or ability after there is an interaction of learning in school, and that ability must be able to be applied by students in social life. Learning model is a term used to describe the implementation of the teaching and learning process from beginning to end. In the learning model already reflects the application of an approach, method, technique or tactic of learning as well. Other researchers with learning models are conceptual frameworks that describe systematic procedures for organizing learning experiences to achieve specific goals [20] accessed from a link [21]. From the understanding that has been explained, that the learning model must be able to make students achieve the success of learning itself. Models of learning in schools From the various learning models mentioned above, all learning models are good, but in reality, every teacher does not practice them correctly. If the teacher is good at using the learning model, students will prove successful in mastering the knowledge they learn. There are studies that reveal that research learning models that affect learning outcomes, show the ability of students who are quite significant for those who use modeling and those who do not. As research on the use of taking and give learning models can improve students' ability to understand teaching material quickly and accurately [23,24]. All learning models are good and are very useful for teaching if mastered by the teacher himself. However, the existing learning model does not have enough impact to foster a sense of entrepreneurship in students. Therefore, the Self Designed Project learning model is made for vocational teachers, especially those who teach productive subjects, one of which is about tune-up engine in light vehicle expertise program. The concept is the implementation of production-based learning one of which is the method in which students are invited to participate in production planning. Beginning student learning is introduced to tangible products taken from industry (existing engines in the industry) then students are taught how to plan maintenance or product repairs. The results of this planning are called learning outcomes (evident learning). Physical learning outcomes in the form of product planning documents that later the product must be implemented. The steps of the Self Designed Project learning model are as shown below.
Figure 1.
Steps for learning the self-designed project [19].
The self-designed project learning steps are made to make students have the desire to be entrepreneurs after getting tune-up engine lessons with the teacher using this self-designed project learning model. Before starting learning using this alternative learning model, students are initially given a pre-test on entrepreneurship, after that only learning uses this alternative learning model. This self-designed project learning model in its preparation must be done in a Group Discussion Forum (FGD) because it is for compiling entrepreneurial material related to the practice of tune-up engines. This activity involved teachers who were able to practice tune-up engines and entrepreneurial teachers and then involved professional certification bodies (LSP) and industry. The results of this FGD are as learning material when the two teachers will deliver the material in accordance with their subjects.
The design of the learning model developed is expected to be an alternative learning that can improve entrepreneurial abilities. The scenarios of this learning alternative are:
Design
3.1.1.1. Learning objectives. Improving student entrepreneurial competence in productive subjects in light vehicle engineering tune up engines through their own skills using workpieces and procedures according to industry standards.
Learning materials
Changes in learning management include 1) the rational need for learning conditions such as industry conditions, 2) a general description of work in industry, 3) an overview of the workforce of vocational graduates in industry, 4) a description of a junior technician, 5) a product evaluation system work in industry and 6) Discipline, work ethic and productivity. Ability to plan work and design products which include the compilation of 1) The importance of the work to be done 2) The function of work 3) examples of work in accordance with Industry SOP, 4) products, 5) Facilities/equipment, 6) The process of work (Work steps), 7) Cost budget plan 8) Target consumers, and 9) Implementation schedule. Work on the results of work planning including 1) working with machines, 2) doing occupational safety and health, 3) using appropriate tools and materials and 4) carrying out quality control measures.
Learning activities.
This learning model activity begins with preparations which include administration preparation, subject matter, workpiece preparation, work safety equipment preparation, and equipment preparation. The implementation of this model starts with preparation and continues with the next step, which are: Creating school conditions into working conditions in the industry, the teacher invites students to learn like working in the industry. Explain the steps to plan making work which includes the preparation of 1) The importance of the work to be done 2) The function of work 3) examples of work in accordance with Industry SOP, 4) products, 5) Facilities/equipment, 6) The process of work (Steps work), 7) Cost budget plan 8) Target consumers, and 9) Implementation schedule. Guiding students to work on projects that have been designed.
Implementation scheme.
Scheme of the implementation of alternative learning can be seen in the picture below:
Main activities
Preliminary stage.
Step 1, students act as workers accepting/choosing the type of product to be worked on. Workers inspect products that must be serviced. Core stage.
Step 2, workers make plans for product improvement including the preparation of 1) the state of the product to be repaired 2) The function of the product/service, 3) Materials that must be repaired, 4) Facilities/equipment, 5) The process of repair (Work steps), 6) Cost budget plan and 7) Implementation schedule.
Closing stage. The teacher is responsible for the entire learning program, observing and evaluating learning outcomes, learning processes and programs.
After the learning process uses this self-designed project learning model, the teacher will provide a posttest in the form of a questionnaire so that they can see the extent to which students' desire to become entrepreneur's increases. The self-designed project learning model has been proven to increase student entrepreneurship in research that uses the self-designed project learning model in the field of research revealed that, on average, students' personal entrepreneurship on every aspect has increased even though based on the International Management System is still in the medium category. Based on the results of the measurement, there are two components that still constrain students' progress in entrepreneurship, namely to bear the risk, and self-confidence [19]. According to research, giving spirit to become entrepreneur, is to do exercises in their fields so as to make a habitat in accordance with their fields and abilities [18]. Entrepreneurial ability is one factor to develop an entrepreneurial spirit, such as being independent, being brave to take risks, being able to seize opportunities, be creative and innovative. The same research revealed that at this stage students experience real activities as workers/entrepreneurs who are able to apply entrepreneurial materials that have been given previously. It is expected that students' entrepreneurial abilities will be better because they have experienced real activities compared to making previous work. The results of the study said, there was an increase in the competence of the experimental class group that fostered the formulation of integration material on the application of learning self-designed project learning [19].
Entrepreneurship
The word "entrepreneurship" comes from the 17th-century French Entrepreneur word "entreprendre" which means someone who is risking a new company. Looking at entrepreneurship in modern languages, saying it is the art or science of innovation and risk-taking for the sole purpose of generating profits in business [25]. Whereas, an entrepreneur is a person who assumes the main risk of creating additional wealth by making time equity and/or career commitment to provide value to a product or service [26]. The product or service may be new or different, but the value is added by an entrepreneur [27]. It has been proven that there is one study that states, entrepreneurship education has developed rapidly among universities in Taiwan. The number of universities offering relevant entrepreneurship courses has increased from 18 in 2003 to 89 in 2007, which is a 56% increase in the total number of universities. Meanwhile, the number of entrepreneurship programs has also grown rapidly from 102 in 2005 to 145 in 2007. In addition, in 2009, nine universities had opened modular entrepreneurship courses and brought the total to 550 relevant entrepreneurship courses offered in Taiwan [28]. In 2011, the number of technical universities that provided entrepreneurship education reached 45 and the number of relevant entrepreneurship courses grew to 110. Among them, 1/4 of the universities listed entrepreneurship programs as required credits [28][29][30]. The level of entrepreneurial ability in Indonesia is still low when compared to countries in the Asia Pacific region. The ratio between the number of entrepreneurs compared to the population of Indonesia is only 1:83, while the Philippines 1:66, Japan 1:25, even Korea is less than 20. Judging by the ratio of entrepreneurs internationally, the ideal ratio is 1:20 [31]. To reduce unemployment one way that can be done is to develop the spirit of entrepreneurship as early as possible. This is because a nation will advance if the number of entrepreneurs is at least 2% of the population. In 2010 Indonesia had around 400,000 entrepreneurs = 0.18% of the population. If a formula for 2% of the population is needed to achieve prosperity, then Indonesia must now have around 4,600,000 entrepreneurs [32].
Synthesized from all the scientists' opinions cited above, this study uses the definition of entrepreneurship as "the situation of school graduates, especially vocational schools currently in Indonesia, which are the most unemployed, with entrepreneurship learning believed to be able to reduce the unemployment rate in Indonesia and by student entrepreneurship is able to open their own business with the knowledge that has been learned at school. Therefore, the learning model at school must always be angels with entrepreneurship because that way students will get used to the entrepreneurial environment so that it is expected that students will want to become entrepreneurs in accordance with the relevant industrial world ".
Conclusions
Nowadays everyone must be demanded to have a creative and innovative person in the work both in the business world or in the industrial world. In the world of work, competition is increasingly fierce because of the incompatibility of industry needs with existing school graduates. Entrepreneurship is a solution to overcome this problem, many research findings have revealed that entrepreneurship education is important to reduce unemployment in various countries, especially developing countries. With the existence of entrepreneurship education or learning models based on increasing entrepreneurship in vocational schools such as self-designed project, learning models are expected to continue to be developed, so that it can have a positive impact and especially the success of the objectives of the vocational school itself. An alternative learning model, the self-designed project, has been applied in one of the studies and has been proven to provide students with an increase in entrepreneurship. Research that uses the selfdesigned project learning model has been applied to the machining expertise in vocational high schools, it is hoped that further research can apply it to different fields, one of which is automotive. | 2020-05-21T09:17:49.197Z | 2020-04-01T00:00:00.000 | {
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88697639 | pes2o/s2orc | v3-fos-license | First survey of the Passalidae ( Coleoptera , Scarabaeoidea ) species from Reserva Ecológica de Guapiaçu ( REGUA ) , Cachoeiras de Macacu , RJ , Brazil
We present the details of a survey with species of passalid conducted in the Reserva Ecológica de Guapiaçu (Cachoeiras de Macacu, Rio de Janeiro State) together with illustrations for each species and an identification key. The study includes material collected between May 2010 and October 2013. We identified 11 species in three genera and two tribes (Passalini and Proculini). Passalini comprised two genera, Passalus with six species, and Spasalus with one species, representing 71.42% of all the species encountered. Proculini was represented by only one genus Veturius, with four species, representing 28.57% of the species surveyed. Nine species were recorded for the first time from Cachoeiras de Macacu municipality.
INTRODUCTION
The family Passalidae is included in the superfamily Scarabaeoidea and contains about 1,000 species in two tribes (Passalini and Proculini), of which at least 50% have been identified in America (Fonseca and Reyes-Castillo 2004;Boucher 2006;Mattos and Mermudes 2014).Passalidae is a morphologically homogeneous group.Species of this family live in decaying trunks and members of the colony show subsocial behavior, with complex acoustic communication between adults and larvae (Schuster 1983;Mattos and Mermudes 2014).Furthermore, these species play an important role as primary decomposers in tropical forests and can act as possible conservation bioindicators in priority areas (Schuster 1984;Schuster et al. 2000;Fonseca and Reyes-Castillo 2004).However, only few studies on the systematics of this family have been conducted in the Atlantic Forest biome, which could be because of the small number of researchers in Brazil (Santos-Silva 2000; Mattos and Mermudes 2013;Mattos and Mermudes 2014).
The Atlantic Forest biome has great endemic species richness and actually represents only 7.5% of the original area (Myers et al. 2000).Thus, studies that contribute to the knowledge of biodiversity in the Atlantic Forest biome are highly relevant for conservation and management strategies.This study presents an inventory of passalid diversity in the Atlantic Forest area within the Reserva Ecológica de Guapiaçu (REGUA), and to our knowledge, is the first study of coleopteran species in the reserve.Thus, we expand the knowledge of the diversity of Passalidae in one of the last remaining conserved areas of the Atlantic Forest in the municipality of Cachoeiras de Macacu.
Study site
Reserva Ecológica de Guapiaçu (REGUA) is a nongovernmental organization that protects about 6,000 ha of the Atlantic Forest, in the municipality of Cachoeiras de Macacu (22°25ʹ09.9ʺS, 042°46ʹ13ʺ W), Rio de Janeiro state, Brazil (Figure 1).The main objective of REGUA is to conserve the remaining forests in the Guapiaçu valley in southeastern Brazil, and more than 80% of the land is part of the Parque Estadual dos Três Picos (Pimentel and Olmos 2011).
Data collection
Field collecting was conducted between May 2010 and October 2013 in the REGUA, and from all marked trails in the conservation unit.We considered the samples collected into cold (April to September) and hot (October to March) periods, totaling ten samples.The insects were collected manually after inspection of decaying fallen logs by using knives and axes.A four-member group conducted twice-daily sampling, 4 h each in the morning and afternoon.A sample set
Passalus (Passalus) denticollis
Material examined.Brazil: Rio de Janeiro, Cachoeiras de Macacu, Reserva Ecológica de Guapiaçu, 1 specimen, 11.VII.2012, 60 m, Silveira col., I. Mattos det. 2012;Trilha Amarela, 3 specimens, 14.V.2011, 8 m, Mermudes et al. col., I. Mattos det. 2011;Trilha da Schincariol, 2 specimens, 14.V.2011, 47 m, Mermudes et al. col., I. Mattos det. 2011;Trilha da Cerca, 1 specimen, 20-23.V.2011, 35 m, Mermudes et al. col., I. Mattos det. 2011;Trilha Marrom, 1 specimen, 14.V.2011, 63 m, Mermudes et al. col., I. Mattos det.2011; Trilha São comprised three consecutive collection days, totaling 24 h per sample set.Illustrations were made using a Leica MZ7.5 stereomicroscope fitted with a drawing tube.The sampling effort was assessed by verifying the accumulation curve of the species constructed with the observed richness and number of specimens collected in the study area.Passalid richness was estimated by three nonparametric estimators: the estimated richness obtained by Chao I is a function of the ratio of the number of observed species represented by a single individual (singletons) and the number of observed species represented by two individuals (doubletons).The other estimator used was ICE, which is a coverage estimator that focuses on species found in ≤ 10 sampling units; and Jack I employed the number of species that occur only a single sample, as based on the incidence (Magurran 2004).The analyses were carried by EstimateS Win8.20, with (Colwell 2006).All specimens were deposited in the Collection José Alfredo Pinheiro Dutra of the Universidade Federal do Rio de Janeiro.
RESULTS
The passalids that we found mainly represent a subset of those known from the more extensively surveyed Atlantic Forest as reported by Fonseca and Reye-Castillo (2004) who listed the passalids from Brazil, and more recently Mattos and Mermudes (2014) who listed Brazilian passalids from a continental Island in Rio de Janeiro state (Ilha Grande).We found eleven species of passalids reported by these authors from other localities (Fonseca and Reye-Castillo 2004;Mattos and Mermudes 2014).
In this study, we report eleven passalids species in three genera which belonged to two Neotropical tribes of the Passalinae (Table 1).The species accumulation curves were based on the three different estimators Chao I, Jack I and ICE reached an asymptote when all species have been observed to 10 samples.These results represent more than 85% of the richness estimate and is significant with the sampling effort applied (Figure 2).Dalman, 1817, Figure 8 Passalus (Pertinax) convexus Dalman, 1817: 333;Luederwaldt, 1931: 114;Fonseca & Reyes-Castillo, 2004: 15 (cat.).
Figure 2 . 6 3
Figure 2. The accumulation curve of Passalidae species.Sobs: Total number of species observed in all samples.Singletons: single individual or case of rare species.Doubletons: two individuals or abundant species.See Supplemental Materials on-line.
Table 1 .
Number of Passalidae species from the Rio de Janeiro (RJ) state and Reserva Ecológica de Guapiaçu (REGUA). | 2019-04-01T13:09:08.358Z | 2016-06-04T00:00:00.000 | {
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119423145 | pes2o/s2orc | v3-fos-license | On the thermocapillary motion of deformable droplets
In studies on Marangoni type motion of particles the surface tension is often approximated as a linear function of temperature. For deformable particles in a linear external temperature gradient far from the reference point this approximation yields a negative surface tension which is physically unrealistic. It is shown that H.Zhou and R.H.Davis J. Colloid Interface Sci., n.181,60,1996 presented calculation where the leading deformable drop moved into a region of negative surface tension. With respect numerical studies the restriction of the migration of two deformable drops is given in terms of the drift time.
the motion of the surrounding liquid by viscous traction. Then, the droplet or bubble will move in the direction of decreasing interfacial tension. It is necessary to note that the normal component of the capillary forces arising during the motion may deform the shape of a particle (2). Young, Goldstein and Block (1) and others have shown that in the limit of high surface tension (undeformed spherical particle) its motion is controlled by surface tension gradients only. Note that the motion of a deformable particle also depends on the surface tension itself.
If the particle moves with constant velocity the transformation of a laboratory coordinate system to a coordinate system moving with the particle frame will essentially simplify the solution (2), (3), (5). Let us denote the particle coordinate system moving with the droplet velocity U by O ′ and the laboratory coordinate system by O respectively. We consider the coordinate transform from O to O ′ in the case of a drop moving in the uniform external temperature gradient Ae x (2), see Fig.1. For an arbitrary point F we obtain, where i = 1, 2 correspond to the inner and outer liquid phase, respectively, V is the fluid velocity, T ′ denotes the difference between the temperature T in O and a undisturbed temperature AUt at the center of O ′ , R is a radius vector which points from O to F and t is the time.
In the limit of an infinitely large surface tension the normal stress boundary condition is not modified under the above transformation [8]. However, in the case of finite surface tension, this boundary condition requires special attention. Usually, γ is assumed to be linearly dependent on temperature or on concentration is linearized (6), where ∂γ/∂T is a constant and T 0 and γ 0 correspond to the reference values of temperature and surface tension, respectively. Note that for many cases ∂γ/∂T < 0. Due to the transformation of T the surface tension γ(R, T ) is also transformed in the moving coordinate system, The surface tension γ ′ is time dependent now. Recall that the surface tension must be positive (6), From [3] and [4] follows an upper bound of the drift distance Ut in system O or an upper bound of the time of particle migration in the moving system O ′ .
Ignoring the above restrictions results in the appearance of a negative surface tension in the course of the particle migration in finite time and thus may lead to a physically unrealistic behavior of the particle. This restriction is relaxed in the case of the undeformed drop (1), (4) and (3) where the normal stress boundary condition is always satisfied. However, this is not true when the surface tension has a finite value. We noted that in the literature on thermocapillary migration of drops and bubbles no attention was paid to this point. For example, Zhou and Davis (7) first considered the problem of axisymmetric thermocapillary migration of two deformable viscous drops . The authors assumed a linear dependence of surface tension on temperature. In terms of (7) we have where T (x s ) is the temperature at a point x s on the interface and T 0 (x r ) is a reference temperature. In an attempt to obtain a solution which is independent of the choice of (7). For more details see (2) and (4).
Zhou and Davis (7) give for the dimensionless surface tension in the moving coordinate system γ(x s ) = 1 − qT (x s ), [6] whereγ = γ/γ 0 is the dimensionless surface tension, q = aA(−∂γ/∂T ) is the dimensionless rate of change of the interfacial tension due to temperature variation, T (x s ) = (T (x s ) − T 0 (x r ))/(aA) is the dimensionless temperature difference and a is the radius of the first drop. It can readily be seen that Eq. [6] defines surface tension which is positive for any time or migration distances. As we showed before, the correct transformation of the linear approximation [5] leads to a negative surface tension in finite time. The previous conclusion that physically acceptable solutions must be restricted by migration time contradicts Eq. [6].
Let us derive the correct form of the transformed surface tension in terms of (7). The problem of the migration of two droplets is evolutionary and it must be accomplished by a kinematic condition applied on the droplets' surfaces. The transformation from the laboratory coordinate system to the particle coordinate system are given by (5): The migration velocity of the droplet now depends on time and therefore the migration distance Ut on the right hand side of [3] is given as an integral term, In terms of (7) we have for the dimensionless surface tension [10] The integral term in Eq.
[10] changes the scenario of a numerical calculation. The surface tension changes with time and it is necessary to keepγ positive.
We shall now proceed to estimate the time when the surface tension of some point x r on the leading drop will not satisfy [4]. For simplicity let us stay in the laboratory coordinate system, forγ = 0 we obtain the relation 1 qT (x s ) = 1, whereT (x s ) = (T (x s ) − T 0 (x 0 ))/(aA) and x 0 is a reference point in system O. It is readily seen that the dimensionless length of a spatial frame is given by X = 1 q . Then the maximum transformation distance of the leading drop is the difference between X and the initial position x r . For the case of equal material parameters considered by (7) we have a = 1 and the surface separation distance on the axes is ∼ 1. Hence the length of the drops' drift is also ∼ 1. Let us assume that the lower bound of the velocities for moving deformable drops is the velocity of non-deformable drops. For slightly unequal drops and a large separation distance between their centers the velocities are nearly the same and equal to the Young-Bratukhin value of 0.133.. Following Eq.
[12] in (7) we normalize this value with 2/15 because for inner and outer liquids the viscosity and the thermal diffusivity are equal. From this normalization procedure we obtain that the migration velocities are ∼ 1. As a result the critical value of the migration time is ∼ 1.
We developed a numerical code for solving the problem of the motion of two deformable viscous drops in an external temperature gradient (9). Restrictions [4] were considered in the laboratory coordinate system O. In Fig.2 we plotted the evolution of the minimum separation distance d between the droplets' surfaces in time. We chose a = 1, α = 0.5, q = 0.2 in terms of (7), where α is the droplets radii ratio. The dotted curve confines the physical region where [4] is satisfied. The curves 1 − 10 correspond to different initial separations. Curve 2 is in agreement with the results given by Zhou and Davis (see Fig.4 in (7)) and with the asymptotics for the non-deformable drops (8).
Our computations show that the patterns of drops deformations are similar to those described by (7) but correspond to smaller separation distances d. Note that our analysis is restricted by [4] while the results of (7) lie in the physically unrealistic region. For initially spherical drops and an initial separation distance d = 0.01 Fig.3 | 2019-04-14T03:11:59.414Z | 2001-02-10T00:00:00.000 | {
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16159799 | pes2o/s2orc | v3-fos-license | Gum Arabic as a Cause of Occupational Allergy
Background. Gum arabic is a potential sensitizer in food industry. Methods. We examined 11 candy factory workers referred to examinations due to respiratory and skin symptoms paying attention to exposure and sensitization to gum arabic. Skin tests, pulmonary function tests, and respiratory provocation tests were carried out as indicated by the symptoms and findings. Results. Occupational asthma, caused by gum arabic was diagnosed in 4/11 candy factory workers and two of them had also occupational contact urticaria and one had occupational rhinitis. One of them had oral symptoms associated with ingestion of products containing gum arabic. Conclusions. Airborne exposure to gum arabic may cause sensitization leading to allergic rhinitis, asthma, and urticaria.
Introduction
Gum arabic, or gum acacia is mainly derived from Acacia senegal tree. As a nontoxic material it is used as an emulsifier, a thickening agent, and as a stabilizer in foods, with E-code E414 [1]. It is useful in many kinds of foodstuffs because of its very low viscosity, complete solubility in water, and absence of any taste or odour. Due to technical properties gum arabic can be used in multiple applications like in pharmaceutical industry, lithography, and cosmetics.
Gum arabic is comprised of sugars and glucuronic acid residues in a long chain of galactosyl units with branched oligosaccharides attached to a polypeptide backbone. Protein content of gum arabic varies from 1 to 2%. IgE antibodies against polypeptide chains in gum arabic have been described to elicit asthma in occupational exposure [2]. Occasional cases of occupational asthma in printers [3], candy factory [4,5], and pharmaceutical industry workers [2] have been described. Although gum arabic is extensively used in food industry ingestion of it is a rare cause of immediate allergy symptoms [1].
We describe several cases of occupational asthma caused by gum arabic among candy factory workers.
Patients.
Eleven candy factory workers with respiratory and/or skin symptoms were referred to the Allergy Unit of Turku University Central Hospital (Table 1).
Workplace Description in the Candy
Factory. This Finnish candy factory was a major producer of multiple goodies in Finland since 1910. Gum arabic was an important ingredient in many candies. In making soft pastilles, gum arabic was dissolved in water as kibbles without making dust in the air. Hard boiled candies, instead, were covered with spray dried gum arabic inside a rotating drum. Dry, powdered gum arabic, packed in 25 kg bags, was poured by workers into the drums. Cornstarch, used to cover liquorice, also made dust in the air.
Working clothes were a short-sleeve jacket and/or T-shirt and pants. The workers, who had to do cleaning used rubber gloves. Other protective equipment was not required but respiratory masks were available.
These patients were referred during the last operation year of the factory. In the preceding year the production of [6]. The patients were defined to have bronchial hyperresponsiveness if the provocative dose of histamine diphosphate causing a 15% fall in FEV1 (PD15) was 1.6 mg or less. Serial peak expiratory flow measurements (PEF) were carried out in every two hours during the awaketime for a minimum of two weeks period at work and home including at least two periods of free days [7]. PEF record was considered compatible for occupational asthma if there was at least 20% diurnal variation in two working days and less variation in free days and suggestive if there was not over 20% variation but the variation was clearly higher on working days. PEF record was not compatible with occupational asthma if no clear difference were found between working and free days. The fraction of nitric oxide in the exhaled air (eNO) was measured with Niox Mino portable device according to the instructions of the manufacturer.
Specific Bronchial Provocation
Test. Specific bronchial provocation tests were performed in the Finnish Institute of Occupational Health in Helsinki with powdered gum arabic and by using lactose powder as a referent test. The provocation tests were done in a 8 m 3 challenge chamber according to international guidelines [8]. In both the active and the referent tests, the patient sat in the chamber for 30 minutes with the powder bowl in the front of her.
The powder was dispersed in the air with compressed air once in every one to five minutes. PEF and FEV1 values were monitored for 24 hours after each challenge test with a pocketsize spirometer (One Flow, Sti Medical, Saint-Romans, France). A >20% fall in FEV1 or PEF values was considered as significant. The patient was also followed for clinical symptoms and lung auscultation.
Skin Prick Tests (SPT)
. SPTs were carried out with commercial common environmental allergens including birch, grass and mugwort pollens, cat, dog and horse epithelium, house dust mite, molds and latex (ALK-Abelló A/S, Hosholm, Denmark and concerning birch and timothy from February 2006 to October 2006 Allergopharma, Reinbek, Germany), and with gum arabic (Caesar & Loretz GmbH D-40721) 1 : 10 (w : v) in physiologic saline using a commercial one-peak lancet and prick-prick method. Depending on the exposure in the working place own powdered gum arabic and cornstarch and carmine red colour, all moisturized with saline, were also tested with SPT. Histamine dihydrochloride 10 mg/mL (ALK-Abello) was used as a positive control and the diluent (Soluprick, ALK-Abello) as a negative control. The largest diameter and the diameter opposite to it were measured at 15 min. A reaction was interpreted as positive when the mean of the wheal diameters was at least 3 mm greater than that of the negative control.
Cutaneous Exposure Test.
Open cutaneous application test was done on a skin area about 5 cm in diameter on the volar surface of the arm. Gum arabic powder (5 g) moisturized with saline was applied and gently removed at 15 min for the reading of the reaction. In addition to erythema an appearance of one or more wheals was interpreted as a positive reaction.
Patch Tests.
Patch testing was carried out to one patient with eczematous skin disease according to standardized guidelines [9]. The allergens were derived from Chemotechnique (Vellinge, Sweden), and the application time was 48 hours. The final interpretation of the test reactions was done at 96 hours.
Serological
Tests. Gum arabic specific IgE (Immuno CAP f297, Phadia) was measured in patients with suspected occupational asthma.
Definition of Occupational Asthma.
The subject was defined to have occupational asthma due to gum arabic if the asthmatic symptoms worsened at the working place, there was positive skin prick test or specific IgE to gum arabic and a compatible PEF record with occupational asthma and/or positive challenge test. The aim was to confirm all cases by placebo-controlled bronchial challenge tests. One patient was not tested because her PEF recording was compatible with occupational asthma and she had strong oral symptoms of ingested gum arabic.
Diseases of Candy Factory Workers.
Six candy factory workers had occupational allergic disease (Table 1). Four patients had occupational asthma caused by gum arabic. Concomitant occupational contact urticaria was verified by the cutaneous exposure test in 2/4 of them and occupational rhinitis together with asthma in one of them in the specific bronchial provocation test. One other patient had occupational allergic contact dermatitis caused by thiuram chemicals. One patient had occupational urticaria caused by allergy to carmine red used in candies. There were no positive SPT reactions to cornstarch. No work-related allergies were found in five patients. Their symptoms were not clearly related to work and they were not sensitized to work-related allergens. One of them had atopic asthma, one had laryngitis probably caused by reflux disease and smoking and, one had rhinitis which was found not to be related to work. Two patients had chronic urticaria not related to work.
Occupational Asthma due to Gum Arabic in Four Candy
Factory Workers. The workers with occupational asthma had been doing the same work for 10 to 21 years (mean 14.8 years) and experienced symptoms of the respiratory tract and skin for 1.5 to 10 years (mean 5.1 years). The characteristics Table 2. Table 2. of four candy factory workers with occupational asthma due to gum arabic are seen in Table 2. Figure 1 presents the change in PEF and Figure 2 the change in FEV1 in the challenge tests with lactose (negative control), gum arabic 10%, and gum arabic 100% in patients 1, 2, and 4. Table 2) with occupational asthma started re-education. The three other patients with occupational asthma continued to work in the factory until the production ended a few months later. They avoided exposure to gum arabic. Six months after the work in the candy factory had ended patient 3 (in Table 2) was free of symptoms without asthma medication. She, however, experienced angioedema when ingesting gum arabic containing foods. In patients 1, 2, and 4 asthma was in control with medication, and they did not experience symptoms associated with gum arabic ingestion.
Outcome of the Patients with Occupational Diseases. Patient 1 (in
The patients with occupational skin diseases; caused by carmine red in one and by rubber chemicals in the other; were symptom-free when avoiding those allergens.
Discussion
Occupational asthma due to IgE-mediated allergy to gum arabic was diagnosed in four candy factory workers. Only one corresponding case has been reported in another Finnish candy factory [4], although the use of gum arabic in food industry is extensive [1]. Even though the occurrence of occupational allergy due to gum arabic is rare, it is possible that there is underreporting of the symptoms. In this study we did not survey the workers in the factory for sensitization to gum arabic and associated symptoms because of the approaching closure of the factory. We do not know whether more workers were sensitized to gum arabic and whether there were mildly symptomatic subjects who had not contacted a doctor.
Rhinitis is known to increase the risk of asthma by 3 to 5 times [10], and patients with occupational rhinitis have an increased risk of developing asthma [11]. Our patients had also rhinitis and skin symptoms. They had experienced symptoms for a variable, but rather long time before contacting the doctor. They probably did not contact the doctor until they had developed asthmatic symptoms.
All cases of occupational asthma in this factory were caused by gum arabic. In different candy factories carmine, pectin [12], milk, egg, nuts, seeds [13], spices, flavours, guar gum, and cornstarch may cause occupational allergy or nonspecific respiratory symptoms. In this study one worker, sensitized to carmine, was diagnosed to have work associated urticaria with minor respiratory symptoms. The workers who were diagnosed to have occupational asthma due to gum arabic had most symptoms at the working place when handling gum arabic powder.
SPTs with cornstarch were negative. Airborne cornstarch evidently caused mucosal irritation. We do not know whether exposure to cornstarch powder increased the symptoms due to gum arabic like airborne cornstarch seems to increase symptoms of latex allergy [14].
Latex must also be considered in candy factory workers as a cause of occupational urticaria and dermatitis, rhinitis, and asthma. In this study thiuram chemicals in rubber gloves had caused allergic contact dermatitis in one worker but all workers had a negative SPT to latex.
There is an exposure-response relationship between exposure to protein allergens such as wheat flour and alphaamylase in bakeries and the development of occupational sensitization and symptoms [15,16]. The production of this candy factory was concentrated on hard pastilles before closing the factory which increased the exposure to gum arabic. Increasing exposure to gum arabic probably caused the symptoms. The workers reported most symptoms in situations where exposure to gum arabic powder was the highest.
The sensitization route of these workers was the respiratory tract and/or the skin. Sensitization through the respiratory tract or skin to a food allergen may lead to the Journal of Allergy 5 subsequent development of symptoms during oral exposure as has been reported for sensitization to egg in bakery and confectionery workers [17], for lupine seeds in legume laboratory workers [18], for carmine in a worker engaged in dye manufacturing [19] and for fish [20]. Only one of these candy factory workers with occupational asthma reported oral symptoms associated with ingested gum arabic. Despite her asthma relieved after discontinuation of the exposure, the oral symptoms remained.
We have shown that gum arabic may cause occupational allergic rhinitis and asthma with urticaria symptoms. In this study the cases of occupational asthma in the candy factory appeared when there was an increase in the exposure. None of the patients had any previous atopic disease. Working methods which produce less powder and respiratory and skin protection are recommended. Early diagnosis of occupational allergy due to gum arabic is important in order to prevent the development of asthma. | 2014-10-01T00:00:00.000Z | 2011-05-19T00:00:00.000 | {
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202778152 | pes2o/s2orc | v3-fos-license | Off-Grid Systems
The term sustainability is frequently associated with our future cities. However, in the most remote and unprivileged corners of the world off-grid systems have already established a new dimension for sustainability. With no infrastructure nor grid connections, off-grid systems offer a way to be self-sustainable and thrive with the least resources. If dealt with proper planning and caution, off-grid systems can be beneficial on a large scale and can also contribute to local integrated networks. Off-grid systems exist in various forms such as electricity generation, bioenergy, off-grid housing, etc. As sustainability is of global interest the issue of off-grid is reviewed globally in remote locations of India and Jordan. The latest advancements in technology are considered. Social, economic, and environmental sustainability are central themes of consideration. This paper displays how off-grid systems are advantageous in terms of the basic needs of life e.g. shelter, energy, and food. Included are a wide array of crucial case studies in different continents. The main case studies include biogas plants, and solar electricity in India and Zaatari refugee camp in Jordan.
Introduction
The term sustainability can be misunderstood as to how far we are prepared to sacrifice our present needs and lifestyle in order to meet the demands of the future. However, sustainability can be achieved by meeting our needs today and keeping the promises of the future alive simultaneously. One of the most significant testimonies to this fact is off-grid systems. The idea has been implemented and verified. It has also been widely accepted by government organizations, local communities and as well as NGOs (Louie, Dauenhauer, Wilson, Zomers, & Mutale, 2014).
The popularity of off-grid systems has emerged from the imbalance between supply and demand. It is a noble approach towards development being independent of any initiative from higher or external authorities. In places where there is negligence from the government or development has not reached yet with all its fruits, off-grid is critical for self-help development. Off-grid provides scope for sustainability in all three aspects e.g.-social, economic, and environmental. Furthermore, it also helps to maintain a balance between them.
One of the world's biggest concern at present is sustainable energy production. The off-grid system offers possibilities in and not limited to electricity from solar power, hydropower and wind power systems, self-supply of water and sanitation, energy generation from biogas, food production, etc. Apart from these, there is a considerable amount of innovative systems which often go unnoticed due to the small scale or remoteness of the location. In the last thirty years, remarkable acceleration has been seen in the pace of off-grid improvements in the sunny southern hemisphere. Globalization has helped technology and innovation to spread across borders. The remote areas of India comprise one of the lower economical sections of the country. Therefore, the need to promote off-grid projects is essential to meet the demands of the people. The case studies mentioned here clearly state that the small scale off-grid projects are creating a difference in the lives of the people. If government support is given on a large scale then definite progress can be made on a vast scale.
In remote areas of India off-grid systems are used mostly in electricity generation and cooking. The previous uses of wood caused air pollution and health hazards from the cooking smoke. In many present off-grid systems utilization of charcoal improves the situation environmentally and health-wise. The biogas is used widely in domestic and community-scale in rural areas. The small-scale implementation off-grid systems in the villages in India is a recent but widely accepted phenomenon. It generates the opportunity for cheap production and social employability. Being a religious country India also witnesses waste to energy and solar systems in some major temples of the south.
Another important case is the discussion on refugee camps in Jordan. It deals with the whole lifecycle of the product of 10 years or more. The construction was started in 2011 and finished within 7 months. It has been able to provide all the facilities such as energy, water, etc. to the refugees. With no infrastructure and network in the desert, it has offered a home to 80,000 people with the help of a limited budget from the UN. The project developed through many steps like supplying water through reservoirs, installing solar panels, etc.
The need for energy in our society is ever increasing. We are having more awareness and usage of renewable energy resources. However, 85% of our energy consumption (Harvard, 2012) comes from burning fossil fuel (oil, coal, and gas). Off-grid is one of the newest and most innovative approaches in this area. The term off-the-grid (OTG) is referred to the system which is independent of the grid infrastructure such as electricity, water et al. The concept is becoming popular mainly for two reasons. Sustainability and independent energy generation. Research and application have included single houses to large communities, ranged from energy generation to distribution. Off-grid can be considered as an effective solution to meet the demand of our climate change goals.
The practical infeasibility and financial availability of grid extension into remote areas assign a remarkable contribution to the off-grid energy generation (Sen & Bhattacharya, 2014). Roughly, the cost of electricity generation depends upon the efficiency of the off-grid model. A third of increased demand in world energy comes from India (International Energy Agency, 2017). In India, HOMER or Hybrid Optimization Model for Electric Renewables is a limited and popular field of research. In many parts of the country, the Integrated Energy Renewal System (IERS) is suggested (Kanase-Patil et al., 2010) to meet the demand for electricity and cooking. India is large as a continent consisting of remote areas where till date energy from the traditional grid has not been made available.
In other developing countries the usage of the off-grid model is popular especially in electricity generation and cooking. Electricity is one-third part of energy consumption. Others are transportation and cooking etc. In southern Asia observations on off-grid has shown a mixed trend (Palit & Chaurey, 2011) in various countries. The bridging of the financial gap between affordability and electricity cost (Mainali & Silveira, 2011) can be addressed by off-grid. Solar Energy is the most popular in all off-grid systems. It includes various applications such as solar lighting system, heating system, lanterns, cooking, etc. In climates like India where there is an abundance of solar power, it is not difficult to understand the wide implementation of PV based off-grid systems. In the numerous remote locations in India solar off-grid lighting systems (SOLS) i.e. Solar Lanterns (SLs) and Solar home lighting systems (SHS) (Choragudi, 2013) are now possible. These systems provide the opportunity to have lighting systems without the cost of electricity production in remote areas of the Indian subcontinent.
However, the scope of off-grid in developing regions is often limited (Bhattacharya, 2012). Major issues being a lack of resources, infrastructure, and poor technology. From the electricity generation to storage the off-grid model demands efficiency. There is a considerable amount of scope in improvement in off-grid systems.
Off-Grid Housing -Electricity generation a major factor Introduction
Around 1.6 billion people in the world will be forced to live without electricity, not because of the non-availability but due to the dispersed population living in the off-grid area. Therefore the concern of producing electricity in these areas become a relevant area of discussion (www.world statistics.com).
The major source of energy production in remote areas is from fossil fuels. The energy produced by this is not efficient enough to cater to the needs of the population and also this produces a lot of carbon dioxide (CO2) and Carbon Monoxide (CO) resulting in an increase in the greenhouse effect. This definitely provides a motivation to look for alternatives that do not emit green-houses gases. The challenge is to find a solution for the off-grid areas without disturbing the ecosystem.
Remote Areas of India
There are approximately 649,481 villages in India (List of villages in India, 2018). Although there is a universal electrification policy adopted by the Indian government still, one-third of the populated areas are without electrification. The census states that 93 % of urban households and 55% of the rural household currently receive electricity (India Village Directory). Therefore India needs an alternative solution to cater to this problem. Looking towards the possible options to provide electrification to such a vast country with almost 1.4 billion people, off-grid housing definitely stands out to be the best possible solution.
Off-Grid Projects Bioenergy Gasifier
"Biomass is an industry term for getting energy by burning wood, and other organic matter. Burning biomass releases carbon emissions but has been classed as renewable energy in the European Union and United Nations legal frameworks, because plant stocks can be replaced with new growth" (Page, 2016).
Biomass has a high potential energy source in India, it enjoys great support from the state and the Centre. Currently, India is a leading country in Bioenergy. Gosaba Island gasifier is an epitome of how biomass energy can be utilized to change the lives of people in remote areas in India.
Case: Gosaba Island Gasifier
Gosaba Island is an island in West Bengal's Sundarban region and was deprived of electricity as it was not economically feasible to extend power from the grid to these widespread islands. West Bengal Renewable Energy Development Agency (WBREDA) took the initiative to provide electricity to the island. The Vadodara based Ankur Scientific energy technology in collaboration with WBREDA opened a biomass plant in the village. The island that was once lit with kerosene lamps gave way to the electric bulbs and soon the island became a small town.
Case: Turning Destructive Pine to Productive gas; Kumaon Valley
AVANI-a voluntary organization helped the people of Kumaon valley in Uttarakhand to produce electricity by the use of pine needles. The organization aims to produce about 14.65MW of electricity from the biomass (Avani, 2014). The state has the opportunity to electrify the area through the gasifier which is more accessible and cheap.
The gasifier volatize the pine needles into a producer gas which is a combination of the combustion gas. The gas is passed through the filters consisting of the sawdust and fine cloth to remove the impurities from it, the resultant gas is used to run the diesel engine which generates electricity.
Benefits from the Gasifier
The pine based gasifier guarantees to benefit the common household kitchen by allowing them to use charcoal effectively as it is one of the byproducts (10% of the residue) of the gasifier, this allows for a smoke-free household eliminating pollution and lung diseases. Charcoal produced by the 120 KW gasifier is sufficient to meet the household demands of at least 100 homes in the area. The villagers can pay for the charcoal or can collect the pine needles and get it in exchange. This project aims at increasing the employment growth in the area and as well as providing them with electricity and also a smoke-free alternative to cooking food. The efficient charcoal is also considered as a substitute for the LPG gas which is very expensive and limited in India. Therefore the project actually aims towards the off-grid solution leading towards sustainability. According to the estimates, the pine gasifier has the potential to generate electricity in the whole Himalayan region thus meeting the need of 1.4 families (Avani 2014).
Case: Hybrid Vermicompost Biodigesters
Karnataka is a developed state of India. Bengaluru, the capital of Karnataka is known as the silicon valley of India. However, the rural areas of this state are still deprived of modern technology and survive on open-fire cooking, which is a threat to the environment. The women generally collect wood from the forests to support their traditional cooking. SKG Sangha (SKGS), a non-profit organization of Kolar district of Karnataka has pledged to provide safe and clean cooking gas to the people of this district and also generate a source of income by selling the fertilizers made from the biogas as a byproduct. The biogas is prepared by cow dung, it consists of an underground brick-built digester, an inlet at the ground level to feed the digester with new feedstock and two separate outlets to collect biogas and to remove the residue" (Sangha, S 2013). The galvanized steel and HDPE pipes are used to transport the gas from the plant to the kitchen stoves.
The biogas has the capacity of producing up to 4 3 gas/day from an input of 50-100 kg cow dung. The liquid residue produced from the plants accounts for 36 to 72 tons and can be used directly in the fields but to transform the liquid fertilizer to the organic matter SKGS use the Vermiculture technique. This technique involves the storing of the liquid residue into a compost and then athe ddition of fibrous materials to decompose it for at least 3 weeks. After the decomposition is completed, the earthworms are added to the mixture. The top layer of the worm casts are used as vermicompost (Sangha, S 2013). Benefits of the Project: • Smoke-free household generated decreasing the respiratory problems for women. • Saves a lot of time for women as they search for wood is a tiresome activity. • Generates employment for the people of rural areas. • The vermicompost replaces the chemical fertilizers • Increases the soil fertility and the water retention of the soil.
Case: Jatropha plant, an emerging option for rural electrification
'Ranidhera' village in Chhattisgarh is one of the electricity-deprived villages. The electricity was generated after the 'Winrock International India' started an innovative project of producing electricity by the extraction of biofuel from the seeds of the Jatropha plant. The project was collaboratively supported by the British High Commission, the Swiss Agency for Development and Cooperation and the Ministry of New and Renewable Energy. The aim of the project was to electrify around 6000 remote areas of the country.
Case: Tirupathi, The Green Temple
'Tirumala Tirupathi Devasthanam' is the richest temple in the world. It has the largest number of devotees visiting on a single day. The temple is located in the southern part of India in Andhra Pradesh and has been trying to adopt renewable sources of energy for meeting its everyday requirements. The temple has a wastewater treatment plant that recycles and purifies the water. The canteen of the temple provides free mineral water to discourage plastic bottles and they also use solar energy for cooking. The solar cooker was installed in 2002 and has the capacity to prepare 50,000 kg of rice along with curry for 15000 persons in one cycle. The solar technology is installed by the Gujrat based company 'Gadhia Solar' and the HTT Gmbh. of Germany (Gadhia Solar).
The installation cost of the system is around 11 million rupees. The expenses were covered by the temple as well as the Union Ministry of Non-Conventional Energy sources. The solar cooker needs no modification and has a lifespan of 25 years.
Introduction
Politics play a major role in today's communities, and lately, the world has been facing critical situations and wars, unfortunately, all of that creates instability in the existing communities where conflicts occur, resulting in large populations forced to immigrate to safer areas and build their new communities. One of the latest conflicts is in Syria in 2011, which so far has forced millions to evacuate their homes and become homeless or refugees. Many refugee's camps have been established in remote areas to host those people and try to secure basic life needs for them, but with lack of resources, infrastructure, and communications it was a huge challenge; water supply, food production, energy, waste treatment, medication, education and others in the built-up camps (Fisher, 2016).
Zaatari Camp in Jordan is one of the largest camps for Syrian refugees around the world which was established in 2012 covering an area of 5.3 sq.km in northern Jordan, near to Al Mafraq which is a desert area with lack of water resources, food supply, and energy. The challenge was to secure these needs for more than 80,000 refugees. The population is growing, at least for the next 15 years, with some refugees in the camp for a lifetime, no temporary solutions would be practical or even acceptable. Off-grid housing is a convincing approach in such situations, a need to develop a self-standing sustainable community to host all these people for years. Tents were pitched as a place for them to live in temporarily which will be replaced by better housing options later on, such as shelter programs supplying donated caravans, cabins & built-up units (Fisher, 2016 Refugees (UNHCR), with an average of 80 births per week, while statistics show that 19.9 % of the population are children below 5 years' old who need intensive care (Fisher, 2016).
Water Supply
Water is essential for any living creature and it is one of the main needs for humans, without water or even lack of it no life can be assured, so organizations along with the Jordanian government had to implement a plan to secure water supply for the camp. As an initial temporary plan water was supplied by trucks to the camp, although It was expensive and time-consuming and can be delayed or interrupted but it was the only solution at that time till the drilling of the two new internal boreholes near the camp are completed provides 3.2 million liters of drinking water per day (UNHCR, reliefweb, 2017). Also work is ongoing to improve the water supply in villages surrounding Zaatari, which will benefit both Syrian refugees and host communities. In addition to some expansions in MAFRAQ pipeline which feeds the nearest city to the camp will benefit 25,000 Syrian refugees and local residents (Bhai 2018).
The results and goals of these projects are that "People living here will get double the amount of water, better pressure and more reliable. The work we're doing here will not only help the Syrian refugees in the camp but also serve the people in the surrounding communities," says Mr. Hameed. Water distribution presently takes place via a fleet of approximate 82 trucks delivering water to the communal public and private water tanks (Bhai, 2018).
Energy
Electricity as a power supply is one more requirement to establish a new residential area, provides an essential lifeline for camp residents, from lighting shelters to preserving food and maintaining hygiene, "Light is absolutely essential," said Anne-Marie Grey, the executive director and CEO of USA for UNHCR, in an interview. "When you go into a camp, you realize how it's a safety issue as much as a right to light or a right to energy issue" (UNHCR 2017). As stated before, in the case of Zaatari camp the challenge was to provide enough power to supply all population in that remoted area, using generators and some connections from the local network was defiantly a temporary solution; as it was costly and insufficient; especially with a monthly bill of 800,000 $ (Pyper 2015), so UN started considering the idea of constructing a renewable power supply project to serve the area's needs.
The UNHCR's three-year Energy Strategy 2015-2018 plan was to build a solar plant which will be the largest ever built in a refugee camp, providing clean and much-needed power to the Syrian refugees in the camp. According to UNHCR "The plant will reduce annual carbon dioxide emissions from the camp by 13,000 metric tons per year, equivalent to 30,000 barrels of oil. It will also deliver annual savings of around US$5.5 million, which UNHCR -the UN Refugee Agencywill be able to reinvest in vital humanitarian assistance" (UNHCR 2017). (Luck, 2017) Briefly, The 40,000 photovoltaic panel solar plant was constructed on the outskirts of the camp with an approximate area of 0.8 square km, arranged in rows hundreds of meters. According to UNHCR "the 12.9-megawatt peak solar photovoltaic plant was funded by the Government of Germany with a cost of 15 million euros (US$ 17.5 million)" and will be operational by the end of 2017 (Luck 2017).
Community and Social Services
The Zaatari camp hospital services include primary health care, maternity care, dental care, mental health, and nutritional care. Community centers provide psychosocial support & recreational activities (UNHCR, reliefweb, Feb 2017).
The Zaatari Camp schools have over 20,000 school-aged children enrolled. Textbooks and school supplies are distributed to all children in the camp. (UNHCR 2014).
Waste Management
Waste management considers two main categories; wastewater and solid waste, in order to maintain a healthy hygienic life in the camp it is necessary to develop a plan to control, dispose 10 and even recycle general waste. According to UNHR approximately 2,100m3 of sludge is collected by desludging trucks every day; regularly 28-50 tons of unsegregated solid waste is collected every day. A wastewater treatment plant was established to treat approximately 80% of the wastewater generated in the camp. Currently, all houses are connected to septic tanks, which means that desludging trucks make the rounds of the camp every day to empty septic tanks. In order to increase the efficiency of the camp's sanitation system, the next step, as from next year, will be to connect all the septic tanks to the main pipeline that will take all the waste to the treatment plant. (Aljaradin 2016). For solid waste, some efforts have been done so far, it is collected by trucks and transferred to external solid waste facilities, in order to consider recycling an approach to start separating the solid waste; plastic, paper, cardboard, glass & metal has been taking place (Saidan M, 2016).
Turn Waste into Energy
An approach to turn food and animal wastes into clean and safe fuel and fertilizer, although eliminating pathogens from the biowaste is a challenge due to it's potential to cause illness and attract disease-carrying animals, it has been possible by involving educated biogas technicians and closed-loop farmers. This will provide energy and even a chance to residents for learning, sharing and implementing clean and renewable energy solutions within Zaatari Refugee Camp. Studies proved that this approach will reduce fossil fuel usage and indoor air pollution by properly managing food waste (Initiative, 2016).
Conclusion
Unfortunately, the off-grid systems are sluggish and unable to meet the demand of the rapidly growing energy need (Louie, Dauenhauer, Wilson, Zomers, & Mutale, 2014). The scale of these is also tiny compared to Cities. We need more ideas, innovation, attention, knowledge, and attention to make these systems beneficial for our future 'Sustainable cities'. The small-scale implementation of off-grid systems in rural areas of India brings much hope but is not sufficient to cater to the massive demand for energy of growing India. The government needs to come up with innovative measures to accommodate the benefits of these systems into our future cities. The economies of scale of the systems have to grow critically to be comparable to cities. Technological advancement should be adopted to integrate the on-going systems to the main grid for achieving the optimum sustainable outcomes.
The refugee camp is a unique example of off-grid systems. However, this model is crucial to study as a remedy for crisis (Natural or political) situation of the cities. It shows a quick response in developing a sustainable community which can be an effective lesson dealing with natural disasters in cities. However, one has to think about larger case solutions more apt for a sustainable city. The situation is highly undesired as a permanent or even a temporary state in any city. | 2019-09-17T02:40:24.820Z | 2019-09-02T00:00:00.000 | {
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252646416 | pes2o/s2orc | v3-fos-license | The Retrograde Memory for News Events Test (RM-NET) and the relationship between news event memory and performance on standard neuropsychological tests
Novel tests of semantic memory (SM)—for example, memory for news events (NE; news facts) or famous personalities—are useful for estimating the severity of retrograde amnesia. Individuals with mild cognitive impairment exhibit relatively intact SM/language on traditional neuropsychological tests but exhibit consistent impairment on novel tests of SM, suggesting novel SM tests are dissimilar from traditional SM tests. To identify the relationship between NE memory and traditional cognitive measures, older adults (N = 51) completed a traditional neuropsychological battery and the Retrograde Memory News Events Test (RM-NET; a new test that robustly measures NE memory across the adult life span with high temporal resolution), and the relationship between performance on these tests was examined. Total RM-NET scores were more closely aligned with episodic memory scores than SM scores. The strength of the association between NE scores and episodic memory scores decreased as the age of NE memory increased. Tests of news events appear to reflect performance on traditional tests of episodic memory rather than SM, especially when recent news events are tested.
Traditional neuropsychological tests are used to diagnose or characterize the nature of cognitive impairments in individuals, such as those with Alzheimer's disease, frontotemporal dementia, mild cognitive impairment (MCI), Huntington's disease, Korsakoff syndrome, or epilepsy.These tests tap into abilities that reflect different domains of cognition, such as episodic memory (EM), semantic memory (SM)/language, attention/processing speed, and executive functions.Many of these patient groups also exhibit impairment on novel tests of SM, such as news event memory or memory for famous personalities (Sanders and Warrington 1971;Kopelman 1989;Sadek et al. 2004;Milton et al. 2010;Irish et al. 2012;Smith 2014;Langlois et al. 2016).
Novel tests of SM, such as tests of notable news events or famous personalities, may detect impairment better than traditional neuropsychological tests of SM (Venneri et al. 2016).For example, individuals with MCI who do not typically exhibit impairment on traditional semantic tests (e.g., tests of language, category and letter fluency, and object naming) are consistently impaired on tests of news event memory or memory for famous personalities (Flicker et al. 1987;Murphy et al. 2008;Leyhe et al. 2009aLeyhe et al. , 2010;;Seidenberg et al. 2009;Irish et al. 2010;Barbeau et al. 2012;Thomann et al. 2012;Smith 2014).Although these novel semantic tests reveal consistent impairment, it is unclear how performance on these tests relates to performance on the wider array of neuro-psychological tests used to identify cognitively impaired individuals in the clinic.Therefore, these novel measures merit broader examination to identify which cognitive domains they reflect and whether they reflect new information about SM not measured by the traditional tests used clinically to identify cognitive impairment.
One way to understand the relationship between novel SM tests and traditional EM tests is to examine the brain structures that support performance on the two types of tests.By definition, news event memory is SM because it reflects memory for facts and knowledge about the world.Accordingly, like other fact memory, memory for news facts is impaired by damage to lateral temporal cortices (Bayley et al. 2005;Gilboa et al. 2005;Bright et al. 2006).Like other facts, news facts also typically lose information about the context in which they were learned (i.e., the time and place of the learning event).Therefore, according to the standard concepts described by Tulving (1983), news facts do not represent medial temporal lobe (MTL)-dependent EM because they do not retain information about the context of the learning event.Indeed, theories of memory consolidation agree that retrieval of recent, but not remote, SM depends on structures in the MTL (Marr 1971;Squire and Alvarez 1995;Nadel et al. 2000;Moscovitch et al. 2005).Thus, recent news event memory scores are additionally dependent on the MTL, which could drive an association between performance on news event tests and performance on other MTL-dependent tests (e.g., traditional EM tests of new learning).
Although SM and EM have distinct definitions and phenomenology (Tulving 1983), more contemporary theories suggest that EM and SM are more likely part of a continuum rather than distinct entities (Renoult et al. 2012(Renoult et al. , 2019;;Irish and Vatansever 2020).Indeed, the neural substrates of EM and SM retrieval are highly overlapping (Dede and Smith 2016;Renoult et al. 2019;Irish and Vatansever 2020).Specifically, a common network is thought to support memory retrieval (parahippocampal cortex, middle temporal gyrus, ventral-lateral parietal cortex, and midline prefrontal and parietal regions), but depending on the retrieval requirements/memory content, the network differs in the degree that it involves regions more specific to EM (hippocampus) or more specific to SM (anterior temporal lobe and inferior frontal gyrus) (Renoult et al. 2019).This work is in line with the earlier idea that memory retrieval in everyday life involves dynamic interplay between these two types of memory (Tulving 1983).Consistent with these ideas, there is a continuum of SM that depends on the characteristics of the memory itself.Traditional SM tests assess memory for knowledge learned long ago (e.g., object knowledge and language), and this type of information is re-encountered frequently across the life span (common words, objects, and language use).In contrast, the novel SM tests most sensitive to mild brain injury reflect knowledge learned more recently and not reencountered frequently (e.g., news events from the recent past that received limited news coverage).In this framework, novel SM tests likely tap into a mixture of EM and SM that better captures the memory retrieval network affected in mild cognitive impairment.
The relationship between novel measures of SM and performance on traditional neuropsychological tests has not been comprehensively examined.Across the five studies that have examined these relationships, there was variability in which domains were examined and the types of novel semantic tests used.As a whole, there were few consistent findings.Leyhe et al. (2010) used a 20-item test for news events from the previous 60 yr and found that mean performance across MCI and AD groups was significantly correlated with performance on a global measure of cognition (Mini-Mental State Examination [MMSE]) but was not correlated with individual tests of attention/processing speed or executive function (other domains were not examined).Johnson and Klingler (1976) used a 70-item test for news events from the previous 30 yr and found that performance for individuals with normal cognition (ranging from young to older adults) was significantly correlated with performance averaged across several standard tests of verbal and nonverbal EM (other domains were not examined).Seidenberg et al. (2009) used a test of famous personalities who came to prominence in the recent past (10 items) or remote past (10 items) and found that the number of semantic details reported by individuals with normal cognition or MCI was also significantly correlated with traditional measures of verbal EM (other domains were not tested).De Simone et al. (2020) also found significant correlations with measures of EM and executive function for their sixitem recall and recognition test of news events spanning 45 yr.However, when Langlois et al. (2016) examined individuals with MCI or AD using a 40-item test for news events from the previous 45 yr, they did not detect a significant relationship with standard measures of verbal EM.Instead, they found significant correlations between news event memory and individual measures of SM (pyramids and palm trees test and the information subscale from the WAIS; other domains were not examined).
Only two of these studies examined these relationships as a function of the age of memory (Seidenberg et al. 2009;Leyhe et al. 2010).Measures of global cognition (Leyhe et al. 2010) and verbal EM (Seidenberg et al. 2009) were significantly correlated with novel measures of SM from both recent (1-to 10-yr-old) and remote (40-to 55-yr-old) time periods.
Thus, when identifying relationships with neuropsychological tests, there is variability in the types of novel SM tests used, in the number of time periods examined, and in the number of test items used to estimate retrograde memory.These relationships have never been examined systematically with a novel SM test that has high temporal resolution across the entire adult life span and where performance for each time period is estimated by more than a handful of questions.In addition, there has been no comprehensive analysis of these associations using robust estimates of five standard cognitive domains.Typically, a single neuropsychological test was used to estimate ability for a cognitive domain, even though using more than one test per domain increases the reliability of assessing a cognitive domain (Anastasi and Urbina 1997;Palmer et al. 1998;Loewenstein et al. 2007).
We describe the creation of a novel SM test of notable news facts (Retrograde Memory News Events Test [RM-NET]) that spans the entire adult life span (70 yr, separated into 3-to 5-yr time periods) (see Table 1).We examined behavioral findings from the RM-NET that reflect different elements of news event memory (e.g., accuracy, confidence, and response times).We also examined measures obtained from the RM-NET posttest that reflect incidental encoding during the RM-NET for events from the last 30 yr (see Table 2), as well as subjective reports of the amount of semantic knowledge available and the presence of concomitant autobiographical memories associated with the news event.Because of the relatively separate traditions of examining clinical populations using news events tests or standard neuropsychological tests, we asked how performance on the RM-NET relates to performance on traditional neuropsychological tests.Based on prior findings, we hypothesized that RM-NET memory accuracy would significantly predict performance on measures of EM.Furthermore, because news event memory is fact memory (i.e., a type of SM), we also predicted that RM-NET memory accuracy would significantly predict performance on measures of SM.Therefore, we tested how performance on the RM-NET was related to performance on standard neuropsychological tests in five cognitive domains: EM, SM/language, executive function, attention/processing speed, and visuospatial function.Because there appears to be a continuum of memory retrieval spanning EM to SM, another goal was to evaluate the relative effect sizes between RM-NET memory accuracy and these two domains.To obtain reliable estimates of ability for each domain of cognition, four to seven measures were used to estimate each domain.Finally, due to the dependence of recent news event memory and EM on the MTL, we hypothesized that the strength of the relationship between RM-NET memory accuracy and EM would decrease as memory age increased.
Effects of covariates on variables of interest
The covariates are described in the Materials and Methods in "Identifying Relevant Covariates for Primary Analyses."Table 3 shows descriptive information for covariates and variables of interest, and Supplemental Table S1 shows significance values when using covariates to predict variables of interest.The RM-NET interval (the relative number of days that elapsed between when the first participant was tested and when each subsequent participant was tested) (see the Materials and Methods) significantly predicted RM-NET total accuracy, total confidence, and total response times.
In addition, sex significantly predicted RM-NET total response times.These covariates were included in analyses of RM-NET components.Sex, education, total medical health burden, and total mental health burden significantly predicted one or more of the cognitive domain composite scores.None of the other covariates significantly predicted any of the variables of interest.In order to make the regression models comparable, all these significant covariates were included in the primary analyses.
Descriptive information about the RM-NET components and the RM-NET posttest components
Descriptive information about the sample, cognitive domain composite scores, and components of the RM-NET appear in Table 3.
Estimated marginal means and SEM (corrected for covariates) for RM-NET accuracy, confidence, and response times appear in Figure 1 for each time period.There were no significant changes in these components across the 13 time periods (P-values > 0.13).Analysis of RM-NET posttest components revealed no significant changes in knowledge judgments (P = 0.406) or the number of autobiographical memories (P = 0.072) across the eight time periods.
For autobiographical memories, this trend indicated an increasing number of autobiographical memories for older news events.Note that because reports of autobiographical memories were so rare (mean = 0.28 trials/time period) and they were not obtained for all time periods from the RM-NET, we did not investigate autobiographical memories further.There was a significant linear decrease in subsequent memory accuracy across the time periods (F (1,50) = 5.26, η p 2 = 0.097, P = 0.026).
Identifying relationships between total RM-NET memory accuracy and performance on traditional neuropsychological tests
We asked how performance on news events test relates to performance on traditional neuropsychological tests.We answered this using two approaches: (1) a theoretical approach and (2) a datadriven approach.For the theoretical approach, individual neuropsychological tests were grouped according to the primary domain of cognition they were thought to reflect (see the Materials and Methods), creating five cognitive domain composite scores.We carried out bivariate correlation analysis between RM-NET total accuracy scores and the cognitive composite scores to obtain estimates of effect size.The distributions of the RM-NET scores and domain composite scores appear in Figure 2, A and B, respectively.The largest effect sizes were observed for EM composite scores (r = 0.425, P = 0.002) and attention/processing speed composite scores (r = 0.453, P = 0.001).Smaller (and nonsignificant, P-values > 0.01) effect sizes were observed for SM/language (r = 0.311, P = 0.026), executive function (r = 0.309, P = 0.027), and visuospatial function (r = 0.336, P = 0.016).To account for the possible influence of covariates, multiple regression was also used to obtain measures of effect size (B [unstandardized beta coefficients ± SEM] and β [standardized beta coefficients]).RM-NET accuracy scores were used to predict each of the five cognitive composite scores (including significant covariates from Supplemental Table S1).Results from these analyses are concordant with the results from the correlational analysis (see Table 4; Fig. 3).These findings suggest that the RM-NET primarily reflects the EM and attention/processing speed domains of cognition measured by traditional neuropsychological tests, and these findings were robust to the inclusion of covariates.
Next, we used a data-driven approach to determine which individual neuropsychological tests were associated with RM-NET accuracy scores.Because there were so many neuropsychological tests, we opted for an exploratory factor analysis in lieu of bivariate correlations.We carried out the factor analysis on all the traditional neuropsychological test Z-scores and the RM-NET total accuracy scores.This method empirically identified clusters of these tests where performance varied together.The important finding was which factors the RM-NET loaded on mostly strongly and which individual tests were contained in these factors.We found that eight factors accounted for a significant amount of variance among the tests for a total of 73% cumulative variance explained.The RM-NET loaded highest on the first (0.355), fourth (0.432), and fifth (0.491) factors.The first factor explained 29.8% of variance and was composed of the EM tests (except for visual reproduction immediate recall) and the digit span sequencing test.This factor primarily reflected verbal and nonverbal EM, as well as verbal short-term memory and attention.The fourth factor explained 6.6% of variance and was composed of digit span forward and digit span backward tests.This factor reflected verbal short-term and working memory.The fifth factor explained 5.7% of variance and was composed of the RM-NET, the Multilingual Aphasia Exam token test, and the Clock Drawing command test.This factor appeared to reflect long-established SM.Thus, the RM-NET was most strongly related to individual neuropsychological tests that reflected verbal and nonverbal EM, verbal short-term and working memory, and long-established SM.
Identifying whether the relationship between RM-NET memory accuracy and episodic memory changes with memory age
The hypothesis that the relationship between RM-NET memory accuracy and EM changes as a function of memory age was tested by carrying out regression using memory age to predict the effect sizes between RM-NET memory accuracy for each time period and the single EM composite score (shown in Fig. 4A).We found that memory age significantly predicted effect sizes such that higher memory age was associated with lower effect size (Table 5; Fig. 4A).For the other domains of cognition, there were no significant effects of memory age on effect sizes (Table 5; Fig. 4B-E).
Discussion
The RM-NET was developed to robustly measure news event memory across the entire adult life span with high temporal resolution (Fig. 1).We examined relationships between RM-NET memory accuracy scores and robust measures of performance in five cognitive domains as estimated by traditional neuropsychological tests.Testing was carried out in older adults without dementia to provide ample variability in these measures (Fig. 2) and to obtain measures of the strength of the associations (effect sizes).RM-NET accuracy was most highly associated with composite scores derived from traditional measures of EM and attention/processing speed (Fig. 3).Medical and mental health burden reflects the total number of comorbidities.Frequency following news ranges from 1 (never) to 4 (frequently).Number of news sources ranges from 0 to 7. Confidence rating scale was from 1 (pure guess) to 4 (definitely sure).The knowledge rating scale was from 1 (no information) to 10 (a lot of information about the news event topic).Autobiographical memory reflects the number of news events (out of 160) that were accompanied by autobiographical memories.The RM-NET interval reflects the relative number of days that elapsed between when the first participant was tested and when each subsequent participant was tested.
RM-NET accuracy was most highly associated with individual neuropsychological tests that reflected verbal and nonverbal EM, verbal short-term and working memory, attention, and long-established SM/language.The strength of the relationship between RM-NET accuracy and EM decreased as a function of the age of the news event memory (Fig. 4A).This pattern was not observed for other domains of cognition (Fig. 4B-E).
The RM-NET and episodic memory RM-NET memory accuracy was strongly associated with EM scores on the traditional neuropsychological tests.The finding that EM scores were related to measures from novel SM tests (e.g., news events or famous names) is consistent with three earlier studies.These studies examined news event memory in young to older adults (Johnson and Klingler 1976) or older individuals with normal cognition, MCI, or AD (De Simone et al. 2020), or they examined memory for famous names in older adults with normal cognition or MCI (Seidenberg et al. 2009).These studies did not include co-variates for demographic characteristics when examining these relationships; therefore, the associations could have been influenced by such factors (e.g., sex).Our findings demonstrate that significant relationships with EM can be detected even when the influence of demographic characteristics are taken into account.
We also observed that the effect size of the association between EM and RM-NET memory accuracy decreased as memory age increased (Fig. 4A), a pattern that was not observed for other domains of cognition (Fig. 4B-E).Because EM and recent news event memories depend on the integrity of the MTL (Kapur and Brooks 1999;Manns et al. 2003;Bayley et al. 2006), our findings likely reflect shared dependence on these brain structures, as well as more overlap between the more extended neural systems that support both EM and SM retrieval (Dede and Smith 2016;Renoult et al. 2019;Irish and Vatansever 2020).With regard to the idea that memory retrieval represents a continuum between EM (which contains highly detailed information related to the context of the learning event) and SM (which has lost this information), our findings support the idea that recent news event memories and remote news event memories lie on different parts of this continuum.
These ideas may also explain why Seidenberg et al. (2009) also found significant correlations between a measure of EM (delayed free recall of a word list) and a novel measure of people who became famous in the recent past (1-10 yr ago).Unlike the current findings, they also found a significant correlation between EM and famous personalities from the remote past (40-55 yr ago), and their effect sizes were similar for recent and remote memory (Pearson correlation coefficient values = 0.40).This difference between the findings of these studies may pertain to the different response requirements associated with the novel SM tests.The RM-NET data reported here reflect forced-choice recognition memory for news events where each test item was scored as either correct or incorrect.In contrast, Seidenberg et al.'s (2009) test allowed four opportunities to report semantic information about the famous personalities (i.e., reason they are well known, known works/accomplishments, names of other people associated with the individual, and history and background of the individual), resulting in a score from 0 to 12 for each name the participant previously identified as being famous.Recollection of this additional information for Seidenberg et al.'s (2009) test may be more reliant on EM, regardless of the age of the memory.The RM-NET and semantic memory/language
A B
Although RM-NET accuracy scores were most highly related to EM and attention/processing speed, there was a trend for them to be related to SM (P = 0.058) (Table 4).In addition, factor analysis revealed that the RM-NET was grouped with a test of oral comprehension/language test (token test).Although the other test it was grouped with, the Clock Drawing command test, is considered to primarily reflect visuospatial functions, performance on this test also appears to be influenced by long-established semantic knowledge regarding the appearance of a clock (Rouleau et al. 1996;Leyhe et al. 2009b).Thus, these two neuropsychological tests likely reflect a correspondence between the RM-NET and tests sensitive to impairments in basic language comprehension and recall of long-established semantic knowledge.
Given that novel SM tests are more sensitive to mild impairment than traditional semantic tests (Seidenberg et al. 2013;Orlovsky et al. 2018), it is important to consider why this is the case and why it may not be surprising that RM-NET accuracy was only weakly (and nonsignificantly) associated with the SM composite scores.As described earlier, traditional SM tests typically assess memories that are very long established and re-experienced (relearned) frequently, whereas novel SM tests do not.These qualities of novel SM tests are the same ones that are associated with making information particularly vulnerable to disruption.Because novel SM tests differ from traditional SM tests in these two characteristics, it may explain why novel tests are sensitive to mild brain injury/disease (e.g., MCI) and why performance on the RM-NET is not highly associated with performance on these traditional tests.
It is also relevant that traditional and novel SM tests also lie on different parts of the SM continuum, with traditional SM tests solidly at the SM pole and reflecting highly conceptual entities, word knowledge, and language use, while novel semantic tests do not.In this way, the RM-NET shares characteristics with other types of SM that depart from the traditional concept of SM.For example, Petrican et al. (2010) found empirical evidence for the idea that as news event memories age, the memory traces lose the additional contextual information about how they were learned, which is thought to reflect EM.Moreover, personal SMs (e.g., knowledge about your primary school) that are accompanied by spatiotemporal and perceptual details (Renoult et al. 2012;Grilli and Verfaellie 2014;Grilli and Verfaellie 2016), as well as estimates of recollectionlike knowledge about famous people (Waidergoren et al. 2012), can make these types of SM more similar to EM and make them MTL-dependent.
The RM-NET and attention/processing speed RM-NET memory accuracy was also strongly associated with attention/processing speed scores (Fig. 3), and the strength of this association did not change with memory age (Fig. 4).This finding suggests that there were similar demands on attention/processing abilities for recent versus remote memories.Factor analysis also revealed that the RM-NET was highly associated with individual attention/processing speed tests that reflected verbal short-term and working memory and attention.Therefore, even though the RM-NET was timed (individuals had only ∼13 sec to read and answer the question), which could have inflated relationships with processing speed measures, the strong relationship with attention/processing speed appears to be driven by measures of attention, not processing speed.This idea is in line with findings from another study that found no significant relationship between performance on an untimed news event memory test and a traditional measure of processing speed (Leyhe et al. 2010).
Limitations
This study had several limitations.First, because most of the data were acquired in the context of a functional neuroimaging study (which will be reported separately), the RM-NET was administered in a timed format and participants could not see their fingers when making responses.This procedure may have inflated the association between RM-NET accuracy and performance on cognitive domains (e.g., attention/processing speed, executive functions, or visuospatial function).Alternatively, these components could be retained if an assessment of these additional cognitive domains was desired.Second, it took ∼2 yr to acquire these data and forgetting occurred across this interval (i.e., a significant effect of the date of testing on total RM-NET accuracy).To adapt to this challenge, we included the date of testing as a covariate in the analyses.Finally, the RM-NET takes 1 h to administer, precluding its widespread clinical utility.It would be fruitful to develop a shortened version of the test, fine-tuned to detect clinically useful outcomes (e.g., distinguishing between normal cognition and MCI).
Summary
We describe the RM-NET, which is a comprehensive measure of memory for news facts acquired across the entire adult life span.The test can be given in two formats: recognition memory and free recall.Recognition memory scores from the RM-NET were measured and primarily reflected traditional neuropsychological tests of EM, with news facts from the recent past more strongly associated with EM than facts from the remote past.A measure of incidental encoding of the RM-NET items also provides a measure of anterograde EM.In addition to EM, verbal working memory and short-term memory, as well as attention and long-established SM, also likely contribute to success on news events tests.
Although the RM-NET was only weakly related to SM composite scores, and scores shared variance with one measure of oral comprehension/language, news event memory appears to reflect a type of SM not effectively measured by the traditional SM tests commonly used to detect cognitive impairment in the clinic (fluency and confrontational naming).This is likely due to the test's reliance on memory of news facts that have had less opportunity for relearning and that vary in the age of the memories queried.
Novel SM tests, such as the RM-NET, continue to provide promise and opportunity for detecting mild impairments in cognition when traditional SM tests cannot.In addition, computerized administration and scoring of the RM-NET can occur without a trained administrator, increasing the ease and efficiency of identifying individuals with mild cognitive impairment.had not lived in the United States for most of adulthood; ineligible for MRI; left-handed (because functional neuroimaging was obtained for these participants); traumatic brain injury or head injury with loss of consciousness >30 min; strokes or transient ischemic attacks; chronic disorders of the lung or heart; seizures or other neurological conditions such as Parkinson's disease or dementia; type I diabetes or uncontrolled type II diabetes; general anesthesia in the previous 4 mo; chemotherapy or full-body radiation for cancer treatment; diagnosed with schizophrenia, bipolar disorder, or psychotic disorders; untreated major depression or exhibiting moderate to severe depression symptoms; or currently enrolled in an alcohol/drug treatment program.Exclusion criteria were identified via telephone interview prior to enrollment, except for depression symptoms, activities of daily living, MMSE, reading ability, and blood pressure, which were measured at the first visit.Participants were included in the study regardless of whether they exhibited impairment on the standard neuropsychological tests used to estimate ability in the five cognitive domains that were examined (see "Traditional Neuropsychological Assessment," below).Therefore, these participants likely included individuals with normal cognition or mild cognitive impairment.
Criteria used for exclusion
Participants were considered to have impaired activities of daily living if they obtained a scaled score of ≤40 (low functioning) on the "health and safety" or "managing money" subscales of the Independent Living Scales Test (Loeb 1996) or if they obtained a 3. Memory age indicates the age of the news event memory relative to the testing date.Memory age significantly predicted episodic memory effect sizes (A; higher memory age was associated with lower effect size) but did not predict effect sizes for the other cognitive domains (B-E).score of ≤8 on the 15-item Functional Assessment Questionnaire (Pfeffer et al. 1982).They were considered impaired on the MMSE if they received a score <25 (Folstein et al. 1975).Reading ability was considered to be impaired if their sum of correct responses was less than two standard deviations below norms on the Wide Range Achievement Test (WRAT4) Blue Word Reading Test.They were considered to have moderate depressive symptoms if they obtained a score of ≥7 on the 15-item Geriatric Depression Scale (Sheikh and Yesavage 1986).Normal blood pressure was determined by using the age-and sex-adjusted values from the American Heart Association (Whelton et al. 2018).Blood pressure readings were obtained in visit 1 or visit 2 or reported by the participant (i.e., from a measurement obtained from another source, such as a recent doctor's visit).Finally, participants were excluded after the first visit if they could not successfully complete a news events practice test that simulated how the test was administered in visit 3. Unsuccessful performance on the practice test was evident if participants were unable to respond to 19 recognition memory news event questions within the 12.8-sec time window after two attempts at the practice test (see "Procedure," visit 3).
No individuals were excluded for exhibiting impaired activities of daily living, impaired MMSE score, or impaired reading ability after the first or second visit.Sixteen individuals were deemed ineligible after the first or second visit due to moderate or severe depression symptoms (n = 3), uncontrolled high blood pressure (n = 2), or inability to complete the news events practice test (n = 1), or because they developed a condition found in our exclusion criteria prior to completing RM-NET (n = 1) or because they withdrew from the study prior to completing RM-NET (n = 5).Because part of the testing occurred in the MRI scanner (see "Procedure," below), we also excluded participants due to brain abnormality (e.g., brain tumor) detected on structural MRI (n = 2) or MRI claustrophobia or incompatibility (n = 2).The remaining 51 participants (19 women) completed the study.
Traditional neuropsychological assessment
The Mini-Mental State Examination (MMSE) was administered as a general measure of cognition.We also completed a comprehensive neuropsychological assessment using 26 measures to assess cognitive ability in five domains (four to seven tests/domain): EM, SM/ language, executive functions, attention/processing speed, and visuospatial functions.The measures for each domain were as follows: Episodic memory.Verbal memory: CVLT-II: trials 1-5 total recall, recognition (d ′ ), and long-delay free recall; and WMS-IV (or -R): logical memory immediate recall (sum of stories A and B) and logical memory delayed recall (sum of stories A and B).Nonverbal memory: WMS-IV visual reproduction: immediate recall (sum of items 1-5) and delayed recall (sum of items 1-5).
Semantic memory/language.DKEFS verbal fluency: letter (sum of correct responses) and category (sum of correct responses), Multilingual Naming Test (sum of correct responses with or without semantic cue), and Multilingual Aphasia Exam token test (sum of correct responses).
Executive functions.WAIS-IV digit span: backward (sum of correct items); DKEFS verbal fluency switching (total switching accuracy), trail making: condition 4 (Total Time); and Wisconsin card sorting-64 (number of categories completed and number of perseverative errors).
Attention/processing speed.DKEFS trail making: condition 2 (total time); WAIS-IV digit span sequencing (sum of correct items): digit span forward (sum of correct items) and digit vigilance test (total time and number of errors).
Visuospatial functions.Clock Drawing: command and copy; MMSE overlapping pentagons item; WASI-II block design (sum of correct items); and WMS-IV visual reproduction copy (sum of items 1-5).
Retrograde memory news events test (RM-NET)
A 231-item test was constructed to create a reliable measure of news event memory for the entire adult life span.The procedures used to carry out pilot testing during the creation of the test were approved by the Institutional Review Boards at the University of California at San Diego or the Veterans Affairs San Diego Healthcare System.Construction of the test occurred in four phases.
Phase 1: identification of time periods and refinement of test items available from prior tests.Data collection with the RM-NET was to begin in 2018, so that year was identified as the starting point of the test.Three-year time periods were created for the most recent 15 yr to obtain high temporal resolution of memories as they transition from being hippocampus-dependent to hippocampusindependent (Kapur and Brooks 1999;Manns et al. 2003;Bayley et al. 2006).Five-year time periods were created for the more remote years (see Table 1) going back to 1948, so that memory would be queried from 2017 until the time participants were ∼15 yr old (anticipated age range of participants = 65-90 yr of age).In addition, we included 20 items per time period for the most recent 30 yr (to allow for FMRI analysis [to be reported in a separate publication]) and eight to 10 items for more remote time periods to obtain reliable estimates of older memories.Next, we identified suitable test items (recall and recognition questions) from the 314 items available from previous studies (Smith et al. 2010;Smith 2014).Specifically, there were 314 items covering events that occurred between 1931 and 2005.To identify the most suitable items to retain for detecting subtle impairment in SM, items associated with enduring events (news facts that are repeatedly encountered across the lifetime; e.g., what happened to the World Trade Center in New York City) were eliminated.Enduring events can be identified objectively when individuals who did and who did not live through the event obtain similar accuracy scores.Pilot testing of young adults (N = 24, aged 18-24 yr) via Amazon's Mechanical Turk was carried out in 2017 to obtain accuracy scores for questions from 1948 to 1997, when these individuals were <15 yr of age.Test items were eliminated if the young adults exhibited recall accuracy similar to that of the older adults (N = 21, aged 65-89 yr) (from Smith 2014).The remaining items were thought to represent transient events, associated with a single or limited encoding period, resulting in a reliable estimate of memory age.
Phase 2: creation of test items for events that occurred between 2006 and 2017.The investigators followed the same rules for selecting topics of interest as were used to select topics for previous versions of the test (Squire 1974;Manns et al. 2003;Smith and Squire 2009;Smith et al. 2010;Smith 2014).Test items were created for events that were likely to receive transient media coverage (transient events; e.g., South Korea impeaching their first female president) and attempting to exclude topics that were likely to become enduring events.Similar to how events were selected for these previous news events tests, the events were identified from year-end summaries of notable events and reflect different genres of events (e.g., entertainment, sports, politics, crime, and human interest).Unlike previous studies, we obtained this information by searching the Internet and reviewing reputable news sources (e.g., "2015 year in review") instead of reviewing published periodicals.We attempted to select events in the same way for each year and so that events from year to year would be expected to have experienced the same extent of initial exposure.Events of regional interest were avoided so that the test would have more widespread utility.Recall and recognition memory versions of the test were constructed by creating seven to eight questions associated with events from each year.For the recognition test, foils were first fashioned by creating plausible incorrect answers.
Pilot testing (N = 10 adults) was carried out to identify poorly worded questions and to generate more plausible foils for the recognition test (i.e., by using their incorrect answers from the recall portion of the test as alternative choices in the recognition test).See Table 2 for examples of new test items.
Phase 3: matching test difficulty with previous news event tests.The test difficulty was assessed and adjusted so that RM-NET accuracy scores matched accuracy scores from successful news event memory publications (Cohen and Squire 1981;Kopelman 1989;Manns et al. 2003).To achieve this, pilot testing of the available 165 questions for events that occurred between 2017 and 1988 (N = 24 older adults, aged 65-76 yr) was carried out via Mechanical Turk.The mean accuracy for each 5-yr time period was compared with the accuracy for the same 5-yr time period from the previous publications (i.e., accuracy from events that occurred 1-5 yr prior to testing).Questions were eliminated from each time period until the mean accuracy matched the mean accuracy from previous tests.Specifically, if the accuracy was higher than previous tests, questions with high accuracy were eliminated, and if the accuracy was lower than previous tests, questions with low accuracy were eliminated.Five questions were eliminated in this phase, leaving 160 questions for the events from 2017 to 1988.
Phase 4: creation of a surprise recognition memory posttest for the RM-NET to measure anterograde episodic memory.To assess incidental encoding ability during the news event test, we created a follow-up test that measured subsequent memory for the specific topics of the news event questions (Smith and Squire 2009).For the 160 questions for news events that occurred 1-30 yr before testing , we created a three-alternative, forced-choice recognition memory test that queried the specific topic asked about earlier.Foil options were created so that memory of the general news event topic would not be sufficient to guide memory judgments.Instead, participants had to remember the details of what the question had asked about for each event (see Table 2 for examples).Based on pilot testing (N = 24 older participants obtained via Mechanical Turk), accuracy on this test was 90% correct after a 20-min delay.By administering the 160-item posttest, one can obtain a robust measure of EM in addition to the robust measure of SM provided by the RM-NET itself.
News habits questionnaire
It may be important to know the extent to which participants follow the news when interpreting performance on the RM-NET (Johnson and Klingler 1976;Howes and Katz 1988;Kapur et al. 1999).Accordingly, participants were asked how often they follow the news to obtain a measure of general exposure frequency (never = 1 point, rarely = 2 points, somewhat often = 3 points, and frequently = 4 points).Next, participants were asked to rank seven news sources in sequential order by how often they used them to follow the news (1 = most used, 7 = least used, and 0 = never used).These news sources were television, word of mouth, radio, periodicals, news websites (e.g., BBC.com and CNN.com), news repository websites (e.g., Buzzfeed news and Yahoo news), and/or social media (e.g., Facebook and Twitter).The total number of news sources was computed by adding up the number of sources that were selected, irrespective of their ranked order.
Procedure
All procedures were approved by the Institutional Review Board at the Veterans Affairs San Diego Healthcare System.The experiment took place across four visits.Participants were first assessed with a comprehensive neuropsychological battery (visits 1 and 2).If neuropsychological testing had been completed elsewhere within 12 mo of administration of the RM-NET (visits 3 and 4), these data were used, reducing the number of tests administered in visit 2. A medical history questionnaire was also completed in visit 2. The recognition memory portion of the RM-NET was administered between March 2018 and April 2020.Test items that occurred between 2017 and 1988 were administered inside the MRI scanner (visit 3), while test items that occurred between 1987 and 1948 were administered outside the MRI scanner (visit 4) using similar methods.
Prior to completing the RM-NET, participants were given the opportunity to become familiar with the task by completing a 16-item practice version of the RM-NET and odd/even judgment test (see below).Participants completed five blocks of the task, where each block consisted of recognition memory judgments followed by confidence judgments for 32 news event questions (four items from each of the eight time periods covering 2017-1988) (see Table 1) using an MRI-compatible four-button response box (Current Designs).The order of the news event questions was counterbalanced across participants.Because data were collected during an FMRI experiment, limited time was allowed for responses.Each news event question was presented for 12.8 sec, during which time participants made a four-choice recognition memory judgment (the question remained on the screen for the full duration regardless of the participant's response).Because participants could not see their hands during the test, they used a finger-button map (presented next to the news event question) to indicate whether their response was A (index finger), B (middle finger), C (ring finger), or D (pinky finger).After the 12.8 sec had elapsed, participants then had 3.2 sec to provide a four-choice rating to indicate their confidence that they had selected the correct answer to the news event question (4 = definitely sure [index finger], 3 = probably sure [middle finger], 2 = somewhat sure [ring finger], or 1 = pure guess [pinky finger]).
In between news event questions, participants were presented odd/even judgment trials.For these trials, a single-digit number (between 1 and 8) was presented and participants had 3.2 sec to make their even (index finger) or odd (pinky finger) judgment.A variable number of odd/even judgment trials occurred after each news event trial (zero to seven odd/even trials).The odd/even judgments facilitated analysis of brain activity, which will be reported separately.For all questions, participants were allowed to change their responses so long as the reselection was made within the time allotted and their last response was taken as their final answer.
About 20 min after completing the first 160 test items, participants began the 160-item posttest to obtain additional information about each news event (i.e., news events from 2017 to 1988).Participants were asked questions about each news event.First, they were asked to identify the specific topic they had been asked about earlier (see Table 2).This surprise recognition memory test reflects incidental subsequent memory for the RM-NET content (EM).Next, they were shown the news event question and asked to indicate on a 10-point scale the depth of their knowledge about the news event (1 = none and 10 = a lot).They were instructed to make this judgment regardless of whether they believed they knew the answer to the news event question.This component provides a measure of the quality of the news event memory to facilitate neuroimaging analysis.Finally, participants indicated whether or not they had a specific autobiographical memory associated with the news event using the definition of an EM from the Autobiographical Memory Interview (i.e., a score of three on this measure) (Kopelman et al. 1989); for example, if they could report specific details about the time and place when they learned about the event and not whether they simply remembered hearing it on the radio or seeing it on television.This component provides information about whether episodic memories accompany the semantic, news event memories.Note that very few such events were reported by participants (see Table 3), limiting discussion of findings for this variable.Eprime software (Psychology Software Tools, Inc.) was used to administer the test.Responses were made using a computer keyboard and unlimited time was given.Participants typically took between 60 and 120 min to complete the posttest.
At the final visit (visit 4, 29.4 d ± 38.5 d after visit 3), participants completed the remaining items from the RM-NET, seated at a table using a laptop computer.The test was comprised of up to 71 questions about news events from 1987 to 1948 (see Table 1).News events that occurred when participants were <15 yr of age were not administered.The test was administered using the same methods that were used for visit 3 (inside the MRI scanner), except that an external keyboard was used to make the responses.To make the testing experience more similar to the experience in the MRI scanner, participants were not allowed to view their hand to make the memory judgments.Instead, the external keyboard was placed out of view.
Data analysis
There were two primary objectives.First, we examined the relationship between performance on the RM-NET and performance on traditional neuropsychological tests.Second, we examined whether the strength of the relationship between the RM-NET and EM (i.e., the effect size) decreased as memory age increased.Variables of interest were created prior to carrying out group-level parametric statistics.Descriptive statistics and exploratory graphing were used to assess natural data distributions, normality, and homogeneity.Means and standard deviations are reported, unless explicitly noted otherwise.Analyses were carried out using SPSS version 27.
RM-NET components
For each of the 16 time periods, mean news event memory accuracy, confidence, and response times were computed (2017-1948, 1-70 yr before testing) (see Table 1).News event memory accuracy reflected the percentage of correctly answered news event questions relative to the total number of questions in the time period.The same method was used for confidence and response time components.For trials where participants failed to provide a response for a news event question within the allotted time (12.8 sec), the trial was counted as incorrect, no confidence measure was assigned, and a response time of 12.8 sec was assigned.Next, we computed total accuracy, confidence, and response time scores across the entire adult life span by averaging the mean scores from each time period that was available for each participant , and so on until reaching the last time period where the participant was 15 yr of age).In this way, each time period received equal weighting regardless of how many questions were available for the time period.For the three most remote time periods (see Table 1), data were only available from the oldest participants (<65% of the sample); therefore, these time periods were excluded from subsequent analyses as a function of time period.
For news events that occurred between 2017 and 1988 (1-30 yr before testing), additional information was available from the posttest: subsequent memory accuracy, amount of knowledge reported, and presence of autobiographical memories.For these components, mean scores were computed for each time period in the same way as described above, except that there were only eight time periods represented instead of 13.Total mean scores were also computed for these variables by averaging the mean scores across the time periods.
Cognition domain composite scores
Performance on individual tests from the neuropsychological test battery were converted into Z-scores based on published norms.Composite scores (Z-scores) were computed for each participant and domain by averaging the individual Z-scores for the tests in that domain, resulting in five composite scores representing the five cognitive domains assessed.Normative data for all but three tests were developed by the University of California at San Diego Alzheimer's Disease Research Center normal cohort or elsewhere (e.g., National Alzheimer's Coordinating Center Uniform Data Set; Mayo older American normative studies [Heaton et al. 2004;Steinberg et al. 2005]).For three measures, norms were not available and performance was rated as impaired or unimpaired according to published methods.For both Clock Drawing command and Clock Drawing copy, Z-scores were based on the mean and SD reported in Rouleau et al. (1996).The drawing for overlapping penta-gons was taken from the MMSE and scored on eight criteria (Jefferson et al. 2002), where a score of three or more errors was considered impaired.For WMS-IV visual reproduction copy and Wisconsin card sorting number of categories completed, scoring was based on the normed percentile ranking, and a score less than the 16th percentile was considered impaired.In order to include these five tests in the cognitive domain composite scores, impaired scores on these measures were converted to a Z-score of −1.1.This value was selected because performance of more than one standard deviation below norms has been used as a cutoff to identify mild cognitive impairment (Jak et al. 2009;Bondi et al. 2014).Unimpaired scores on these three measures were converted to a Z-score of 0. For the Wisconsin card sorting task, the lower Z-score from its two measures was used for the executive function composite score.
Identifying relevant covariates for primary analyses
Participant characteristics, such as age, education, and news habits, can sometimes affect performance on news events tests (Warrington and Sanders 1971;Johnson and Klingler 1976;Kapur et al. 1999).Therefore, prior to carrying out the primary analyses, we used stepwise, multiple regression to identify covariates that significantly predicted dependent variables of interest (i.e., RM-NET components and cognitive domain composite scores) (see Table 3).Covariates that were significant predictors (P < 0.05) for any variables of interest were included as covariates in the primary analyses.Estimated marginal means and SDs are reported and reflect scores adjusted for covariates.
The participant characteristics evaluated included traditional demographic characteristics (age, gender, education, and race/ethnicity), medical health and mental health comorbidity burden (see "Comorbidities," below), and news habits (sum of frequency of news exposure and number of news sources).Finally, because all participants were administered the same RM-NET items during the 2-yr data collection interval, each participant had a unique duration between the date of testing and the years in which the news events occurred.To take this test interval into consideration and to control for possible effects of forgetting over that interval, we created a covariate (RM-NET interval) for each participant.The RM-NET interval reflected the number of days that elapsed between when each participant was tested relative to when the first participant was tested (i.e., the relative dates of visit 3).
Comorbidities
Due to the small sample size and wide variety of comorbidities and medications reported by participants, composite measures of comorbidity burden were created following the method developed by Charlson et al. (1987).The measures were modified so they would reflect the disorders queried in the self-reported medical history questionnaire but without assigning weightings based on disease severity.Composite scores for total medical health burden and total mental health burden were computed separately for each participant.Each composite score reflected the sum of the reported comorbidities.For comorbidities that comprised multiple subconditions (listed in parentheses below), the comorbidity was counted if at least one of the subconditions was reported.
The total mental health burden composite reflected five comorbidities (scale 0-5): posttraumatic stress disorder (PTSD), substance abuse (substance abuse, history of alcohol/drug treatment, or history of nicotine abuse), mood disorders (depression, taking antidepressant medication, anxiety, or taking antianxiety medication), sleep disorders (sleep apnea or sleep disorder), and mental health treatment (history of mental health treatment).
Primary analyses
Identifying relationships between RM-NET memory accuracy and performance on traditional neuropsychological tests We sought to answer the question of how performance on news events tests relates to performance on traditional neuropsychological tests.We hypothesized that performance on the RM-NET would significantly predict performance on tests of EM and SM/ language.To test these hypotheses, we used two approaches: (1) a theoretical approach and (2) a data-driven approach.For the theoretical approach, bivariate Pearson correlations were computed between RM-NET memory accuracy scores and each cognitive domain composite score.To correct for the possible influence of covariates, we also carried out hierarchical, multiple regression analyses using RM-NET memory accuracy to predict mean performance in each domain of cognition (composite Z-scores for EM, SM/language, executive functions, attention/processing speed, and visuospatial functions).Relevant covariates were entered into the model followed by RM-NET memory accuracy.Each cognitive domain composite score served as the dependent variable.Although our goal was to report relative effect sizes (correlation coefficients or beta coefficients), probability values are also reported, correcting for multiple comparisons across each of the five domains (P < 0.05/5 = P < 0.01).
We also carried out a data-driven approach because it is possible that strong relationships between individual neuropsychological tests and the RM-NET could be concealed when averaging across individual tests to create the cognitive domain composite scores.Due to the large number of tests, bivariate correlational analysis was not used.Instead, we carried out an exploratory factor analysis that included all standard neuropsychological test Z-scores as well as RM-NET accuracy scores.The principal component method was used for extraction of factors, and factors with eigenvalues >1 were retained.The goal was to identify which factors the RM-NET loaded on most strongly according to rotated factor loadings.
Identifying whether relationships between RM-NET memory accuracy and episodic memory change with memory age
We tested the hypothesis that the effect size between news event memory and EM changed with the age of the news event memory (memory age).First, for each of the 12 time periods, a regression model (same as described above) was used to obtain the effect sizes (unstandardized beta coefficients) when using the RM-NET time period accuracy score to predict the EM composite score.Next, multiple regression was used to test whether the age of the news event memory predicted these 12 effect sizes.The age of the news event memory variable was created by taking the first number from each time period (1-to 3-yr time period = 1, 4-to 6-yr time period = 4, 7-to 9-yr time period = 7, and so on), resulting in a variable with 12 values.We then tested for a linear relationship between this variable and the 12 unstandardized beta coefficients.The robustness of this analysis was tested by permuting the RM-NET accuracy scores across participants 1000 times and then repeating the regression analysis described above.The number of times that total variance accounted for (R 2 ) of the permuted data exceeded the R 2 of the nonpermuted data was counted and taken to reflect the probability of observing changes in the effect sizes that could have occurred by chance.The analysis of time periods was limited to news events from the last 50 yr (12 time periods).For comparison, we also carried out this analysis for the other domains of cognition.
Figure 2 .
Figure 2. Descriptive measures (N = 51) for RM-NET accuracy scores (A) and cognitive domain composite scores (B).Violin plots illustrate medians (horizontal lines) and distributions (outer curved lines).Below the median is the first quartile (25th percentile, bottom white rectangle) and above the median is the third quartile (75th percentile, top white rectangle).Cognitive domain composite scores reflect mean Z-scores for four to seven tests relative to published norms.
Figure 1 .
Figure1.Mean accuracy scores (top), confidence judgments (middle), and response times (bottom) for the RM-NET for news events that occurred between 1 and 55 yr prior to testing (memory age) for 51 older adults.Performance did not change across time periods according to linear or power functions (P-values > 0.17).Means and SEMs were adjusted for the effects of covariates.
Figure 3 .
Figure 3. Relative effect sizes of the association between RM-NET accuracy scores and cognitive domain composite scores.RM-NET accuracy scores significantly predicted episodic memory and attention/processing speed composite scores.The Y-axis reflects the unstandardized beta coefficients (±SEM) from the regression equations (including covariates).(*) P < 0.05, corrected for multiple comparisons.
Figure 4 .
Figure 4. Effect sizes for the association between RM-NET accuracy scores for each time period and a single episodic memory domain composite score.Y-axis as in Figure3.Memory age indicates the age of the news event memory relative to the testing date.Memory age significantly predicted episodic memory effect sizes (A; higher memory age was associated with lower effect size) but did not predict effect sizes for the other cognitive domains (B-E).
Table 1 .
Time periods of the Retrograde Memory News Events Test (RM-NET) Years before testing represents the approximate age of the memories queried.Testing occurred between March 2018 and April 2020.News events from the eight time periods spanning 1-30 yr before testing were queried in the RM-NET posttest.
Table 2 .
Example questions from the Retrograde Memory News Events Test (RM-NET)
Table 3 .
Descriptive statistics for participant characteristics and variables of interest
Table 4 .
Multiple regression results using cognitive domain composite scores to predict mean RM-NET accuracy scores
Table 5 .
Multiple regression results where the age of the news event memory predicted the effect size between RM-NET accuracy scores and cognitive domain composite scores P-values of <0.01 were considered statistically significant after correcting for multiple comparisons. | 2022-10-02T06:16:22.938Z | 2022-09-30T00:00:00.000 | {
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21728845 | pes2o/s2orc | v3-fos-license | Laparoscopic adhesiolysis: not for all patients, not for all surgeons, not in all centres
ASBO is a common cause of emergency surgery and the use of laparoscopy for the treatment of these patients is still under debate and conflicting results have been published, in particular regarding the high risk of iatrogenic bowel injury. In fact, although over the last few years there has been an increasing enthusiasm in the surgical community about the advantages and potential better outcomes of laparoscopic management of adhesive small bowel obstruction (ASBO), recently published studies have introduced a significant word of caution. From 2011 in our centre, we have started to systematically approach ASBO in carefully selected patients with a step-by-step standardized laparoscopic procedure, developed and performed by a single operator experienced in emergency laparoscopy, collecting data in a prospective database. Inclusion criteria were: stable patients (without diffuse peritonitis and/or septic shock with suspicion of bowel perforation), CT scan findings consistent with a clear transition point and therefore suspected to have a single obstructing adhesive band. Patients with diffuse SB distension in the absence of a well-defined transition point and suspected to have diffuse matted adhesions (based on their surgical history and radiological findings) should be initially managed conservatively, including gastrografin challenge. Up to date, 83 patients were enrolled in the study. The rate of iatrogenic full-thickness bowel injury was 4/83 (4.8%); two of these cases were managed with simple repair and the other two required bowel resection and anastomosis. Conversion to open was performed in 3/4 of these cases, whereas in one a repair of the full-thickness injury was completed laparoscopically. All the iatrogenic injuries were detected intraoperatively and none of the reoperations that occurred in this series were due to missed bowel injuries. At 30 days follow-up, none reported incisional hernias or SSI or death. With the described accurate selection of patients, the use of such standardized step-by-step technique and in the presence of dedicated operating surgeons with advanced emergency surgery laparoscopic expertise, such procedure can be safe and feasible with multiple advantages in terms of morbidity and LOS. A careful preoperative selection of those patients who might be best candidates for laparoscopic adhesiolysis is needed. The level of laparoscopic expertise can also be highly variable, and not having advanced surgical expertise in the specific subspecialty of emergency laparoscopy, ultimately resulting in performing standardized procedures with proper careful and safe step-by-step technique, is highly recommended.
Dear Editor,
We read with great interest the study by Behman et al. on laparoscopic adhesiolysis [1]. Despite increasing enthusiasm for laparoscopic management of adhesive small bowel obstruction (ASBO), this study introduces significant caution and raises serious concern for the proposed laparoscopic approach to ASBO due to a reported higher risk of bowel injury from a population-based analysis of more than 8500 patients. We share the same concerns in recommending to all general and acute care surgeons in every centre, a routine laparoscopic approach for all patients with ASBO. In fact, in this manuscript a routine laparoscopic approach seems to have been used regardless of patients' characteristics and surgeons' expertise.
Firstly, careful preoperative selection of those patients who might be best suited for laparoscopic adhesiolysis is needed; the level of laparoscopic expertise may be highly variable, and having advanced surgical expertise in the specific subspecialty of emergency laparoscopy [2], leading to standardized procedures with a safe step-by-step technique, is highly recommended before undertaking such procedures.
We have started to systematically approach ASBO in carefully selected patients from 2011 with a step-by-step standardized laparoscopic procedure, developed and performed by a single operator experienced in emergency laparoscopy, collecting data in a prospective database [3]. Up to May 2017, we have treated a prospective consecutive series of 83 cases, all operated by the same single operator with a standardized step-by-step technique developed by the same surgeon. We are also involved as co-investigators of the Finnish trial on laparoscopic vs open adhesiolysis for ASBO [4]. From our previous experience in the field, we have developed and adopted a well-defined protocol for laparoscopic management of ASBO. The selection of patients must be accurate and only stable patients (without diffuse peritonitis and/or septic shock with suspicion of bowel perforation) having CT scan findings consistent with a clear transition point and therefore suspected to have a single completely obstructing adhesive band should be considered for the laparoscopic approach [5]. Patients with diffuse SB distention in the absence of a well-defined transition point and suspected to have diffuse matted adhesions (based on their surgical history and radiological findings) should be initially managed conservatively, including a Gastrografin challenge [6].
In detail, the inclusion and exclusion criteria in our experience were: Inclusion criteria: -adult patients; -informed consent; -initial CT diagnosis of complete ASBO with an identifiable transition point and an anticipated single obstructing band with completely collapsed distal small bowel loops; -and/or with a radiological/clinical evidence of failure of a NOM trial with hyperosmolar WSCM via NGT.
Exclusion criteria: -hemodynamic instability and preoperative shock; -diffuse peritonitis and/or evidence of severe intra-abdominal sepsis; -high suspicion of gangrenous/perforated bowel; -high probability of diffuse and dense matted adhesions (e.g. multiple previous laparotomies ≥ 3 with intraoperative findings of diffuse dense and matted adhesions); -preoperative diagnosis of any other cause of complete mechanical SBO than adhesions (i.e. carcinomatosis, hernias, cancer, intussusception, biliary ileus, etc.).
The following were NOT exclusion criteria from diagnostic laparoscopy in our experience, although these are risk factors for conversion after an initial diagnostic laparoscopy: -previous midline laparotomy was not considered an absolute contraindication; -suspicion of bowel strangulation and/or volvulus and/or bowel ischaemia without gangrene or perforation; -localized clinical peritonitis; -CT findings of free abdominal fluid; -CT or AXR findings of small bowel distension with a size > 4 cm; -CT additional findings such as SB faeces sign, SB thickening, and mesenteric oedema/vascular engorgement.
Careful interpretation of the CT scan findings may be useful in identifying good candidates for a laparoscopic approach. We also recommend that only fully trained and experienced laparoscopic surgeons should attempt this technique. The correct surgical technique is of paramount importance to avoid bowel injuries. From our experience, once a laparoscopic approach is decided, we recommend not to use Veress needles or blind insertion of the first port in close proximity to the previous scars. We believe that a safe entry to the abdomen can be best obtained by either inserting the first Hasson trocar in the left flank with open access or by using a blunt dilating tip, optical trocar entering the abdominal wall at the level of the Palmer point, under direct vision (step 1- Fig. 1) [7]. Once the pneumoperitoneum has been gradually established, the surgeon must assess if there is adequate room for good vision and further safe trocar placement. If not, we would recommend timely conversion. Inadequate vision mandates withdrawing from any attempt to manipulate the bowel using laparoscopic instruments.
Further tips and tricks derived from our experience are not to immediately search for the transition point and for the strangulating band by manipulating the distended and fragile bowel loops, but rather to start with identification of the caecum, then starting to explore from the collapsed distal ileal loop in a distal-to-proximal fashion (step 2- Fig. 1). Only collapsed loops should be manipulated. Grasping the mesentery rather than the bowel wall helps to run the bowel without traumatizing the bowel (step 3- Fig. 1). Usually, the distal collapsed loops can be run relatively easily and explored until reaching what we used to name the "sentinel loop", a collapsed loop which is fixed and stuck, giving the feeling to the operator that running the bowel cannot be continued further proximally. Usually, this is the location for the transition point; by carefully grasping only the mesentery of the dilated loop adjacent to this point, the level of transition between the proximal distended bowel and the distal collapsed loop is identified and often the single obstructing band can be seen (step 4- Fig. 1). The advice at this point is to gently underpass the band with the aid of blunt manoeuvres, for example using the suction device first and/or spreading the two branches of an atraumatic grasper. These manoeuvres allow one to visualize and isolate the band and at the same time to obtain a little space from the adjacent bowel loops. Once a window is obtained, the band can be carefully and easily cut using cold scissors over the guidance of the two open branches of the atraumatic grasper which are spread and used in a fashion of a right angle instrument (step 5- Fig. 2). A further strong recommendation is to avoid any use of energy-based dissection, either monopolar or bipolar, during the procedure. Thermal injuries may evolve to delayed perforation only several days afterwards.
Any bleeding during lysis of adhesions is generally minor and often self-limiting. Gauze compression may help and any persistent bleeding can be dealt with at the end of the operation, after releasing the obstruction and before finishing the procedure. Finally, adhesiolysis should be limited to only the obstructing band or to the adhesions that need to be divided to reach the transition point. Further extensive adhesiolysis is unnecessary, potentially harmful and should therefore be avoided. Careful observation for any serosal tears over the loops which were manipulated may allow intraoperative laparoscopic simple repair of these, thus preventing postoperative fistula or leak. Full-thickness injury usually mandates conversion. In our previously mentioned prospective single-operator series (unpublished data), the current rate of iatrogenic full-thickness bowel injury is 4/83 (4.8%); two of these cases were managed with simple repair and the other two required bowel resection and anastomosis. Conversion to open was performed in 3/4 of these cases, whereas in one a repair of the full-thickness injury was completed laparoscopically. All the iatrogenic injuries were detected intraoperatively and none of the reoperations that occurred in this series were due to missed bowel injuries.
With the described accurate selection of patients, the use of such standardized step-by-step technique [8] and in the presence of dedicated operating surgeons with advanced emergency surgery laparoscopic expertise and experience in the field of laparoscopic adhesiolysis, such procedure can be safe and feasible with multiple advantages in terms of morbidity and LOS [9][10][11].
In the study by Behman, patient selection criteria are not specified since the study is a population-based cohort study where data have been collected from health administrative records. However, some degree of patient selection emerges from the study, since patients undergoing laparoscopic procedures were younger, had fewer comorbidities and were cared for at larger hospitals.
We therefore agree with the authors' conclusions that surgeons should approach laparoscopic adhesiolysis with a high level of awareness and use strategies to mitigate the risks. We suggest that laparoscopic adhesiolysis is not for all patients and may not be for all surgeons. Ideally, laparoscopic adhesiolysis should be only performed in high volume centres with specific expertise in emergency laparoscopy, especially in tertiary referral centres with availability of operating room and laparoscopic equipment at all times, and with highly trained surgical and OR staff.
As investigators of the current Finnish-Italian LASSO trial4, we eagerly await the results and will hopefully clarify further details about safety and outcomes of the laparoscopic treatment of ASBO.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Research involving human participants and/or animals All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.
Informed consent For this type of study informed consent is not required.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/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. Fig. 2 Step 5: gently underpassing the band with the aid of blunt manoeuvres using the suction device first and/or spreading the two branches of an atraumatic grasper (a, c). These manoeuvres allow visualization and isolation of the band whilst obtaining a little space from the adjacent bowel loops. Once a window is obtained, the band can be carefully and easily cut using cold scissors over the guidance of the two open limbs of the atraumatic grasper which are spread and used in the fashion of a right angle instrument (b, d) | 2018-05-21T21:28:04.234Z | 2018-05-08T00:00:00.000 | {
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224308452 | pes2o/s2orc | v3-fos-license | Induction of cytotoxicity by Bruguiera gymnorrhiza in human breast carcinoma (MCF‐7) cell line via activation of the intrinsic pathway
Breast cancer is among the frequently occurring cancer worldwide. The foremost underline aim of this study was to determine the growth inhibitory effect along with mechanistic study of a Bruguiera gymnorrhiza extract on MCF‐7. The cytotoxicity activity was determined by using the MTS assay. Butanol extract exhibited the maximum cytotoxicity activity against the MCF‐7 cells with IC50 of 3.39 μg/mL, followed by diethyl ether and methanol extract (IC50 at 16.22 μg/mL and 37.15 μg/mL, respectively) at 72 h. The DeadEndTM Colorimetric Apoptosis Detection System confirmed the induction of apoptosis (via DNA fragmentation) in MCF‐7 cells. Both butanol and diethyl ether extracts of B. gymnorrhiza significantly increase the caspase‐3 level. However, the diethyl ether extract induced higher caspase‐9 levels compared to caspase‐8, suggesting that the intrinsic pathway was the major route in the process of apoptosis. Thin‐layer chromatography profiling demonstrated the presence of phenolic, terpene, and alkaloid compounds in crude methanol, diethyl ether, and butanol extracts. The phytochemicals present in the extracts of B. gymnorrhiza might have the potential to be a future therapeutic agent against breast cancer.
INTRODUCTION
Breast cancer is among the frequently occurring cancer in men and women. [1] In 2018, approximately 15% of all cancer deaths were due to breast cancer among women. [2] The most common treatments available for breast cancers are chemotherapy, radiotherapy, and surgical treatment. However, chemotherapy and radiotherapy treatments yield adverse side effects which can be overcome by using targeted drug delivery systems and potential phytochemical-based drugs. [3][4][5] Based on the World Health Organization report, a major percentage of the world's population utilizes plant-derived medicines for health care. [2] Plant-derived medicines or natural drugs, which form the basis of traditional medicine, have been used for centuries by different cultures. The significance of medicines of natural origin has satisfactorily understood in the pharmaceutical industry. [6] Various Mangrove species produce secondary metabolites with various bioactive properties such as insecticidal, antibacterial, antidiarrheal, cytotoxic, and antiviral. [7][8][9][10][11][12][13][14][15][16] The main purpose of a therapeutic drug in cancer treatment is to kill the cancer cell without harming normal cells. In cell death mechanism, apoptosis gains much importance due to safely triggering the suicidal process in cancer cells without affecting normal cells. Apoptosis is an extremely regulated mechanism that shows a distinct role in the process of cell growth, death, and development. Cells undergoing apoptosis possess morphological and biochemical alterations such as condensation of chromatin material and cytoplasm shrinkage, phosphoserine exposure, and DNA fragmentation. [17] Therefore, in this study, the cytotoxicity effects and cell death mechanisms of the extracts of mangrove plant Bruguiera gymnorrhiza on MCF-7 were investigated. Bioactive compounds present in B. gymnorrhiza show an essential part in inhibiting the cell growth apoptosis mechanism by the activation of caspases, thus potentially developed as one of the candidates of chemotherapeutic agents with minimal side effects.
Cytotoxicity assay
The cytotoxicity of extracts was determined by using CellTiter 96 AQueous One Solution Proliferation assay (MTS) as a formerly reported method. [20][21][22] The MCF-7 cell line was cultured in ISO-certified Animal Cell Culture Laboratory, (MS/ISO/IEC 17025:2015 SAMM No. 796), Institute of Marine Biotechnology. The absorbance was observed at 490 nm using a plate reader (enzyme-linked immunosorbent assay Multiskan, Thermo Fisher, USA).
DNA fragmentation through TUNEL system (late apoptosis)
DNA fragmentation study was performed by using the DeadEnd TM Colorimetric Detection System (Promega, Madison US). The MCF-7 cells were cultured in an 8-well slide chamber and then incubated in 5% (v/v) CO 2 incubator at 37°C for 24 h. The experiment was performed according to a previously reported method. [15,20] The dark stain of fragmented DNA represents the induction of apoptosis, which was observed by an inverted microscope (Olympus, Selangor Malaysia).
Caspase assay
The activation level of caspases 3/7, Caspase-8 and Caspase-9 was determined by using Caspase-GloTM Assay (Promega, USA). The assay was performed according to a previously described method. [20,23] The breast cancer cell line was treated with active partition extracts of B. gymnorrhiza at a concentration of IC 50 at 72 h and incubated for several time points (0 h-36 h) at 37°C. The reading of each sample was measured using a luminometer at Optical density (OD) of 490 nm.
Thin-layer chromatography profiling
Thin-layer chromatography (TLC) analysis was done on TLC silica gel plates (60 F254) to identify the presence of phytochemicals in the active extracts of B. gymnorrhiza. The B. gymnorrhiza extract was diluted in a mandatory amount of solvent using a previously reported method. [18,24] The developed plates were air-dried, heated, and observed under ultraviolet light at both 254 nm and 365 nm as well as anisaldehyde and Dragendorff's derivatized reagents.
Statistical analysis
GraphPad Prism software (GraphPad Software, Inc San Diego, CA) was used to calculate the growth inhibition (IC 50 ). Significant differences were expressed by using analysis of variance and (Dunnett's) test using SPSS software version 20.0 (IBM SPSS, USA, New York, US).
Cytotoxicity effect of the Bruguiera gymnorrhiza extracts on MCF-7
In this study, the cytotoxicity effects of the B. gymnorrhiza methanol, diethyl ether, and butanol extracts on the MCF-7 cell line were investigated. This study demonstrated that B. gymnorrhiza methanol, diethyl ether, and butanol extracts of B. gymnorrhiza produced time-dependent increased inhibitory effects on MCF-7 cells. Interestingly, the significant inhibitory effect was observed when cells were treated with methanol, diethyl ether, and butanol extracts at concentrations of 12.50 μg/mL and 6.25 μg/mL [ Figure 1a-c]. However, diethyl ether and butanol extracts only showed a significant cytotoxicity effect at concentrations of 3.12 μg/ mL and lower concentrations. B. gymnorrhiza butanol extract showed the highest cytotoxicity effects with the IC 50 value of 3.39 μg/mL, followed by diethyl ether extract (16.22 μg/ mL). However, B. gymnorrhiza methanol extract showed an inhibitory effect above 30 μg/mL (37.15 μg/mL). The diethyl ether and butanol extracts of B. gymnorrhiza produce cytotoxicity similar to previous studies. [25][26][27] Therefore, diethyl ether and butanol extracts were selected for further mode of cell death investigation.
The apoptotic effects of Bruguiera gymnorrhiza extracts on MCF-7 cell line
The DNA fragmentation in MCF-7 was observed by the dark stain in the nucleus of the cancer cell. 1% (v/v) dimethyl sulfoxide was considered negative control and DNAase as positive, according to data shown in our previous study. [13] Interestingly, dark brown stained nuclei were observed in cells treated with the diethyl ether and butanol extracts over the 36-h treatment period, as revealed in Figure 2a-f. These results strongly denote that both extracts of B. gymnorrhiza killed MCF-7 cell line through apoptosis. The induction of apoptosis is the foremost active strategy to inhibit cell growth and acts as an important effective therapeutic target in drug development. [17,[28][29][30][31] The effects of Bruguiera gymnorrhiza extracts on caspase-3, 8, and 9 protein levels in MCF-7 cells The significant pathways responsible for the activation of apoptosis involve activation in caspase expression and activation. [17] The expression level of caspase-3 [ Figure 3a] significantly increased in cells that were treated with diethyl ether extracts at various treatment periods (9,12,16, and 20 h) and reached its peak at 24 h (2.75 folds) and with butanol extract at 16, 20, and 24 h, with the highest level at 16 h (3.25 folds). Interestingly, the level of caspase-8 [ Figure 3b] increased at 12 h by 2.7 folds, reaching its peak at 24 h by 5.92 folds in diethyl ether extract-treated cells. The significant increase in the protein level of caspase-9 [ Figure 3c] was also noticed, with the highest activity produced when the cells were treated at 1 h (10 folds). Apoptosis is chiefly controlled by caspases; aspartate-specific cysteine proteases involve in the process of apoptosis (function as both initiators and executioners). [17,32] Caspase activation is triggered by two major signaling routes: (i) the extrinsic-death receptor and the (ii) intrinsic-mitochondrial pathways. [33] The induction of apoptosis by anticancer agent might be possible by extrinsic pathway and intrinsic pathway or by both pathways activation. [34]
Thin-layer chromatography
The results showed the separation of compounds spotted on the TLC plate in different solvent system ratios [ Figure 4a and b]. The results indicate that the extracts contained conjugated carbon double bonds (C = C) and alkaloid. The presence of brown spots on a yellow background indicates the presence of organic compounds. The dark orange, violet, and gray spots represent alkaloids, phenol, and terpenes, respectively. The presence of potential phytochemicals in Xylocarpus moluccensis (mangrove species) possessing cytotoxic activity similar to previously reported studies. [14,35,36] In our previous study, [14,15] it was reported that Xylocarpus sp. (mangrove) successfully induces the process of apoptosis in HeLa cells. Xylocarpus moluccensis induces apoptosis through activation of the extrinsic pathway in HepG 2 cell lines. However, in the present study, the major activation route was intrinsic along with the extrinsic pathway, which suggested that mangrove species have the potential to induce apoptosis with both extrinsic and intrinsic pathways. The difference might be due to a particular mechanism activated in specific cell lines. The phytochemical study further confirms the presence of potential phenolic, terpene, and alkaloid compounds in crude methanol, diethyl ether, and butanol extracts. The active compounds present in B. gymnorrhiza might have the potential to be a future therapeutic agent. Further study of isolation of pure compounds from B. gymnorrhiza will be our next priority to study the underlying clear mechanism.
CONCLUSION
One of the significant discoveries in cancer research was investigating the cell death mechanism exerted by anticancer chemotherapy. In this study, the extracts of mangrove plant B. gymnorrhiza showed significant cytotoxicity effects with IC 50 values at 72 h -the induction of apoptosis was confirmed by DNA fragmentation with the underlying mechanism, mainly due to intrinsic pathways. TLC profiling showed that the extracts contain alkaloid, phenolic, and terpenoid compounds. Thus, from this study, it is understood that there is enormous potential for mangrove extracts prepared from B. gymnorrhiza to develop as chemotherapeutic agents in cancer treatment.
Financial support and sponsorship
The research work was supported by Strategic Research Grant, Universiti Malaysia Terengganu (Vot No. 55197). | 2020-10-19T13:27:28.652Z | 2020-10-01T00:00:00.000 | {
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14838925 | pes2o/s2orc | v3-fos-license | Learning to Generate Reviews and Discovering Sentiment
We explore the properties of byte-level recurrent language models. When given sufficient amounts of capacity, training data, and compute time, the representations learned by these models include disentangled features corresponding to high-level concepts. Specifically, we find a single unit which performs sentiment analysis. These representations, learned in an unsupervised manner, achieve state of the art on the binary subset of the Stanford Sentiment Treebank. They are also very data efficient. When using only a handful of labeled examples, our approach matches the performance of strong baselines trained on full datasets. We also demonstrate the sentiment unit has a direct influence on the generative process of the model. Simply fixing its value to be positive or negative generates samples with the corresponding positive or negative sentiment.
Introduction and Motivating Work
Representation learning (Bengio et al., 2013) plays a critical role in many modern machine learning systems. Representations map raw data to more useful forms and the choice of representation is an important component of any application. Broadly speaking, there are two areas of research emphasizing different details of how to learn useful representations.
The supervised training of high-capacity models on large labeled datasets is critical to the recent success of deep learning techniques for a wide range of applications such as image classification (Krizhevsky et al., 2012), speech recognition , and machine translation . Analysis of the task specific representations learned by these models reveals many fascinating properties (Zhou et al., 2014). Image classifiers learn a broadly useful hierarchy of feature detectors rerepresenting raw pixels as edges, textures, and objects (Zeiler & Fergus, 2014). In the field of computer vision, it is now commonplace to reuse these representations on a broad suite of related tasks -one of the most successful examples of transfer learning to date (Oquab et al., 2014).
There is also a long history of unsupervised representation learning (Olshausen & Field, 1997). Much of the early research into modern deep learning was developed and validated via this approach (Hinton & Salakhutdinov, 2006) (Huang et al., 2007) (Vincent et al., 2008) (Coates et al., 2010) (Le, 2013). Unsupervised learning is promising due to its ability to scale beyond only the subsets and domains of data that can be cleaned and labeled given resource, privacy, or other constraints. This advantage is also its difficulty. While supervised approaches have clear objectives that can be directly optimized, unsupervised approaches rely on proxy tasks such as reconstruction, density estimation, or generation, which do not directly encourage useful representations for specific tasks. As a result, much work has gone into designing objectives, priors, and architectures meant to encourage the learning of useful representations. We refer readers to Goodfellow et al. (2016) for a detailed review.
Despite these difficulties, there are notable applications of unsupervised learning. Pre-trained word vectors are a vital part of many modern NLP systems (Collobert et al., 2011). These representations, learned by modeling word co-occurrences, increase the data efficiency and generalization capability of NLP systems (Pennington et al., 2014) (Chen & Manning, 2014). Topic modelling can also discover factors within a corpus of text which align to human interpretable concepts such as art or education (Blei et al., 2003).
How to learn representations of phrases, sentences, and documents is an open area of research. Inspired by the success of word vectors, Kiros et al. (2015) propose skipthought vectors, a method of training a sentence encoder by predicting the preceding and following sentence. The representation learned by this objective performs competitively on a broad suite of evaluated tasks. More advanced training techniques such as layer normalization (Ba et al., 2016) further improve results. However, skip-thought vectors are still outperformed by supervised models which directly optimize the desired performance metric on a specific dataset. This is the case for both text classification arXiv:1704.01444v2 [cs.LG] 6 Apr 2017 tasks, which measure whether a specific concept is well encoded in a representation, and more general semantic similarity tasks. This occurs even when the datasets are relatively small by modern standards, often consisting of only a few thousand labeled examples.
In contrast to learning a generic representation on one large dataset and then evaluating on other tasks/datasets, Dai & Le (2015) proposed using similar unsupervised objectives such as sequence autoencoding and language modeling to first pretrain a model on a dataset and then finetune it for a given task. This approach outperformed training the same model from random initialization and achieved state of the art on several text classification datasets. Combining language modelling with topic modelling and fitting a small supervised feature extractor on top has also achieved strong results on in-domain document level sentiment analysis (Dieng et al., 2016).
Considering this, we hypothesize two effects may be combining to result in the weaker performance of purely unsupervised approaches. Skip-thought vectors were trained on a corpus of books. But some of the classification tasks they are evaluated on, such as sentiment analysis of reviews of consumer goods, do not have much overlap with the text of novels. We propose this distributional issue, combined with the limited capacity of current models, results in representational underfitting. Current generic distributed sentence representations may be very lossy -good at capturing the gist, but poor with the precise semantic or syntactic details which are critical for applications.
The experimental and evaluation protocols may be underestimating the quality of unsupervised representation learning for sentences and documents due to certain seemingly insignificant design decisions. Hill et al. (2016) also raises concern about current evaluation tasks in their recent work which provides a thorough survey of architectures and objectives for learning unsupervised sentence representations -including the above mentioned skip-thoughts.
In this work, we test whether this is the case. We focus in on the task of sentiment analysis and attempt to learn an unsupervised representation that accurately contains this concept. Mikolov et al. (2013) showed that word-level recurrent language modelling supports the learning of useful word vectors and we are interested in pushing this line of work. As an approach, we consider the popular research benchmark of byte (character) level language modelling due to its further simplicity and generality. We are also interested in evaluating this approach as it is not immediately clear whether such a low-level training objective supports the learning of high-level representations. We train on a very large corpus picked to have a similar distribution as our task of interest. We also benchmark on a wider range of tasks to quantify the sensitivity of the learned represen-tation to various degrees of out-of-domain data and tasks.
Dataset
Much previous work on language modeling has evaluated on relatively small but competitive datasets such as Penn Treebank (Marcus et al., 1993) and Hutter Prize Wikipedia (Hutter, 2006). As discussed in Jozefowicz et al. (2016) performance on these datasets is primarily dominated by regularization. Since we are interested in high-quality sentiment representations, we chose the Amazon product review dataset introduced in McAuley et al. (2015) as a training corpus. In de-duplicated form, this dataset contains over 82 million product reviews from May 1996 to July 2014 amounting to over 38 billion training bytes. Due to the size of the dataset, we first split it into 1000 shards containing equal numbers of reviews and set aside 1 shard for validation and 1 shard for test.
Model and Training Details
Many potential recurrent architectures and hyperparameter settings were considered in preliminary experiments on the dataset. Given the size of the dataset, searching the wide space of possible configurations is quite costly. To help alleviate this, we evaluated the generative performance of smaller candidate models after a single pass through the dataset. The model chosen for the large scale experiment is a single layer multiplicative LSTM (Krause et al., 2016) with 4096 units. We observed multiplicative LSTMs to converge faster than normal LSTMs for the hyperparam-eter settings that were explored both in terms of data and wall-clock time. The model was trained for a single epoch on mini-batches of 128 subsequences of length 256 for a total of 1 million weight updates. States were initialized to zero at the beginning of each shard and persisted across updates to simulate full-backpropagation and allow for the forward propagation of information outside of a given subsequence. Adam (Kingma & Ba, 2014) was used to accelerate learning with an initial 5e-4 learning rate that was decayed linearly to zero over the course of training. Weight normalization (Salimans & Kingma, 2016) was applied to the LSTM parameters. Data-parallelism was used across 4 Pascal Titan X gpus to speed up training and increase effective memory size. Training took approximately one month. The model is compact, containing approximately as many parameters as there are reviews in the training dataset. It also has a high ratio of compute to total parameters compared to other large scale language models due to operating at a byte level. The selected model reaches 1.12 bits per byte.
Experimental Setup and Results
Our model processes text as a sequence of UTF-8 encoded bytes (Yergeau, 2003). For each byte, the model updates its hidden state and predicts a probability distribution over the next possible byte. The hidden state of the model serves as an online summary of the sequence which encodes all information the model has learned to preserve that is relevant to predicting the future bytes of the sequence. We are interested in understanding the properties of the learned encoding. The process of extracting a feature representation is outlined as follows: • Since newlines are used as review delimiters in the training dataset, all newline characters are replaced with spaces to avoid the model resetting state.
• Any leading whitespace is removed and replaced with a newline+space to simulate a start token. Any trailing whitespace is removed and replaced with a space to simulate an end token. The text is encoded as a UTF-8 byte sequence.
• Model states are initialized to zeros. The model processes the sequence and the final cell states of the mL-STM are used as a feature representation. Tanh is applied to bound values between -1 and 1.
We follow the methodology established in Kiros et al. (2015) by training a logistic regression classifier on top of our model's representation on datasets for tasks including semantic relatedness, text classification, and paraphrase detection. For the details on these comparison experiments, we refer the reader to their work. One exception is that we use an L1 penalty for text classification results instead of L2 as we found this performed better in the very low data regime. Table 1 shows the results of our model on 4 standard text classification datasets. The performance of our model is noticeably lopsided. On the MR (Pang & Lee, 2005) and CR (Hu & Liu, 2004) sentiment analysis datasets we improve the state of the art by a significant margin. The MR and CR datasets are sentences extracted from Rotten Tomatoes, a movie review website, and Amazon product reviews (which almost certainly overlaps with our training corpus). This suggests that our model has learned a rich representation of text from a similar domain. On the other two datasets, SUBJ's subjectivity/objectivity detection (Pang & Lee, 2004) and MPQA's opinion polarity (Wiebe et al., 2005) our model has no noticeable advantage over other unsupervised representation learning approaches and is still outperformed by a supervised approach.
Review Sentiment Analysis
To better quantify the learned representation, we also test on a wider set of sentiment analysis datasets with different properties. The Stanford Sentiment Treebank (SST) (Socher et al., 2013) was created specifically to evaluate more complex compositional models of language. It is derived from the same base dataset as MR but was relabeled via Amazon Mechanical and includes dense labeling of the phrases of parse trees computed for all sentences. For the binary subtask, this amounts to 76961 total labels compared to the 6920 sentence level labels. As a demonstration of the capability of unsupervised representation learning to simplify data collection and remove preprocessing steps, our reported results ignore these dense labels and computed parse trees, using only the raw text and sentence level labels.
The representation learned by our model achieves 91.8% significantly outperforming the state of the art of 90.2% by a 30 model ensemble (Looks et al., 2017). As visualized in Figure 2, our model is very data efficient. It matches the performance of baselines using as few as a dozen labeled examples and outperforms all previous results with only a few hundred labeled examples. This is under 10% of the total sentences in the dataset. Confusingly, despite a 16% relative error reduction on the binary subtask, it does not reach the state of the art of 53.6% on the fine-grained subtask, achieving 52.9%. We conducted further analysis to understand what repre- sentations our model learned and how they achieve the observed data efficiency. The benefit of an L1 penalty in the low data regime (see Figure 2) is a clue. L1 regularization is known to reduce sample complexity when there are many irrelevant features (Ng, 2004). This is likely to be the case for our model since it is trained as a language model and not as a supervised feature extractor. By inspecting the relative contributions of features on various datasets, we discovered a single unit within the mLSTM that directly corresponds to sentiment. In Figure 3 we show the histogram of the final activations of this unit after processing IMDB reviews (Maas et al., 2011) which shows a bimodal distribution with a clear separation between positive and negative reviews. In Figure 4 we visualize the activations of this unit on 6 randomly selected reviews from a set of 100 high contrast reviews which shows it acts as an online estimate of the local sentiment of the review. Fitting a threshold to this single unit achieves a test accuracy of 92.30% which outperforms a strong supervised results on the dataset, the 91.87% of NB-SVM trigram (Mesnil et al., 2014), but is still below the semi-supervised state of the art of 94.09% (Miyato et al., 2016). Using the full 4096 unit representation achieves 92.88%. This is an improvement of only 0.58% over the sentiment unit suggesting that almost all information the model retains that is relevant to sentiment analysis is represented in the very compact form of a single scalar. Table 2 has a full list of results on the IMDB dataset.
Capacity Ceiling
Encouraged by these results, we were curious how well the model's representation scales to larger datasets. We try our approach on the binary version of the Yelp Dataset 25 August 2003 League of Extraordinary Gentlemen: Sean Connery is one of the all time greats and I have been a fan of his since the 1950's. I went to this movie because Sean Connery was the main actor. I had not read reviews or had any prior knowledge of the movie. The movie surprised me quite a bit. The scenery and sights were spectacular, but the plot was unreal to the point of being ridiculous. In my mind this was not one of his better movies it could be the worst. Why he chose to be in this movie is a mystery. For me, going to this movie was a waste of my time. I will continue to go to his movies and add his movies to my video collection. But I can't see wasting money to put this movie in my collection I found this to be a charming adaptation, very lively and full of fun.
With the exception of a couple of major errors, the cast is wonderful. I have to echo some of the earlier comments --Chynna Phillips is horribly miscast as a teenager. At 27, she's just too old (and, yes, it DOES show), and lacks the singing "chops" for Broadway-style music. Vanessa Williams is a decent-enough singer and, for a non-dancer, she's adequate. However, she is NOT Latina, and her character definitely is. She's also very STRIDENT throughout, which gets tiresome. The girls of Sweet Apple's Conrad Birdie fan club really sparkle --with special kudos to Brigitta Dau and Chiara Zanni. I also enjoyed Tyne Daly's performance, though I'm not generally a fan of her work. Finally, the dancing Shriners are a riot, especially the dorky three in the bar. The movie is suitable for the whole family, and I highly recommend it.
Judy Holliday struck gold in 1950 withe George Cukor's film version of "Born Yesterday," and from that point forward, her career consisted of trying to find material good enough to allow her to strike gold again. It never happened. In "It Should Happen to You" (I can't think of a blander title, by the way), Holliday does yet one more variation on the dumb blonde who's maybe not so dumb after all, but everything about this movie feels warmed over and half hearted. Even Jack Lemmon, in what I believe was his first film role, can't muster up enough energy to enliven this recycled comedy. The audience knows how the movie will end virtually from the beginning, so mostly it just sits around waiting for the film to catch up. Maybe if you're enamored of Holliday you'll enjoy this; otherwise I wouldn't bother. Grade: C Once in a while you get amazed over how BAD a film can be, and how in the world anybody could raise money to make this kind of crap. There is absolutely No talent included in this film -from a crappy script, to a crappy story to crappy acting. Amazing...
Team Spirit is maybe made by the best intentions, but it misses the warmth of "All Stars" (1997) by Jean van de Velde. Most scenes are identic, just not that funny and not that well done. The actors repeat the same lines as in "All Stars" but without much feeling. Figure 5, we observe a "capacity ceiling" where the test accuracy of our approach only improves by a little over 1% across a four order of magnitude increase in training data. Using the full dataset, we achieve 95.22% test accuracy. This better than a BoW TFIDF baseline at 93.66% but slightly worse than the 95.64% of a linear classifier on top of the 500,000 most frequent n-grams up to length 5.
The observed capacity ceiling is an interesting phenomena and stumbling point for scaling our unsupervised representations. We think a variety of factors are contributing to cause this. Since our model is trained only on Amazon reviews, it is does not appear to be sensitive to concepts specific to other domains. For instance, Yelp reviews are of businesses, where details like hospitality, location, and atmosphere are important. But these ideas are not present in reviews of products. Additionally, there is a notable drop in the relative performance of our approach transitioning from sentence to document datasets. This is likely due to our model working on the byte level which leads to it focusing on the content of the last few sentences instead of the whole document. Finally, as the amount of labeled data increases, the performance of the simple linear model we train on top of our static representation will eventually saturate. Complex models explicitly trained for a task can continue to improve and eventually outperform our approach with enough labeled data.
With this context, the observed results make a lot of sense.
Sentiment fixed to positive Sentiment fixed to negative
Just what I was looking for. Nice fitted pants, exactly matched seam to color contrast with other pants I own.
Highly recommended and also very happy!
The package received was blank and has no barcode. A waste of time and money.
This product does what it is supposed to. I always keep three of these in my kitchen just in case ever I need a replacement cord.
Great little item. Hard to put on the crib without some kind of embellishment. My guess is just like the screw kind of attachment I had.
Best hammock ever! Stays in place and holds it's shape. Comfy (I love the deep neon pictures on it), and looks so cute.
Other Tasks
Besides classification, we also evaluate on two other standard tasks: semantic relatedness and paraphrase detection. While our model performs competitively on Microsoft Research Paraphrase Corpus (Dolan et al., 2004) in Table 3, it performs poorly on the SICK semantic relatedness task (Marelli et al., 2014) in Table 4. It is likely that the form and content of the semantic relatedness task, which is built on top of descriptions of images and videos and contains sentences such as "A sea turtle is hunting for fish" is effectively out-of-domain for our model which has only been trained on the text of product reviews.
Generative Analysis
Although the focus of our analysis has been on the properties of our model's representation, it is trained as a generative model and we are also interested in its generative capabilities. Hu et al. (2017) and Dong et al. (2017) both designed conditional generative models to disentangle the content of text from various attributes like sentiment or tense. We were curious whether a similar result could be achieved using the sentiment unit. In Table 5 we show that by simply setting the sentiment unit to be positive or negative, the model generates corresponding positive or negative reviews. While all sampled negative reviews contain sentences with negative sentiment, they sometimes contain sentences with positive sentiment as well. This might be reflective of the bias of the training corpus which contains over 5x as many five star reviews as one star reviews. Nevertheless, it is interesting to see that such a simple manipulation of the model's representation has a noticeable effect on its behavior. The samples are also high quality for a byte level language model and often include valid sentences.
Discussion and Future Work
It is an open question why our model recovers the concept of sentiment in such a precise, disentangled, interpretable, and manipulable way. It is possible that sentiment as a conditioning feature has strong predictive capability for language modelling. This is likely since sentiment is such an important component of a review. Previous work analysing LSTM language models showed the existence of interpretable units that indicate position within a line or presence inside a quotation (Karpathy et al., 2015). In many ways, the sentiment unit in this model is just a scaled up example of the same phenomena. The update equation of an LSTM could play a role. The element-wise operation of its gates may encourage axis-aligned representations. Models such as word2vec have also been observed to have small subsets of dimensions strongly associated with specific tasks (Li et al., 2016).
Our work highlights the sensitivity of learned representations to the data distribution they are trained on. The results make clear that it is unrealistic to expect a model trained on a corpus of books, where the two most common genres are Romance and Fantasy, to learn an encoding which preserves the exact sentiment of a review. Likewise, it is unrealistic to expect a model trained on Amazon product reviews to represent the precise semantic content of a caption of an image or a video.
There are several promising directions for future work highlighted by our results. The observed performance plateau, even on relatively similar domains, suggests improving the representation model both in terms of architecture and size. Since our model operates at the byte-level, hierarchical/multi-timescale extensions could improve the quality of representations for longer documents. The sensitivity of learned representations to their training domain could be addressed by training on a wider mix of datasets with better coverage of target tasks. Finally, our work encourages further research into language modelling as it demonstrates that the standard language modelling objective with no modifications is sufficient to learn high-quality representations. | 2017-04-06T09:48:20.000Z | 2017-04-05T00:00:00.000 | {
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244399080 | pes2o/s2orc | v3-fos-license | A study of Irrigation Water Pollution By Some Heavy Metals in Baghdad Governorate
A study of irrigation water was conducted Baghdad city to find out extent of its pollution by some heavy metals (Pb, Cd, Ni, Co, CU, Cr, Zn and Fe). Water samples were collected randomly from different sources (river, well and stream). Results showed that the concentration of studied heavy metals were as follows: Lead between 0.43-11.75 mg L-1, Cadmium between 0.01-0.95 mg L-1, Nickel between 0.008-0.46 mg L-1, Cobalt between Nil - 0.185 mg L-1, Copper is between 0.326 - 1.58 mg L-1, Chromium is between Nil-0.068 mg L-1, Zinc 0.398-1.182 mg L-1, as for Iron between 0.794 - 3.253 mg L-1, and high concentrations of heavy metals were in all samples, The most sites were higher than a critical limits permitted by the International Food and Agriculture Organization.
Introduction
Iraqi water pollution problem is one of the big problems that started to appear and increase, which necessitated serious thinking about finding ways to combat it and reduce its effects [1]. Water pollution is any physical, chemical or biological change in quality of water, directly or indirectly, that negatively affects living organisms or makes water unfit for drinking and required uses and is one of the main problems that most countries of world face due to the expansion of industrial activity. Water pollution greatly affects human life and living organisms, as many factories deliberately dispose of their waste and products of their industries such as oil derivatives, factory waste, city waste, chemical fertilizers, pesticides and diseasecausing organisms, diseases and radioactive materials by throwing them into water, such as oceans, seas and rivers, so that water becomes less suitable for natural uses for drinking and agriculture. Heavy metals are present in very low concentrations not exceeding 50 µL -1 , when these waters are far from sources of pollution, but these concentrations may increase due to their proximity to sources of pollution [2]. [3], explain in their study on assessing water pollution with heavy metals of Al-Quds power station in Baghdad, the presence of Lead, Nickel and Manganese contamination amounted to 29.6, 59.1 and 910 µgL-1 and was higher than the permissible limits according to [4].
Heavy metals are considered dangerous environmental pollutants and their danger lies in their bio-cumulative characteristic in the bodies of living organisms that feed on plants cultivated in polluted soils for reasons related to factors of geological weathering of soil because of excessive use of chemical fertilizers and agricultural pesticides, most of the time it is the result of irrigation with water polluted with waste of factories, and sewage wastes [5]. The high traffic density of vehicles also plays an important role in increasing the concentrations of heavy metals in soils of internal road sites of Baghdad city due to the emissions of vehicles, and they took the following order in terms of the height of concentration: Zn> Ni >Pb> Cd [6]. [7] estimated concentrations of some heavy metals in water and sediments of Tigris River, explained the high concentration of lead in some of measured samples, and they attributed this to subtraction of large quantities of liquid waste towards river at south of city, and its concentrations in summer season are more than in the winter due to evaporation and sedimentation factor, as well as the discharge of industrial wastewater and others. [8] indicated that water of Diyala River was contaminated with lead and cadmium elements with high concentrations exceeding the permissible limits of [9] and poor water quality as its class C 4 S 1 is low in Na and very high in salinity, and a significant increase of the Lead in shoot system at the flowering stage, plant was contaminated with Pb, as the concentrations reached 21.75 and 42.92 mg pb. kg -1 dry matter successively. [10], on the effect of using Danvili Valley water in city of Mosul on contamination of soils with heavy metals showed that it is bad for agricultural purposes, depending on assessment of its physiochemical properties and concentration of heavy metals zinc, cadmium, lead, copper and iron, its mean concentrations in water samples were 7.07, 2.27, 8.87, 4.87, 6.70 mg L -1 .
Materials and Method
Water samples (of river, stream and well) were collected from twelve sites in city of Baghdad shown in figure 1. by 2.5 L plastic containers after washing them twice with water sample and adding drops of toluene to prevent bacterial growth, Chemical properties of water were estimated as shown in Table 1, concentration of dissolved heavy etals in water has also been estimated according to methods contained in [11] as follows: pH using a pH-meter type (HACH) model (HQ411d) , electrical conductivity (EC) was measured using EC-meter type (HACH) model (EC71). dissolved ions of calcium and magnesium were determined by titration with fresnite. sodium and potassium was determined using a flame photometer model (AFP 100), chloride estimated by titration with 0.0141 standard silver nitrate, carbonate and bicarbonate assessed according to the methods in [12] Mediated titration with 0.01 N sulfuric acid. sodium adsorption ratio (SAR) values were calculated according to the formulas given in [13] as SAR = Na / √ (Ca + Mg)/2 mmolL -1 .
Water class was determined according to [14]. Heavy metals ions (Fe, Zn, Cr, Cu, Co, Ni, Cd, and Pb) were estimated in water samples after filtering the samples to get rid of impurities using Atomic Absorption Spectrophotometer (AAS) type Shimadzu model (AA7000). Figure 2. Lead concentration (mg L -1 ) in water samples of study sites (Permissible limit for irrigation is 5 mg L -1 ) [15]. Figure 5 indicates concentration of Co in water samples, as the highest value of Co concentration was in Diyala Bridge area, which was 0.185 mg L -1 , while lowest value was in Rashid and Yusufiya regions, whose value was imperceptible Nil mg L -1 . Figure 6. Cu concentration (mg L -1 ) in water samples of study sites (Permissible limit for irrigation is 0.2 mg L -1 ) [15]. Figure 7 indicates concentration of Cr in water samples, as highest value of Cr concentration was in Diyala Bridge region, which was 0.068 mg L -1 , while lowest value was in areas of Rasheed, Al-Dora, Abu Gharib, Al-Turath area, Karrada, and Al-Jadriya, whose value was imperceptible to Nil mg L -1 . Figure 9 shows Fe concentration in water samples, as the highest value of Zn concentration was in Diyala Bridge area, which was 3.253 mg L -1 , while lowest value was in Abu Ghraib area 0.794 mg L -1 .
Figure 9.
Fe concentration (mg L -1 ) in water samples of study sites (Permissible limit for irrigation purposes is 5 mg L -1 ) [15]. Results showed that lead concentration exceeded permissible limit according to classification of FAO (1994), which is 5 mg L -1 in Diyala Bridge area, while rest of sites were within the permissible limit. As for Cadmium concentration, it exceeded permissible limits in all water samples for study sites, which amount to 0.01 mg L -1 , concentration of Ni and Co exceeded permissible limit (0.2 and 0.05 mg liter -1, respectively) in Diyala Bridge area, while rest of sites were within the permissible limit, Cu concentration exceeded permissible limit in all study sites. As for concentration of Cr, Zn and Fe, all sites were within permissible limit (0.1, 2.0, 5.0 mg L -1 , respectively). Water pollution in general and Diyala River in particular, may be attributed to exacerbation of all kinds of pollutants inside Al-Rustumiya station for heavy water purification, as a result of its poor treatment and contamination, including industrial and non-industrial pollutants. However, wastewater (sewage) resulting from filtering process is a major factor in pollution. As it poses a threat to public health because of pollutants it contains, and residents of neighboring areas may use this water for multiple purposes, including agriculture or drinking. [16], showed presence some heavy metals ions in water of Tigris River, as concentrations of Lead reached 0.01-0.27mg L -1 , Cd from imperceptible to 0.026 mg L -1 , Ni ranged from imperceptible to 0.079 mg L -1 , Co ranged from imperceptible to 0.096 mg L -, Cu from imperceptible to 0.053 mg L -1 . ,Cr from imperceptible to 0.757 mg L -1 and Zn from imperceptible to 0.942 mg L -1 ,thus the river water is contaminated and Lead had highest concentration, followed by Ni, Cd, Co, Cr, Cu and Zn, they attributed this to density and population activity in area, frequent congestion of cars and burning of fuel, which leads to an increase in release of heavy metals. | 2021-11-19T20:53:25.876Z | 2021-11-01T00:00:00.000 | {
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235758082 | pes2o/s2orc | v3-fos-license | Varicella-zoster virus early infection but not complete replication is required for the induction of chronic hypersensitivity in rat models of postherpetic neuralgia
Herpes zoster, the result of varicella-zoster virus (VZV) reactivation, is frequently complicated by difficult-to-treat chronic pain states termed postherpetic neuralgia (PHN). While there are no animal models of VZV-induced pain following viral reactivation, subcutaneous VZV inoculation of the rat causes long-term nocifensive behaviors indicative of mechanical and thermal hypersensitivity. Previous studies using UV-inactivated VZV in the rat model suggest viral gene expression is required for the development of pain behaviors. However, it remains unclear if complete infection processes are needed for VZV to induce hypersensitivity in this host. To further assess how gene expression and replication contribute, we developed and characterized three replication-conditional VZV using a protein degron system to achieve drug-dependent stability of essential viral proteins. Each virus was then assessed for induction of hypersensitivity in rats under replication permissive and nonpermissive conditions. VZV with a degron fused to ORF9p, a late structural protein that is required for virion assembly, induced nocifensive behaviors under both replication permissive and nonpermissive conditions, indicating that complete VZV replication is dispensable for the induction of hypersensitivity. This conclusion was confirmed by showing that a genetic deletion recombinant VZV lacking DNA packaging protein ORF54p still induced prolonged hypersensitivities in the rat. In contrast, VZV with a degron fused to the essential IE4 or IE63 proteins, which are involved in early gene regulation of expression, induced nocifensive behaviors only under replication permissive conditions, indicating importance of early gene expression events for induction of hypersensitivity. These data establish that while early viral gene expression is required for the development of nocifensive behaviors in the rat, complete replication is dispensable. We postulate this model reflects events leading to clinical PHN, in which a population of ganglionic neurons become abortively infected with VZV during reactivation and survive, but host signaling becomes altered in order to transmit ongoing pain.
Introduction
The human neurotropic herpesvirus, varicella-zoster virus (VZV), causes varicella (chickenpox) upon primary infection and herpes zoster (shingles, HZ) following reactivation from a latent state that was established during primary infection [1]. HZ will occur in about one-third of unvaccinated individuals in their lifetime and remains a major public health concern, owing to an expanding aged demographic that are at risk of HZ disease and morbidities. Incidence and disease severity increase significantly with advancing age and/or immune impairment [2]. While incidence of both primary and reactivated disease can be greatly reduced by vaccination [3][4][5][6], most adults remain at risk for HZ because they harbor latent, wild-type VZV within sensory ganglia, and uptake of HZ vaccines in the US is far from optimal [7]. In many countries, use of HZ vaccines is minimal or non-existent [8,9], despite providing reasonable rates of protection from HZ and its debilitating consequences. Therefore, research continues into improved treatment and prevention strategies for HZ.
Pain is the most common complication of HZ and is one of the largest contributors to its morbidity. Pain may occur before rash development (prodrome), but more than 80% of individuals over 60-years with HZ will experience pain during and/or after the rash that requires prescription medication treatment [10]. A significant fraction of HZ patients will progress to debilitating chronic pain states known as postherpetic neuralgia (PHN), defined by many as pain lasting more than three months after the typical dermatomal rash of HZ has resolved [11]. Symptoms of PHN commonly include allodynia, defined as hypersensitivity to normally innocuous stimuli that frequently persists after stimulus removal. PHN may also include increased sensitivity to thermal stimuli (hyperalgesia) [12]. PHN incidence rates have been reported to be as high as 20-30% of those with HZ, with greater severity linked to certain dermatomes, rising age, and decline of immune status [8]. Because clinical PHN lasts well beyond visible signs of skin disease, it may not reflect continuous viral replication but rather tissue damage sustained during active HZ disease that propagates ongoing pain signaling. Clinical observations suggest VZV triggers numerous and long-lasting changes indicative of neuropathic damage within innervating sensory tissues [13]. Further, the severity of PHN directly correlates with preceding HZ symptom severity [11], suggesting a "more damage, more pain" scenario. A key difference between acute and chronic pain is response to antiviral treatment. The antiviral acyclovir can often effectively limit acute HZ and associated pain when delivered in a timely manner [14], and treatment during acute HZ may also reduce the duration of subsequent PHN [15]. However, administration of antivirals during PHN is ineffective in most cases [16], reinforcing the concept that chronic pain signaling is not a consequence of ongoing VZV replication. The processes involved are not entirely clear and the mechanisms governing the transition from acute to chronic pain states remain ill-defined. Notably, HZ involves extensive intra-ganglionic spread to result in the infection of large numbers of neurons and nonneuronal cells within the reactivating ganglion [17]. These mediate virus delivery to the periphery by many neurons, and also cause inflammation both at the ganglia and at the skin that contribute to pain [18,19]. Postmortem studies of cadaver ganglia with ongoing HZ at time of death reveal extensive ganglionitis and tissue damage [20,21]. Many complex mechanisms underlying PHN pain have been proposed but few have been experimentally substantiated [12,21,22].
The lack of a reliable small animal model of VZV reactivation and HZ disease has precluded the study of VZV reactivation-driven pain. Rodent models show little to no clinical presentation after infection. Even non-human primate models using the closely related simian varicella virus (SVV) are difficult to employ for HZ and pain studies due to inefficient virus reactivation. Even then, animals do not routinely develop signs of PHN. Several groups have used the rat to investigate VZV latent states [23][24][25]. It was then demonstrated by multiple groups that Wistar and Sprague-Dawley rat strains inoculated with VZV at the footpad develop prolonged signs of pain that could serve as preclinical models for exploring mechanisms and treatment strategies for PHN [22]. The models involve subcutaneous inoculation of cell-associated VZV into the rat hind footpad [26][27][28][29][30][31][32] or more recently, the whisker pad [33][34][35][36][37][38]. While animals show no outward signs of skin infection, inflammatory response, or disease, they develop nocifensive behaviors lasting several weeks. It has never been thoroughly resolved if VZV productive replication occurs within inoculated rats and if this is a requirement for nocifensive responses. Work from our group indicated that VZV did not replicate in rat primary cell cultures, suggesting VZV replication in vivo is unlikely [32]. One in vivo study indicated that VZV-induced hypersensitivity in rats was unresponsive to acyclovir administration [27]. Our group reported that rats inoculated with UV-inactivated VZV did not develop long-term nocifensive behaviors, suggesting a requirement for viral gene expression in the development of pain behaviors [31]. VZV has been shown to induce subtle changes in host gene expression within infected ganglia [32]. Ganglionic sections of rats with hypersensitivity show sporadic staining for the major VZV transcriptional regulatory protein, IE62 [28][29][30]34]. Taken together, these results suggest that VZV may initiate abortive infections in the rat that nevertheless induce nocifensive behaviors and hypersensitivity.
Here, we further addressed requirements of VZV replication and gene expression in development of pain indicators in the rat PHN models. We developed and used novel VZV mutants that show conditional replication, depending on the stability of specific essential proteins containing a "degron" domain fused to either the amino-or carboxy-termini of the target protein (Fig 1) [39]. Turnover of degron-protein fusions is regulated by a cell permeable drug, trimethoprim (TMP), that stabilizes the degron domain and prevents proteasomal degradation. We targeted VZV proteins that have essential roles at the beginning and end of the lytic herpesvirus gene expression cascade. All herpesviruses express their genes in an ordered manner in which immediate-early (IE or α) genes are expressed first, followed by early (E or β) genes that encode proteins that generally act to replicate viral DNA, and then late (L or γ) genes whose proteins are generally involved in virus assembly and egress [40]. VZV IE gene encoded Overview of VZV BAC development with a degron insertion and TMP-dependent protein turnover. (A) A target gene in the VZV BAC is engineered by recombining a degron sequence with an interrupting kanamycin resistance cassette (kan r ) that allows for positive selection in E. coli GS1783 as detailed in the methods. A second induced recombination event in conjunction with expression of the homing endonuclease I-SceI results in markerless excision of the kan r cassette, so that the degron coding protein is fused to the target gene ORF. BACs are then transfected into human TRPE cell monolayers in the presence of the stabilizing ligand trimethoprim (TMP) to yield infectious VZV. (B) In the presence of TMP (top), TMP (green) is thought to bind the degron and prevent ubiquitin (Ub, red) ligation, thus stabilizing protein and halting turnover. In the absence of TMP (bottom), Ub is ligated to the degron, and the entire protein is targeted for degradation by ubiquitin-proteasome pathway. Created with BioRender.com.
proteins have regulatory functions that subsequently control the rest of the viral gene expression program [40][41][42][43][44]. Using a degron domain from the E. coli dihydrofolate reductase (DHFR), we developed three conditionally replicating VZV recombinants. These were used in conjunction with a cell-complemented VZV deletion mutant that did not express the essential ORF54 gene encoding the capsid portal protein [45]. The VZV recombinants were then assessed for their ability to induce behavioral hypersensitivities in rats when inoculated in the presence or absence of TMP. We show that VZV blocked at late stages of assembly and full productive replication in the rat still induced prolonged hypersensitive behaviors, establishing that productive replication is not required. In contrast, rats inoculated with conditionally replicating VZV with a degron attached to IE regulated proteins only developed hypersensitivity when the inoculates were supplemented with TMP to stabilize the targeted degron proteins. This suggested that the expression of essential IE transcriptional regulatory proteins IE4 and IE63 was required for the stimulation of persistent pain behaviors in the rat. These data are consistent with a hypothesized mechanism in which a limited VZV gene expression program in the rat results in altered host neuronal pain signaling. We discuss that this may occur in patients with PHN, in which an abortive VZV infection process occurs during HZ within sensory neurons that survive reactivation but go on to signal pain.
Ethics statement
All animal studies were performed in accordance with protocols approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC, protocol #18022168). This protocol meets the standards for humane animal care and use as set by the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals.
Cells and viruses
VZV parental Oka (pOka) is a wild-type varicella isolate from which the current live attenuated vaccines were derived [46]. It has been established that pOka can induce both mechanical and thermal hypersensitivity in rat models of PHN [31][32][33][34][35][36][37][38] and was used as a positive control in all studies. Recombinant viruses were derived from a pOka bacterial-artificial chromosome (BAC) which contains a self-excisable BAC replicon [47] that was subsequently corrected for two spurious mutations in the VZV ORF40 and ORF50 genes and contained an N terminal GFP reporter fused to ORF23 [48].
Human telomerase (Tert) immortalized RPE-1 (TRPE, ATCC CRL4000) cells were grown in Dulbecco's Minimal Essential Media (DMEM, Gibco 10569-010) supplemented with 10% fetal bovine serum (FBS, R&D Systems S11150) and an antibiotic/antimycotic mixture (Caisson ABL02). TRPE cells were used for reconstitution of virus from BAC DNAs, VZV propagation, and stock preparation. VZV stocks were made as previously detailed [31]. Briefly, TRPE monolayers grown to 80-90% confluency at 37˚C were infected, incubated 48-72 hours at 34˚C, and underwent trypsinization and re-plating when needed, until >60% of cells showed visible cytopathic effect, or 95%+ fluorescence positivity where relevant. Virus infected cell stocks were generated by trypsin digestion, concentrated by low-speed centrifugation, then resuspended in cell freeze media (DMEM, 20% FBS, 10% DMSO) and subjected to slow freeze at -80˚C, followed by liquid nitrogen storage. Conditional VZV mutants were derived and grown in TRPE cells with the media additionally supplemented with 100 nM TMP (Sigma T7883) [39]. Prior to storage, all conditional VZV samples were washed extensively in cold DMEM without TMP to remove residual drug prior to freezing. Aliquots from liquid nitrogen storage were titrated to assess VZV cell-associated infectious center formation (taken as titer) by dilution and plaque assay in permissive conditions on ARPE-19 cells (ARPE, ATCC CRL2302) using supplemented DMEM as noted above, and TMP when necessary.
Generation of recombinant VZV
Recombinant VZV ORF54Δ and its complementing ARPE19-ORF54 (A54) cells were detailed previously [45]. Recombinants VZV ORF4nDHFR, VZV ORF63cDHFR, and ORF9cDHFR were constructed by two-step Red-mediated scarless recombination methods as previously described [49], utilizing the VZV pOka BAC that contains green fluorescent protein (GFP) fused to the N-terminus of the minor capsid protein encoded by ORF23 (HSV VP26 homolog), as described previously [48]. Mutagenesis was performed in the GS1783 E. coli strain (gift of Dr. Gregory Smith, Northwestern University, IL) containing the heat shock-inducible (42˚C) λRed recombination system and an L-arabinose inducible I-SceI restriction enzyme. A transfer plasmid for the 480-bp degron domain derived from the E. coli DHFR gene [39] was generated by the insertion of the I-SceI kanamycin resistance (kan r ) cassette flanked by 40-bp homologous sequences used for the scarless removal of the selective marker ( Fig 1A). PCR primers (Table 1) were designed to anneal at the end of the degron domain sequence and extend the cassette by 40-bp sequences homologous to the targeted insertion sequence in the VZV BAC. The fragment was amplified using the proofreading polymerase PrimeSTAR GXL (Takara Biochemicals, R050A). For generation of VZV ORF63cDHFR, the coding sequence of ORF70 (duplicated ORF63 gene) was replaced by a PCR amplified ampicillin resistance cassette before kan r removal from ORF63cDHFR, and co-selected by growth on plates supplemented with chloramphenicol (to maintain the BAC) kanamycin and ampicillin. All BACs were subjected to extensive characterization by digestion with multiple restriction enzymes to ensure no deletions of BAC DNA sequences, and all in frame gene-degron fusions were verified by Sanger sequencing of PCR amplified fragments across the respective junctions. Infectious virus was reconstituted by the transfection of the recombinant BACs using Lipofectamine 3000 (ThermoFisher) on TRPE cells grown in the presence of 100 nM TMP. VZV were passaged 3-5 times in permissive growth media (supplemented DMEM + 100 nM TMP) to allow self-excision of the BAC sequence, and master stocks were prepared and titrated as previously described. All mutant VZV used in these studies were grown from low passage master stocks with minimum of 8-10 additional passages. Integrity of the inserted DHFR sequences were confirmed by Sanger sequencing across the 5' and 3' insert junctions of both the BACs and of the resultant viruses.
DNA of recombinant viruses generated from the BACs was Southern blot analyzed by extraction of nucleocapsid VZV DNA from 5 x 175 cm 2 infected cell flasks showing >70% cytopathic effect using the procedure detailed previously [50]. 1ug of DNA from each VZV recombinant DHFR virus and VZV from the VZV pOka BAC was assessed by restriction digestion with multiple enzymes including KpnI (S1 Fig) and SphI (S2 Fig), subjected to agarose gel electrophoresis, transferred to a nylon membrane (Millipore INYC00010), and then probed to identify the fragments that contain the degron element. The hybridization probe was generated by PCR amplification with oligos homologous to the DHFR degron using the primers 5'-CCTGGTTTAAACGCAACACC and 5'-GTGAGAGTTCTGCGCATCAG to amplify a 474-bp product, which was then labeled with a Biotin DecaLabel kit (ThermoFisher K0651). Probe hybridization (10 ng/mL) was completed overnight at 42˚C and detected by binding fluorescent IRDye 800CW Streptavidin (LI-COR 926-32230) for 1-hr at room temperature at a 1:10,000 dilution. The blot was then imaged on a LI-COR Odyssey IR in linear range.
Virus growth analysis
Growth of VZV recombinants modified with degrons were completed using an infectious focus assay as previously detailed [51] with modifications. Briefly, sub-confluent TRPE monolayers were plated in 12-wells (~3.5x10 5 cells/well) and infected with cell-associated, pretitrated VZV at approximately 100 plaque forming units (PFU) per well, in duplicate. Infections were carried out in media containing 100 nM TMP (permissive) or no TMP (nonpermissive) conditions that was then maintained throughout the time of incubation. At times indicated, infected cells monolayers were harvested by trypsinization of the infected cultures and then replating dilutions of the cell suspensions in duplicate on ARPE cells under permissive conditions to quantify the number of infectious cells able to initiate formation of a plaque. Wild-type VZV pOka served as positive control. Titrated infections were fixed at 5-dpi, stained with crystal violet, and plaques were counted on a dissection microscope. Biological sample duplicates and technical duplicates were averaged and graphed.
Immunoblotting
Immunoblotting methods were previously described [52] and performed with minor modifications. Briefly, sub-confluent TRPE monolayers in 6-well plates (~1 x 10 6 cells/well) were infected with 1x10 4 PFU VZV-TRPE cell-associated virus (1:100 infected to uninfected cells) and incubated in DMEM with or without 100 nM TMP for 24-, 48-, and 72-hours post-infection. Cells were harvested by washing twice in ice-cold 1x PBS and removed by mechanical dislocation into 1x PBS containing protease (ThermoFisher Halt) and phosphatase (Roche PhosSTOP) inhibitors. After concentration by centrifugation at 12,000xg at 4˚C for 5 minutes, cells were resuspended in 100 μl 1x PBS with 2x protease inhibitors, to which 100 μl of 2X SDS PAGE lysis buffer was added for a final sample volume of 200 ul. Samples were briefly probesonicated, heated to 95˚C for 5 min, then loaded onto precast 4-15% acrylamide gradient SDS-PAGE gels (Bio-Rad Criterion) and run at 65V until completion. Proteins were transferred by electrophoresis to a polyvinylidene difluoride membrane (Millipore Immobolin-FL 00010) overnight at 15V, and membranes blocked overnight at 4˚C using LI-COR "Intercept" Blocking Buffer. Blots were incubated with dilutions of primary antibodies at 4˚C for 4-24 h in diluted blocking buffer solution containing 0.1% Tween-20, washed extensively in the same buffer, and further incubated with secondary species-specific antibodies linked to near IR dyes (LI-COR, IRDye 680/800) at 1:20,000 dilution for 1-h at room temperature, washed, and imaged on a LI-COR Odyssey IR in a linear range.
Fluorescent microscopy
For cell localization studies, TRPE monolayers prepared on 4-well chambered slides (Sigma-Aldrich Nunc Lab-Tek II C6807) were infected with 50 or 10 PFU VZV per chamber and incubated 4-days under TMP permissive and nonpermissive conditions at 34˚C. Monolayers were fixed by incubation for 20-min in 4% paraformaldehyde at room temperature, washed in 1X PBS, and blocked overnight in 10% heat-inactivated goat serum (HIGS) in PBS. Samples were incubated in HIGS-PBS diluted primary antibodies overnight, washed, and incubated with Alexa Fluor-coupled secondary antibodies for 1-h at room temperature. After a final 10-minute incubation with DAPI, washed chambers were separated from the slides and coverslips mounted. Slide imaging was performed on an Olympus IX83 using a 60X (N.A. 1.25) oil objective in a linear range. These images were processed using Olympus CellSens software and Ima-geJ, and comparative images were processed equally. Plaque sizes were determined by imaging VZV infectious centers at 4-dpi prepared under permissive and nonpermissive conditions and grown at 34˚C. Monolayers were fixed by 20-min incubation in 4% paraformaldehyde at room temperature, washed in PBS, and stored at 4˚C in PBS until imaged. All viruses, except for pOka, produce a GFP fused ORF23 protein (ORF23p) that served to identify VZV infectious centers by fluorescent microscopy. To image pOka plaques under similar conditions, infected samples were probed with a primary antibody to ORF23p and detected with Alexa Fluor 488 secondary antibody as described in the localization analysis method. Images containing individual plaques were each acquired under identical acquisition settings with Cell Sens software on an Olympus IX83 microscope with a 10X (N.A..030) air objective. Images were exported from Olympus CellSens software and analyzed in Metamorph (Version 7.7, Molecular Devices, San Jose, CA). Data was reported as area in pixels (1 px = 1.024 μM).
Nucleic acid analyses of animal DRG tissues by RT-qPCR
Messenger RNA transcripts encoding ORF4, ORF62, ORF63, or DHFR sequences were quantified from rat DRG following footpad inoculation and tissue harvest as detailed previously [32], using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and TaqMan probes (S1 Table). Briefly, male Sprague-Dawley rats (n = 24) were divided into four groups for inoculation (pOka, VZV ORF4nDHFR, VZV ORF63cDHFR, and uninoculated) and sub-divided into three timepoint groups for post-infection harvest at 4-, 5-, and 7-dpi. Rats (n = 6/group) received injections of 2x10 5 PFU VZV into the glabrous footpad. At each timepoint, rats (n = 2/group) were necropsied, L4, L5, L6 DRG were micro-dissected and snap frozen in liquid nitrogen until nucleic acid purification. RNA was purified by mechanically disrupting tissues in TRIzol (ThermoFisher) reagent with a PT1200E tissue homogenizer (Kinematica, Bohemia, NY) for 10-s at~75% power. RNAs were dissolved in nuclease free H 2 O, DNase-treated (ThermoFisher EN0521) and converted into cDNA using a High-Capacity RNA-to-cDNA kit (Applied Biosystems 4387406). cDNAs were analyzed by thermocycling (95˚C, 60˚C, 40X) in an Applied Biosystems StepOne Plus qPCR system with PrimeTime Gene Expression Master Mix (IDT 1055770). Gene expression was detected by TaqMan assay and relative expression values were calculated by the 2 -ΔΔCt method with comparison to rat GAPDH (Applied Biosystems Rn01775763_g1) and displayed as fold-change over GAPDH (equal to 1). Data are averaged results from two identical qPCR assays. ORF62 and ORF63 TaqMan primer sets (S1 Table) have been described [56].
Animal studies
Male Sprague-Dawley rats (Charles River Laboratories) weighing 200-250 grams were acclimated to housing and mechanical and thermal behavioral measurement conditions for 1-2 weeks until establishing a consistent behavior baseline that was in accordance with historical records of previous studies. Animals falling outside the established parameters were excluded. All inoculations and behavioral assessments were blinded so the behavioral response recorder was ignorant of the inoculum source and injection location. Cell-associated VZV for animal inoculation was prepared by fast thawing pre-titrated virus aliquots stored in liquid nitrogen, followed by centrifugation at 150xg at 4˚C for 10 minutes, an ice-cold 1x PBS wash to remove residual freeze media, and resuspension in 1x PBS for a final concentration of 4x10 6 PFU/ml. Washed, intact cell-associated virus was maintained on ice for no more than 1-h prior to foot or whisker pad inoculation. Uninfected cell equivalents were prepared identically. When TMP was included in the inoculum, virus infected cells were resuspended at the appropriate concentration in ice-cold 1x PBS supplemented to 500 nM TMP.
In the footpad model, inoculations were carried out as detailed previously [31] with slight modifications. Briefly, gently restrained rats were subcutaneously inoculated with 25-50 μl containing 2x10 5 PFU VZV into the glabrous skin of the left or right rear footpad. Whisker pad inoculations were as detailed previously [34] using isoflurane anesthetized rats whereupon virus was injected subcutaneously in the right or left whisker pad. Animals were monitored for recovery and returned to housing. Mechanical and thermal responses of the footpad were performed as detailed previously [31]. Briefly, mechanical allodynia (MA) at the footpad was assessed using a set of calibrated von Frey monofilaments (Stoelting Company, Wood Dale, IL). Animals were focus-distracted by presenting fruit cereal throughout the procedure [57]. Response measurement began with the 10-gram (evaluator size 5.07) filament and measured six times each on the injected (ipsilateral) and non-injected (contralateral) glabrous rear footpads through a metal grid stage. The monofilaments were applied using the "Up-Down" method [58]. A maximum von Frey filament weight of 180-grams was employed. Data was calculated as 50%-gram weight threshold and presented as withdrawal threshold average in grams. Thermal hyperalgesia (TH) measurements utilized a Hargrave's apparatus (IITC, Woodland Hills, CA) and followed the Hargrave's method after a minimum 24-h rest period following MA measurements [59]. Briefly, rats were placed on a stable 32˚C glass stage in a partitioned acrylic box and allowed to acclimate for 10-minutes prior to measurement. A concentrated light source at consistent distance from under the glass stage was applied to the center area of each rear footpad, and the time to withdrawal for each rear footpad was recorded in seconds, with a maximum cut-off time of 30-seconds to avoid tissue damage. Each footpad was assessed four times in sequential order, testing only the ipsilateral or contralateral footpad per pass, allowing several minutes between measurements of a single footpad to avoid residual sensitivity issues. Data is presented as the average withdrawal time per footpad in seconds.
For assessment of facial affective pain responses, the Fuchs place-escape-avoidance-paradigm (PEAP) method was used as previously described [34,60]. Briefly, rats were placed in a 30 x 30 x 30-centimeter clear, acrylic box in which half was made opaque by attaching a black cloth to the exterior. Rats were allowed to acclimate to box for 10-minutes, during which rats unanimously chose to remain on the dark side. A 60-gram (evaluator size 5.88) von Frey monofilament was applied to the left or right whisker pad, depending on the rat's location in the box, every 15-seconds for a total of 30-minutes. Monofilament was applied to the ipsilateral (inoculated) whisker pad if the rat's head was on the dark side of the enclosure and applied to the contralateral whisker pad if rat's head was on the clear side. Uninoculated rats chose to remain on the dark side, while inoculated rats showed reduced time on the dark side if the von Frey monofilament stimulation was noxious. A positive nocifensive response is defined by preference for the clear side of the box after repeated stimulation of the ipsilateral whisker pad on the dark side. The measurements are presented as time spent on the dark side in 5-minute binned increments over a 30-minute period.
Statistics
All statistical tests were performed on Prism 9 software (GraphPad, La Jolla, CA). Error bars are represented as standard deviation (SD), or standard error of the mean (SEM) as noted in the figure legends. Unpaired T-tests with Welch's correction were performed on the plaque size data (Fig 2D). Two-way ANOVAs were performed for each behavioral assessment (Figs 7-11) with a Bonferroni post-hoc correction.
Development and characterization of conditionally replicating VZV with degron fusion to essential VZV proteins
The hypothesis that a VZV limited gene expression profile was required for the development of nocifensive behaviors in the rat models of PHN was only partly answered by our previous studies [32]. In that work, VZV was shown to infect primary rat cell cultures and undergo some virus gene expression, but VZV did not spread beyond the first round. We reasoned that such cultures may not represent the many cell types that could be potentially infected and support VZV in vivo in rats and showing a lack of VZV growth in all rat tissues in vivo is a difficult task. We considered that a more definitive approach to test our hypothesis would be to evaluate rats inoculated with VZV mutants that are genetically unable to proceed past certain stages of the virus replication cycle. While recombinant VZV with essential gene mutations have been described [47,61,62], their growth requires complementation and for reasons unclear, genetically stable VZV-permissive lines harboring VZV genes have proved to be more difficult to generate than those used for the development of HSV-1 mutants. Complementation of previously detailed VZV deleted for ORF9 [47] and ORF4 [63] was achieved by infection with high titer baculovirus containing a CMV-IE promoter-driven VZV gene, with concurrent treatment with sodium butyrate to inhibit type I histone deacetylases. This approach that was not amenable to apply to the rat models of PHN and did not yield the high titers required in the models. Therefore, we adopted an alternative approach in which replication-conditional VZV were made using a protein degron fusion system [39]. The 480-bp coding degron motif used was derived from E. coli DHFR and was added by recombination to fuse to several target VZV genes in frame within the virus. The DHFR degron facilitates protein turnover unless it is stabilized by the cell-permeable ligand TMP (Fig 1A). In the absence of TMP, the protein is turned over as a result of ubiquitin binding to the degron, which initiates proteasome-mediated turnover of the fused protein ( Fig 1B). The system overcame the need to generate complementing cells and was successfully exploited to show that ORF63 protein is essential for SVV growth [64]. Unexpectedly, VZV with the degron fused to ORF62 (with concurrent deletion of the reiterated ORF71) encoding the major transcriptional regulator protein IE62 were found to be not TMP-growth conditional. Several additional VZV BACs with the degron fused to candidate VZV proteins involved in DNA replication (some evaluated as both amino and carboxyl fusions) did not yield functional virus from 3 separate transfections of 4-6 independently developed BAC constructs per virus (Table 2). This suggested that the degron addition was not compatible with some essential protein functions and that not all VZV genes can be analyzed by the degron approach.
Three VZV recombinants were subsequently found to show TMP-dependent growth, one being VZV with the degron inserted at the 3' end of ORF9. This ORF encodes ORF9p, an essential phosphorylated late-expressed protein (orthologous to HSV-1 VP22) that predominantly localizes to the cytoplasm [65] and interacts with several structural proteins at the trans-Golgi network (TGN) during late infection [66]. Evidence suggests that ORF9p has key roles in tegument formation and secondary envelopment [67]. This VZV (VZV ORF9cDHFR) replicated similar to wild-type pOka VZV when grown in media supplemented with 100 nM TMP but showed a near 2-log reduction in the number of infected cell progeny by 48-h when TMP was withheld from the growth media (Fig 2A). Growth of VZV pOka was not influenced by the presence or absence of TMP. A similar result was found for VZV with the degron attached to the 5' end of ORF4, which has been reported to be expressed as an immediate-early gene [41]. ORF4 is an essential gene and its protein (IE4) regulates VZV gene expression at the post transcriptional levels that involve the nuclear export of viral intronless mRNA [68,69]. IE4 has nuclear and cytoplasmic distribution in infected cells and interacts with SR nuclear shuttling proteins [69]. The virus (VZV ORF4nDHFR) showed a slight reduction of growth in the presence of TMP when compared to wild-type pOka virus (Fig 2B), but when grown in the absence of TMP, highly reduced progeny virus yields were detected at day 1 and longer times. This suggested that the addition of the degron to the protein may have a subtle effect on IE4 function but did not impair its essential functions. The third growth conditional VZV contained the DHFR degron domain inserted at the 3' end of ORF63, in a similar manner to that done previously with SVV IE63 [64]. ORF63 encodes the protein IE63, which has essential viral and host gene regulatory functions [42]. Since ORF63 lies within the reiterated genome sequences and is duplicated as ORF70, the BAC was deleted for ORF70 by replacing it with an ampicillin resistance cassette (amp r ). The resulting virus (VZV ORF63cDHFR) showed significantly reduced virus replication and production of virus progeny over a 4-day time course in the absence of TMP, with approximately 2/3 rd to 1.2-log difference without TMP compared to virus growth with TMP ( Fig 2C).
The three viruses were characterized to verify the expected degron insertion and its stability in the virus. This included DNA sequencing of a PCR amplified fragment spanning the fusion of each virus to confirm the in-frame fusion, and analyses of viral DNA obtained from nucleocapsids obtained from virus grown in the presence of 100 nM TMP in the media. The DNA was subjected to gel analyses and Southern analysis using a DHFR degron-specific probe sequence. This was particularly important to characterize VZV ORF63cDHFR and show that ORF63 had replaced ORF70 by homologous recombination and removed the BAC replicon sequences (S1 Fig). The degron-specific probe hybridized to a large DNA fragment of the KpnI digested VZV genomic DNA for ORF9cDHFR and ORF4nDHFR, but for VZV ORF63cDHFR, two KpnI digested DNA fragments showed a 480-bp increase in size and hybridized the DHFR probe. These bands were of the sizes expected for not only ORF63, but also for its replacement of the ampicillin cassette in the ORF70 BAC used to derive VZV ORF63cDHFR. The blots and fragment sizes also demonstrated the expected recombinational removal of the BAC replicon from the virus, as originally designed [47]. The virus DNAs were likewise assessed by digestion with SphI (S2 Fig), which gave a novel fragment for each virus that confirmed the correct insertion of the degron sequence at the target site. We next addressed growth by plaque size formation for the three degron viruses in parallel to pOka under identical conditions in the presence or absence of TMP (Fig 2D). Approximately 30 plaques per virus per condition were imaged for each virus/condition and analyzed by integrated morphometry analysis. Plaques formed by pOka and the parental BAC derived VZV (parent) were identical when grown with or without TMP, establishing that TMP does not affect WT VZV growth; parent plaque size trended to be slightly smaller than those formed by wild-type pOka on TRPE cells. VZV plaques formed by the degron-containing viruses showed a similar marginally reduced average plaque size under permissive conditions compared to pOka, but under nonpermissive conditions, the plaques were barely detectable and involved only a few cells in the absence of TMP for VZV ORF9cDHFR and ORF4nDHFR. VZV ORF63cDHFR showed greatly reduced plaque size but appeared to show a slightly leaky growth restricted phenotype as seen in the timed growth curve analyses (Fig 2C).
We next assessed protein production by the three conditional viruses over a growth period of 72-h, in the presence or absence of TMP. Infections were initiated at low multiplicity (1 infected to 100 uninfected cells) so that multiple rounds of VZV infection could occur. For VZV ORF9cDHFR infections, protein levels made in the continued presence of TMP were similar to that made by wild-type VZV (pOka) (Fig 3), but in the absence of TMP, VZV protein accumulation was dramatically reduced in VZV ORF9cDHFR infections. ORF9cDHFRp showed the expected size increase of about 18 kDa as a result of degron addition, and almost no unfused ORF9 protein in VZV ORF9cDHFR infections was detected. Of note, multiple forms of ORF9p and ORF9cDHFRp were detected, consistent with previous reports of this being a phosphoprotein [67]. For infections initiated with VZV ORF4nDHFR, the expected size increase due to the addition of the degron was apparent, and TMP-dependent protein production increased over a 72-h infection in contrast to pOka, but little in the absence of TMP (Fig 4). VZV proteins from ORF29p and gE were greatly reduced in VZV ORF4nDHFR infections grown in the absence of TMP, consistent with impaired viral spread and amplification, and the TMP-dependent loss of infectivity in growth curves (Fig 2B). Infections initiated with VZV ORF63cDHFR resulted in expression of the larger IE63cDHFR in the presence of TMP as expected (Fig 5), and it was the predominant form made by the recombinant virus. Reduced levels of protein accumulated under nonpermissive conditions, although some accumulation of IE63cDHFR occurred in the absence of TMP over time. The increase in accumulation of other VZV proteins is consistent with a slightly leaky phenotype for this virus.
Further characterizations were carried out to address if the degron addition had an influence on subcellular localization of the fused proteins. The nuclear localization (NLS) and/or
PLOS PATHOGENS
Requirements for varicella-zoster virus-induced pain nuclear export signals (NES) have been previously located for each protein [65,[70][71][72]. The cellular localization of IE4nDHFR, ORF9cDHFRp, and IE63cDHFR were compared to the unmodified proteins made by wild-type pOka infected cells, all grown in the presence of TMP. Images were acquired from edges of 2-dpi individual plaques showed that, in general, protein localization for each DHFR fused protein was similar to the distribution of the native proteins (Fig 6). IE4 and IE63 from pOka localized to both nuclear and cytoplasmic compartments, with IE4 showing a more predominantly cytoplasmic distribution in most cells. IE63 was more nuclear localized in cells at the edge of plaques, which represent earlier stages of infection as compared to distributions at plaque centers. ORF9p was seen in both nuclear and cytoplasmic compartments in both DHFR and wild-type VZV infected cells. The similar distribution of the DHFR and wild-type proteins for each virus suggests that degron addition does not greatly affects subcellular distribution of the proteins. We next sought to determine if these genes were expressed at detectable levels in DRG of inoculated rats. Previous studies have found that VZV transcripts [25,73] and proteins [26,28,30] can be detected by in situ hybridization or immunohistochemistry in ganglia after inoculation. We used a set of described PCR primer/probes to detect ORF62 and ORF63 [56], and additional sets to identify ORF4 and the DHFR degron domain by RT-qPCR. The TMPdependent virus stocks were generated as detailed in the methods and were the same used throughout all animal studies. Titrated virus was inoculated into rat footpads. The L4, L5, and L6 DRG of animals harvested at days 4-, 5-, and 7-dpi was used to prepare total RNA and then converted to cDNA for assessment of VZV gene expression in DRG (S3 Fig). We were unable to obtain statistically significant increases in gene expression for the probed VZV genes. This indicates that levels of transcripts for ORF62, ORF4, and ORF63, or the DHFR sequence are
PLOS PATHOGENS
Requirements for varicella-zoster virus-induced pain
Fig 6. Localization of degron fused viral proteins compared to wild-type VZV proteins in infected cells. (A) Images
show the edges or small regions of plaques formed by wild-type VZV (pOka), VZV ORF9cDHFR (top), VZV ORF4nDHFR (middle), or VZV ORF63cDHFR (bottom) at 2-dpi. Protein cellular distribution in fixed cells was imaged to represent the distribution seen in the cultures after staining with antibodies to ORF9p, IE4, or IE63. The 'target protein' column indicates the specific protein probed, as noted in the lower left corner of the column. The center column shows DAPI stained nuclei, and the rightmost column shows a merged panel of target protein too low for consistent detection by this approach in rats infected with wild-type VZV, VZV ORF4nDHFR, and VZV ORF63cDHFR. The lack of significance when compared to relative changes in GAPDH is similar to our previous study [32] and is generally consistent with sparse detection of VZV transcripts by in situ hybridization studies. Despite low detection levels for VZV ORF63cDHFR infected rats, the similar pattern that formed in the detection of ORF63 and DHFR, which should identify the same transcript, may suggest these data are consistent with the ORF63 transcript being detectable in rat ganglia at low levels [25].
Analyses of rats inoculated with VZV ORF9cDHFR indicate productive replication is not required for VZV-induced chronic hypersensitivity behaviors in rats
We next used the conditionally replicating VZV to assess how reducing viral gene expression and replication influenced VZV induction of prolonged nocifensive behaviors in rat models of PHN. All TMP-dependent VZV were generated in cells supplemented with TMP. All viruses were managed identically for consistency, with the TMP-supplemented inoculant resuspended in PBS containing 500 nM TMP instead of PBS alone. Animals were then inoculated into the rear footpad as detailed previously [31], in a random left/right manner (n = 6) so the inoculated paw and the nature of the inoculant was blinded from the behavioral assessors. Rat groups received equivalent infectious units of VZV ORF9cDHFR containing 500 nM TMP, the same virus without TMP, wild-type pOka as the positive control, or uninfected cell equivalent as the negative control. Development of mechanical allodynia (MA) and thermal hyperalgesia (TH) was assessed over a period 74 days (Fig 7). As predicted, pOka inoculated animals developed mechanical hypersensitivity responses by day 14, consistent with previous studies [26][27][28][29][30][31][32]. Hypersensitivity was significant at multiple time points when compared to the contralateral (uninfected) footpad or rats that received uninfected cells, which all developed no significant hypersensitivity over the course of the study. Importantly, VZV ORF9cDHFR induced significant hypersensitive states lasting over the testing period similar to that seen for pOka inoculated animals, whether or not a bolus of TMP was administered with the virus in the inoculate. Mechanical responses lasted several weeks ( Fig 7A) and began to wane towards the end of this study, so that hypersensitive responses at 66-dpi were no longer significant for the pOka and VZV ORF9cDHFR groups compared to controls. Measurements of thermal hypersensitivity followed a similar pattern, though the withdrawal time difference between positive and negative thermal responses was more subtle, so that significance in the differences was not seen for every time point. By 18 dpi, significant differences were detected in all VZV inoculated groups when compared to the uninfected cell group or contralateral paw measurements (Fig 7B). The variability in thermal response is consistent with previous studies [26,28,29,31]. An average withdrawal time under 7-seconds was observed in hypersensitive rats, while responses for negative control animals remained above 10-seconds throughout most measurements in the 74-day study. Thermal hypersensitivity also waned during the later stages of the study. Given that VZV ORF9cDHFR replication is TMP-dependent and would be unable to spread when inoculated in the absence of TMP, we take these results to indicate that the development of hypersensitivity behaviors in the rat footpad model does not require productive replication, but is more the result of a single round, non-productive infection after inoculation. We also evaluated the ability of these viruses to induce affective pain responses in the rat facial model (Fig 8). Rats were inoculated at the whisker pad in the same groups as the footpad experiments, and rats were assessed for nocifensive behaviors in a fully blinded manner using the Fuchs' PEAP assay [34,60]. This physiological test evaluates animal behaviors resulting from noxious stimuli, in which rats show reluctance to locate to preferred locations if the stimulus evokes higher levels of pain or sensitivity. Animals receiving uninfected cells showed no behavioral indicators of aversion to stimulation of the inoculated whisker pad and remained predominantly on the dark or preferred side of the enclosure over the course of the experiment. However, stimulated animals that received pOka showed a considerable reduction of time spent on the dark side of the enclosure. Such behaviors began to return to baseline, by 28-dpi and were no longer significant. While VZV-induced affective pain indicators at the whisker pad were shorter-lasting than the mechanical and thermal hypersensitivities detected at the footpad, the behavior of rats inoculated at the whisker pad with different viruses were consistent with the footpad data, in that VZV ORF9cDHFR induced hypersensitivity responses when inoculated with or without TMP. These data support the hypothesis that VZV ORF9cDHFR retains the ability to induce hypersensitivity with or without conditional replication and is consistent with the conclusion that ongoing replication of VZV is not required for the induction of pain behaviors.
Analyses of VZV ORF4nDHFR indicates production of the VZV IE4 is required for development of VZV-induced hypersensitivity
A similar set of studies were performed in the footpad and facial models to examine how VZV ORF4nDHFR stimulates hypersensitivity when replication is permitted or prohibited. In footpad model studies (Fig 9), responses indicating mechanical hypersensitivity developed by 22-dpi in pOka inoculated rats. The timing of onset of hypersensitivity was later than that seen in the ORF9cDHFR study, but such variability has been seen previously [26,28,31,32]. No measurable hypersensitivity developed in rat footpads injected with uninfected cells or developed in the contralateral uninoculated footpads. In contrast to the results obtained from animals inoculated with VZV ORF9cDHFR, hypersensitivity developed in rats that received VZV ORF4nDHFR supplemented with 500 nM TMP, but animals did not develop hypersensitivity if TMP was not included in the inoculum. Rather, animals showed withdrawal responses similar to uninfected cell equivalent groups (Fig 9A). Hypersensitivity in animals inoculated with VZV ORF4nDHFR with TMP was detected throughout the entirety of the assessment period (the study was terminated at 55 dpi). Thermal hypersensitivity responses followed a similar pattern (Fig 9B) so that by 24-dpi, significant hypersensitivity was seen at select times in rat groups that received pOka or VZV ORF4nDHFR containing TMP. Quicker withdrawal times did not develop in contralateral footpads, footpads inoculated with uninfected cells, or in footpads that received VZV ORF4nDHFR without TMP supplementation. Rat groups that developed hypersensitivity remained significant at most measurement times until to the end of the experiment when compared to the negative control. We did see some significant responses in the contralateral footpad at certain timepoints, but this is not unusual and has been observed at random and on occasion in previous thermal hypersensitivity assessments. However, these contralateral measurements did not form a consistent pattern of continued hypersensitivity, as observed on the ipsilateral side, and generally resembled the responses of the negative control group. In the rat facial model of affective pain (Fig 10) the pOka and VZV ORF4nDHFR with TMP groups were found to spend significantly more time on the light side of the enclosure at 7-dpi when compared to the uninfected cell equivalent group. Consistent with the footpad studies, animals that received VZV ORF4nDHFR without TMP continued to spend a majority of time on the dark side as seen for the uninfected cell group. The trend continued over the course of the 5-week experiment, at which time nocifensive behaviors of the hypersensitive groups waned, as seen previously [34]. Post-hoc analysis indicates significance for the pOka group during all measurement groups, while the VZV ORF4nDHFR TMP supplemented group lost significance during the final measurement timepoint. At no point did the VZV ORF4nDHFR without TMP group show any significant indication of hypersensitivity. We highlight that these results contrast with the responses of animals receiving VZV ORF9cDHFR, in which pain behaviors developed with or without TMP. The results suggest that the degron mediated removal of IE4 prevents the development of pain behaviors and suggests that IE4 is necessary for the induction of pain responses after VZV inoculation.
Analyses of additional VZV mutants confirms the requirement for gene expression, but not full viral replication for the development of pain behaviors in rats
We sought to confirm the contrasting outcomes of the VZV ORF9cDHFR and ORF4nDHFR in the rat footpad model through the analyses of additional VZV recombinants containing mutations in different genes. We evaluated animals inoculated with VZV ORF63cDHFR in the same manner (Fig 5). In parallel, we examined the responses in the footpad model of animals that were inoculated with a recently described recombinant VZV that is deleted for expression of ORF54 (VZV Δ54S) [45]. ORF54 encodes the portal protein involved in the packaging of viral DNA into preassembled capsids in the nucleus, and VZV lacking ORF54 cannot replicate beyond the initial round of replication in non-complementing cells. To grow VZV Δ54S, an ARPE-19 based complementing cell line was used (A54). Following footpad inoculation, the behavioral responses of rats receiving these viruses and controls were assessed for mechanical ( Fig 11A) and thermal hypersensitivities (Fig 11B). In these studies, the negative control group was divided into two, with one group of rats inoculated with uninfected A54 (n = 3) and the second with uninfected TRPE (n = 3) cells. The behavioral responses of the two uninfected cell controls were indistinguishable from each other (and thus combined in the graph) or historical uninfected cell controls, indicating that the complementing cell line did not induce a significant pain response. pOka infected cell inoculated animals developed significant mechanical hypersensitivities from 18-dpi onwards that lasted through to the end of the study (Fig 11A). Thermal hypersensitivities for pOka inoculated animals also trended towards a response, although the measurements in this study were not significant at times other than 40 days (Fig 11B). Animals that received VZV ORF63cDHFR with a bolus of TMP at the footpad developed significant mechanical hypersensitivity at a time similar to those animals receiving pOka, but animals receiving the virus without TMP did not develop any significant hypersensitivity behaviors and showed responses similar to those of animals receiving uninfected controls at all timepoints. This result indicates that the production of IE63 is critical for the induction of a prolonged mechanical hypersensitivity response in the rat model. In contrast, significant and long-lasting mechanical pain responses developed in animals inoculated with the genetic mutant VZV Δ54S. While this virus was administered with its complementing cells, the subsequent infection of any cells in the rat host would not be expected to progress to infectious virus production. These studies further support our previous conclusions involving VZV ORF9cDHFR (Figs 7 and 8) and ORF4nDHFR (Figs 9 and 10) and denote that hypersensitivity in the rat models of PHN require early infectious processes and the expression of regulatory proteins, but that this occurs during a single and abortive round of infection in the rat host.
Discussion
In this work, we developed and exploited novel VZV mutants to dissect how components of the VZV infectious process contribute to the development of nocifensive behaviors in rat models of PHN. The data establish that presence of two VZV regulatory proteins, IE4 and IE63, are required for the development of hypersensitivity, but that production of infectious progeny virus in cells of the rat host is dispensable. The results imply that development of hypersensitivities in rats inoculated with VZV are the consequence of a single round, abortive infection in cells of the rat host but require some level of viral gene expression. These data also suggest that hypersensitivities are not the result of a reaction (immune or otherwise) to the VZV-associated cell antigens injected but require VZV to initiate an infection in cells of the rat host. The studies have implications for the induction of hypersensitivity and PHN in humans: we speculate that the partial VZV expression program could occur in human ganglia after an HZ event, in which infected but surviving neurons have a destabilized host neuronal homeostasis that leads to prolonged signaling of pain that may underlie PHN. This is the first report of conditionally replicating VZV mutants, and was achieved by adopting a protein degron system that had been developed to study the consequences of protein turnover and removal in eukaryotic cells [39]. Viable VZV with ORFs 4, 9, or 63 containing the 160-amino acid degron were TMP growth regulated, so the degron addition had minimal overall effect on the function of the targeted proteins. Each virus showed similar levels of growth and protein production over time compared to wild-type VZV in growth-permissive conditions and formed plaque sizes that were only slightly reduced under permissive conditions. The degron did not strongly influence intracellular localization of the fused proteins, consistent with only a modest, if any influence on protein function. In the absence of TMP, each virus showed severely limited capacity in growth, viral spread, and protein production, with ORF4 and 9 degron viruses showing tighter regulation than VZV ORF63cDHFR. This system was previously applied to study of the role of SVV ORF63 [64]. The degron system could clearly be useful to evaluate additional VZV essential genes. It has the advantage of permitting growth of mutant viruses in any permissible cell type. It also circumvents the need to derive complementing cell lines expressing the VZV gene-of-interest in trans in order to propagate VZV with a deleted gene, which has generally appeared to be more difficult for VZV in the limited human cell lines that support its growth. Previously developed VZV lacking ORF9 [47] and ORF4 [63] were grown by complementing the absent gene in cells that had been transduced with high-titer baculoviruses expressing a cytomegalovirus IE promoter driven VZV gene, which then required treatment with sodium butyrate to inhibit type-1 histone deacetylase activities and the chromatin-mediated silencing of the baculovirus. We attempted to prepare the VZVΔORF4 virus detailed previously [63], but could not obtain titers sufficient to exploit in the rat PHN models. The baculovirus approach is also complicated by the potential of the baculovirus itself and the sodium butyrate treatment required for transgene expression to affect behavioral responses [74][75][76]. The lack of success discouraged our pursuit of the VZVΔORF9 virus detailed previously [47]. VZV lacking the duplicated ORF63 and ORF70 has been reported by one group to replicate without complementation [77], but others report that deletion of ORF63 and ORF70 abrogated virus growth [78,79]. As far as we are aware, no ORF63 complementing cell line or system has been described. However, a caveat is that the conditional replication strategy is unlikely to be applicable to all VZV genes. Our attempts to target three DNA replication proteins did not result in viable virus ( Table 2), suggesting that the degron addition interfered with essential protein functions. We also found that degron addition may not regulate protein stability, as found for VZV containing the degron added to the major transcriptional regulator encoded by ORF62 and ORF71. The reason for a lack of regulation is not clear, but it could be due to accessibility of the degron tag to ubiquitin-ligases as a consequence of cellular compartmentalization. For such genes, VZV deletion virus may require more classic complementation methods, and there has been some recent success in developing ARPE-19 based cell lines such as used here to grow the VZV Δ54S [45]. We are currently extending the degron system to evaluate additional VZV genes involved in DNA replication, to ask if VZV blocked at the DNA replication stage are able to induce hypersensitivity responses.
The data show that productive VZV replication in the rat PHN model is not required to cause VZV-induced pain in those models. This fits with our suspicion that the high species specificity of VZV prevents viral replication in vivo at some post-entry phase in rats. While rats have long been used as both models of pain and as models of latency, VZV permissivity in the rat has never been fully resolved. Numerous studies report the detection of viral transcripts and some proteins in VZV infected rat ganglia that were hypothesized to reflect the VZV latent state [25,26,28,32,73,80,81]. The long-term pain behaviors have been useful to examine potential pain alleviating drugs [26][27][28][29]31,82,83]. Dalziel et al. (2004) found that that pain induced by VZV infection was not alleviated by a 10-day treatment with systemic acyclovir (ACV) administration to rats [27]. ACV inhibits herpesvirus DNA replication, and blocked pain responses generated in rats receiving HSV, suggesting these two related herpesviruses induced pain by different mechanisms. Our work showed that UV-irradiation of the VZV infected cell inoculate (to reduce infectivity by more than 2-logs) prevented development of most pain behaviors [31]. However, we reasoned that neither approach was definitive in determining if VZV replication was required for induction of pain behaviors. The minimum inhibitory concentration of ACV for VZV is considerably higher than for HSV and the results of Dalziel et al. may have reflected insufficient levels to block VZV. UV-irradiation may have caused considerable damage to the inoculum. However, our finding that two mutants unable to fully replicate under our inoculation conditions (VZV ORF9cDHFR and VZV Δ54S) allows us to conclude that potential ongoing VZV productive replication, should it occur in rats, is not needed for development of nocifensive behaviors. Both mutants would likely be blocked at late stages of infection and express most VZV genes; ORF9p is essential and primarily involved in tegument assembly and secondary envelopment [47,67], while VZV lacking ORF54 would not be able to package DNA into capsids. Though VZV Δ54S was administered in complementing cells, the virus produced would be unable to form assembled virions in cells of the rat. A54 cells induced responses that were similar to uninfected cells in the same experiment and in historical studies [30][31][32][33][34]. Thus, the rat pain models appear to reflect a nonpermissive host in which abortive infection by VZV is sufficient for pain indicators.
In contrast, studies with the ORF4 and ORF63 conditionally replicating VZV establish that some VZV gene expression is essential for the induction of mechanical hypersensitivities. ORF4 is expressed as an IE gene based on classic cycloheximide-actinomycin D reversal experiments after cell-free VZV infections [41,68,69]. The protein has post-transcriptional regulatory activities suspected to be involved in nuclear export of intronless mRNA, in a manner similar to HSV ICP27 [84]. Removal of this protein from the infectious process would likely severely limit downstream VZV gene expression programs, as most VZV lytic transcripts are not spliced. Similarly, ORF63 was shown to be IE expressed using cycloheximide-actinomycin D approaches, and studies indicate it is a regulatory protein that is critically involved in early infectious processes [42]. Rats inoculated under conditions of TMP-permitted replication with each virus developed long-lasting hypersensitivities, establishing that the viruses themselves were not defective, but under nonpermissive replication conditions, neither induced significant behavioral responses. The data solidifies that some VZV genes and proteins with regulatory function are needed in rats for prolonged pain indicators. Presumably, VZV lacking IE4 would not shuttle intronless VZV messenger RNA to the cytoplasm for translation [69]. What the consequences of ORF63 are on the rest of the VZV expression program is less clear. The lack of any response without permission to replicate also indicates that antigen load in the inoculate, and components contained in the infecting virus tegument and capsid, are not sufficient to drive the signaling processes associated with hypersensitivity.
At this stage, there are some questions that remain to be resolved. We do not yet know whether the production of IE4 and/or IE63 are the actual drivers of the pain responses, or if their roles are to permit the expression of downstream genes that induce nocifensive behaviors. The resolution of this would require the study of additional mutants, such as a mutant that would enable us to prevent DNA replication in the rat host. Based on our previous work, where we did not see DNA replication in primary rat cell cultures, we would predict that a VZV with conditional degron-controlled essential DNA replication protein might show the same type of hypersensitivity induction seen for VZV ORF9cDHFR and induce hypersensitivity without DNA replication. Such mutants are being developed. It is also not yet clear as to what tissues the VZV limited expression program is needed to induce the hypersensitivity responses. We hypothesized that VZV proteins are produced within a few neurons of a sensory ganglion that innervate the site of inoculation, and that these trigger altered neuronal signaling. However, studies to determine significantly increased expression of VZV transcripts in rat ganglia have not been successful here and in a previous report beyond 5-7 days [32]. This indicates that VZV gene expression within innervating neurons is low and could be transient. Others have reported the detection of IE62 and IE63 in ganglia obtained after in vivo inoculation at sparse levels [24][25][26]73] and gene array studies suggest there are some changes in the respective ganglia [32]. We postulate that further studies of transcripts at the single cell level of the ganglia may allow the correlation of host with viral gene expression, but we considered them outside of the scope of the current work. It is even possible that the essential components of VZV gene expression is in non-neuronal cell types, such as glia and support cells proximal the sensory nerve endings at the periphery. These studies are planned or in progress.
Taken together, the data are consistent with the hypothesis that a partial gene expression program is required and sufficient for the induction of hypersensitivity in rat models of PHN. We speculate that this may be quite relevant to clinical HZ and the development of PHN, despite the fact that PHN follows reactivation whereas the rat model reflects events after a primary inoculation. However, when VZV reactivates in sensory ganglia, it probably starts within one or a small fraction of neurons, usually within a single ganglion. VZV then undergoes intraganglionic spread by cell-cell fusion, spreading to other neurons that are, therefore, newly infected [85,86]. These deliver virus to the periphery via innervating axons that terminate throughout the dermatome. There is neuropathic damage in many neurons and a reduction of nerve fibers in the afflicted dermatome [13]. Ganglionitis has been proposed to partly account for acute pain associated with HZ [18]. However, the ganglionic replication of VZV is usually limited, probably because of intrinsic, innate, and VZV-specific adaptive immune responses. We know that in HSV ganglionic infection models, ganglia resident CD8+ T cells limit virus gene expression and can suppress active neuronal replication through non-cytolytic means [87,88]. As such, the neuron survives, despite having initiated some viral gene expression. We propose this occurs on a much larger scale in the human ganglion hosting the HZ reactivation event, and clinical PHN may reflect the activity of neurons that have been infected, have made some VZV proteins (that induce host cellular changes) but then become subjected to multiple noncytolytic effectors, such as mediated by ganglion resident VZV-specific T cells that halt the full infectious program in a non-cytolytic manner. We postulate that a limited viral protein expression program (and/or the immune effectors targeting them) in surviving neurons may undergo altered host gene expression programs that involve genes of pain signaling. We are currently examining how individual VZV gene products may induce development of PHN-like behaviors in the rat models and the host expression programs that result. This may identify mechanisms that can be therapeutically targeted to limit the development of PHN.
Supporting information S1 Table. Primers for RT-qPCR. TaqMan primer/probe sets used in RT-qPCR analysis of VZV-infected rat DRG. 6-FAM (6-carboxyfluorescein). BHQ1 (black hole quencher 1). (DOCX) S1 Fig. KpnI restriction enzyme analysis of viral nucleocapsid DNA and Southern blot with DHFR-specific probe to show expected insertion sites and VZV ORF63cDHFR homologous recombination to ORF70 within the TR s region. Southern blots (left) are aligned with the ethidium bromide-stained 1% agarose gel electrophoresis image of separated fragments following KpnI digestion of purified VZV nucleocapsid DNA from each DHFR degron inserted virus, or from virus derived from the parental VZV BAC (right). The DHFR probe generated by PCR predominantly hybridized to the same large DNA gel fragment for VZV ORF9cDHFR and ORF4nDHFR. For VZV ORF63cDHFR, two DNA fragments seen in other viruses (and not hybridizing the DHFR probe) increased in size by 480-bp and both hybridized the DHFR probe, with the larger representing ORF63 and smaller representing ORF70, respectively. A map of the regions of the DNAs for the expected KpnI DNA fragments in each virus is shown below the gel images with the expected fragment size indicated with and without the DHFR sequence insertion. The green vertical bar in the lower diagram for VZV ORF63cDHFR represents the position of the insertion of the BAC mini-F sequence (~8 kb) that self-excises with virus derivation and passage. A minor low abundance DNA fragment hybridizing the DHFR probe of~6000-bp in size is present in every virus and was judged to be due to non-specific hybridization. Southern blot images were acquired on LICOR Odyssey in linear range. Created with BioRender.com. (TIF)
S2 Fig. SphI restriction digestion and Southern blot analysis to show DHFR degron insertion.
Southern blotting of 1% agarose DNA-separated SphI digested fragments with a DHFRspecific probe (left) and the ethidium bromide-stained VZV nucleocapsid DNA after gel electrophoresis (right). A map of the DNA fragments is shown at the bottom for each virus DNA as predicted from insertion at the correct sites for each virus. The map shows the predicted DNA fragment size with and without the degron sequence insertion. The sizes of a DNA ladder are shown in the composite image. The blots reveal that the degron insertions for each virus result in the increase of specific DNA fragment by 480-bp that then are the main fragments hybridizing the DHFR probe as predicted. Two~6000 bp fragments hybridizing the DHFR probe at low levels for VZV ORF4nDHFR DNA are of sizes expected from partial digestion products at low levels in which the expected fragment is not restriction digested from the adjacent SphI DNA fragment. Created with BioRender.com. (TIF) S3 Fig. Detection of ORF4, ORF62 and ORF63 transcripts in rat DRG tissues after inoculation of wild-type VZV or VZV containing DHFR degrons. L4, L5, L6 DRG were isolated from VZV pOka (top), ORF4nDHFR (middle), or ORF63cDHFR (bottom) inoculated rats at 4-, 5-, and 7-dpi and used to prepare total RNA. RNA was then quantified and analyzed by TaqMan probes for expression of ORF62 (blue), ORF4 (red), ORF63 (green), and DHFR (yellow) transcripts compared to naïve uninoculated animals. RNA quantification was then normalized and analyzed by the 2 -ΔΔCt method relative to GAPDH. The dotted line (= 1) represents no change over GAPDH control. Data represents two similar experiments combined and averaged. | 2021-07-08T06:16:33.798Z | 2021-07-01T00:00:00.000 | {
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246863945 | pes2o/s2orc | v3-fos-license | NuSTAR and AstroSat observations of thermonuclear X-ray bursts with short-recurrence times in 4U 1636$-$536
We report results from the spectro-timing analysis of the atoll source 4U 1636$-$536 observed with NuSTAR and AstroSat during its hard spectral state. In three observations of 207 ks total exposure, we identify 31 thermonuclear X-ray bursts including five doublets and a triplet. Recurrence time as short as 3.8 minutes is seen in one of the doublets. To the best of our knowledge, this is the shortest recurrence time known for this source. Our time-averaged spectroscopy during the bursts indicate the presence of an additional powerlaw or a blackbody component in a few cases, perhaps due to varying temperatures during bursts or plausible deviation from ideal blackbody behavior, however, it is difficult to probe this using the time-resolved spectroscopy owing to limited statistics. Time-resolved bursts are well fit using an absorbed blackbody model with temperatures varying between 1.7 and 2.2 keV. Burst oscillations around 581 Hz are detected with 3$\sigma$ confidence during the decay phase in two of the X-ray bursts. One of the burst oscillations is seen at 582 Hz, a frequency observed during the 2001 superburst in this source.
Introduction
Thermonuclear (Type I) X-ray bursts are sudden surges in X-ray emission from an accreting neutron star in a Low-Mass X-ray Binary (LMXB). They result from the unstable burning of hydrogen and helium on the surface of the neutron star. The sharp increase in Xray intensity is followed by an exponential decay. The rise occurs in ∼0.5-5 s and the decline in ∼10-100 s (Lewin, 1977;Lewin ., 1993;Galloway ., 2008;Bhattacharyya, 2010Bhattacharyya, , 2021. X-ray bursts have been found to recur on timescales of hours or days. This recurrence need not be at regular intervals. Burst occurring within 45 minutes of the preceding burst is conventionally labelled as short waiting time (SWT) burst (Boirin ., 2007;Keek ., 2010). Recurrence time as short as 3.3 minutes has been observed in the 11 Hz pulsar, IGR J17480−2446 (Motta ., 2011;Linares ., 2011Linares ., , 2012. Several models have been proposed to explain the occurrence of short recurrence bursts (Fujimoto ., 1987;Boirin ., 2007;Keek ., 2010). Recently, Keek & Heger (2017) proposed that a fraction of the fuel from the previous outburst is transported by convection to the ignition depth, producing a short recurrence time burst. Beri . (2019) found that the recurrence timescales observed match that obtained by Keek & Heger (2017) but the predicted profile of triple X-ray bursts do not match that observed.
Narrow but high-frequency (∼300-600 Hz) features either during the rising/decay phase or in both phases have been observed in the power spectrum of many thermonuclear X-ray bursts. These features are termed as burst oscillations and arise from brightness asymmetry modulated by the rotation of the neutron star. Hence, oscillation frequency matches the stellar spin frequency (see e.g., Strohmayer ., 1996;Watts, 2012;Bhattacharyya, 2021).
4U 1636−536 is a neutron star LMXB with a mainsequence companion star, located at a distance of ∼6 kpc (Galloway ., 2006). On the basis of the track traced in the color-color diagram (CCD), 4U 1636−536 is classified as an atoll source (Schulz ., 1989). As the source transitions through CCD from a hard state to a soft state, the accretion rate is believed to increase (Bloser ., 2000). A prolific X-ray burster, 4U 1636−536 shows all kinds of SWT bursts viz. doublets, triplets and even quadruplets (Ohashi ., 1982;Ped-ersen ., 1982;Keek ., 2010;, 2019) during the hard state (see e.g., Beri ., 2019). This source exhibits burst oscillations at ∼581 Hz, the spin frequency of the neutron star (see e.g., Zhang ., 1997;Strohmayer ., 1998;Bilous & Watts, 2019;Roy ., 2021). Although burst oscillations are predominant during the soft state, they also occur during the hard state (see e.g. Galloway ., 2008;Galloway & Keek, 2017). For 4U 1636−536 the shortest recurrence time observed is ∼5.4 minutes (see Linares ., 2009;Beri ., 2019). Here in this paper, we report timing and spectral study of 4U 1636−536 using data from AstroSat and NuSTAR observatories. This paper is organised as follows. The observational data and procedure are described in the following section (Section 2). Section 3 is focused on the details of spectral and timing analysis. In Section 4, we discuss the results obtained.
NuSTAR
The Nuclear Spectroscopic Telescope Array (NuSTAR; Harrison . (2013)) consists of two detectors: Focal plane modules A and B, or FPMA and FPMB), operating in the energy range 3-78 keV. The detectors have a time resolution of 10 s and a dead-time of ∼2.5 ms (Bachetti ., 2015). 4U 1636−536 was observed with the NuSTAR on April 27, 2019 (labelled Obs 1 in Table 1). Mondal . (2021) have used the same observation for performing a broadband spectroscopy during the persistent emission of the source. We use the NuSTAR data analysis software (NuSTARDAS v1.8.0) distributed with HEASOFT v6.26.1 and the latest calibration files (CALDB v20201101) for data reduction and analysis. The calibrated and screened event files are generated using nupipeline v0.4.6. A circular region of radius 100 centered at the source position is used to extract the source light curve and spectrum. Background light curve and spectrum are extracted from the same-sized radial region, away from the source. The task nuproducts is used to generate the spectra and response files.
AstroSat/LAXPC
Large Area X-ray Proportional Counter (LAXPC) unit onboard AstroSat consists of three detectors: LAXPC10, LAXPC20, and LAXPC30, covering the 3-80 keV band with a time resolution of 10 s and a dead-time of approximately 43 s. We reinvistigate two of the AstroSat observations (labelled Obs 2 and Obs 3 Table 1) using the LAXPC software (laxpcsoftv3.0) A signal-to-noise ratio of 3 is used as a filter for both the light curves. (Antia ., 2017). These observations have also been reported in Roy . (2021) and Kashyap . (2021). Since LAXPC30 had ceased operation due to gain instability, it is not used for Obs 2 and Obs 3. Besides, we only use data from LAXPC20 for Obs 3 as LAXPC10 had very low gain during this observation.
MAXI-GSC and Swift-BAT Light Curves
The hard X-ray transient monitor, Swift / Burst Alert Telescope (BAT), observes ∼ 88% of the sky daily with a detection sensitivity of 5.3 mCrab and a time resolution of 64 seconds in the energy band 15-50 keV (Krimm ., 2013). The Gas Slit Camera (GSC: Mihara . (2011)) onboard the Monitor of All-sky X-ray Image (MAXI: Matsuoka . (2009)) covers ∼ 85% of the sky per day with a detection sensitivity of 15 mCrab in the 2-20 keV band . We use the MAXI and Swift-BAT light curves of 4U 1636−536 to infer its spectral state (Figure 1). Both the light curves of 4U 1636−536 are binned with a binsize of 1 day. A signal-to-noise ratio of 3 is used as a filter. If the source is observed close to the peak of the MAXI light curve in the 2-20 keV band, it suggests a soft spectral state of the source. On the other hand, if the source is observed at times close to the peak of the BAT light curve in the 15-50 keV band, a hard spectral state is indicated. From Figure 1, it is clear that Obs 1 and Obs 3 are in hard state. However, the state of Obs 2 is not obvious.
Color-Color Diagram
To know the spectral state of the NuSTAR and AstroSat observations, we plot the color-color diagram ( Figure 2) of all the RXTE observations of 4U 1636−536 as given in (Altamirano ., 2008b) and the location of the observations studied in this paper. For the RXTE observations, hard and soft colors are taken as the 9.7-16.0/6.0-9.7 keV and 3.5-6.0/2.0-3.5 keV count rate ratios, respectively. The Obs 1 colors are normalized by the corresponding Crab Nebula color values that are closest in time (Obs ID / Obs Date: 10502001010 / 2019-05-09) to correct for the gain changes. For Obs 2 and Obs 3, the nearest AstroSat Crab observations (Obs ID / Obs Date: 9000002026 / 2018-04-08 and 9000002364 / 2018-09-13) are used for normalisation. The definition of hard and soft colors for both NuSTAR and LAXPC is same as that of RXTE. The counts from both FPMA and FPMB are added to obtain the color values of the NuSTAR observation.
The color-color diagram of atoll sources has an island branch and a banana branch which are, respectively, spectrally harder and softer states (Altamirano ., 2008a). The length of the curve, parametrizes the location of the source on the diagram. is normalized so that it varies from 1 to 2.5 as shown in Figure 2 (see, e.g. Méndez ., 1999), and indicates the mass accretion rate (Hasinger & van der Klis, 1989). We find that all the observations belong to the hard spectral state of the source with ∼ 1.3-1.4.
Energy-resolved burst profile
We find 15 thermonuclear X-ray bursts (labelled as B1-B15) in the NuSTAR observation (see Figure 3). Their energy-resolved profiles indicate that all bursts are significantly detected up to 20 keV. To illustrate this, we show here the burst profile of one X-ray burst (see Figure 4). We use three narrow energy bands, viz. 3-6 keV, 6-12 keV, 12-20 keV. In some bursts, such as B1, B7, B10 and B15, a dip is found in the 3-6 keV or 6-12 keV or both bands during the burst peak.
In the two AstroSat observations, 16 bursts are found altogether, 14 of which are in Obs 3 alone (see Figure 3). Bursts B22, B28 and B31 show a dip during the peak. These bursts (B16-B31) are significantly detected up to 25 keV (Roy ., 2021;Kashyap ., 2021). We measure the rise time ( rise ) for all the bursts. Rise time is defined to be the time it takes the burst flux to reach 90% of the peak flux from a time (start time) when the flux was 25% of the peak value (Galloway ., 2008). The rise time is found to lie between 2.5 and 8.0 s.
Five doublets and one triplet
Among the 15 X-ray bursts, we find multiple shortrecurrence bursts (see Figure 5) viz. two doublets, D1 (bursts B2 and B3) and D2 (bursts B11 and B12) and one triplet T1 (bursts B5, B6 and B7). To estimate the recurrence time or wait time between two X-ray bursts, we take wait time to be the separation between their respective peak times (Boirin ., 2007). The wait time between B2 and B3 in doublet D1 is 3.8 minutes, whereas that between B11 and B12 in doublet D2 is 10.4 minutes. The wait time between B4 and B5 in triplet T1 is 8.2 minutes, and that between B5 and B6 in the same triplet is 10.9 minutes. The rise time for the SWT bursts in these events varies from 2.5 to 3.5 s.
We also find three doublets in Obs 2 and Obs 3. The pair of bursts B16 and B17 in Obs 2 is a doublet (D3), the wait time time being 23.9 minutes. In Obs 3, the doublets are: bursts B18 and B19 (D4) with wait time 26.9 minutes, and bursts B23 and B24 (D5) with wait time 25.4 minutes. The rise time for the shortrecurrence bursts in the two observations lies in the range 3-4 s.
Time-averaged spectroscopy
For performing time-averaged spectroscopy, we use 100 s preburst emission as the background. The burst duration is taken up to the point where the count rate drops to 1/ of its peak value. The 3-20 keV burst spectra are fitted in XSPEC version 12.10.1f (Arnaud, 1996) with an absorbed blackbody model i.e. tbabs*bbodyrad (see Figure 6). The tbabs component accounts for the interstellar extinction. The hydrogen column density parameter, H , in tbabs is set to 0.25 × 10 22 cm −2 (Asai ., 2000). The component bbodyrad has the parameters: color temperature, bb,∞ , and normalization, bb,∞ = ( bb,∞ / 10 ) 2 , where bb,∞ is the apparent blackbody radius in km and 10 is the source distance in units of 10 kpc. To account for cross-calibration between FPMA and FPMB, a multiplicative constant component is included in the model. This constant is fixed to 1 for FPMA and allowed to vary for FPMB. The uncertainties correspond to 90% confidence level. The 2 of the spectral fit ranges from 0.94 to 1.06. The temperature ( bb,∞ ) is in the range 1.6-2.0 keV. The radius, bb,∞ lies between 2.3 and 6.8 km. The unabsorbed flux in the 3-20 keV band, estimated using the cflux convolution model, varies from 0.19 to 1.19 ×10 −8 erg cm −2 s −1 . The cross-calibration constant is found to vary in the range 0.95 to 1.11 except for burst B7 for which the constant is found to be 0.77. The best-fit spectral parameters are shown in Figure 7.
We try adding an additional blackbody component to the spectra and found a significant improvement in the spectral fits. We also try replacing the second blackbody component with a powerlaw and in certain cases found a better fit. However, double blackbody model is not well constrained in most cases. Table 2 gives the best-fit values for selected bursts, and the F-test probability values to indicate the significance of the additional component. The given three bursts of which B4 is the brightest, indicate the presence of an additional component.
The idea that the persistent emission can evolve during an X-ray burst was explored in detail by Worpel . (2013Worpel . ( , 2015. They proposed a dimensionless parameter to quantify the same. In this scheme, following Roy . (2021), we fit the preburst emission of 100 s with the model 'tbabs*(bbodyrad+constant*nthcomp)' and then keep the best-fit parameters constant while fitting the burst spectra. The fitting parameters are listed for three bursts in Table 2. We find that the spectral fit does improve with 2 ranging from 0.75 to 1.05. The temperatures are in the range 1.5-2.0 keV. The radii range from 1.9 to 6.6 km. The parameter values lie between 1.5 and 4.1. The unabsorbed flux (3-20 keV) varies from 0.33 to 1.28 ×10 −8 erg cm −2 s −1 .
Time-resolved spectroscopy
Since photospheric radius expansion (see e.g., Tawara ., 1984;Galloway ., 2008) can cause apparent dips in some light curves (Grindlay ., 1980;Bhattacharyya & Strohmayer, 2006;Bult ., 2019), we perform time-resolved spectroscopy to check for the same in the bursts observed with NuSTAR. For this, we extract the spectra for the five intervals as shown in Figure 4 for the bright bursts (B1-B15 except B3 and B7) observed with NuSTAR. The spectra are grouped using grppha to ensure a minimum of 25 counts per bin. We use the spectrum of 100 s preceding the burst to serve as the background spectrum for all the five intervals. The resulting burst spectra are fitted in XSPEC with the model: tbabs*bbodyrad along with a multiplicative constant component to account for cross-calibration between FPMA and FPMB.
We also use the method (see § 3.5) for the bright bursts. The peak temperatures are in the range 1.7-2.2 keV. The maximum radii range from 5.4 to 7.4 km. The peak bol varies from 0.8 to 2.0 ×10 −8 erg cm −2 s −1 . (148) The parameter values lie between 0.1 and 8.4. For the case of AstroSat observations (Obs 2 and Obs 3) time-resolved burst spectroscopy has been reported in Roy . (2021) and Kashyap . (2021), therefore, we do not perform spectral analysis of the same. For the doublets (D3, D4 and D5) observed with AstroSat, none of the bursts show characteristics of radius expansion in the time-resolved spectroscopy.
The characteristic timescale, , for the bursts is calculated as = b / pk where b is the burst fluence and pk is the peak flux during the burst (see e.g., Galloway ., 2008). For the bursts in Obs 1, this timescale is estimated to be 11-20 s. The values for the bursts in D3, D4 and D5 lie in the range 11-15 s.
Power Spectrum: Burst Oscillations
We search for <1024 Hz (Nyquist frequency) oscillations in each of the 15 bursts in Obs 1. Events from both the FPM detectors are combined to reduce the effect of deadtime. The events (photon arrival times) in the 3-20 keV energy band are sampled at the Nyquist rate of 2048 Hz. We perform fast Fourier transform (FFT) of successive 1 s segment (shifting the 1 s time window) of the input barycentre-corrected merged event file corresponding to the burst time interval. The FFT scan is repeated with the start time offset by 0.5 s. A clear signal around 581 Hz is seen during two of the bursts in the Leahy-normalized (Leahy ., 1983) power spectrum. In such bursts, we examine the region that show oscillation and attempt to maximize the measured power, , by varying the start and end points of the segment in steps of 0.1 s and trying segment lengths of 1 s and 2 s within a time window of 3 s (20+10=30 overlapping segments). Thus, the number of trials is 30. In both cases, oscillations are found in the decay phase at a confidence level of above 3 . The results are summarized in Table 3. The dynamic power spectra are shown in Figure 8.
A similar search is performed for the bursts in Obs 3 as well using events from LXP20 alone. No clear evidence of burst oscillation is found in them. Previously, Roy . (2021) reported burst oscillations in B16 in Obs 2 during the rising phase. We reiterate that result in Table 3.
To establish the significance of detection of oscillations in burst B2, we carry out Monte Carlo simulations. We generate Poisson-distributed events for the same window size (i.e. 2 s). A deadtime of 2.5 ms is used and implemented by removing any event that occurs within 2.5 ms after a previous event. The initial number of events is chosen such that after the deadtime correction, the number of remaining events is equal to the actual counts within Poisson fluctuations. Two such sequences of events are created to represent the two independent NuSTAR detectors, and then merged. We generate 50000 trial windows and calculate corresponding FFTs. The chance probability of occurrence of the observed signal is obtained by counting the number of trial windows with Leahy powers at or above the observed Leahy power. We find three windows with at least one signal above this power in the frequency range 579-582 Hz. Thus, we estimate the chance probability to be 6 × 10 −5 which implies a significance of 4.0 since = √ 2 erf −1 (1 − ) where is the chance probability. For B10, we use a window size of 1 s and obtain a significance of 4.1 from the chance probability of 4 × 10 −5 .
Lomb-Scargle periodogram (Lomb, 1976;Scargle, 1982;Horne & Baliunas, 1986), as implemented in the PERIOD program distributed with the Starlink Software Collection (Currie ., 2014) is also used to estimate the spin frequency. We obtain consistent values of spin period using this method and the uncertainty estimated corresponds to the minimum error on the period, as given in Table 3.
In Figure 9, we show the folded pulse profile for burst B2. The oscillations are modelled with the function: + sin 2 . The rms fractional amplitude is given by /( √ 2). The best frequency found with pulse profile modelling (Chakraborty & Bhattacharyya, 2014;Roy ., 2021) is 582.00 Hz and 578.81 Hz for bursts B2 and B10, respectively. Due to inadequate statistics, we do not study the energy dependence of the burst oscillations found in the NuSTAR observation.
Discussion and conclusion
In this work, we present the spectro-timing study of 4U 1636−536 observed with NuSTAR and AstroSat. As seen from the BAT and MAXI light curves and from the color-color diagram, the source is in a hard spectral state. This is consistent with that observed by Mondal . (2021) (for Obs 1) and Kashyap . (2021) (for Obs 2 and Obs 3) who have used the same observation and found that their spectroscopy results favour the hard/low state of the source.
The NuSTAR light curve (Obs 1) shows the presence of 15 X-ray bursts including two doublets and one triplet while the AstroSat observations (Obs 2 and Obs 3) show three doublets. So far, six triplets have been reported from this source, including one observed with AstroSat (Beri ., 2019). These authors reported a shortest recurrence time of 5.5 minutes in that triplet, very close to the 5.4 minutes recurrence time reported in a doublet by Linares . (2009). The wait times between two bursts in a triplet seen in this study conform to the wait times in triplets reported in Beri . (2019). The 3.8 minutes recurrence time in a doublet found in our work is the shortest recurrence time reported for this source. To the best of our knowledge, until now only three sources are known to have such a short recurrence time of 3-4 minutes. Keek . ., 2012), however, it is considered to be an exceptional source. In the full MINBAR sample of SWT bursts, half of the events arise from a single source, IGR J17480−2446. SWT bursts generally occur at all mass accretion rates, but for some individual sources such as IGR J17480−2446, they may be restricted to a ., 2020, for details). Keek & Heger (2017) suggest that bursts igniting in a relatively hot neutron star envelope leave a substantial fraction of unburnt fuel at shallow depths. This fuel is brought down to the ignition depth by opacity-driven convective mixing on the observed timescale of minutes and produce a subsequent burst. Although recurrence timescales observed during these SWT X-ray bursts could be explained by the model proposed by Keek & Heger (2017) we do not find any bumps before a follow-up burst in T1 as predicted by these authors. Such differences were also noted by Beri . (2019). Boirin . (2007) noted that all sources that show SWT bursts have a fast spinning neutron star ( spin > 500 Hz) suggesting the requirement of rapid rotation for multiple-burst events and the importance of rotation-induced mixing processes. They also proposed a waiting point in the chain of nuclear reactions such that a decay reaction with half life similar to the short recurrence times temporarily halts the nuclear burning. However, Keek & Heger (2017) observed that a considerable spread in the recurrence times (3-45 minutes), even for SWT bursts from a particular source, disfavoured this scenario. One plausible explanation to the existence of the short recurrence bursts could be based on the spreading layer model as suggested in Grebenev & Chelovekov (2017). However, these authors also note that further refinement in the model is needed for the complete understanding of the existence of SWT X-ray bursts.
Our time-averaged spectroscopy shows that absorbed blackbody model provides a good fit for the burst spectra yielding an emitting radius ( bb,∞ ) of ∼2.3-6.8 km and blackbody temperature ( bb,∞ ) of ∼1.6-2.0 keV. Similar studies have been carried out by Degenaar . (2016) for 4U 1636−536 SAX J1747.0−2853 and their radius and temperature values are in good agreement with ours. However, there is scope for an additional model component such as powerlaw, perhaps due to varying temperatures during bursts or plausible deviation from ideal blackbody behavior. We also analyze using the method and find that the spectral fit improves. The values suggest a substantial contribution from the persistent emission. Our time-resolved spectroscopy do not show any evidence of photospheric radius expansion (see also Roy ., 2021;Kashyap ., 2021). However, we would like to add a caveat that the possibility of radius expansion cannot be ruled out due to limited statistics. In most of the bursts, the values are 5 close to the burst peak suggesting enhanced persistent flux at this stage.
The actual emitting radius ( bb ) and blackbody temperature ( bb ) are given by bb = bb,∞ · 2 /(1 + ) and bb = bb,∞ · (1 + )/ , respectively (Sztajno ., 1985), where (1 + ) = (1 − 2 / 2 ) −1/2 is the surface gravitational redshift, is the color factor which accounts for the scattering of burst photons by the electrons and the frequency-dependent opacity of the neutron star atmosphere, and / is the neutron star radiusto-mass ratio. We do not apply the correction factors for the measurement of a real value of neutron star radius.
Bursts which are helium-rich, are expected to have shorter characteristic timescales (<10), fast rise times ( 2) and peak flux exceeding the Eddington limit (see e.g., Galloway ., 2008). The characteristic timescales of 11-20 s, long rise times of 2.5-8 s and the lack of any radius expansion signature indicate that the bursts are hydrogen-rich bursts. This is consistent with the observation that the sources exhibiting shortrecurrence bursts are all hydrogen-accretors. In our best knowledge, till date, no SWT burst has shown radius expansion (see e.g., Galloway ., 2008;Keek ., 2010;Keek & Heger, 2017).
It is generally believed that the highest burst rates on average are found for the sources with burst oscillations. However, it may be a selection effect as not all bursts exhibit burst oscillations, and therefore, a high burst rate means observation of many bursts and hence burst oscillation detections. Oscillations seen in this study are consistent with the range 578-582 Hz given in Bilous & Watts (2019) although most of the burst oscillations from this source are seen at 580-581 Hz. We detect a strong signal at a frequency (582 Hz) similar to that observed during one of the superbursts in 4U 1636−536, albeit with a much higher fractional amplitude (see Table-3) compared to ∼0.01 observed in the superburst. In the case of accretion-powered pulsations in intermittent AMXPs, the pulsation frequency is ∼0.5-1 Hz above the burst oscillation asymptotic frequency with fractional amplitude of up to a few percent (see recent review by Bhattacharyya, 2021). Therefore, 582 Hz pulsations observed during the 2001 superburst are believed to be accretion-powered (see Strohmayer & Markwardt, 2002, for details). Given a higher value of fractional amplitude of the oscillations detected in our study and considering an uncertainty up to 0.4 Hz (Muno ., 2002), the higher value of burst oscillation frequency cannot be ruled out.
During the decay phase of two bursts in Obs 1, the oscillations are found with ∼ 3 confidence. Decay phase oscillations are generally described through the cooling wakes (Cumming & Bildsten, 2000) or the surface oscillation modes (Heyl, 2004). Cooling wake is the temperature asymmetry due to cooling of the neutron star's surface, spreading across the star in a finite time after an X-ray burst. High amplitude decay oscillations (>10%) are usually explained using this model (Mahmoodifar & Strohmayer, 2016). Surface mode is the asymmetry in the neutron star ocean or atmosphere excited during an X-ray burst. The oscillation amplitudes are typically ∼10%. It may be plausible that both the models contribute to the evolution of burst oscillations (Roy ., 2021).
Summary
• In 207 ks of observation of 4U 1636−536 made during its hard spectral state with NuSTAR and AstroSat, we find five doublets and one triplet. The recurrence time of 3.8 minutes in one of the doublets is the shortest recurrence time reported for this source.
• Our time-averaged spectroscopy shows that an absorbed blackbody model provides a good fit. The temperatures and radii measured during each of the bursts in Obs 1 are consistent with measurements reported using similar approach.
• From time-resolved spectroscopy, no radius expansion could be inferred in any of the burst. The characteristic timescales and the rise times suggest that the composition of the bursts is primarily hydrogen.
• Burst oscillations are found in the decay phase of two of the bursts observed with the NuSTAR. One of these bursts is part of a doublet. The oscillations are found near the known spin frequency (581 Hz) of the source. A 582 Hz burst oscillation is seen in one of the bursts.
has made use of the NuSTAR data analysis software (NuSTARDAS) jointly developed by the ASI science center (ASDC, Italy) and the California Institute of Technology (Caltech, USA). The data for this research have been provided by the High Energy Astrophysics Science Archive Research Centre (HEASARC) and the Indian Space Science Data Centre (ISSDC). We thank the anonymous referee for important inputs towards the improvement of the paper. | 2022-02-16T06:48:06.955Z | 2022-02-15T00:00:00.000 | {
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249234320 | pes2o/s2orc | v3-fos-license | Diapocynin neuroprotective effects in 3-nitropropionic acid Huntington’s disease model in rats: emphasis on Sirt1/Nrf2 signaling pathway
Background and Aim Huntington's disease (HD) is a rare inherited disease portrayed with marked cognitive and motor decline owing to extensive neurodegeneration. NADPH oxidase is considered as an important contributor to the oxidative injury in several neurodegenerative disorders including HD. Thus, the present study explored the possible neuroprotective effects of diapocynin, a specific NADPH oxidase inhibitor, against 3-nitropropionic acid (3-NP) model of HD in rats. Methods Animals received diapocynin (10 mg/kg/day, p.o), 30 min before 3-NP (10 mg/kg/day, i.p) over a period of 14 days. Results Diapocynin administration attenuated 3-NP-induced oxidative stress with significant increase in reduced glutathione, glutathione-S-transferase, nuclear factor erythroid 2-related factor 2, and brain-derived neurotrophic factor striatal contents contrary to NADPH oxidase (NOX2; gp91phox subunit) diminished expression. Moreover, diapocynin mitigated 3-NP-associated neuroinflammation and glial activation with prominent downregulation of nuclear factor-Кβ p65 and marked decrement of inducible nitric oxide synthase content in addition to decreased immunoreactivity of ionized calcium binding adaptor molecule 1 and glial fibrillary acidic protein; markers of microglial and astroglial activation, respectively. Treatment with diapocynin hindered 3-NP-induced apoptosis with prominent decrease in tumor suppressor protein and Bcl-2-associated X protein contents whereas the anti-apoptotic marker; B-cell lymphoma-2 content was noticeably increased. Diapocynin neuroprotective effects could be attributed to silent information regulator 1 upregulation which curbed 3-NP-associated hazards resulting in improved motor functions witnessed during open field, rotarod, and grip strength tests as well as attenuated 3-NP-associated histopathological derangements. Conclusion The present findings indicated that diapocynin could serve as an auspicious nominee for HD management. Graphical abstract
Introduction
Huntington's disease (HD) is a devastating autosomal inherited neurodegenerative disease. It is distinguished by a triad of motor dysfunction, psychiatric disruption, and cognitive dysfunction. Neuronal loss especially in the striatum of HD patients is the major feature of the disease as the striatum is involved in motor control as well as learning functions (Wiprich and Bonan 2021). HD prevalence is about 5 cases in 100,000 people (Illarioshkin et al. 2018). The available treatments for HD provide only symptomatic relief with numerous undesirable side effects; thus, it is essential to investigate safer and more effective therapies that can halt the disease progression ).
Administration of 3-nitropropionic acid (3-NP), a selective striatal neurotoxin, is reported to mimic neuropathological changes of HD as it inhibits succinate dehydrogenase enzyme leading to oxidative stress (Damiano et al. 2010;Zuccato et al. 2010). Moreover, 3-NP promoted microglial activation evidenced by the prominent increase of ionized calcium binding adaptor molecule 1 (Iba1)-immunoreactive cells, along with augmented release of inflammatory cytokines that contribute to neurological dysfunction and striatal neuronal death . Nicotinamide dinucleotide phosphate (NADPH) oxidase (NOX), a crucial source of reactive oxygen species (ROS), was witnessed at high levels in the striatum of HD patients especially NOX2 isoform. Moreover, NOX activity as well as ROS-associated neuronal death were prominently decreased upon treatment of HD patients with NOX inhibitors (Valencia et al. 2013). The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is implicated in the downregulation of nuclear factor-Кβ (NF-Кβ) and its downstream inflammatory cytokines which contribute to many neurodegenerative disorders such as HD (Memet 2006). Administration of 3-NP is associated with Nrf2 downregulation leading to augmented oxidative stress, neuro-inflammation, and apoptosis . Silent information regulator 1 (Sirt1) is involved in the induction of Nrf2 transcriptional activity as well as the expression of its downstream genes as those related to reduced glutathione (GSH), the well-known redox scavenger (Ren et al. 2019). Additionally, neuroprotective effect of Sirt1 was correlated with induction of brain-derived neurotrophic factor (BDNF) expression (Tian et al. 2020). BDNF is a principle neuroprotective factor that is reported to induce neurogenesis and combat apoptosis, in addition to its imperative role in synaptic plasticity (Xia et al. 2010). Moreover, Sirt1 is known to impede tumor suppressor protein (P53) activity along with the inhibition of its downstream genes expression such as Bcl-2-associated X protein (BAX), thus hindering apoptosis (Rahman and Islam, 2011;Ren et al. 2019). 3-NP model of HD is associated with prominent decrease in Sirt1 and BDNF expression which contributes to neuronal damage witnessed in this model (Sayed et al. 2020). It is also reported that 3-NP is implicated in P53 upregulation with increased expression of pro-apoptotic proteins as BAX triggering apoptotic cell death (Gopinath et al. 2011;Gonchar et al. 2021).
Diapocynin is an oxidative derivative of the naturally occurring agent apocynin, the most commonly used investigational NOX inhibitor. Compared to apocynin, diapocynin is found to possess higher lipophilicity and greater potency to inhibit NOX activity (Luchtefeld et al. 2008;Kanegae et al. 2010). It is supposed to inhibit NOX by hindering the migration of the enzyme cytosolic subunits to the membrane as well as their binding with membrane components, and thus preventing the enzyme activation and the subsequent generation of superoxide anion and the other hazardous ROS (Stolk et al. 1994). Diapocynin has shown anti-oxidant, anti-inflammatory, and neuroprotective properties against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease and d-galactose/ovariectomy rat model of Alzheimer's disease primarily via NOX inhibition (Ghosh et al. 2012;Ibrahim et al. 2020). Apocynin has been found to exhibit powerful anti-oxidant and anti-inflammatory effects in various in vitro and in vivo models of neurodegenerative diseases including rotenone-induced Parkinson's disease (Gao et al. 2003), transgenic mouse model of Alzheimer's disease (Lull et al. 2011), focal or global cerebral ischemia (Gu et al. 2005;Jackman et al. 2009), familial amyotrophic lateral sclerosis model (Harraz et al. 2008), pilocarpine model of epilepsy (Pestana et al. 2010), and ketamine-induced psychosis (Behrens et al. 2007). Moreover, apocynin was found to attenuate motor alterations assessed as circling behavior and to mitigate the striatal neuronal damage in HD rat model induced by intra-striatal injection of quinolinic acid (Maldonado et al. 2010). Therefore, the present study was performed to explore the possible neuroprotective effects of diapocynin in 3-NP model of HD focusing on the involvement of Sirt1mediated signaling pathways.
Animals
Adult male Wistar rats weighing 180 ± 20 g were purchased from the animal facility of Faculty of Pharmacy, Cairo University (Cairo, Egypt). They were acclimated to the laboratory circumstances for 2 weeks before the study beginning. Rats were kept under controlled environmental conditions of constant temperature (23 ± 2 °C), humidity (60 ± 10%), and light/dark (12/12 h) cycle. Food and water were freely permitted during the experiment. This study was carried out in line with the principles of The Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011) and was approved by the Ethical Committee for Animal Experimentation of the Faculty of Pharmacy, Cairo University, with permit number PT 3162. All efforts were exerted to reduce the animal suffering through the experimental duration.
Drugs and chemicals
Diapocynin and 3-NP (Sigma-Aldrich Chemical Co., MO, USA) were dissolved in 1% DMSO and saline, respectively Ibrahim et al. 2020). The doses were freshly prepared every day.
Experimental design
Animals were randomly categorized into four groups (15 rats each) as follows: Group 1 received normal saline intraperitoneally as well as 1% DMSO orally and served as control group. Rats in group 2 received diapocynin orally at a dose of 10 mg/kg/ day. The dose of diapocynin was chosen based on prior studies which have utilized diapocynin or its parent drug "apocynin" at a comparable dose range in D-galactose/ovariectomy (Ibrahim et al. 2020) as well as transgenic mouse models of Alzheimer's disease (Lull et al. 2011), and in chronic cerebral hypoperfusion model of vascular dementia (Choi et al. 2014). Group 3 was injected with 3-NP (10 mg/ kg/day; i.p.) (Ross et al. 2014). In group 4, rats were administered diapocynin 30 min before 3-NP injection (Joseph et al. 2017). Drugs were given for 14 days and rats were subjected to the behavioral tests on the 15th day. Animals were then euthanized, and brains were dissected with striata being separated. Brain samples were processed for the histopathological and immunohistochemical examinations in addition to the estimation of biochemical parameters in the striatal region.
Behavioral assessment
Open field, rotarod, and grip strength tests were used to measure the animals' motor functions 24 h after the last day of injection and they were conducted in the same order with a 2-h resting interval between the tests. ANY-Maze video tracking software (Stoelting Co, USA) was used to record the rats' movement in the open field apparatus.
Open field test
The animal's impulsive locomotor functions were detected using a square wooden box (80 × 80 × 40 cm) apparatus with red walls and black polished floor having 16 equal squares distributed by means of white lines. Rats' behavior was recorded using an overhead camera located in the sound isolated room where the test was carried out. Each animal was kept in the center of the box to spontaneously explore the field for 3 min. The subsequent parameters were measured during the assigned time; distance traveled, mean speed, immobility time in addition to distance traveled and time active in the central squares and the corners as well as the number of rearing (Avila et al. 2010;Abdel Rasheed and Ibrahim 2022).
Rotarod test
This test is performed to assess the animals' motor co-ordination and balance. Each animal was subjected to 5-trial days before the beginning of the experiment. Animals showing the capability to remain on the rod (120 cm long and 3 cm in diameter with 20 rpm rotation speed) for 5 min were enrolled in the current investigation. The fall off latency was detected on the test day following the open field analysis (Sayed et al. 2020).
Grip strength test
The latency of gripping a horizontal wire was considered as an indirect measure of grip strength. Each rat was permitted to hang using its forepaws on a steel wire; 2 mm in diameter, 35 cm in length, which was strained horizontally at a height of 50 cm over a cushion support. The time taken by each animal while it is still holding the wire was recorded (Elbaz et al. 2018).
Enzyme-linked immunosorbent assay
Striata were dissected and homogenized to prepare 10% homogenates in saline. Afterwards, centrifugation was carried out and the protein content was assessed in the supernatants according to Lowry et al. (1951). The supernatants were also used to assess the following parameters, each according to the instructions of its corresponding ELISA kit; GSH (BlueGene Biotech CO., LTD, ShangHai, China), glutathione-S-transferase (GST), Nrf2, BDNF, and inducible nitric oxide synthase (iNOS) (My BioSource Inc., San Diego, CA, USA). Moreover, ELISA kits were also used to measure striatal contents of P53 (AFG Bioscience LLC, Northbrook, IL, USA), B-cell lymphoma-2 (Bcl2) (Cusabio, Wuhan, China), and BAX (Wuhan Fine Biotech Co., Wuhan, China).
Western blot analysis of Sirt1 and NF-Кβ p65
Following protein extraction from striatal tissues, equal protein volumes were segregated according to their molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane by the aid of a semi-dry transfer apparatus (Bio-Rad, Hercules, CA, USA). Membranes were placed in 5% blocking solution composed of non-fat milk to prevent non-specific binding. Afterwards, they were incubated overnight at 4 °C on a roller shaker with the subsequent primary antibodies: anti-β-actin (1:1000; Cat. No. PA5-16914), anti-Sirt1 (1:1000; Cat. No. PA5-20964), and anti-ρS536-NF-Кβ p65 (1:1000; Cat. No. PA5-17782) (ThermoFisher Scientific, MA, USA). Afterwards, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany). Finally, chemiluminescence was identified via the Amersham detection kit as designated by the manufacturer and subjected to X-ray film. A scanning laser densitometer (Biomed Instrument Inc., CA, USA) was used to quantify the target proteins and the results were normalized against β-actin protein expression.
Quantitative real-time PCR analysis of NOX2 subunit (gp91phox)
Using RNeasy Mini kit (Qiagen, Venlo, Netherlands), total RNA was separated from striatal tissues and its purity was assessed spectrophotometrically by 260/280 nm. The reverse transcription of the extracted RNA into cDNA was developed by the aid of Reverse Transcription System (Promega, Leiden, Netherlands) according to the kit's instructions. The gene expression of gp91phox was measured by the means of quantitative real-time PCR using SYBR Green Master Mix (Applied Biosystems, CA, USA). Table 1 displays the sequences of primers used. The relative expression of target genes was attained by the help of the 2 −ΔΔCT formula with β-actin being a reference gene (Livak and Schmittgen 2001).
Histopathological examination
The whole brains were dissected and fixed in 10% neutral buffered formalin for 72 h. Samples were processed in serial grades of ethanol, cleared in xylene, and then they were infiltrated and embedded into Paraplast tissue embedding media. 5μm thick serial sagittal brain sections were cut by a rotatory microtome for demonstration of striatal regions in different samples and mounted on glass slides. Tissue sections were stained by Hematoxylin and Eosin (H&E) as a general staining method for microscopic examination using light microscope (Culling 2013). Nissl staining was carried out to identify surviving neurons with Nissl's granules as it indicates healthy tissues. Sections were deparaffinized and rehydrated with xylene and graduated alcohol series. Afterwards, they were stained in Nissl stain for 5 min and air dried at room temperature for 1 h. After being dipped in alcohol for a while, the sections were cleared with xylene, cover slipped and examined microscopically. Average number of intact neurons was estimated from six randomly selected areas for each section using the Leica Qwin software (Leica Microsystems GmBH, Wetzlar, Germany). Neurons with a visible nucleus as well as an apparent whole outline were designated as normal ones (Culling 2013).
Immunohistochemical examination of Iba1 and GFAP
According to the manufacturer's protocols and instructions, immunohistochemical staining for Iba1 and glial fibrillary acidic protein (GFAP) was performed. Antigen retrieved brain tissue sections were blocked by 3% hydrogen peroxide for 15 min then they were incubated with the primary antibodies; anti-GFAP (Cat. No. 13-0300; Thermo Scientific Co., MA, USA; 1:100) and anti-Iba1 antibodies (Cat. No. ab108539; Abcam, MA, USA; 1:100) overnight at 4°C. Afterwards, they were washed with phosphate-buffered saline then incubated for 20 min with biotinylated link antibody and peroxidase-labeled streptavidin (Dako, Carpinteria, CA, USA). The reaction was visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB Substrate Kit, Vector Laboratories Inc., Burlingame, CA, USA). Sections were counterstained with hematoxylin, dehydrated, and cleared in xylene then examined via light microscope.
For immunohistochemical quantitative analysis, six random striatal non overlapping fields were scanned and analyzed for determining the positive mean area percentage of GFAP immunoexpression and the mean number of Iba1-positive microglial cells in each immunostained tissue section. All morphological examinations, photographs as well as quantitative analysis were recorded using Leica Application system modules for histological analysis (Leica Microsystems GmbH, Wetzlar, Germany).
Statistical analysis
The study results were illustrated as mean ± S.E.M. The data were analyzed by the means of one-way analysis of variance test (one-way ANOVA) followed by Tukey's multiple comparison test for all parameters excluding the rearing frequency which was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparison test and presented as median (max-min). Statistical analysis was implemented using GraphPad Prism software (version 6) with the level of significance being fixed at p < 0.05.
Diapocynin attenuated oxidative stress induced by 3-NP injection in rats
Rats which received 3-NP injection displayed augmented oxidative stress with significant increase in gene expression . Administration of diapocynin to normal animals did not induce any significant changes in oxidative stress markers, as compared to normal control group (Fig. 3).
Diapocynin inhibited neuro-inflammation induced by 3-NP injection in rats
3-NP injection induced marked increase in iNOS striatal content along with significant upregulation of NF-Кβ p65 by 4.6-and 6.5-fold, respectively, as compared with control group. Treatment with diapocynin ensued a noticeable inhibition in NF-Кβ p65 expression (64%) as well as marked reduction in iNOS content (44%) in comparison with 3-NP group, F(3, 20) = 167.1 and 127.4, respectively (p < 0.0001). Normal rats which received diapocynin showed no significant changes in the formerly mentioned parameters, as compared to the normal control animals (Fig. 4).
Diapocynin mitigated the exaggerated apoptosis induced by 3-NP injection in rats
The striatal contents of P53 as well as BAX which are key players in the apoptotic pathway were markedly increased in 3-NP-injected rats by 4.3-and 9.2-fold, respectively, with regards to normal control animals. However, the antiapoptotic; Bcl2 content was significantly reduced upon 3-NP injection by 74%, in comparison with control group. Diapocynin mitigated 3-NP-induced apoptosis with noticeable decrease in P53 (50%) and BAX (50%) contents on the contrary to Bcl2 increased striatal content by 2.5-fold, F(3, 20) = 121.6, 80.09, and 51.81, respectively (p < 0.0001). Diapocynin administration in normal animals did not induce significant changes in the previously mentioned apoptotic parameters, in comparison with the control group rats (Fig. 5). . Statistical analysis was done using one-way ANOVA followed by Tuk-ey's post hoc test. * p < 0.05 vs. control, @ p < 0.05 vs. 3NP group. 3-NP 3-nitropropionic acid, GSH reduced glutathione, GST glutathione-S-transferase, Nrf2 nuclear factor erythroid 2-related factor 2, BDNF brain-derived neurotrophic factor
Diapocynin diminished 3-NP-induced alterations in Sirt1 protein expression
3-NP injection was accompanied with prominent downregulation of Sirt1 protein expression by 70% as compared with the control group. Treatment with diapocynin mitigated 3-NP-provoked Sirt1 depression leading to significant increase in its protein level by 2.5-fold, F(3, 20) = 141.1 (p < 0.0001). Normal animals which received diapocynin revealed no significant changes in the expression of Sirt1 in comparison with the normal control group (Fig. 6).
Diapocynin attenuated 3-NP-induced histopathological alterations in striatum
Striatal tissues were examined by H&E in addition to Nissl stain which was used to determine the number of intact neurons. Photomicrographs of normal animals demonstrated normal morphological features of striatal regions with many records of apparent intact neurons of different sizes with intact subcellular and nuclear details (black arrows), in addition to showing intact intercellular brain matrix with minimal records of reactive glial cells infiltrates. Diapocynin administration in normal animals revealed almost the same records as the control group without abnormal histological alterations. 3-NP-injected rats demonstrated a focal area at anterior lateral border of striatal regions showing many records of darkly stained and pyknotic degenerated neurons (red arrows). Photomicrographs of 3-NP group also revealed significant neuronal loss and moderate edema of brain matrix accompanied with many reactive microglial cells infiltrates in lesion core and higher reactive astrocytic infiltrates allover striatal regions and around core lesion borders (arrow heads). Treatment with diapocynin induced significant neuroprotection with abundant records of apparent intact neurons (black arrows) and few scattered degenerated cells (red arrows) along with intact intercellular brain matrix with significantly fewer reactive glial cells infiltrates (arrow heads) (Figs. 7 and 8).
Discussion
The current investigation emphasized the neuroprotective effects rendered by diapocynin, a selective NOX inhibitor, against 3-NP neurotoxicity model of HD. It was previously stated that 3-NP injection provokes striatal neurodegeneration resulting in prominent dysfunction in animals' grip strength and locomotor activities as well as motor co-ordination (Sayed et al. 2020). Correspondingly, in the present study, 3-NP-injected rats displayed diminished grip strength, impaired motor co-ordination, and disrupted locomotor function in grip strength, rotarod, and open field tests, respectively. Remarkably, diapocynin administration improved 3-NP-injected rats' grip strength and locomotor behavior in the previously mentioned corresponding tests as well as their ability to stay for a longer time during the rotarod assessment. In context, diapocynin was previously reported to significantly restore locomotor and motor co-ordination impairments in MPTP-induced Parkinson's disease mouse model (Ghosh et al. 2012). It also rescued in each group with each bar with vertical line illustrating the mean ± S.E.M. of 3 rats per group. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. * p < 0.05 vs. control, @ p < 0.05 vs. 3NP group. 3-NP 3-nitropropionic acid, GFAP glial fibrillary acidic protein motor dysfunction induced in organophosphate-intoxicated rats (Putra et al. 2020).
In the current study, 3-NP-injected animals exhibited a state of oxidative stress as indicated by prominent reduction in GSH and GST contents contrary to increased gp91phox; NOX2 subunit expression. In fact, the mitochondrial toxin 3-NP is reported to penetrate the blood brain barrier and produce surplus of ROS leading to pathological symptoms simulating that associated with HD (Gonchar et al. 2021). The increased oxidative state and neuronal degeneration triggered by 3-NP injection might be attributed, in part, to the obvious suppression of BDNF/Nrf2 anti-oxidant machinery as reported herein. Nrf2 is known to modulate the production of GSH and its related enzymes as GST; thus, it could overcome the augmented oxidative stress associated with 3-NP (Vasconcelos et al. 2019). Moreover, Nrf2 inhibits NOX activity, so it hinders ROS production via this fundamental enzyme in ROS generation (Benarroch 2017). Nrf2 is also reported to induce transcription of BDNF, an important neurotrophic factor which is involved in neuronal proliferation, differentiation, and survival as well as memory and learning functions (Huang et al. 2020;Yao et al. 2021). Meanwhile, BDNF is implicated in Nrf2 translocation and consequent activation leading to repaired redox hemostasis (Bouvier et al. 2017). It was previously reported that BDNF protein levels are reduced in HD animal models (Ferrer et al. 2000;Duan et al. 2003). A prominent decrease of GSH as a result of Nrf2 downregulation was also previously detected in 3-NP model in rats (Sayed et al. 2020). Concurrently, HD is associated with augmented NOX2 activity leading to massive ROS generation and cell death (Valencia et al. 2013). On the other hand, administration of diapocynin in the present study alleviated 3-NP-associated oxidative hazards with significant enhancement in the previously mentioned anti-oxidant defenses, namely BDNF, Nrf2, GSH, and GST, whereas gp91phox expression was noticeably reduced.
Nrf2 signaling pathway is also reported to inhibit neuroinflammation induced by the redox sensitive transcription factor; NF-Кβ which contributes to neuronal injury linked to 3-NP injection (Brandes and Gray 2020). NF-Кβ is also known to induce microglial activation with consequent release of pro-inflammatory mediators as iNOS which promotes cell death via nitric oxide deleterious hazards (Napolitano et al. 2008). In accordance with these findings, 3-NPinjected animals, in the present investigation, have shown prominent upregulation of NF-Кβ p65 along with increased iNOS content while upon treatment with diapocynin, redox status was improved with reduced iNOS striatal content owing to NF-Кβ p65 downregulation.
Sirt1, a nicotinamide adenine dinucleotide-dependent enzyme, is a critical key player in the regulation of oxidative stress, inflammation, and apoptosis (Ren et al. 2019). It is reported that Sirt1 can inhibit NF-Кβ p65 activation thus, combatting its associated inflammatory milieu (Yeung et al. 2004). Additionally, Sirt1 is involved in apoptosis regulation via inhibition of P53 with consequent downregulation of the apoptotic protein; BAX (Vaziri et al. 2001). Moreover, Sirt1 induces the expression of Nrf2 anti-oxidative pathway, consequently inhibition of Sirt1 is implicated in neuroinflammation, apoptosis as well as oxidative stress (Huang et al. 2015). Interestingly, Sirt1 can also modulate neuroinflammation via reducing astrocyte activation with subsequent decrease in GFAP; a well-known marker of astrogliosis (Shaheen et al. 2021). Additionally, Sirt1 was reported to inhibit microglial activation and its deleterious inflammatory cascade (Li et al. 2020). In the current study, rats receiving 3-NP have shown increased NF-Кβ p65 expression as well as P53 up-leveling with consequent elevation in BAX content while Bcl2 content was obviously reduced. These findings could be attributed to down regulation of Sirt1 and Nrf2 inhibition detected in that group. Diapocynin enhanced Sirt1/Nrf2 expression with consequent downregulation of NF-Кβ p65 in addition to decreased P53 level associated with marked elevation in the anti-apoptotic Bcl2 content contrary to decreased pro-apoptotic BAX striatal content. Besides, Sirt1 downregulation was accompanied by prominent increments of Iba1-positive microglial cells as well as GFAP immunoreactivity which indicate microglial and astrocytic activation witnessed in 3-NP group, respectively. However, diapocynin administration attenuated glial reactivity as a reflection of dismounted gliosis and inflammation through Sirt1 induction. Thus, diapocynin diminished 3-NP-associated oxidative stress, inflammation, gliosis, and apoptosis via augmenting Sirt1/Nrf2 pathway.
Finally, 3-NP is implicated in apparent pyknosis with marked decrease in surviving neurons evidenced by Nissl stain (Gao et al. 2015). Animals receiving 3-NP, in the present investigation, have shown noticeable neuronal pyknosis with marked increase in darkly stained degenerated neurons which indicates an evident decline in neuronal survival. Interestingly, 3-NP-instigated histopathological anomalies were mitigated upon diapocynin administration as indicated by noticeable increase in the number of intact neurons.
Conclusion
The present study highlights the neuroprotection rendered by diapocynin in 3-NP-instigated HD model in rats proposing Sirt1/Nrf2 pathway modulation as a key player in its beneficial action against oxidative stress, neuro-inflammation, and apoptosis. Consequently, diapocynin is a profound nominee for HD management. | 2022-06-02T06:22:53.642Z | 2022-05-31T00:00:00.000 | {
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15418691 | pes2o/s2orc | v3-fos-license | Sensor Deployment for Network-like Environments
This paper considers the problem of optimally deploying omnidirectional sensors, with potentially limited sensing radius, in a network-like environment. This model provides a compact and effective description of complex environments as well as a proper representation of road or river networks. We present a two-step procedure based on a discrete-time gradient ascent algorithm to find a local optimum for this problem. The first step performs a coarse optimization where sensors are allowed to move in the plane, to vary their sensing radius and to make use of a reduced model of the environment called collapsed network. It is made up of a finite discrete set of points, barycenters, produced by collapsing network edges. Sensors can be also clustered to reduce the complexity of this phase. The sensors' positions found in the first step are then projected on the network and used in the second finer optimization, where sensors are constrained to move only on the network. The second step can be performed on-line, in a distributed fashion, by sensors moving in the real environment, and can make use of the full network as well as of the collapsed one. The adoption of a less constrained initial optimization has the merit of reducing the negative impact of the presence of a large number of local optima. The effectiveness of the presented procedure is illustrated by a simulated deployment problem in an airport environment.
Introduction
Imagine a scenario where a toxic gas is spreading in an area or a building and safe paths have to be found to evacuate people. Or think of an airport environment where people moving through rooms and corridors has to be surveilled in order to detect and avoid terroristic actions. Or consider the need of measuring environmental quantities, such as temperature or humidity, on wide areas to the aim of improving theoretical models or making more accurate weather forecast.
There is a great number of situations that would greatly enjoy the use of network of sensors. Indeed, many of the previous tasks are difficult, or impossible, to be accomplished by a single sensor. The employment of a large number reconfigurability owned by a network of moving sensors turn out very useful.
A general tendency in robotic networks is to have sensors (agents) endowed with the same computational and sensing capabilities. This choice increases the overall robustness of the network, but usually calls for distributed coordination algorithms. Having equal sensors, indeed, naturally leads to define optimization and coordination algorithms based on local observations and local decisions ( [13,25,18]). Many of the algorithms proposed in the previous section involve the solution of a global optimization problem requiring a complete knowledge of the environment and of sensors' distribution. The solutions to p-center, pdispersion and p-median problems proposed in [5,7,6], instead, are all spatially distributed, with the meaning that each sensor requires only the knowledge of positions of its neighbors (or even less if it has a limited sensing radius). This fact allows a distributed implementation where each sensor computes its next movement without centralized coordination.
Other solutions to the area-coverage problem look at sensors like particles subject to virtual forces or potential fields. The compositions of suitably defined attractive and repulsive forces is then used to make the network behaving in the desired fashion (spread sensors, avoid obstacles, keep connectivity, etc.). Representative for this kind of approach are the algorithms presented in [17,33], or in [24], where also secure connectivity issues are considered. In [16], instead, it is raised the relevant problem of power consumption in wireless networks and three energy-efficient algorithms are presented for sensors' deployment.
Network-like Environments and Paper Contributions
In this paper, we focus on network-like environments as there are surveillance or monitoring problems where such a kind of model can provide a more suitable description.
Network models represent a natural choice whenever environments have an intrinsic network structure. It is the case, for instance, when sensors have to be optimally deployed over a network of roads to monitor vehicular traffic, or in a river network to measure temperature or pollutants concentration. Even some location-allocation problems can involve networks. Consider, for instance, the case where useful facilities (i.e. schools, hospitals) have to be located in the interior of a network of roads, which is the source of a nuisance (i.e. noise, pollutants), with the goal of minimizing its harmful impact on them (see [8] for the case with one facility); or the dual problem of locating obnoxious facilities (i.e. dumps, industrial plants, mobile phone repeater antennas) reducing the hazard on the network.
Most notably, we think that a network-like model can provide an effective and compact description for complex environments, focusing only on major features and abstracting from those geometrical details which are less important for the deployment problem. The coverage of nonconvex environments with holes or obstacles, for instance, is a challenging task ( [3]), which can enjoy significantly the use of a reduced network-like model. Environments with a complex structure, accounting for a large number of variously sized and shaped rooms, passages, forbidden areas and obstacles, can be reduced to a set of connected paths where the sensing task is more requested or where sensors are forced to pass. An airport is a very representative example of this kind of environments. In this case people moving throughout the airport can be aptly compared with a network flow and focus can be on paths more than on corridors, halls and lounges.
Many of the problems introduced in previous sections have been formulated even for a network-like environment ( [30,31]), but they usually consider a finite discrete set of demand points located on the network's nodes and try to optimize the locations of facilities w.r.t. some objective function accounting for the distance from them. An important thread of works for the deployment of sensors both on plane and on a network is represented by the papers [19,20,21]. In such works, the authors propose powerful greedy algorithms that provide a constant approximation of the optimal solution. Their method, however, aims at solving a global static deployment problem, considering a finite set of demand points and allowing sensors to jump among positions.
In this paper, instead, we consider a deployment problem where sensors use local information to dynamically solve the optimization problem while they are moving in the environment. The constraints induced by the environment to the motion of sensors are explicitly considered. More precisely, we address a generalized p-median problem involving omnidirectional sensors with potentially limited sensing radius and we extend the formulation presented in [6,7,25] to network-like environments. The task is to find sensors' positions that optimize an objective function defined on the network and accounting for the sensor's features and preferential areas. This is a mixed problem, since the network is considered embedded in the plane (it is a continuous set of demand points) and the planar euclidean norm is used to measure the distance between sensors and network.
The core of the cited formulation and of our solution is a discrete-time gradient ascent algorithm based on Voronoi partitions and aiming at maximizing the objective function. It is a well known fact, however, that such a kind of algorithms can get stuck early in local optima, especially when sensors are forced to move in an over-constrained environment like a network. Moreover, the local maximum found by the algorithm is often greatly related to the initial sensors' position.
For these reasons, we present a novel two-step procedure performing an initial coarse optimization, whose purpose is to provide a good starting point for a second finer optimization. The first step can be carried out off-line, either by a central unit, or by each sensor individually (without doing real movements). The impact of local optima is reduced by allowing sensors, in the initial optimization, to virtually move in R 2 and to vary their sensing radius arbitrarily. In order to reduce the complexity of this phase, sensors can be initially clustered and the optimization problem solved for the clusters' centers. After that, a desired number of sensors is spread close to clusters' centers and the sensors' positions thus found are projected on the closest edges of the network. The projected positions are then used in the second optimization, where sensors are constrained to move only on the network. The second step can be performed on-line, in a distributed fashion, by sensors moving in the real environment.
The use of a two-step procedure is motivated also by the very nature of some surveillance tasks, such as for instance airport surveillance, where a large number of individuals (sometimes referred to as mass objects [15]) are monitored. In these cases, sensors can solve the first step optimization using imprecise or estimated information and keeping still; then they can move to reach the final projected positions using planned routes compatible with the network. After this initial deployment, sensors can change their positions by dynamically solving a distributed optimization problem (second step) based on real measures taken from the (potentially varying) environment.
It is worth noting that the present procedure can be used to solve both static and dynamic deployment problems. Moreover, the first step deserves attention by its own, since it provides a solution to those problems involving facilities located in the interior of the network mentioned at the beginning of this section.
Another contribution of this paper is the introduction of a simplified model of the network (similar to the discretization in [14]) called collapsed network and consisting of finite many points. It is obtained by decomposing each segment of the original network in one or more sub-segments and collapsing each subsegment in its barycenter. This model allows a coarser but faster optimization, since computations with barycenters are remarkably less than those needed by the full network. Collapsed network, hence, is intended mainly for fastening the first optimization, but can be used profitably also for the second step. Indeed, it turns out particularly useful in practical implementation involving hardware with limited computational capabilities.
As mentioned above, our work is related to that of [6,7,25]. In particular, the first step of the optimization, allowing sensors to move in R 2 , could be regarded as a specialization of the problem described in [7]. However, the different topology induced by the network introduces issues related to the explicit computation of the gradient and to the convergence of the maximization algorithm, which deserve special solutions. A relevant difference is that the gradient of the objective function presents discontinuity points caused by barycenters on the boundary edge of two neighboring Voronoi cells. Such barycenters can change allocation during sensors' motion, inducing abrupt variations to the value of the objective function associated to each cell. This fact prevents a classical convergence proof for gradient algorithms, hence we consider our proof as a minor contribution of the paper. Some results about convergence may alternatively be derived by using the method of Kushner and Borkar ([22,1]) of stochastic approximation to deal with our differential inclusion.
The outline of the paper is as follows. In section 2 the mathematical definitions of sensors, network and Voronoi covering are introduced along with the objective function to be maximized to solve the deployment problem. Section 3 is devoted to the introduction of the collapsed network and to the formulation, and proof of convergence, of a gradient ascent algorithm to solve the first step (subsection 3.1) and the second step (subsection 3.2) of the optimization procedure. Section 4 addresses the solution of the second step optimization involving the full network. Finally, a network describing an airport environment is used in section 5 to illustrate by simulations the effectiveness of the proposed optimization procedure. Conclusions and future research directions are reported in section 6.
Preliminaries and Problem Formulation
In this section we introduce the mathematical framework to describe the sensors, the network and its Voronoi covering.
Definition 1 Given two points p 1 , p 2 ∈ R 2 , with p 1 = p 2 , s 12 = [p 1 , p 2 ] ⊂ R 2 is the segment joining p 1 and p 2 and s o 12 = (p 1 , p 2 ) is the open segment between them. We define length of a segment s 12 as ℓ(s 12 ) = p 2 − p 1 , where · is the Euclidean norm; barycenter of a segment s 12 the point b(s 12 ) = 1 2 (p 2 + p 1 ) ∈ s 12 ; partition of a segment s = [p 1 , p 2 ] in k sub-segments, the set of segments {s i } i=1,...,k given by . . , n} i = j}, such that: Definition 3 Given a network N and a set of points P = {p 1 , . . . , p m } ⊂ N , the Voronoi covering of N generated by P with respect to the Euclidean norm is the collection of sets V N i (P) i∈{1,...,m} defined by is the i-th cell of the usual Voronoi partition of R 2 generated by P. The previous definition is about a covering and not a partition since neighboring cells can have a nontrivial intersection: a portion of a segment can belong to the shared edge of two cells V i (P) and V j (P).
We adapt the framework provided in [6] to describe the sensors' and network features. Each sensor is modeled by the (same) performance function f : R + → R, that is a non-increasing and piecewise differentiable map having a finite number of bounded discontinuities at R 1 , . . . , R N ∈ R + , with R 1 < . . . < R N . We can set R 0 = 0, R N +1 = +∞ and write In order to model regions of the network with different importance, we can use a density function φ : N → R + , which is bounded and measurable on N . Given g(q)dq) the sum of the linear integrals of g over the segments of N (respectively V N i (P)) using an arclength parameterization. With these functions we can define the multi-center function H : (2) 6 We can also provide an alternative expression for (2) based on the Voronoi covering induced by P as follows (3) is not null if and only if there exists a non trivial segment s ⊆ s ij ∈ S such that s ⊂ ∆ N hk for some i, j ∈ {1, . . . , n} and h, k ∈ {1, . . . , m}.
Deployment over a Collapsed Network
In a collapsed network each segment of the original network is decomposed in one or more sub-segments and each sub-segment is collapsed in its barycenter. Chosen a value for r guaranteeing a good approximation, we can build the r-collapsed network C N r as follows: Definition 5 (r-Collapsed Network) Given a network N = (V, S) and r > 0, ∀s ∈ S consider its partition in k s = ℓ(s) r sub-segments s i (having at most length r) and the associated set of barycenters {b (s i )} i=1,...,ks . We define the r- The multi-center function must be re-defined since the integration domain is now a discrete set represented by the barycenters. Hence we set where φ be are suitable (density) weights assigned to barycenters.
Sensors Moving in R 2
In this section we solve the deployment problem for a collapsed network and sensors moving in R 2 , which constitutes the first step of our optimization procedure. Also for the multi-center function (4) we can provide an alternative expression using the Voronoi covering. We need the following definition 1 : Definition 6 Given an r-collapsed network C N r for some r ∈ R + and a set of points P = {p 1 , . . . , p m } ⊂ R 2 , the Voronoi covering of C N r generated by P with respect to the Euclidean norm is the collection of sets V 1 For this definition similar remarks as Remark 4 also apply.
7
We define also the boundary of a Voronoi cell as and, in order to simplify the problem, we make the following assumption With this assumption the multi-center function (4) can be written also as or, setting dist(q, P) = min i∈{1,...,m} q − p i , as Remark 8 H(P) is not globally Lipschitz as f (·) is not a continuous function. However, if f (x) is continuous and piecewise differentiable with bounded derivative, then H(P) is globally Lipschitz. In order to prove the global Lipschitz continuity, let us consider two sets of points P = {p 1 , . . . , p m } ⊂ R 2 and Proof. The continuous differentiability of H on R 2 m \ D C N r m is a straight consequence of the same property of f (·) on R 2 \ D C N r . As concerns the gradient, 8 using (6) we have In the hypothesis of Assumption 7, we have that the index set I be = argmin i∈{1,...,m} ( b e − p i ) has cardinality 1. Hence, ∀b e such that whereby the thesis follows using the definition of V C N r h (P). The sensors' location-allocation problem can be addressed by means of a gradient-like algorithm. If a continuous time implementation is looked for, the following fictitious dynamics would be associated to the sensors' positionṡ Unfortunately, this dynamics conveys some problems. It is well defined as long as Assumption 7 and Theorem 9 are fulfilled, but these hypotheses are, in fact, too stringent for the algorithm to work properly. Indeed, they would require the evolution of the sensors to avoid any position in the discontinuity set and the barycenters not to enter or exit the Voronoi cells where they are at the initial time instant. First of all we can reduce the analysis to continuously differentiable functions to avoid issues related to the existence of the gradient. Still the relaxation of Assumption 7 induces some problems on the definition of the gradient. Barycenters on a boundary edge of a Voronoi cell belong to all the cells sharing that edge. All sensors' positions producing these configurations are discontinuity points for ∇H(P). Roughly speaking, the gradient takes different values depending on which cell the shared barycenters are assumed to belong to. This fact makes the equation (8) a set of differential equations with discontinuous right-hand side.
The problem of shared barycenters can be solved by adding a lexicographic criterion to the definitions based on the euclidean distance. Indeed, with this criterion barycenters on boundary edges are allocated univocally to the sensor having the lower index (w.r.t. the Lexicographic Order (L.O.)) among sharing sensors. This fact allows us to define a genuine Voronoi partition, no longer a covering, whose generic cell is given by (compare with Definition 6): We must now define a generalized (lexicographic) gradient of H, ∇ l H(P), we use the classical formula given by (7). ∀b e ∈ ∂V which is formally equal to the formula (7) provided by Theorem 9.
The differential equation using this new definition for the gradient, however, does not imply the existence and uniqueness of the solution, and this proof may turn out to be complex due to special sensors' and barycenters' configurations. Moreover, the formula (10) accounts only for the infinitesimal perturbations of sensors' position not inducing any barycenters to enter or exit Voronoi cells, hence changing their allocation.
In order to simplify the convergence proof and to provide an algorithm which is more suitable for a realistic implementation, we consider here a discrete-time version of the gradient algorithm. In our case, the discrete-time implementation can overcome the problems with discontinuity gradients due to the properties of the function H and its discontinuity points, as it is shown by the following theorem.
Theorem 10 Consider the following discrete-time evolution for the sensors' positions where the h-th component of ∇ l H is given by (10) and H : R 2m → R as in (5).
If f (·) has locally bounded second derivatives, then, for suitable δ k , P (k) lies in a bounded set and i) H(P (k) ) is monotonically nondecreasing; ii) P (k) converges to the set of critical points of H.
Proof. It is easy to see that there exists a ball B ⊇ N such that, if p i ∈ ∂B, then ∂ l H(P) ∂pi points inside B. Thus the fact that P (k) is bounded for δ k sufficiently small, easily follows. According to the definition in equation (9) of a Voronoi cell using the lexicographic rule, we can define H pi (P). We must prove that H pi (P (k+1) ) ≥ H pi (P (k) ) for ∀p i ∈ P (k) obeying the discrete-time evolution (11) and that ∃p i ∈ P (k) such that H pi (P (k+1) ) > H pi (P (k) ) if P (k) does not belong to the set that is the cost H pi (·) computed by using the new sensors' positions P (k+1) but the old allocation of barycenters to sensors, i.e. the Voronoi partition generated by P (k) . Therefore, we write where the (generalized) gradient is computed by means of the lexicographic assignment based on the Voronoi partition generated by P (k) . If the Voronoi partitions generated by P (k+1) and P (k) coincide (same barycenters' allocation), then H pi (P (k+1) ) = H u pi (P (k+1) ) and we can assert directly that H pi (P (k+1) ) ≥ H pi (P (k) ) or H pi (P (k+1) ) > H pi (P (k) ) if does not belong to the set of critical points of H. It is worth noting that similar remarks about the strict inequality apply also in what follows and they will not be repeated. If the barycenters' allocation is different, H pi (P (k+1) ) may be smaller than H u pi (P (k+1) ) and we cannot say anything about its relation with H pi (P (k) ). This fact is due to the presence of barycenters changing allocation during the sensors' evolution, hence the cells involved in the change cannot be considered independently. Let us consider for simplicity only one barycenter b e ∈ V C N r j (P (k) ) and suppose that at step k + 1 b e ∈ V C N r i (P (k+1) ). With this assumption no barycenters can enter or exit the union of the two cells but b e . We have In other words, H pi (P (k+1) ) is grown w.r.t. the ideal value H u pi (P (k+1) ) due to the allocation of b e to p (k+1) i , whereas, H pj (P (k+1) ) is decreased w.r.t. H u pi (P (k+1) ) by the contribution that b e would have given if it were allocated to p (k+1) j even at the step k + 1. It is worth noting that if b e changes allocation from p (P (k+1) ) and, being i < j, the allocation is induced by the lexicographic rule). Therefore, due to the monotonicity of f , There exist some constants c ij k , δ ij k > 0 such that H pi (P (k+1) ) + H pj (P (k+1) ) ≥ H pi (P (k) ) + H pj (P (k) ) + δ ij k c ij k + o (δ ij k ) 2 .
Extending the same reasoning to more complex configurations involving more than one common boundary edge and more than two neighboring cells, we can say that ∃c k , δ k > 0 such that This proves assertion i). Assertion ii) can be proved by exploiting the results of Section 3.2 of [6]. More precisely, using the fact that P (k) is bounded and f has locally bounded second derivatives, then there existsδ > 0 such that we can choose δ k ≥δ. Then we conclude, using Proposition 3.4 of [6], that assertion ii) is true.
Remark 11 (Distributed Implementation)
The use of a gradient ascent algorithm based on a Voronoi partition, allows us to solve not only a static deployment problem, but also a dynamic one. As shown in [6], this kind of algorithms is spatially distributed, with the meaning that the i-th sensor needs only to know the position of its neighbors in order to determine the boundary of its cell and, hence, to compute ∂Hp i ∂pi . For the same reason the i-th sensor can choose the value of the step-size δ i k independently of the other sensors simply performing locally a classical line search algorithm. This property makes the algorithm suitable for a spatially distributed implementation.
The independence in the choice of the step-sizes δ i k is obviously preserved in each period k, as long as a synchronous implementation is considered. In this case sensors have access to a global clock, or perform a synchronization algorithm. At the beginning of the the k-th period (instant t k ), all sensors are idle, build their Voronoi cells and compute their gradients and step-sizes, then they move until, at most, the end of the period (instant t k+1 ). If, instead, an asynchronous implementation is considered, further hypotheses are necessary to ensure that independence is preserved. Unfortunately, the discontinuity of the gradient prevent us from using the results of [32]. But, if a sensor has the capability to detect when its neighbors start and stop moving and when a new sensor joins the neighborhood, the asynchronous algorithm presented in [7] (Table IV), can be applied, thus automatically recovering the independence.
Remark 12
In the previous theorem, for sake of simplicity, we did not consider degenerate configurations where different sensors have the same position (p i = p j for i = j). But it can be proved that if the initial positions of sensors are not degenerate, sensors can always choose a suitable δ i k to avoid the occurrence of these configurations.
Sensors Moving on the Network
This section is devoted to the case of sensors constrained to move on the network and sensing a collapsed network. Therefore, these results are suitable for an implementation of the second step of our procedure on hardware with limited computational capabilities.
We still assume f (·) to be a continuously differentiable function and we make use of the lexicographic criterion for the barycenter allocation. As concerns sensors' motion, however, we cannot use directly the gradient since the sensors have to remain on the network. We must consider now the directional derivative of H along the edges of the network.
Following the guidelines of the previous section, the following theorem can be proved.
Theorem 13 Given a network N = (V, S) and the related r-collapsed network C N r , the multi-center function H is continuously differentiable almost everywhere on N m . In particular, on each open segment s o ij such that s ij ∈ S, given the unit vector w ij such that s ij · w ij = s ij , the directional derivative in p h ∈ s o ij along w ij is The directional derivative is a multivalued function on the vertices of the network as more than one edge can share the same vertex, but we need a univocal definition. Hence, we fix a choice rule such that the directional derivative in a vertex is given by the maximum among all the derivatives defined for each possible direction that does not lead the sensor out of the network. If all the directional derivatives in a vertex point outward the network, then the derivative is set equal to zero. (15) we define the directional derivative of H(P) in any point p h ∈ N as follows
Definition 14 Given the set
given by (14) ∀p We can now define the discrete-time gradient-like algorithm.
Theorem 15 Consider the following discrete-time evolution for the sensors' positions P (k+1) = P (k) + δ k DH(P (k) ), (17) where the h-th component of DH is given by (16) and H : N m → R as in (5). If f (·) has locally bounded second derivatives, then, for suitable δ k , P (k) lies in a bounded set and i) H(P (k) ) is monotonically nondecreasing; ii) P (k) converges to the set of critical points of H.
Proof. The proof is essentially the same of the Theorem 10, except for the use of the derivative (16) in place of the gradient (10). In particular equality (12) becomes for p i moving along w hl .
Deployment over a Full Network
In this section we consider a more accurate version of the second step of the optimization procedure, namely sensors constrained on the network and sensing the full network. To start with, let us define the boundary of a Voronoi cell as ∂V N i (P) = {q ∈ N | q − p i = q − p j , ∃p j ∈ P}, and the instantaneous discontinuity set of f (·) as With the orthogonality assumption the expression (3) simplifies to Theorem 17 Given a network N = (V, S) if Assumption 16 holds, the multicenter function H is continuously differentiable almost everywhere on N m . In particular, on each open segment s o ij such that s ij ∈ S, given the unit vector w ij such that s ij · w ij = s ij , the directional derivative in p h ∈ s o ij along w ij is Proof. Consider the gradient of H(P) in the form (18) Let us consider the second term of (20) where ∆V N ih (P) if and only if p i ∈ N GD (p h , P). 2 Let us consider now the first term of (20) where ∆V N hh (P) . . . , p h + ε, . . . , p m }). Now we want to prove that the sum of the second term of (20) and the last term of (22) is null. First of all, recall that the sum in the second term of (20) can be limited to the cells in the neighborhood of the h-th cell, namely ∀i ∈ I h with I h = {j ∈ {1, . . . , m} | p j ∈ N GD (p h , P)}. This fact implies that Moreover it can be easily seen that ∆V N ih (P) + ⊂ ∆V N hh (P) − and ∆V N ih (P) − ⊂ ∆V N hh (P) + ∀i ∈ I h . Indeed, any segment s ∈ ∆V N ih (P) + is such that s ∈ V N i ({p 1 , . . . , p h + ε, . . . , p m }) and s / ∈ V N i (P), and, for any infinitesimal perturbation of p h , it is possible only if s ∈ V N h (P) and The conclusion follows from the fact that lim ε→0 ∆V N hh (P) + ∪ ∆V N hh (P) − = ∂V N h (P) and ∀q ∈ ∂V N h (P) q − p i = q − p h . As concerns the second-last term of (22), recalling again that lim ε→0 ∆V N hh (P) + ∪ ∆V N hh (P) − = ∂V N h (P) and the Assumptions 16, we can write where diam(N ) max p,q∈N q − p . The same argument holds for the term with ∆V N hh (P) − , hence we have with M h (P) the number of segments in V N h (P) and I k = 1] for s k we can apply the Theorem 19 in appendix. Recall that in this case ν (x, q) = q − x , hence the equation γ k (t) − p h − R α = 0 may have at most two zeros at t k α,1 and t k α,2 ∀α ∈ {1, . . . , N }. It is worth noting that assumptions 16 iii) and iv) play here the same role of assumptions i) and ii) in Theorem 19. Therefore, from the definition of f (·), equation (1), we have the thesis.
In order to define a gradient-like algorithm, also in this case, we must relax Assumptions 16. First of all, focus on the orthogonality assumption. It has been introduced to avoid the presence of entire segments in the boundary of a cell, because these configurations induce problems in the definition of the gradient (they represent points on which the gradient may assume different values). Even in this case we opt to use the lexicographic rule in order to univocally assign a segment on the boundary to only one cell, and, again, we consider a discretetime dynamics for the gradient-like algorithm.
Using the lexicographic rule, we re-define the Voronoi cell as follows and verify that the expression (18) for H(P) is still formally correct. We remove the orthogonality hypothesis by adding to (19) an I k term for each segment entirely included in the boundary of a Voronoi cell. This fact does not change the expression (19), since, with the new definition V N h (P), M h (P) accounts now also for segments on the boundary.
The relaxation of the other assumptions would imply some discontinuities in the integration domain induced by the discontinuities of the function f . These discontinuities, without additional assumptions, would prevent us from guaranteeing H(P) to be monotonically nondecreasing along the evolution of P given by the gradient dynamics. Hence, we assume now f to be continuous and piecewise differentiable. Being f continuous, the second term in I k in (19) is null.
As made in the previous section, the directional derivative must be univocally defined on the vertices. To this aim, we use the expression (16) given in Definition 14, but with reference to the formula (19) for the directional derivative in a point in the interior of a segment. Using these definitions we can state the following theorem.
Theorem 18 Consider the following discrete-time evolution for the sensors' positions P (k+1) = P (k) + δ k DH(P (k) ), where the h-th component of DH is given by (16) and D wij H(P)[p h ] by (19) and H : N m → R as in (18). If f (·) has locally bounded second derivatives, then, for suitable δ k , P (k) lies in a bounded set and i) H(P (k) ) is monotonically nondecreasing; ii) P (k) converges to the set of critical points of H.
Proof. As long as sensors' configurations not violating orthogonality assumption are considered, the gradient is smooth and the proof is canonical. In the case of discontinuity points, segments belonging to the boundary of a cell can change allocation during sensors' motion. Hence, we can proceed as in the proof of Theorem 10 and 15 replacing barycenters with segments. In particular, being s h a segment changing allocation, equations (13) are now replaced by Again, due to the fact that any point
A case study
In this section we apply the proposed two-step optimization procedure to a network representing a wing of the Amsterdam Schiphol airport. The network is made up of 63 vertices and 87 segments and the density function φ is the sum of The 50 sensors to be deployed have performance function f (x) = 1 2 1 − tanh where R is a parameter considered as variable in the first step and as fixed in the second step of the optimization. Even if the previous function describes sensors with an infinite sensing radius, they will be represented as shaded circles of radius 7 8 R to emphasize that the performance function assumes values lesser than 0.01 for larger distances.
First step
The first step of the optimization is performed on a collapsed network with collapsing factor r = 0.3 (see the small dots along the grey network in fig. 2a,-c)). Sensors are grouped in 10 clusters of 5 elements each, and each cluster is represented as a single sensor. Clusters set initially R = 10 and decrease linearly its value up to 1 during the simulation (compare fig. 2-a) with fig. 2-c)). As apparent by the flows in fig. 2-b), clusters are allowed to move in R 2 .
It is important to recall that, both the variation of the sensing radius and the unconstrained motion of sensors are allowed in the first step as it is performed off-line. This step makes use of the algorithm described in section 3.1 and is thought to provide a good starting point for the second step. However, if sensors are initially located on the network as in fig. 2-a), they can execute the first step independently, using partial or rough information of the environment, without moving, and then plan a route on the network to reach the previously computed final positions. Since final positions can be not on the network (see fig. 2-c)), they must be projected on it to be reachable. Anyway, this projection has to be performed before the second step to provide a valid starting point.
Second step
The second step considers a full network with sensors having fixed radius R = 1 and initially deployed in the positions shown in fig. 3-a). Such positions are obtained by spreading randomly 5 sensors close to each cluster center and projecting them on the closest segments of the network. Sensors now can take real measures from the environment and perform the optimization on-line, while moving, according to the algorithm described in section 4. They are constrained to move on the network as shown in fig. 3-b). Final positions (see fig. 3-c)) show how sensors, originally clustered, diffused to better cover preferential areas (see fig 1).
Conclusions and Future Works
This paper focused on the problem of optimally deploying sensors in an environment modeled as a network. An optimization problem for the allocation of omnidirectional sensors with potentially limited sensing radius has been formulated. A novel two-step optimization procedure based on a discrete-time gradient ascent algorithm has been presented. In order the algorithm to not get stuck early in one of the many local minima, in the first (off-line) step, sensors are allowed to move in the plane. Moreover, a reduced model of the environment, called collapsed network, and sensors' clustering, are used to speed up the first optimization. The positions found in the first step are then projected on the network and used in the second (on-line) finer optimization, where sensors are constrained to move only on the network.
The proposed procedure can be used to solve both static and dynamic deployment problems and the first step alone can provide solutions to locationallocation problems involving facilities located in the interior of the network.
A main future research direction will consider the integration of classical Operative Research methods with the present gradient algorithm. In particular the first optimization could be addressed by adapting methods and heuristics developed for the solution of the multisource Weber problem ( [2]). The aim is to build an overall global optimization technique to solve location-allocation problems of large dimensions with many facilities. Moreover, future research, more related to deployment problems, will consider other sensor's models such as those with limited sensing cone.
A Theorem 19 Let ϕ : R 2 × R + → R be a smooth function w.r.t. its second argument and a non-increasing, piecewise differentiable map with a finite number of bounded discontinuities at R 1 , . . . , R N ∈ R + , R 1 < . . . < R N , w.r.t. its first argument. Let ν : R 2 × R 2 → R + be a continuously differentiable map w.r.t. both its arguments. Let s = [a, b] ⊂ R 2 be a segment and assume that ∂ν(x, q) ∂x With the chosen parameterization of s, the equation ν(x, γ(t)) − R i = 0 may have k i zeros t i,j ∈ [0, 1], j ∈ {1, . . . , k i }. Thanks to i) and ii) the set of zeros t i,j does not change cardinality for x nearx, thus, t i,j depends smoothly on x. | 2010-06-17T18:36:14.000Z | 2010-06-17T00:00:00.000 | {
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"oa_url": "http://arxiv.org/pdf/1006.3542",
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232087065 | pes2o/s2orc | v3-fos-license | Biology of NSCLC: Interplay between Cancer Cells, Radiation and Tumor Immune Microenvironment
Simple Summary The immune system represents an important link for tumor development, tumor control and tumor progression. The tumor immunogenic balance, determined by the prevalently immuno-inhibitory tumor- and conventional radiation-related effects is shifted negatively towards immunosuppression, which can worsen treatment outcome and prognosis. Emerging evidence suggest that those suppressive effects might be converted to an immunostimulative environment that can improve the therapeutic ratio with uses of newer conventional radiotherapy approaches combined with emerging immunotherapy agents. Abstract The overall prognosis and survival of non-small cell lung cancer (NSCLC) patients remain poor. The immune system plays an integral role in driving tumor control, tumor progression, and overall survival of NSCLC patients. While the tumor cells possess many ways to escape the immune system, conventional radiotherapy (RT) approaches, which are directly cytotoxic to tumors, can further add additional immune suppression to the tumor microenvironment by destroying many of the lymphocytes that circulate within the irradiated tumor environment. Thus, the current immunogenic balance, determined by the tumor- and radiation-inhibitory effects is significantly shifted towards immunosuppression, leading to poor clinical outcomes. However, newer emerging evidence suggests that tumor immunosuppression is an “elastic process” that can be manipulated and converted back into an immunostimulant environment that can actually improve patient outcome. In this review we will discuss the natural immunosuppressive effects of NSCLC cells and conventional RT approaches, and then shift the focus on immunomodulation through novel, emerging immuno- and RT approaches that promise to generate immunostimulatory effects to enhance tumor control and patient outcome. We further describe some of the mechanisms by which these newer approaches are thought to be working and set the stage for future trials and additional preclinical work.
Introduction
The World Health Organization (WHO) estimated 1.76 million deaths caused by nonsmall cell lung cancer (NSCLC) in 2018. Lung cancer represents the leading cause of deaths worldwide with NSCLC representing approximately 85% of all lung malignancies [1]. Despite substantial improvements in therapy, 5-year overall survival (OS) for NSCLC does not exceed 25% [2,3]. Prognosis and survival of patients affected by NSCLC are correlated to disease stage, with OS being prognostically favorable by earlier diagnosis and treatment [4,5]. Nevertheless, a significant percent of patients are diagnosed in advanced stage with little chance to be cured.
Chronic inflammation plays a key role in tumorigenesis of NSCLC [6]. Inflammatory factors like cigarette smoking are associated with chronic bronchitis and emphysema, which lead to the development of lung cancer [7]. The process of cellular malignant transformation consists of many steps over long time periods, ranging from pre-carcinogenic chronic inflammation, progressing, if not treated, towards the development of invasive carcinoma and systemic disease [8][9][10]. Some chronically inflamed pre-carcinogenic environments will never result in malignant cell transformation, while others exposed to the same carcinogen will undergo tumorigenesis. This uncertainty of initiating tumorigenesis events highlights the malignant "modulating" role of genetic predisposition in cancer [11] and underscores that inflammation may assist in this process. The profile and status of the inflammatory-changed environment following the chronic carcinogen(s) exposure is exceedingly variable, and its potential for malignant transformation is related to polymorphic immune response genes affected by a diversity of anti-oxidant and DNA repair associated genes [11]. The immune system, in its cell-mediated and humoral form, is deeply involved in generation of an inflammatory environment and is considered to be the first step of tumorigenesis. Chronic exposure of normal cells to carcinogen(s) leads to initiation of immune cell activation with subsequent upregulation of the pro-inflammatory cytokines like Interleukin-1 alpha (IL-1α) and IL-1-β, and production of Cyclooxygenase (COX)-1 and COX-2 immune-regulatory enzymes in epithelial and mesenchymal. This activation of the inflammatory pathways is associated with the development of malignant disease [12][13][14][15]. Additionally, the induction of COX-2 favors increased angiogenesis in an inflammatory environment and helps pave the way for hyper-vascularization needed for tumor development and progression [16][17][18].
In addition to assisting tumorigenesis, the immune system also plays a fundamental role in the defense against cancer by surveillance and identification of foreign or "non-self" from self and assisting with elimination of DNA-damaged cancer cells from the body. However, tumor cells can escape by suppressing the immune system. Furthermore, therapies, especially if they cause immune-suppression, may further aid the escape of the tumor cells from the immune system. Most patients with NSCLC will eventually require radiotherapy (RT) alone (especially those that are medically inoperable) or in combination with systemic therapy (especially those with advanced stage disease). Regardless, there are many reports that show that conventional RT induces an immunosuppression, and thereby can negatively affect the overall survival [19][20][21][22]. Thus, the current immunogenic balance, determined by the inhibitory effects caused by tumor cells themselves and radiation therapy can lead to poor clinical outcomes. There is newer emerging evidence suggesting that immunosuppression is an "elastic process" that can be transformed into an immune-stimulating environment by "correcting" many manipulable components of this triangle radiation, tumor, and immune cells by using combination approaches that change the way RT, chemotherapy, and immunotherapy are used. Significant efforts are being deployed in the development of novel treatment strategies and protocols aimed to convert radiation-and tumor-induced immunosuppression into a predominant immunostimulation by means of immunotherapy, unconventional RT and the combination of these modalities in order to improve the outcomes of NSCLC patients. The aim of the present review is, therefore, to highlight the natural interplay between the NSCLC cells, tumor immune microenvironment and radiation that routinely results in immunosuppression, and how those immunosuppression-related factors could be manipulated for generation of prevalently immunostimulatory effects to improve NSCLC patient's outcome.
The following two sections will focus on evidence indicating the tumor-and radiationrelated immunosuppressive effects, followed by discussions about novel strategies for overcoming and converting these effects into immunostimulative effects.
Tumor-Related Immune Suppression in NSCLC
The interplay between the tumor and immune cells is the subject of an extended and ongoing research. Table 1 represents latest evidence on tumor-related immunosuppressive effects . Tumor cells escape immune surveillance by downregulation of HLA and costimulatory molecules, or by production of immunosuppressive factors and upregulation of immune cell apoptosis inducing molecules [23][24][25][26]. As a consequence, the immune system will "ignore and tolerate" tumor cell proliferation and progression. The presence of tumor-infiltrating lymphocytes (TIL) in cancer cell nests is an independent prognostic factor of survival in various types of cancers including NSCLC [57], and if used properly, could be used to help convert immune cells to fight against cancer. The inefficiency of the immune response against tumor is inversely proportional to tumor growth, being weaker in larger tumors and stronger in smaller tumors [58]. There is a gap in the current knowledge and understanding of the mechanisms behind the immune response against NSCLC, and additional attention to these mechanisms are needed if we are to improve the outcome of patients in the future. Table 1. Tumor-related immunosuppressive effects.
Tumor-Related Effect Consequences
Production of immunosuppressive factors and upregulation of immune cell apoptosis inducing molecules [23,24]. Escape from immune surveillance.
Inhibition of dendritic cells and T-cells.
Tumor-associated antigens overexpression in NSCLC [42,43]. Immune system tolerance and less responsiveness to immune checkpoint blockade. Tumor specific (neo) antigens present on MHC molecules, often downregulated in NSCLC [44,45]. Tumor cells evasion of immune destruction.
Inhibitory effects on signaling pathways involved in T-cell activation and cytokine secretion. Tumor cells mediate a checkpoint/"brake" on T-cell activation and thus anti-tumor immunity, by expressing CTLA-4, a B7 ligand and an inhibitory homolog of CD28 [48,49].
Tumor cells evasion of immune destruction.
Tumor cells expand a local immunosuppressive microenvironment, induce dysfunctional T-cell signaling, and upregulate inhibitory immune checkpoints [51].
Evasion of host immune-mediated surveillance and destruction.
Tumor cells express ligands for PD-1 interacting in that way with surface molecules on CD8+ T-cells; influence the microenvironment via orchestration by cytokines [52].
Tumor cells do not express many neoantigens, and some of those even if expressed might be low immunogenic eliciting only a mild reaction with low affinity antibodies [53].
Cytotoxic lymphocytes unable to recognize tumor cells, inhibited combined cytotoxic reaction together with T-cells.
Release of soluble amino acids tryptophan and arginine within the tumor microenvironment [54,55]. Inhibition of T-cells and NK function, tumor immune tolerance.
Tumor cells express ectonucleotidases CD73 and CD38 which create adenosine from ATP via ADP-AMP [56]. Induction of immunotolerance in cytotoxic lymphocytes. The currently available data comes from peripheral blood or surgically removed NSCLC tumor tissue, with the latter being limited to less than a third of operable NSCLC patients. The vast majority of the patients have an unresectable disease or are inoperable and undergo radio/chemotherapy, and therefore NSCLC tumor tissue in these advanced stage patients is typically unavailable for detailed immunological analysis. However, based on the limited histological and immunological analysis obtained from the available tissue, the main components of a tumor-directed immune response are represented by a complex interaction between various immune cells operating a composite cytokine network that is supported by the surrounding mesenchymal, epithelial and endothelial cells. The immune cells are a large, highly cooperative family consisting of tumor infiltrating lymphocytes (TILs), the tumor associated macrophages (TAMs), the tumor associated neutrophils (TANs), tissue eosinophilia, and T-cell lymphocytes [59][60][61][62][63][64]. The lung antitumor immune response is imitated by activation of the pulmonary antigen presenting cells (APCs), represented by macrophages and dendritic cells [65]. This is a fundamental step towards the beginning of an effective anti-tumor immune response. Following tumor antigen(s) recognition and distinguishing "self" from "non-self", the APCs migrate to the regional lymph nodes and activate the effector immune cells that aid in destruction of tumor cells. These effector immune cells, also known as cytotoxic lymphocytes, include the CD4+ lymphocytes, natural killers, natural killer T-cells, CD8+ lymphocytes and B lymphocytes [66][67][68]. The activation of these cells are enhanced by the secretion of inflammatory cytokines such as IL-12 and Interferon gamma (IFN-γ). These cytokines are released by the activated macrophages, growing tumor cells, and stromal cells surrounding the tumor. Additionally, membrane-receptor induction of programmed death by the cytotoxic lymphocytes also aids cytokine release and apoptosis of tumor cells [69], as the final coordinate anti-tumor response.
For the purpose of effective antigen presentation, a critical role is played by interaction between co-stimulating molecules on antigen-presenting cells and corresponding receptors on cytotoxic lymphocyte [70]. One mechanisms of immune suppression that tumor cells use, are to block this antigen/cytotoxic lymphocyte interaction and prevent the cytotoxic lymphocytes from getting activated against the tumor (Table 1). Several additional tumor-related factors and mechanisms that result in immune suppression have also been described. One of those involved is alterations in signal transduction molecules on effector T-cells leading to the lack of tumor antigen recognition and missing anti-tumor immune response [71]. In this case, increased tumor-related anti-inflammatory humoral factors like IL-10 or Tumor Growth Factor-beta (TGF-β) induce the loss of signal transducer CD3-ε chain (CD3-ε) in TIL. With that, the signaling pathway for T-cell activation is inhibited, the immune response cannot be initiated and results in immunosuppression.
Finally, the stromal cells from the TME also exhibit an important immunosuppressive role through modulation and binding of tumor antigens. By binding tumor antigens, these cells compete with the antigen-presenting cells so that many tumor antigens will be downregulated, resulting in immunosuppression and tumor progression [76][77][78]. By increasing interstitial fluid pressure in the tumor, stromal cells will make significant quantity of tumor antigens to be unavailable and therefore, ignored by T-cells [79]. Besides the tumor and the TME causing immune-suppression, therapies such as convention RT and chemotherapy can also lead to additional immune suppression.
Radiation-Related Immune Suppression in NSCLC
The wide use of RT in the management of NSCLC, especially for locally-advanced, high-volume disease, is associated with radiation-induced lymphopenia. The radiation-induced lymphopenia has been correlated with poor oncologic outcomes. Table 2 summarizes the many radiation-induced immunosuppressive effects. Table 2. Radiation-related immunosuppressive effects.
Direct damage of dendritic cells as professional antigen-presenting cells (responsible for priming of naive T-cells) [81].
Negative impact on T-cell activation leading to immune tolerance.
Systemic immunosuppression leading to poor oncologic outcome. Higher radiation doses to the immune system following RT for stage III NSCLC [82].
Systemic immunosuppression leading to increased tumor progression and death. Radiation-induced depletion of total lymphocytes [83,84].
Reduces tumor control and survival in patients with stage III NSCLC. Upregulation of the transcription of HIF-1α [85].
Upregulation of adenosine [87]. Multiple immunosuppressive effects. Accumulation of regulatory T-cells (related to intrinsic higher radio-resistance and increase of immunosuppressive mediators and cytokines induced by radiation) [87,88]. Immunosuppression.
Multiple immunosuppressive effects. Although RT represents a local treatment, it can add to the cytotoxic effects on the circulating immune component as blood flows through the radiation field whose size, together with prolonged treatment times and increased dose fractionation determines the severity of immune depletion [80]. As for tumor cells and healthy cells, radiation is directly detrimental to all cells located within the radiation field. The immune cells are among the most radio-sensitive cells, being easily damaged and killed by ionizing radiation [92][93][94]. Preclinical evidence confirmed that dendritic cells that act as professional APCs and normally responsible for priming of naive T-cells, are significantly damaged after RT. The RT-induced destruction of these dendritic cells can negatively affect T-cell activation [81]. Additional immune cells that are unintentionally targeted, like dendritic cells, macrophages and B-and T-lymphocytes within the irradiated regional lymph nodes, could also contribute to RT-induced immune-suppression. This "collateral" damage of the peri-tumoral immune cells during RT of tumor cells is a result of trying to treat the target volumes that contain tumor (GTV) and surrounding clinical target volumes (CTV) whereby, tumor is likely to have spread. The collateral damage from scattered irradiation can cause damage to APCs, lymphocytes, supportive mesenchymal and epithelial cells as a bystander consequence of trying to irradiate the tumor volumes. Furthermore, conventional RT volumes are expanded to create larger volumes (i.e., internal target volume (ITV)) to account for the tumor motion due to respiration, and taking into consideration tumor motion in 4D-CT, and the planning target volume (PTV) (to account for the set up error(s) related to the patients daily (re)positioning) (Figure 1). The final target volume for radiation treatment will therefore include much larger area than the one corresponding to the macroscopic lung cancer with significant amount of the peri-tumoral tissue between the GTV and PTV, which will be exposed to the full radiation dose. Thus, there is substantial normal/surrounding peri-tumor microenvironment that is irradiated during conventional irradiation approaches (Figure 1), and could result in destruction of many surrounding immune cells circulating within the irradiated volumes. In order to address the high-risk of subclinical/microscopic disease spread, the clinical target volume (CTV, green contour) is contoured and considered as target for irradiation. Additionally, in order to account for the tumor motion due to respiration, the internal target volume (ITV, orange contour) will also be drawn taking into consideration tumor motion in 4D-CT. Finally, to account for the set-up error(s) related to the patients daily (re)positioning, an additionally larger volume known as the planning target volume (PTV, red contour) is also drawn as the final treating volume for radiation treatment. The yellow arrows indicate the definitive diameter of the final treatment volume in comparison to the macroscopic tumor (GTV). Significant amount of the surrounding peri-tumoral healthy tissue between GTV and PTV will be exposed to the full radiation dose (blue lines), the same that will be delivered to the tumor.
Furthermore, if the regional lymph nodes are also included in the target volumes, irradiation will further extend the radiation-induced immunosuppression to the whole anatomical region where T-cell priming is expected to take place. The bigger the treatment volume during RT, the greater the inhibitory effects of radiation on the immune system. Further, an extended treatment time in terms of normo-fractionated RT (in order of several weeks, typically 5 in case of (neo) adjuvant and 6-7 in case of radical treatment) will additionally increase the radiation-immunosuppressive power. It has been confirmed that radiation-related lymphocytes depletion following RT for NSCLC associates with poor oncologic outcome, indicating that large radiation volumes and multiple daily fractions leads to systemic immunosuppression [19]. Without a doubt, RT destroys tumor cells and results in cure of low-volume, earlier stage lung tumors, with probably no significant immunosuppressive impact on patients' prognosis. However, for those patients whose disease is large and more advanced, and requires radiation volumes that are very large, the radiation-induced immunosuppression might have significantly higher impact and relevance for their prognosis. Furthermore, for these patients with more advanced disease, RT is usually given in combination with chemotherapy and the combined effects of these therapies will further exacerbate systemic inhibitory effect on the immune system. The In order to address the high-risk of subclinical/microscopic disease spread, the clinical target volume (CTV, green contour) is contoured and considered as target for irradiation. Additionally, in order to account for the tumor motion due to respiration, the internal target volume (ITV, orange contour) will also be drawn taking into consideration tumor motion in 4D-CT. Finally, to account for the set-up error(s) related to the patients daily (re)positioning, an additionally larger volume known as the planning target volume (PTV, red contour) is also drawn as the final treating volume for radiation treatment. The yellow arrows indicate the definitive diameter of the final treatment volume in comparison to the macroscopic tumor (GTV). Significant amount of the surrounding peri-tumoral healthy tissue between GTV and PTV will be exposed to the full radiation dose (blue lines), the same that will be delivered to the tumor. Furthermore, if the regional lymph nodes are also included in the target volumes, irradiation will further extend the radiation-induced immunosuppression to the whole anatomical region where T-cell priming is expected to take place. The bigger the treatment volume during RT, the greater the inhibitory effects of radiation on the immune system. Further, an extended treatment time in terms of normo-fractionated RT (in order of several weeks, typically 5 in case of (neo) adjuvant and 6-7 in case of radical treatment) will additionally increase the radiation-immunosuppressive power. It has been confirmed that radiation-related lymphocytes depletion following RT for NSCLC associates with poor oncologic outcome, indicating that large radiation volumes and multiple daily fractions leads to systemic immunosuppression [19]. Without a doubt, RT destroys tumor cells and results in cure of low-volume, earlier stage lung tumors, with probably no significant immunosuppressive impact on patients' prognosis. However, for those patients whose disease is large and more advanced, and requires radiation volumes that are very large, the radiationinduced immunosuppression might have significantly higher impact and relevance for their prognosis. Furthermore, for these patients with more advanced disease, RT is usually given in combination with chemotherapy and the combined effects of these therapies will further exacerbate systemic inhibitory effect on the immune system. The consequences include compromised immune priming, lymphopenia and finally weak anti-tumor immune potential. Indeed, higher radiation doses to the immune system following the definitive RT for stage III NSCLC patients were associated with increased tumor progression and death [82]. One study found that the lower the lymphocyte loss at 6 months after RT (every 100 lymphocytes/mcL), the greater the improvement on PFS and OS. This finding suggested that lymphocyte depletion during RT reduces tumor control and survival in patients with stage III NSCLC [83], while the opposite is also true. Similarly, a secondary analysis of RTOG 0617, including 464 patients affected by stage III NSCLC, found that increased radiation dose to the immune cells was highly prognostic for decreased OS and PFS [84]. These studies suggest that immune cells and organs should be considered as an organ at risk during the radiation treatment planning, and should be spared from radiation, if one is to optimize the peri-tumor immune environment and help convert it from a pro-tumor suppressive environment into an anti-tumor pro-immunogenic environment.
Therapeutic Strategies to Overcome Tumor-Mediated Immune Suppressive Effects in NSCLC
Similar to the melanoma, head and neck, and mismatch repair (MMR)-deficient colorectal cancers, NSCLC tumors are considered "hot" with significant infiltration of tumors by T-cells and high tumor mutation burden (TMB) [95]. However, inflamed TME is not always associated with favorable prognosis. Recently, it has been reported that while inflamed TME is associated with favorable patient outcome in case of lung adenocarcinoma (LUAD) subtype of NSCLC, it is not true for lung squamous cell carcinoma (LUSC) [96]. This difference was attributed to increased expression of immune checkpoint marker expression in immune-inflamed LUSC compared to inflamed LUAD. Moreover, T-cells become exhausted during the attack on the tumor due to constant exposure to the antigens and through immune checkpoint signaling. Therefore, immunotherapy of NSCLC using immune checkpoint inhibitors (ICIs) was started to block this signaling and has become an important cornerstone of NSCLC therapy. However, as explained above, not all NSCLC tumors respond to ICIs as they develop resistance mechanisms due to the constantly evolving interactions between cancer cells and other cells in TME such as other immune cells, cancer-associated fibroblast, and tumor endothelial cells [97]. Therefore, multiple strategies have been developed over time to treat ICI-refractory NSCLC.
The first approach used is the combination of two ICIs, generally anti-PD-1 and anti-CTLA-4 to enhance anti-tumor immune-mediated response. An improved median and 2-year OS over chemotherapy was observed in NSCLC patients treated with nivolumab plus ipilimumab (CheckMate227) [98]. However, treatment-related serious adverse events of any grade were more frequent in the patients treated with combination ICI than with chemotherapy although grade 3 or 4 treatment-related adverse events were similar. In addition to PD-1 and CTLA-4, other immune checkpoints such as LAG-3, TIM-3 and TIGIT have been tested in trials for combination ICI therapies. For example, in a recent CITYSCAPE phase 2 trial, anti-TIGIT, tiragolumab when combined with anti-PD-L1, atezolizumab resulted in a significant benefit in PFS and ORR in PD-L1-positive metastatic NSCLC patients compared to anti-PD-L1 monotherapy [99]. In addition to the combination ICI therapy, ICI treatment as a re-challenge has also been investigated [100]. However, although the responses have been improved following these strategies, tumor-mediated immune suppressive effects still limit the durability and maximal positive outcomes.
In addition to the tumor-mediated increased expression of checkpoints, VEGF and IDO secreted in the TME serve as important immunosuppressive molecules. VEGF promotes hypoxia-mediated neo-angiogenesis in the TME. VEGF inhibitors can restore normal vasculature to enable immune cell infiltration [101] and provide a rationale for their combi-nation with ICIs to improve outcomes. Indeed, in a recent clinical trial, an improved OS was obtained when atezolizumab was combined with doublet chemotherapy and bevacizumab compared to bevacizumab plus doublet chemotherapy [102]. There are multiple studies that are currently testing this concept [103]. IDO1, IDO2 and TDO2 play important role in tryptophan catabolism, a critical metabolic pathway. IDO1 and TDO2 are overexpressed in several cancer types, including NSCLC and are associated with poor prognosis and resistance to immunotherapy [104]. By depleting tryptophan and increasing kynurenine in the TME, these enzymes enhance immunosuppression as Tregs and MDSCs are generated and proliferation and activation of effector T-cells is inhibited [105]. However, a clinical trial in advanced melanoma patients combining IDO1i epacadostat with pembrolizumab failed [106], most likely due to several flaws in the design such as unselected patient population and insufficient dosing [97]. Therefore, IDOi are still being investigated either in combination with ICIs in NSCLC (NCT02460367) or with other combination partners such as RT and STING agonists. Level of another amino acid, arginine that is essential for lymphocyte proliferation and function, is regulated by arginase 1 and 2 (ARG1/2). Similar to IDO, high expression of ARG1/2 has been found in NSCLC [107] and shown to be associated with poor prognosis. These enzymes are mainly released by MDSCs and macrophages in the TME and hamper T-cell function by lowering production of IFNγ, TNF-α, and other inflammatory cytokines [108]. Therefore, therapeutic inhibition of ARG1/2 is being employed to enhance anti-tumor immune responses. A phase I/II study in advanced or metastatic solid cancers (NCT02903914), including NSCLC is currently investigating the anti-tumor effects of a small molecule INCB001158 alone or in combination with pembrolizumab.
Several components of adenosine-signaling pathway such as CD73, A2a receptor (A2aR) are overexpressed in variety of cells in the TME. Multiple molecular pathways, including mTOR, MAPK, HIF1-α, and TGF-β regulate expression of CD73 and in turn adenosine [109]. CD73, an ectonucleotidase, generates adenosine (an effective immunosuppressive molecule) by breaking down extracellular ATP [109]. In accordance, high expression of CD73 is associated with poor outcomes in NSCLC [110]. Similarly, high A2aR expression results in an increased binding of adenosine and leads to accumulation of immunosuppressive Tregs, MDSCs, proliferation of cancer-associated fibroblasts, inhibition of effector T-cells, lowering of PD-L1 expression on tumor cells and other anti-immune inhibitory consequences in NSCLC [111]. Therefore, adenosine signaling pathway has been targeted by inhibiting either CD73 or A2aR alone, or in combination with ICIs to overcome tumor-mediated immunosuppressive effects in NSCLC [97].
Recently, neoantigens that are produced due to mutations in the tumor cells have been identified in NSCLC. Because these antigens are unique to the cancer cells and are generally immunogenic, vaccines containing these antigens have been developed [112] and can exploit the benefits of ICIs [113]. Melanoma-associated antigen (MAGE)-A3 is expressed in approximately 32% of NSCLC [114,115]. However, vaccines containing this antigen did not improve PFS or OS [116]. Thus, the combination of these neoantigen-based vaccines with ICIs may be needed and studies in this direction are required. In this regard, it is important to recognize that the sequencing of vaccine and the ICI is important to achieve optimum results. Recently, it is reported that under suboptimally-primed CD8+ T-cell conditions, PD-1 blockade increases the generation of dysfunctional PD-1+CD38hi cells, leading to anti-PD-1 therapy resistance [117]. Accordingly, it may be speculated that treatments such as RT that act as in situ vaccine may be administered before ICI treatment to achieve improved outcomes.
It is now clearly established that TME-mediated immunosuppressive effects hinder the anti-tumor immune response, which is significantly influenced by the heterogeneity of the TME. Targeting these pathways have been employed either alone or in combination with immunotherapies but with limited success, suggesting that other novel, unconventional strategies such as Stereotactic Body Radiotherapy (SBRT)-based PArtial Tumor irradiation targeting HYpoxic clonogenic cells (SBRT-PATHY) may still have scope to further improve treatment outcomes in NSCLC (discussed in the following paragraph).
Radiation and Immune Stimulation in NSCLC: Bystander and Abscopal Effects
RT, if used appropriately, has a potential to convert a TME into a immuno-stimulative environment that can aid local and distant radiation-induced immune-mediated anti-tumor response [118] and enhance response to immune checkpoint inhibitors [119][120][121]. RT can alter the tumor micro-niche by increasing neo-antigen shedding, increase PD-L1 expression, increase MHC class I expression, and reverse exhausted CD8+ T-cells [120]. RT can also increase infiltration of CD8+ T-cells into the tumor and TME and could potentiate the response [119] and can be thought of as a form of immunotherapy with systemic effects [122]. It can alter tumor cells to increase stimulation or immunogenic cell death pathways within the tumor cells, upregulate MHC molecules within tumor cells, increase release of DAMPs, promote expression of cryptic tumor antigens, and lead to production of immune-stimulatory cytokines and chemokines [85]. RT, if used properly, could also alter APCs to promote infiltration into the tumor cells, improve maturation of APCs, alter the APCs to acquire a more immune-stimulatory phenotype, encourage APCs to uptake antigens and to improve processing cross presentation of antigens by APCs, promotion production of immune-stimulatory cytokines and chemokine production, and aid APCs to enhance migration to regional lymph nodes [85]. At the T-cell level, RT has been shown to increase infiltration into the tumor, especially CD8 and CD4 T-cells, promote production of immune-stimulatory cytokines by the T-cells, help T-cells maintain effector function, and may alter T-regs [85]. Table 3 highlights proposed mechanisms as to why radiation could induce an immuno-stimulatory effect that could enhance response to checkpoint inhibitors.
The above mentioned radiation immunogenic effects are typically observed in the preclinical, experimental conditions but clinically their therapeutic impact remain negligible following the use of conventional RT which is considered to be a weak immunestimulator. In fact, conventional RT usually shows an immunosuppressive character ( Table 2: [19][20][21][22]80,[84][85][86][87][88][89][90][91]). Accordingly, the 5-year overall survival rate of NSCLC patients to RT and chemo-RT remains at a dismal low ranging from 68% for stage IB to <10% for stage IVA-IVB NSCLC [123]. These results suggest that a combination with other strategies such as immunotherapy is essential to enhance the response of these radio-and radiochemotherapy-resistant NSCLCs. The RT would induce activation of the immune system against the tumor cells (as highlighted in Table 3) while its immunosuppressive effects can be reversed by ICIs. Indeed, several clinical trials are currently ongoing to explore the potential of the combined strategy of RT and immunotherapy and have been recently reviewed for both stage III and advanced NSCLC [124]. It is important to note that while in some of these trials, RT and ICIs are being administered concurrently, in others ICIs are given as an adjuvant therapy after RT. The sequence of these therapies are dependent on several factors such as the dose, fractionation, dose/fraction of RT, the type of ICI to be used as well as on the intrinsic properties of the tumor and their response to the first line of therapy as we have reported earlier [121].
In addition to the combination approaches, the immunosuppressive effects of RT could be turned into a pre-dominant immune-stimulatory effect leading ideally to clinically desirable abscopal effect (AE) and bystander effect (BE) using novel, unconventional delivery approaches of RT. BE and AE are tumoricidal non-targeted immune-mediated radiation effects that have a great anti-tumor potential and thus significant clinical relevance. They both represent an out-of-field-extended regression of non-irradiated local (BE) or distant (AE) tumor lesions as a result of an optimally balanced interplay between the radiation-induced, pro-inflammatory and anti-inflammatory cytokines, TME and immune system cells. The characteristics of BE/AE induced by conventional RT are described elsewhere in more detail [125]. To be optimally pro-immuno-stimulative, RT needs to be administered in a way to release enough quantity of hidden tumor (neo) antigens required for a potent immune-stimulation and to maximally spare the loco-regional peri-tumoral immune cells necessary to induce BE/AE. This balance is only occasionally achievable after conventional, normo-fractionated RT, which is a weak inductor of BE and AE. Modern radiation technique needs to be properly adapted and adjusted, if one is to drive the maximum BE and AE effects.
One of the attempts to make such a novel, unconventional RT is SBRT-PATHY, purposefully developed to improve immunogenic potential of radiation, and to act synergistically with the immune system [118]. This approach is fully subordinated to immunostimulation.
The key components of this technique are: (1.) partial tumor irradiation targeting the more immunogenic-hypoxic clonogenic cells, (2.) sparing of the loco-regional immune cells as an organ at risk, and (3.) time-synchronization of irradiation with the homeostatic oscillation of the anti-tumor immune response, giving radiation at certain, individually determined optimal timing corresponding to the most reactive phase of the immune anti-tumor response. Typically, the treatment is given in very short time (1-3 fractions) so as not to interfere much with the functioning of the immune system, using an immunogenic-high radiation dose, in order to be fully stimulative. Although this is an emerging technique and thus only small number of patients have been treated so far, its preliminary results in terms of BE-and AE-induction are encouraging [118]. Most of the patients treated with this approach were affected by unresectable bulky NSCLC, whose immuno-suppressive biological behavior showed to be manipulable resulting in immunostimulation and consequent improvement of treatment outcomes [126]. Indeed, the immunohistochemistry and gene-expression analysis of surgically removed partially irradiated squamous cell and adenocarcinoma NSCLCs following SBRT-PATHY, and non-irradiated but regressing abscopal tumor sites, showed activation of immune system in the radiation-spared TME with very dense infiltration of T-lymphocytes, with more or less pronounced predominance of CD8+ cytotoxic lymphocytes [118]. Furthermore, Apoptosis-inducing Factor (AIF) was highly expressed not only in the partially irradiated NSCLCs, but also in the non-irradiated distant tumor lesions pointing to an induction of tumor apoptosis at all sites (partially irradiated and non-irradiated). Surprisingly and interestingly, the lymphocyte infiltration was absent at non-irradiated distant tumor sites, where AIF was highly upregulated, indicating an alternative radiation-induced activation of apoptosis pathway possibly through cytochrome C [118]. Additionally, analysis of non-irradiated abscopal tumor sites performed by real-time PCR of reverse transcribed mRNAs showed the strongest signals of cell death-regulating signaling molecules IL-6, AIF and TNF-alpha, which had higher expression levels compared to the partially irradiated tumors suggesting an abundance of potentially cell death-inducing signals not only in the partially irradiated NSCLCs but even more so in non-irradiated out of field abscopal sites [118]. These findings indicate that the presence of these signaling molecules at abscopal tumor sites may play an important role in the systemic anti-tumor response modulated by SBRT-PATHY at TME and hypoxic segment of partially irradiated bulky NSCLCs.
A prospective phase I trial is ongoing, currently recruiting patients, aiming to assess the immunogenic potential and optimal timing of SBRT-PATHY for treatment of unresectable bulky tumors of all histologies and organ-sites [127]. Additionally, another prospective phase I/II trial is currently assessing the potential physical and biological advantages of carbon-ions in form of CARBON-PATHY delivered synchronously with an estimated most reactive phase of anti-tumor immune response considering the homeostatic immune oscillations [128]. Moreover, for the purpose of target delineation, CARBON-PATHY is for the first time planned using hypoxia-specific [64Cu][Cu(ATSM)] PET/CT.
Interestingly, it has been reported that certain RT byproducts, such as tumor exosomes can play a role in BE [129]. Radiation-induced BE and the role of tumor exosomes are described elsewhere [120]. More recent data demonstrated that RT can induce the production of tumor exosomes that contain DAMPs and key proteins that play a role in radiationinduced abscopal response [130]. Following irradiation, tumor exosomes can activate dendritic cells and NK cells, and can lead to tumor growth delay via an NK cell dependent pathway in a fashion analogous to irradiation itself [130,131]. These findings showed for the first time the link between tumor exosomes-related BE and NK cell-mediated radiationinduced AE adding to the credibility that RT, if used appropriately, can be used to enhance anti-tumor immunity. Recent animal models are now being developed in NSCLC to better understand radiation-induced immune-related abscopal effects and to find ways to optimally integrate RT with emerging systemic immune checkpoint blockade agents such as the anti-CTLA-4 (ipilimumab) and anti-PD-1 (nivolumab) [131,132]. These models have been looking at RT and anti-CLTA-4 and anti-PD-L1 immune checkpoint combinations, and have suggested synergism of the combination approach [133,134]. Golden et al. [135] reported a proof of principle phase 2 study demonstrating that granulocyte-macrophage colony-stimulating factor along with RT (35 Gray in 10 fractions over two weeks) lead to 27% abscopal response in metastatic NSCLC, breast and thymic cancer patients. Keynote-001 trials demonstrated that in a subgroup of 24 or 97 patients with metastatic NSCLC who received thoracic RT followed by pembrolizumab, there were statistically significant notable improvements in PFS (6.3 vs. 2.0 months, p = 0.008) and OS (p = 0.034) compared to those who did not received RT, with notable increase in pneumonitis in those that previously received thoracic RT (13% vs. 1%, p = 0.046) [136]. Results from a randomized phase II SBRT trial of sequential SBRT and pembrolizumab alone vs. pembrolizumab also demonstrated an improved overall response rate (41% vs. 19%) and median PFS (6.4 months vs. 1.8 months) in favor of the combined SBRT and pembrolizumab approach. Table 3. Radiation-related immunostimulative effects.
Enhance recognition and killing of cancer cells by cytotoxic lymphocytes.
Facilitate the recruitment of effector T-cells to the tumor site.
Increased tumor cells phagocytosis; Promotes pro-inflammatory cytokines release from APCs. Generation of novel peptides and increase of the pool of intracellular peptides presented [120]. Increase the anti-tumor immune response.
DC migration and maturation (increase in efficiency of antigen processing and presentation) Release of pro-inflammatory cytokines and chemokines from APCs. Decrease of CD47 surface expression ("do not-eat-me" signal) [145]. Increase tumor cells phagocytosis.
Enhance recognition and killing of cancer cells by cytotoxic T-cells.
Activation of the cGAS/STING pathway and production of type I IFNs and other pro-inflammatory cytokines (APCs maturation, cross-presentation and T-cell recruitment).
Conclusions
Due to a high incidence and dismal treatment outcome, NSCLC represents one of the major research challenges in the 21st century. While the immune system emerged as an important link in the chain of tumor development, tumor control and tumor progression, the immunogenic balance becomes one of the major focuses of future preclinical and clinical work. In particular, researchers are attempting to shift the prevalently immuno-inhibitory tumor-and radiation-related effects towards a more immuno-stimulative one, with hopes of improving the therapeutic ratio that combines optimal RT in combination with emerging immunotherapy agents. RT, if used appropriately, could aid local and distant radiationinduced immune-mediated anti-tumor response and lead to clinically desirable AE and BE. In one such scenario, being increasingly clinically investigated, RT was shaped in such a way to limit unnecessary irradiation of immune cells at the immediate periphery of visible tumor mass. A novel, unconventional RT (SBRT-PATHY) successfully addressed important aspects deemed necessary for the treatment success: partial tumor irradiation targeting possibly the more immunogenic-hypoxic clonogenic cells, sparing of the locoregional immune cells as an organ at risk, and time-synchronization of irradiation with the homeostatic oscillation of the anti-tumor immune response. Ongoing research with this novel approach will provide a better understanding between the interplay between the host, the tumor, and the various treatment manipulations to render a pro-tumor immunesuppressive environment into an anti-tumor immuno-stimulatory one. This may especially be the case, if novel RT approaches are combined with emerging immunotherapy agents for NSCLC patients.
Patents
Tubin Slavisa. reported an international patent application PCT/EP2019/052164 published as WO 2019/162050. The authors reported no other conflicts of interest. | 2021-03-03T05:22:36.145Z | 2021-02-01T00:00:00.000 | {
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11244403 | pes2o/s2orc | v3-fos-license | The influence of living conditions on adolescent girls’ health
Adolescence is described by the Swedish National Board of Health and Welfare as the healthiest period in life. However, adolescent girls differ in that they self-report that their health decreases with age. The aim of this hermeneutical study was to describe the meaning of living conditions in relation to adolescent girls’ health. Guided by principles of reflective lifeworld research, 15 interviews with adolescent girls were analysed. The result section consists of four narratives with their existential interpretations illustrating different ways of approaching living conditions and their meaning for health and well-being. The narratives are: Approaching everyday life in a balanced way—feeling harmonious; approaching everyday life with ambiguity—feeling confused; approaching everyday life as an intellectual project—striving for control; approaching everyday life as a struggle—feeling forlorn. In addition, a comprehensive understanding was developed by using the lifeworld dimensions: lived body, lived room, lived time, and lived relations. These dimensions may deepen the understanding of important parts of those living conditions which are meaningful for the girls’ health and well-being. By using the dimensions, complex living conditions have been explored and the meaning of different parts clarified. The girls’ thoughts and feelings are often ambiguous and sometimes contradictory, depending on the situation. The health of adolescent girls needs to be understood against the background of their experiences of living conditions. One way to support their health and well-being seems to be to supply them with forums where they can talk about their living conditions.
The Swedish Socialstyrelsen (2009) describes adolescence as the healthiest period in life. However, adolescent girls differ in that they self-report that their health decreases with age. During teenage years, the typical adolescent girl's ability to make choices and take responsibility for her own circumstances in life increases (Hwang & Nilsson, 2011). The environment in which an adolescent grows up may limit his or her potential to make appropriate personal choices and take a stand during the teenage years (Socialstyrelsen, 2009).
According to the World Health Organization (WHO) study, Health Behavior in School-aged Children, living conditions and lifestyle influence how adolescents self-report their health and well-being (Cavallo, Zambon, Borraccino, Raven-Sieberer, Torsheim & Lemma, 2006;Danielson, 2006;Statens Folkhälsoinstitut, 2011). These reports and other studies show that adolescent girls' personal accounts of their health are influenced by elements in their environment, such as having friends (Johansen, Rasmussen, & Madsen, 2006), having support from the surrounding world (Callaghan, 2006), bullying, experiences in school, family structure, their ability to talk to their parents (Currie et al., 2008), disability, and health-compromising habits (Breidablik, Meland, & Lydersen, 2009). Some research has been conducted into the connection between adolescent girls' health and the conditions under which they live. Connections between different factors can be seen, but little research has examined how an adolescent girl understands her own experiences of her living conditions. Johansson, Brunnberg, and Eriksson (2007) argue that relations with family and friends are most important for the mental health of adolescents. Furthermore, responsibility and performance are dynamic processes that also influence adolescent mental health (Landstedt, 2010). Tinnfält (2008) argues that all levels in society influenced the mental health among adolescents.
Health is a phenomenon with multiple meanings. In this study, health is understood in terms of the description of Dahlberg and Segesten (2010). They argue that health is an experience of feeling well and living a good life. Health is a feeling of being able to carry out smaller and bigger life projects. To be able to support the development of adolescent girls' health processes, more knowledge is needed about the meaning of their living conditions, and how significant they are to the girls' health. In an earlier study, Larsson, Johansson Sundler and Ekebergh (2012) found adolescent girls' health to be a complex phenomenon interwoven into their everyday lives. Health and well-being were developed in meaningful situations in the adolescent girl's relationships to others as well as in their ability, which varied for each individual, to manage everyday life. The movements against being well were promoted in contexts that challenged adolescent girls to live and act out their lives, both physical and existential (Larsson et al., 2012). According to Ricoeur (1992), humans live with a unique identity that develops in the community and in interactions with others. Human development, according to Mugerauer (2010), is affected partly by conditions present in biology and partly by the surrounding environment.
The importance of personal living conditions in the development of an adolescent girl's health and well-being seems to be an unexplored area. The aim of this study was to describe the meaning of living conditions in relation to adolescent girls' health.
Method
The study follows the principles of reflective lifeworld research (RLR), based on phenomenological and hermeneutic epistemology, as described by Dahlberg, Dahlberg, and Nyströ m (2008). The RLR approach builds on an interest in the lifeworld, which forms a foundation for understanding human experiences. This approach emphasizes openness to the phenomenon being studied. To be open means that the researcher's attention must be directed toward the studied phenomenon (Dahlberg et al., 2008). Empirical data consisting of 15 interviews collected by the first author (ML) in 2010 focusing on adolescent girls' health were used.
Description of participants and data
The participants were aged between 13 and 19; they all lived in western Sweden in areas ranging from mid-sized communities to rural. The girls' family situations varied in that many had siblings, some lived with both parents, others with their mother, and some alternated between their mother and their father. Some of the girls had separated parents who had new partners. One girl lived alone. Half of the girls went to public school, 7th to 9th grade, and the other half went to secondary school, enrolled in either a theoretical or vocational educational program. One girl had previously lived in other countries in and out of Europe, and another girl had a parent with a different cultural background. The native language of all the girls was Swedish.
The participants decided on a date and place for the interviews, and all interviews were conducted in private rooms. Qualitative interviews (Kvale, Brinkmann, & Torhell, 2009) were conducted to enable personal disclosure focusing on adolescent girls' lived experiences of health in everyday life. The interviews started with the question: ''Can you tell me about your everyday life?'' This initiated a dialog and enabled researcher and participant to become acquainted, where the participants were encouraged to narrate, with examples, of their experiences of health in everyday life. With the aim of being open, questions were formulated in the interviews depending on the situation. Follow-up questions were used to initiate reflections on the participants' health (Dahlberg et al., 2008), for example, How do you mean? How did you feel then? The interviews, which were recorded and transcribed verbatim, lasted between 30 and 80 min, averaging 50 min.
Analysis
The analysis was guided by the principles for reflective lifeworld research (Dahlberg et al., 2008). A hermeneutic interpretive analysis was performed to describe the meaning of living conditions in relation to adolescent girls' health.
First, all data were read with a focus on the meaning of personal living conditions for health experiences. Meaning units were sought and compared to identify similarities and differences. As the analysis proceeded, certain living conditions and their meanings were gathered in patterns. Patterns descriptive for the girls were then structured into themes. The themes were put together in four narratives, the writing which was guided by the method for narrative configuration described by Polkinghorne (1995). This means that events, happenings, and actions are put together and integrated into a temporally structured whole.
Second, the analysis became more interpretative. In this phase, the analysis focused on how the girls experienced their living conditions and how these experiences influenced the girls' health in everyday life. Reasonable interpretations that could clarify the girls' health and living conditions were searched for and formulated in tentative existential interpretations. Ö dman (2007) proposed that an existential interpretation is directed toward meaning, suggesting how humans understand their situations in the world. Tentative existential interpretations and their validity were evaluated in accordance with Ö dman's (2007, p. 120) criteria. First, the source of a valid interpretation should be actual pieces of data. If the interpretation leaves a considerable amount of data unexplained, then the interpretation is viewed as weak. Second, no other interpretations could be found that more implicitly explain the same data, and third, there must be no contradictions in the data supporting an interpretation that is considered valid.
Finally, when the tentative existential interpretations were deemed valid, the analysis proceeded. The existential interpretations were compared and examined to interpret a pattern in the meanings. To deepen the understanding of living conditions in relation to adolescent girls' health, existential interpretations were developed based on the lifeworld dimensions described by Merleau-Ponty (2002) and van Manen (1997). This resulted in a comprehensive understanding of all data of general importance for the phenomenon, which can be seen as an interpreted whole. To uphold an open attitude, the researchers discussed the findings from the interviews during the analysis, and circled between the parts and the whole of the text when discussing the tentative interpretations. Through the analysis, the researchers strived to question their pre-understanding and compared the interpretations of the interview contents, so that the researchers' pre-understanding did not control what appeared in the data (Gadamer, 2004).
Ethical considerations
Written informed consent was obtained from all participants and from the guardians of the participants who were under 15 years, in accordance with Swedish law. To protect the anonymity of the participants in the findings, narratives present a summary of the data instead of quotations. The narratives have fictitious names for the girls. All of the narratives consist of meaning from more than one participant. The study was conducted in accordance with the Helsinki Declaration and was approved by the ethics committee in Gothenburg, Sweden (dnr: 744-09).
Result
The result consists of four narratives with their existential interpretations illustrating different ways of approaching living conditions and their meaning for health and well-being. The narratives are: Approaching everyday life in a balanced way* feeling harmonious, approaching everyday life with ambiguity*feeling confused, approaching everyday life as an intellectual project*striving for control, and approaching everyday life as a struggle*feeling forlorn. Subsequently, the comprehensive understanding follows.
Approaching everyday life in a balanced way*feeling harmonious
Mia lives with her parents and siblings. She describes her family as caring and being there for her; she feels that her mother listens to her, and that she can talk with her parents about anything. Among her friends, she has a well-known core group, and she continuously builds new relationships with others who become her friends, both in and out of school. She enjoys playing sports that challenge her. When in a team, she feels togetherness when contributing and playing together with others. School is a natural part of her everyday life where Mia wants to learn new things. Sometimes she finds the time spent in school to be boring, depending on whether she experiences the school as too tough, even if demanding schoolwork also motivates her learning. When something worries her or makes her downhearted, she handles her feelings through conversations with others or by reflecting on the things that have happened. Mia is often happy and enjoys living her life, without feeling it to be bothersome or strenuous.
Existential interpretation. Mia's living conditions are characterized by balance and harmony. She seems to have stability in the relationships with her family and her close friends, as well as a sense of selfconfidence. Community with others and being in meaningful contexts can be understood as crucial for the balance in her everyday life. The balance and confidence seem to give Mia a safe and secure background for her growth and future development.
Approaching everyday life with ambiguity*feeling confused
Sue is in the middle of her teenage years. Her parents are divorced and with her siblings, she lives one week with her mother and every other week with her father. In her early teenage years, Sue was shy and lonely, without friends. A friend she found with similar experiences helped Sue change, and now she is curious about others. She has friends she cares about. Sue enjoys being in school and in teams. Sue describes herself as moody: one moment she is happy, and in the next she is bad-tempered or sad. She has difficulties handling her mood changes, and she is afraid that they affect her friendships. She often becomes irritated with her mother or siblings where these relationships are often conflict-ridden, which she demonstrates through hot-tempered language. She shares a hobby with her father and they do it together. She enjoys being with her friends, but also needs to be by herself. When on her own, she relaxes through listening to music, reading books, or watching TV.
Existential interpretation. The personal circumstances of Sue's everyday life seem to be confusing and frustrating for her. Even if her life appears balanced, Sue may experience ambiguity between the person she wants to be and not wanting to be a victim of her mood swings. She seems to want to take responsibility for herself and her life, even if she sometimes does not know how to go about it. She can be understood as uncertain concerning choices and how to handle everyday life. The ambiguous circumstances in life seem to take a lot of energy for Sue, and it becomes demanding for her to manage her life while she wants to take care of relationships that are meaningful and important to her.
Approaching everyday life as an intellectual project* striving for control
Lena is in the middle of her teenage years and lives with her parents and siblings. Lena has high demands and expectations of herself. She feels that her parents, teachers, and others of the same age at school also have high expectations of her. She is ambitious, and when she succeeds, she feels she has support from those around her; in contrast, when she performs poorly, she misses this support. She makes detailed plans to reach her targets, especially in school; she is very focused and plans her time in detail. Lena likes discussions, and she wants to have knowledge and find arguments. Few of her classmates or friends have high ambitions like her, and sometimes she even gets angry with them for interfering or disturbing her educational pace. Even if she wants to have friends to share her interests with she finds it difficult and she rarely takes time out to simply to have fun. Lena often thinks that she disturbs her friends and that she is wasting their time. She has experiences from moving frequently in her childhood and memories of old friends who have forgotten about her, as she has gradually been forgetting them with the passage of time.
Existential interpretation. Lena seems to view her life as an intellectual project. She analyses and makes detailed plans for her life but seems to avoid personal disclosure and feelings. Her experiences from leaving close friends behind can be understood as the basis of her emotional wounds. Lena seems to strive for control of herself and her feelings. By distancing herself from feelings, both her own and others, she seems to live her life as a contradictory life project. Her relationships with others appear confusing, as she seems to avoid emotional contact while also seeking it. While living her life as an intellectual project, she seems to be betraying herself. She misses togetherness with others, and appears to replace it with something else.
Approaching everyday life as a struggle*feeling forlorn
Sara is a young adolescent girl living with her mother and her mother's new partner, seeing her father sporadically. Earlier Sara experienced her mother being there for her whereas Sara now often feels that her mother acts unsympathetically and in a controlling fashion, with an attitude of ''knowing best''. Their relationship is conflict-ridden. Sara has been let down several times by others she trusted and is cautious in relationships. Sara has a tough attitude and has been in conflict with some of her classmates, who avoid her so she is excluded from their community. The time she spends in school, which she does not like, is problematic. She has few friends, finds the subjects hard and has failed some of them, and thinks the teachers and the lessons are boring. Sara sometimes visits the school nurse or the welfare officer to have somebody to talk to about her situation, somebody with whom she dares to share her experiences and gain support. Sara sometimes spends her free time with some friends at the youth club, but mostly spends time with her horse, which she loves to take care of.
Existential interpretation. Sara's living conditions appear to make her feel forlorn. She seems desolate, missing community and relationship with others, especially her mother. Her everyday life can be understood as a struggle. Her weak self-confidence and her failures in relationships seem to make her fight for herself and for her existence. Her life has come to be about protecting and defending herself.
Her tough attitude creates an image of being fearless while on the contrary, she seems to be vulnerable and exposed in her loneliness.
Comprehensive understanding
The existential interpretations in the four narratives show how different aspects of living conditions seem important for adolescent girls' health and well-being. To further deepen our understanding of the meaning of living conditions in relation to adolescent girls' health, lifeworld dimensions, as developed by Merleau-Ponty (2002) and van Manen (1997), have been used. These four dimensions are: lived body, lived space, lived time, and lived relations. These dimensions are fundamental for how humans experience the world. 1 By using them, complex living conditions have been explored, and the meaning of different parts clarified. With the help of the lifeworld dimensions, puzzle pieces are created that enclose different living conditions. The shape and size of the puzzle pieces seem to vary, and depending on how they are put together, different patterns emerge. Every adolescent girl's experiences of her health can be understood in light of the pattern she carries. Her health needs to be understood against the background of her experiences of her living conditions, in which the lived body, time, space, and relations seem to be important parts.
The dimensions can be understood as interwoven in experiences, but in the analysis, the dimensions have been distinguished for the nuances of the experiences to be elucidated.
Lived body
The adolescent girl seems to try and challenge her body by acting and taking place in space, to explore who she is. Depending on what she encounters, she creates a picture of herself and how she presents herself. van Manen (1997, p. 103) said: ''In our bodily presence we both reveal something about ourselves and we always conceal something at the same time.'' Living conditions that are supportive and permissive seem to support development and well-being. The lived body feels. Todres, Galvin, and Dahlberg (2007, p. 57) describe it as the ''bodily self 'melts' with love or tense up with fear.'' Emotional expressions are versatile and simultaneously both controllable and uncontrollable, which seems to complicate being in a shared world with others. Adolescent girls seem to search through the lived body, in various ways, to participate in meaningful contexts.
Lived space
The environments in which the girls move between seem to be valued based on the environments' relevance to everyday life. Furthermore, the world around the girls seems not to have one unequivocal meaning but several possible meanings that vary depending on how they fit into the current situation of the girl's life. van Manen says ''the space in which we find ourselves affects the way we feel'' (1997, p. 102). For example, school influences adolescent girls' health in ways which depend on the experiences the girls have in those lived rooms and how those experiences can be handled. van Manen describes that ''lived space is the existential theme that refers us to the world or landscape in which human beings move and find themselves at home'' (1997, p. 102). A girl's movement between environments seems to depend on how she experiences relationships with things and others in the environment, irrespective of the distance.
Lived time
The girls' previous experience and the context in which they live seems to shape their present lives. Merleau-Ponty (2002, p. 478) says that ''what is past or future for me is present in the world.'' The girls live their everyday lives within the background of experience; their previous experience is simultaneously altered by the impact of the present. A narrative can be created when a lived time provides a setting for experiences. In the narrations, the girls' beliefs about the future are intertwined with their present and their past experiences, whether positive or negative. van Manen (1997, p. 104) proposes that ''through hopes and expectations we have a perspective on life to come, or we may have lost such perspective.'' The girls seem to struggle to find parts in their everyday lives that they experience as meaningful, such that they can be understood to be near the breaking point of what they can cope with.
Lived relation
Being part of the family appears to be important for girls, which includes their close friends. In relationships with others, the girls seem to be aware of themselves and how they contribute to, and are influenced by, the context. van Manen (1997, p. 104) proposes that ''as we meet the other, we approach the other in a corporeal way: though a handshake or by gaining an impression of the other in the way he or she is physically present to us.'' The girls are influenced by what they encounter in many ways, including through body language, gestures, vocabulary, and tone of voice. How the girls are met and what they 5 communicate in encounters with others seems to affect the possibility of being in a community as the person they are. Relationships with others instill feelings of confidence that strengthen the girls so their existence stabilizes, and thus is perceived as safe. When relationships with others do not instill confidence, the girls look for other ways to be safe such as for a community that gives meaning to life.
Discussion
The results show that an adolescent girl's living conditions are meaningful aspects that influence her health and everyday life. These parts can also make the girls feeling: harmonious, in control of, confused or forlorn. The girls' thoughts and feelings are often ambiguous and sometimes contradictory. In this study, all the participants were similar superficially in that they lived within families and went to school, which can be perceived as their everyday life. Nevertheless, their living conditions had different meanings and affected their health in varies ways.
The results show that for adolescent girls, relationship with others can be understood as both a possibility for them to feel well, as well as a basis for feelings of loneliness. It seems to depend on which meaning it had for the girl in her current life situation. In adolescence when a girl strives to be independent and free herself from their parents (Allen & Miga, 2010;Hwang & Nilsson, 2011), the relationship she has with her parents and friends seems to be important for her health. Relationships in which girls can be themselves, instill feelings of confidence and stabilizes their existence. Our results is in accord with what Landstedt (2010) means are important for adolescents girls mental health and what Kostenius and Ö hrling (2008) found mutual relationships to be for children's well-being. Our results show that not all adolescent girls have this kind of relationship with their parents and friends, which is in agreement with other research (Currie et al., 2008;Statens Folkhälsoinstitut, 2011). In our study, the girls seemed to need somewhere to talk with somebody about everyday life as a way to enable well-being. The places need to have a low threshold for talking about health, as for example with the school nurse (Golsäter, Sidenvall, Lingfors, & Enskär, 2011;Langaard & Toverud, 2009). It seems important to have places where they can discuss health issues in a respectful and open atmosphere (Golsäter, Sidenvall, Lingfors, & Enskär, 2010), and where they can talk about their everyday life and what it means for them at the moment. These are places where the caregiver needs to be available and have time for individual meetings (Morberg, Dellve, Karlsson, & Lagerströ m, 2006) and make the girls feel that they are participating (Lindqvist, Kostenius, & Gard, 2012).
In Sweden, school nurses invite pupils at 10, 13 and 16-years of age to an individual health dialogue. The school nurses work with a health and lifestyle questionnaire which is the basis for an individual health profile used as a tool in the health dialogues with the pupils (Golsäter et al., 2011;Socialstyrelsen, 2004). The health and lifestyle questionnaire focuses on issues such as school, family, sleep, alcohol as factors for a healthy or unhealthy life. The school nurses use the questionnaire as a structure for the health dialogues and as a starting point, focusing on individual aspects (Golsäter et al., 2011). The health and lifestyle questionnaire is based on risk factors and on ways of decreasing the risk of cardiovascular disease and diabetes mellitus (Golsäter et al., 2011;Lingfors, Lindströ m, Persson, Bengtsson, & Lissner, 2003;Socialstyrelsen, 2004). It may be a risk that the questionnaire guides the health dialogues between the school nurses and the adolescent girls, such that no space is given to what the girls think and feel and that they are deprived of a place to talk about their situation in the way they wants to. The girls also have the youth center, but they are mostly associated with sexual health (Sollesnes, 2010). It seems therefore that adolescent girls have few forums to discuss health and to get help. The participants in our study seemed to have difficulties in understanding the meaning of their own living conditions in relation to their health experiences. This means that caregivers, for example school nurses and nurses at primary health care centers, need to have an attitude of wanting to help the girls sort out experiences in different situations. If caregivers have the lifeworld dimensions as a given structure and work with openended questions that support reflection, it may be that hidden complex patterns can be made visible and the parts made understandable, for both the girl and the caregiver. Through reflections, the girl can be helped to be aware of her thoughts and feelings, how she acts in different places and everyday situations and to clarify where she is in balance or not, which can be a way of supporting her well-being.
The study was conducted in accordance with reflective lifeworld research, and a hermeneutic analysis made it possible to provide suggestions on how the material meanings in the data could be explored and understood. Creating narratives made it possible for the analysis of the events and situations to be combined in new wholes, in which the unique and specific could be preserved. Polkinghorne (2007) argues that the value of narratives is their ability to clarify and give insight into the phenomena with rich detailed and revealing descriptions.
The interpretations should be understood as interpretations related to the data as their origin and should not be taken as claims to truth. Throughout the study, the researchers sought to maintain an open position. Dahlberg et al. (2008) argue that to be open and responsive to the phenomenon in focus is essential for the research to be valid. The researcher's pre-understanding was regularly discussed during the study. Furthermore, an expert in the field who was not involved in the research reviewed the interpretations and findings.
Conclusions and clinical implications
The health of adolescent girls needs to be understood against the background of their experiences of living conditions, in which the lived body, lived time, lived space, and lived relations seemed to be important parts. If caregivers, such as school nurses, use a health and lifestyle questionnaire, it should be used as a basis for a discussion about all aspects of the girl's everyday life, rather than as a checklist. Health promotion work that focuses solely on risk factors may lead to missing other important aspects that influences the adolescent girls' health. One way to support adolescent girls' health and well-being seems to be to supply them with forums where they can talk about their living conditions. | 2016-05-12T22:15:10.714Z | 2012-01-01T00:00:00.000 | {
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197723750 | pes2o/s2orc | v3-fos-license | GAINING A POSITIVE SENSE OF CONTROL: TEACHING THE PRINCIPLES AND PRACTICE OF CONTROL THERAPY IN AN EDUCATIONAL SETTING
Over the past several decades, there has been an exponential growth in psychological theory and research to develop techniques by which individuals can gain a positive sense of control in their lives. One such model is Control Therapy (Shapiro & Astin, 1998; Shapiro, Astin, Shapiro, Soucar, & Santerre, 2010; D. H. Shapiro, Soucar, S. L. Shapiro, & Astin, 2010) which has been studied with diverse clinical populations. This study is a preliminary investigation exploring the application of Control Therapy to an educational setting, through a ten-week course, in which 13 undergraduate students in the United States studied control theory and research, learned about their own “control profile”, and completed an N = 1 self as subject, control-related research project. Scores on the Shapiro Control Inventory (SCI) pre to post all showed movement in the “healthy” sense-of-control direction. N = 1 data showed students were able to effectively match their control profile, self-observation data, and goals to appropriate techniques. Qualitative data showed positive changes on sense of control for 12 out of 13 participants, some quite profound (e.g., “invaluable life lessons and skills” “empowering”). An adverse result was reported by a single student. The paper concludes with limitations of this study and directions for future research.
Finally, there have been efforts to develop ways to measure and assess control. A first generation forced choice test measuring internal and external locus of control was developed by Rotter (1966). A second generation test showing that internal and external locus of control was not either/or, and looking specifically at the dimension of health was developed by the Wallston, Wallston, and DeVellis (1978). A third generation multidimensional Control Profile test was developed to measure 1) sense of control (general and specific domain mind, body, relationships, work, self). 2) modes of control-four scales that reflect cognitive and/or behavioral styles of responding to control-related issues: positive assertive/change mode of control; positive yielding/acceptance mode of control 1 ; negative assertive; and negative yielding. 3) Motivation for control (desire for control: a motivational vector) shows how the student wishes to deal with areas they are not in control of and their level of motivation for addressing areas of concern. 4) Agencey of control-where a person's sense of control comes from (self, other, Other).
The SCI is a theoretically derived, empirically validated instrument and has undergone extensive reliability and validity testing (Shapiro, 1994). For example, the SCI provided incremental validity over Rotter's (1966) internal-external locus of control scale and Wallston et al. (1978) health locus of control scales both for sensitivity (clinical versus normal) and specificity (between clinical groups of depression, generalized anxiety, panic attach, and borderline personality (Shapiro et al., 1993). There was also a correlation of cerebral glucose metabolic rates with sense and modes of control using Positron Emission Tomography (Shapiro et al.,1995). The SCI has also been used in a twenty year follow up study of morbidity and mortality of women with breast cancer (Astin, J. Shapiro, & D. Shapiro, 2013), and a two year study of men at cardiovascular risk involved in a cognitive/ behavioral intervention (Shapiro, Friedman, & Piaget, 1991).
The first time a comparison and contrast of meditation and behavioral self-control 1 A reviewer requested further clarity about the use of the terms yielding, acceptance, and mindfulness. We appreciate the chance to provide further clarity, as often the term mindfulness is used in many different ways. When we use the term mindfulness, we are referring specifically to a meditation technique (from the Vipassana tradition) involving "just noticing" without judgment all that is occurring (cf. S. L. Shapiro & Carlson, 2017). Thus, in Control Therapy, mindfulness is viewed as a self-regulation technique. The yielding accepting mode of control is a construct in the control inventory. Several studies have shown that mindfulness meditation (the technique) can facilitate an increase in quadrant two, positive acceptance construct on the SCI (cf. Astin, 1997;D. H. Shapiro, 1992). A more detailed summary of the control profiles of beginning, intermediate, long term and very long term meditators (20 years), can be found in the SCI Manual (Shapiro, 1994) for each scale, including meditators compared with other clinical and normative populations.
NEGATIVE YIELDING
Too little control (Quadrant 4) strategies was articulated in the literature was (Shapiro & Zifferblatt, 1976;cf. also, Shapiro, 1978) including the assertive/change mode of control and the yielding/accepting mode of control. This work led to the building of efforts toward a unifying theory of control noted above; as well as the matching of a person's control profile, goal, and control enhancing strategies in Control Therapy (Shapiro & Astin, 1998) 2 . Since those initial efforts, other approaches have also sought to combine/integrate meditation and behavioral self control: e.g., Dialectical Behavior Therapy Linehan et al. (2006); Acceptance/ Commitment Therapy (ACT; Hayes, Strosahl, & Wilson, 2016); mindfulness based cognitive therapy (Segal, Williams, & Teasdale, 2002) There are several clinical areas where an impairment of control has been suggested as one of the central features, and hundreds of research studies have shown that individuals have a desire to have a positive sense of control in their lives and feel happier and healthier when they do (see the over 1000 references in Shapiro & Astin, 1998;also Shapiro, Soucar, Shapiro, & Astin, 2010). The principles and practice of Control Therapy has sought to 1) integrate these different constructs at a theoretical level. For a table comparing different constructs of personal control (will, self-control) historically, from the Greek tradition and Thomas Aquinas to William James, see Table 1.1; and for an overview of contemporary control related constructs see Table 1.4 in Shapiro and Astin (1998), Chapter 1); 2) create a means of assessing a person's "Control Profile" so that self-regulation strategies can be matched to a person's control profile and clinical concern to help individuals gain (or regain) a more positive sense of control in their life.
Although research on the efficacy of the principles and practice of Control Therapy has been undertaken with patients having diverse clinical and health care concerns, the model is an educational one. Therefore, this study was a preliminary investigation to explore the promise of this knowledge applied to an educational setting. The class was taught at a public university in the United States to 13 incoming college transfer (third year) students on a first come, first serve basis. Transfer students were selected because this institution has articulated that there are certain especially challenging college "transitions" (e.g., incoming freshmen, transfer students, older returning adults) where individuals may be at increased psychological risk. Teaching a class "Gaining a Positive Sense of Control in your life" to a group of transfer students of diverse ethnic and cultural backgrounds seemed a promising opportunity to explore the potential effects of control theory and practice on students at a potentially stressful transition point in their education.
2 For a discussion of all the reliability and validity studies of the SCI, see Shapiro (1994); The 187-item standardized online test can be found at (www.controlresearch.net) and may be utilized at no charge by educators, researchers, clinicians. The test is in English (and Chinese) on the site, and has been translated into Korean, Hebrew. and Spanish Further, the SCI manual (over 200 pages) summarizes information of the test's initial construction, factor analytic studies with hundreds of subjects, test retest reliability data, alpha reliability and internal consistency of each scale, and over a dozen published validity studies. Likert scoring of each scale (e.g., sense of control and desire for control are six point scales; modes of control are four point scales) is provided, as well as comparisons of different clinical and "normal" groups and how each was assessed.
Participants and Setting
This study evaluated the effectiveness of an undergraduate seminar "Exploring how you gain a sense of control in your life". The class was taught by the first author, a faculty member at this public university, and a Diplomate, American Board of Professional Psychology, Clinical Specialty.
Ten of these students were female and the remaining 3 were male; five were psychology majors, the other majors included engineering, pharmacy, biological sciences, and chemistry. The class was culturally/ ethnically diverse, with four students self-identified as Latino/Latina, and four as Asian Americans (Thai, Philippine, Chinese). The other students were Caucasian. Nearly all students were between the ages of 20-22, with one being 27 years old. Their reasons for taking the class ranged from "interested in topic;" "I wanted to be in a small class to get to know people easily;" "I need to work on some personal issues of control" to "good time slot".
Procedure
Class Content, Confidentiality, Informed Consent. This class was directed toward undergraduates who may or may not be psychology majors. Therefore, the graduate template of a Control Therapy class (Shapiro, 2014) was adapted to an undergraduate level. The classroom reading consisted of the first three modules of the Control Therapy Training Manual which provides a self-directed guide for individuals to learn about Control Therapy by actually engaging in a self-directed project (self as therapist, self as client) 3 . The intention was that the "academics" of the course-e.g., the role of control in different personality theories and psychological constructs; the goal of psychological health in terms of suboptimal, "normal" and optimal control; and different self-regulation modalities-would be grounded in personal experience-a psychology from the inside out.
Self-disclosure and sharing were discussed regarding both in-class discussion and materials prepared for the instructor, including taking responsibility for not sharing more than was comfortable, as well as honoring the confidentiality of what was shared in class. As one reviewer noted, this issue of confidentiality is a critically important point in utilizing the principles and practice of Control Therapy in an educational setting, and the issue of sensitivity to honoring each other's communications in class was strongly emphasized. In addition, The Institutional Review Board (IRB) at the University reviewed and approved this study as exempt research (HS#: 2012-9246), students were assured of anonymity of results, and all students gave informed consent.
Attendance: "Adherence and Compliance." Attendance was required, and if students could not attend a given session, they were asked to contact the instructor. For the ten week class (and 13 participants), there were 130 "class contact times." Students combined missed 9 times for a 93% attendance (and for the nine missed classes, summary notes of the class discussion were provided the student). The standard in this institution is 2 hours of homework minimum for each hour of class. All students reported spending at least two hours outside of class, the majority spent at least four, and two spent ten hours.
Grading. At the end of the term, each student turned in a final paper. Objective criteria for grading was based on how well the student showed proficiency and understanding in the following areas of their final paper: ■ understanding of the different theories of personality, schools of psychotherapy, and how each related to control; ■ an exploration of the role of self-control, self-regulation, self-management, in mental well-being and physical health (wellness/self-care); ■ views of psychological health and its relationship to the construct of control in different personality theories and systems of psychotherapy.
The grade also reflected the student's classroom participation, attendance, and the quality of their wrestling and reflection on their N = 1 papers 4 . This paper included the student's self-evaluation 5 .
Learning about their "control profile". The SCI was used to measure each student's "control profile" at the beginning of class (as a basis for their N = 1 project) and at the eighth week of class to assess changes. Scores are calculated on a standardized scale from 0-100, with a mean of 50. All scores are compared to a "healthy normal" sample 6 (yellow shaded area in Fig. 1) below so the test taker can easily see where their scores fell (the dark blue shaded lines). An example of an SCI profile from a student is illustrated below (Fig. 1). You can see in this profile that the General Domain Sense of Control scores are low; the positive yielding mode (scale 6) is in the positive direction, and negative yielding (Scale 8) is high. Self as source of sense of control (item 13) and other as source of sense of control (item 14) are within the "normal range." Quantitative Group Results. The program IBM SPSS version 20.0 software was used to conduct statistical analyses (Feeney & Kirkpatrick, 2012). A paired samples t-test was used to compare pre and posttest scores, allowing us to reject the null hypothesis at the 0.05 significance level for certain pairs. Participants were tested on the same dependent variable, pre and post: e.g., their overall sense of control. Effect size was also obtained on each variable, and following Cohen (1988), 0.2 was considered a 'small' effect size, 0.5 represents a 'medium' effect size and 0.8 a 'large' effect size.
Qualitative data. Students turned in a final paper, in which they addressed the issues noted above, as well as their own reactions to and exploration of their control profiles on the four topics measured by the SCI: sense of control; modes of control; desire for control, and agency of control. These papers became the basis for the qualitative data. The criteria used to analyze the qualitative data derived from grounded theory (Corbin & Strauss, 1990) and consisted of a) coding and categorizing patterns and themes related to students' control profiles in these four areas; and b) noting support, elaboration, and more in-depth exploration of the quantitative findings.
The narrative data was categorized and coded by the first author into the four groupings noted above, whose reliability and validity have been shown in a dozen previous studies (summarized in Shapiro, 1994, pp. 30-55). Specifically, previous work developed a control content analysis scale to linguistically code terms of importance in Control Therapy and measured by the SCI for these four groupings (Shapiro & Bates, 1990). Work was then published showing how the linguistic coding of these terms in a content analysis sample, using rater reliability, was utilized with outpatient verbal samples (Shapiro, Bates, Greenzang, & Carrere, 1991). In the current study, which was preliminary and heuristic, excerpts from participants' journals were read and grouped by the first author based on the above categories as a way to provide illustrative, descriptive examples of each of the core themes. In so doing, based on face validity, the first author simply grouped student journal comments into categories that were validated in multiple previous studies. There was also triangulation of the data in that the qualitative data elaborated on and provided more detail of the quantitative results.
Quantitative Results: Group Pre-Post Tests and Effect Size
SCI pre-test and post test scores were collected to determine if the educational course improved students' overall sense of control and analyzed as noted above paired samples test, and calculation of effect size (ES). As can be seen from Table 2, there was a shift in the expected, positive direction on all the variables: overall sense of control increased (ES .55), positive sense of control increased (ES .62; p < .10,), negative sense of control decreased (ES .53), positive assertive increased, positive yielding/acceptance increased. Negative assertive (ES .83) and negative yielding (ES .55) both decreased (p < .10). Desire for control significantly decreased (ES .72; p < .05). Self as source of control and others as source of control both increased, the latter significantly (ES .92; p < .05) 7 . In spite of repeated requests, 3 students did not turn in the post test score. Their pre-test scores did not differ from the pre-test scores of those who did turn in the post test.
Quantitative data: N = 1, Example of "matching"
After taking the SCI test, students were given a copy of their control profile, as well as a 20 page report summarizing the results. Based on their pre-test control profile, and the area they wished to explore, a goal was set, baseline data was collected, and control enhancing techniques chosen to match their profile and goal.
Students' self-exploration projects, initial topics: *public speaking anxiety; * when others say you're acting too controlling; *controlling emotions better; *meeting new people; *general control issues; *seeking self-improvement; *feeling out of control everywhere; *time management; *eating behavior; * increasing motivation; *learning to be less passive but stay calm (e.g., in heated arguments); *wanting to sleep better; *body fitness: *Lower movement in negative sense of control is positive (as it is with e.g., negative assertive, negative yielding).
As has been shown in previous research (e.g., Shapiro, Schwartz, & Astin, 1996), there is no one size fits all approach in Control Therapy. N = 1 case studies provide important insights because they show how different control enhancing interventions are tailored and matched to the person's control profile and goal, information that would not necessarily be captured in the pre post group data. Below are two contrasting examples that illustrate this principle. Student 1 chose as an area of concern "eating behavior." Student 2 chose decreasing trying to control others and increasing self-kindness. Student 1. Fig. 2 shows the Control Profiles of Student 1 at Week 1, and the posttest profile of the same student at Week 8.
In the pre-test, for Student 1 the overall sense of control (scale 1) is low. Positive assertive (scale 4) is in the "lower" direction; desire for control (scale 9) is in the normal range; and self as agent/source of control (item 13) is quite low. At Week 8, the overall sense of control has shifted dramatically (scale 1), both positive assertive (scale 4) and positive yielding (scale 5) have increased substantially; and self as a source of control has shifted substantially (item 13) in the positive direction. Interventions were directed toward "binge eating" and included "positive assertive" actions such as removing food from the apartment, not buying junk food; responding to sad and stressful events by writing in a journal when "binge urges" were present; and practicing deep breathing.
Student 2 chose the goal of trying to decrease the desire to always be in control, including trying to "control" other people; and to increase "self-kindness" and "trusting others," wanting to develop the positive/accepting yielding mode of control. This student's profile at Week 1 was different than Student 1 in several ways. The positive sense of control scale was quite high, with a high positive assertive (scale 5) and a high negative assertive (scale 7), and higher desire for control (scale 9). As part of the target intervention the student utilized affirmations "I can do this. I know it is good for me to accept this situation ... I can only control myself." Student 2 also practiced gratitude for friends and family's love and support and forgiveness for those who accidentally or intentionally hurt the student. At the end of the eight week period, Student 2 wrote, "I have increased acceptance and happiness." Comparing Control Profiles of Students 1 and 2: Matching. Both students were able use targeted control-enhancing interventions that matched their control profile and area of concern. Although their interventions were different-tailored to their concern-they both made changes in the positive direction which is reflected in their eight week control profile. Student 1 increased overall sense of control, the positive assertive mode of control, motivation (desire for control) and self as agent. Student 2 showed a substantial increase in positive yielding/ accepting mode of control, and decreased negative assertive, a desire for control and self as source of control, while increasing other as source of control.
These results reflect the importance of matching control enhancing interventions to students' "unique" control profiles and goals.
Qualitative data: Learning Control Therapy-Principles and Practices
As noted, the students' journals as well as their self-exploration projects comprised the study's qualitative data. The criteria used to analyze the qualitative data derived from grounded theory (Corbin & Strauss, 1990) and consisted of a) coding and categorizing patterns and themes related to students' control profiles; b) noting support, elaboration, and more in-depth exploration of the quantitative findings. All students were able to understand and apply the principles and practices of Control Therapy to their lives. All found the "control profile" understandable, and were able to "self-observe" an area, set goals, and
The value of self-reflection about my control profile and process
Twelve of the 13 students found the self-reflection about the control profile and process to be quite helpful. One student did not. These comments are reported below.
-The most precious thing I learned from this class is to think kindly but critically about myself, be honest to myself, develop my own theory of personality, means and supports to take control of my life, make quality decision to make myself a better person living a happier life.
-This class taught me to be self-reflective in a more organized way. This class has taught me so many invaluable life lessons and skills ... I felt like I was on a journey of self-discovery ... how I make sense of the world around me through personality and control, psychological health and control, my views of the nature of the universe and control.
At the start of the class, several students commented that they were not very selfreflective in general, and about "control processes" in particular. For example, a student said "I took this class to satisfy my unit requirement ... but learned how to stay calm (diaphragmatic breathing) when everything else is falling apart. The self-observation was also really helpful: I never paid attention to what triggers my stress, how I get stressed, when I get stressed." Another student noted that reflection was helpful both in learning about obstacles and how to overcome the, but also in developing gratefulness or the positives in one's life. "After taking this class I feel motivated to do well in school and other aspects of my life without being overwhelmed by obstacles. Learning to make time to reflect is something valuable that I learned in class. Reflecting has helped me become more grateful about what I have." Some suggested that even thought it was "right before their eyes", the concept of control was a new concept. For example, one student noted "I was shocked when I realized the main goal of the class was to learn about myself, first because I never had a class like this before but mostly because this class has made me realize how much I did not know about myself. I learned to take make some time for myself and practice reflection. It is because of this reflection, that I learned to be more aware of my feelings and recognize how they affect me and what triggers my emotions, particularly stress. Taking the SCI profile and reviewing the summary of my sense of control was the first step in this learning experience." This comment was similar to others who noted how the class helped them learn to turn inward, and the importance of that in multiple domains of their life. As one student noted: "This class made me more interested in reflecting inwards. I love this aspect of selfcontrol and I wish all classes would go this deep and be as reflective as this class. I would like to integrate what I learned into the rest of my life, how I will proceed with my career, how I handle my career, and how I interact with my career, with others, and my overall approach to life." Again, the above comments were indicative of the view of several other students: -This class taught me techniques to help achieve my personal goals and learn how to gain a sense of control in my life and is something that I can take with me. I feel so happy to have taken this class at this point in my live, and learn about something that will be so useful for the rest of my life.
-I wish this class could continue for another semester that keeps building off this semester's material! I will always remember this class ... the energy I got from taking this class, I will carry this out into my life as best as possible. A great experience, -After weeks of self-assessment, self-exploration and self-evaluation, I feel like I have given myself a valuable opportunity to learn about my weaknesses and myself in a more spiritual way. This project has given me a sense of excitement and adventure because I know that eventually this will guide me in gaining a more positive sense of control in my life under any circumstances. These successes are the ones that I intend to keep with me throughout my life. It is empowering to realize that there is a way to have control of our body and mind.
One negative evaluation, adverse result. One student reported a negative experience (adverse effect) of the class ("boring, irrelevant, not helpful"). As s/he wrote" I felt overwhelmingly stressed with school, and aggrieved that I had to further spend time micro psycho-analyzing ridiculous components of my life to assess whether or not I was 'in control." Based on this student's feedback, it is important to acknowledge that selfreflection, for all its benefits, can be a challenging process, and it is important to remain sensitive to possible "adverse effects" that may be occurring, a point we elaborate on in the discussion section 8 .
Learning about the four modes and self/other agency
Four mode of control. Students expressed that two of the most helpful concepts to them were the ideas of the four modes of control (assertive/change and yielding/acceptance) learning how to balance them as well as the "wisdom" to know when each was valuable and helpful in different situations. As one student said "The most important thing that I learned in this class were the four different modes of control, because I realized I tend to be more negative yielding, and I needed to adjust this because it was affecting how I feel, thus affecting the energy I'd been giving off to others, and ultimately how they react towards me ... Staying centered; keeping the modes in balance ... loving kindness meditation and "tai chi dance" helped in exploring control to give and take in moderation, when to push and pull and how hard or strong." Another student cited a specific example when s/he wrestled with the four modes in an argument with a sibling, and s/he was able to move from negative yielding, to positive assertive and positive yielding without being negative assertive: I had a fight with my sibling and felt really sad and angry and normally I either cry and run away or stand and shout. But this time I practiced the right balance of positive assertive (letting him know how I felt) and positive yielding (accepting the fact that he wasn't intentionally trying to hurt me instead of blaming him for everything ... it felt great to be able to handle a problem in a non-aggressive way ... I'm learning now how to deal with situations and people without getting too hot, overwhelmed.
Students were able to recognize their unique mode control profile, where it was positive, where "a challenge, and learned skills to increase their appropriate use of the two positive modes. One student pointed out how her control story helped her realize why she was so "negative yielding" I always felt that no matter what I did or said the consequence would always be the same and everything that was going to happen was already planned ... I'm pretty sure this was because my grandma repeated to me day after day that my destiny was already set.
This student realized the importance of expanding her repertoire, and learning positive assertive. For others the positive yielding/accepting mode of control, which was a new concept to most students, was highlighted in terms of how important developing this skill was to them, as illustrated by their comments below: -The most important thing I learned is to accept the things that I cannot change/ control ... before I start stressing about something now, I ask myself it it's in the realm of my control or not. If the answer is no, I quickly accept it as is and move on. This empowers me because I don't waste time dwelling on things I that I can't do anything about.
-I've stopped trying to control so much. I used to go insane trying to control things but now I feel a sense of relief when I know there's nothing I can do about a situation or circumstances.
-This class has helped me release active control and accept a more positive yielding approach to my life, the opposite of what I set out to do when I took it Self-other agency. Again, students were able to learn about their own unique profile regarding self and other agency. As noted in Fig. 2, one student felt an important increase in self as agency. Another student noted The biggest change I see is viewing myself as an agent of control. I feel more confident and independent, less helpless when I stress; I believe I can control how I react to situations and am much more aware of my thoughts, feelings behaviors ... I can influence situations and don't have to let my emotions control me; Another student's learning was just the opposite-s/he realized how others could be a positive source of control: I realized through this class that I did not do everything on my own. I had help-from family, friends, even society. This understanding came to me in class. My fellow classmates who were once strangers ... I began to learn more about others. The ideas and thoughts in our discussion opened my mind to more than my own way of thinking. I learned to trust others through this class, and am more open to share my problems with others now.
Learning from the N = 1 projects: Some examples
The N = 1 project comments showed that students felt their efforts toward greater self-control were a "work in progress" but that there was progress.
Eating behavior. For example, a student with changes in eating habits and exercise as a goal, noted "setbacks-motivation seems to run out (exercise program), careful eating discipline seems to drain away" -yet the "trend" after eight weeks was more exercise per week, and better eating habits-decreasing sweets, smaller portions. "I really feel a difference between the person I used to be and the person I am now with regard to changing my eating habits." A different student began with the goal of changing "impulsive eating habits" -but shifted the goal to "increase inner monologue of self-love." S/he used interventions of positive affirmations, mindfulness and diaphragmatic breathing and letting degrading thoughts "flow over a waterfall." As a result of the project, "I spend a great deal of time thinking kind thoughts and thought stopping unkind thoughts. I see a great deal more beauty in myself than when I started and I have a great deal more acceptance as well. The project was from time to time difficult and required a fair amount of effort but after a while it became rather natural." Increasing motivation and study behavior. A student whose baseline was two hours of study per week, set a goal of fifteen hours per week, and wrote in the final evaluation: "I learned that "Control often went missing!" But I averaged nine hours per week. So an improvement." Decreasing negative assertiveness, (over control). Staying clam while expressing a point of view. Another student worked on "my tendency to be negative assertive in my relationships ... found I tend to desire more control over others when it is not my business to regulate their behavior ... still a work in progress ... but making really good progress." A different student felt it important to be more assertive, but wanted to stay calm while doing so.
When I first walked into this class I knew very little of this aspect of myself regarding control. I realized I was quite negative in terms of how I resolved things. But thought the weeks I was able to realize this negativeness and understand and convert it to something more positive. I believe that I have learned to how to gain control of most situations where I believed I was powerless over. For example, I have learned to control my emotions both with my friends and family by not answering back in a hasty or rude manner. Before the class I had trouble remaining calm during arguments. I am now able to be a stronger and more positive individual capable of knowing ways to gain control at any point my life.
Decreasing Stress: self-compassion and forgiveness. Finally, one student who was working on "decreasing stress" noted the self-observation and breathing techniques and mindfulness meditation helped "me connect to myself in a deeper and non-judgmental way. Learning how to be kind to myself during self-observations was particularly new to me. Finding that balance and letting go of the past was essential in order to move on in a positive direction, which involved self-forgiveness. It is through this class that I started to decrease and control my stress level, which has affected me physically and mentally on/off for the past six years." This comment on the positive benefits of the class for stress management was echoed by several other students: -What I learned from this class will remain with me for the rest of my life and honestly changed my outlook on a lot of things. As a result I feel calmer and ready to take on my life.
-I took a lot from this class and hope to apply the techniques I have learned in every stressful or aggravating circumstance. It's already helped me immensely to be more positive yielding especially with my family. I'm still working on dismissing all my negative assertiveness. It's a slow process but I definitely have seen improvements and so has my family. Thank you for those valuable lessons.
*(a three month follow up note): What I learned last quarter in this class has definitely helped me keep my sanity this quarter which has been super challenging, -seeming chaos swirling about (and within) -, but the meditation techniques have been super helpful ... the course had a very positive impact on me.
Journal cover examples
Students were invited to be creative in their journal "cover". Below are examples of the covers from three students' "journals. The one on the left used a quote from Gandhi discussed in the class. The student wrote: "This quote gave me the motivation to improve myself and to know that this improvement would help the world around me ... it instilled in me the idea that I could not help others without first helping myself attain a level of self peace and balance."
Student anonymous evaluations at end of course
Forms were given to each student at the end of the course to turn into the administrative office anonymously to evaluate the class. Of those who completed the anonymous evaluation (8 of 13 all agreed that the important ideas and concepts were clearly presented (87% strongly agreeing). All strongly agreed that students were encouraged to participate in class discussions; all agreed that the instructor was enthusiastic about conducting the course (87% strongly agreed); and all said s/he would feel comfortable seeking help (academic or otherwise from your instructor). Finally, overall teaching effectiveness was rated 4.5 on a 5 point (very good and excellent) scale.
Discussion
This study was a preliminary "evaluation" of the effectiveness of the principles and practices of Control Therapy in the educational setting. The quantitative data is encouraging and the qualitative data is promising in terms of students' self-reported improvement in mind-body connection, ability to adopt stress-management techniques, and capacity for acquiring a positive sense of control in their lives. The process of self-reflective journaling helped students see how actions and thoughts influence one's goals, and when and if one should or should not take active control over a situation.
In terms of quantitative data pre and post, all measures trended in the positive direction: greater positive sense of control, more positive assertive and positive yielding, less negative assertive and negative yielding. In other words, at the end of the course compared to the beginning, students reported themselves as feeling a more positive sense of control, and strengthening positive and reducing negative ways of practicing either assertive or yielding control in various aspects of their lives. We may conclude from these results that students were able to learn principles of control theory and practice and apply them personally. This suggests that the teaching of the principles and practice of control therapy in an educational setting has promising possibilities.
Pre-post, students showed a significantly "lower" desire for control. We interpret this as a positive outcome. The initial pretest score (4.93) for this class is slightly higher than other control profiles of college students in general (4.80), and similar to adult children of alcoholics (4.94; Shapiro, 1994, p. 99). At the end of the class their mean desire for control score was 4.57, quite similar to healthy adult respondents (4.56). Sometimes individuals under stress have a too high desire for control (and fear of losing control). It's possible that as a result of transferring, these students' desire for control scores were quite high, and as they adapted and felt more comfortable-both in general and through exploring these control issues in the class -their desire for control decreased to a more normative range.
Previous research has shown that individuals have unique control profiles and there is no "one size fits all" approach to addressing individuals' control-related needs. (e.g., Shapiro, Lindberg, Daniels, Breuer, & Astin, 1994). This conclusion was borne out in this study as well. Even when students' initial control profiles were quite different, they were able were able to match control enhancing strategies to their unique control profile and goal, to attain successful results.
The process of self-reflection, as noted, was important to the students, helping them learn about their unique control profile, their view of the role of control in personality theory (and goal of psychological health), and how to achieve appropriate use of the assertive/change and yielding/accepting modes of control. As one student noted Before taking this class, I never made time to consciously analyze my priorities or even notice any changes in them. Now, I feel more in touch with myself. I have noticed major changes in the way I perceived major views in life, including my place in the universe or even my theory of humanity. For example, my theory of humanity has changed. At the beginning of class, I viewed humanity in a more simplistic way not considering people's will. Now I take into consideration people's will and the effect of the environment on them. This class has definitely stimulated my thoughts and impacted my personal goals. I have learned so much in this class that I plan to explore many of my areas of weakness in order to slowly achieve balance; I am willing to keep recognizing those areas of strength and to be grateful for them.
Limitations and future directions
There are clearly several cautions and limitations regarding this study. The most obvious one is needing additional studies to determine the generalizability of these findings. For example, this class was a small, self-selected sample at a single institution, with no control group. Therefore, it did not control for motivational variables, differences between these students and students at other universities, or the passage of time. To control for passage of time in future studies one could compare this class to other seminars (e.g., artists of the 1800's) for transfer students matched by age and sex. A further study could use a time lag wait list design by identifying students interested in "exploring how you gain a positive sense of control" and randomly assigning half of them to the class one quarter, and half to the class the next quarter. Pre-post testing could compare intervention group to the wait-list group. This would control for the effects of time, expectation effects, and selfselection. A collaborative multi-institutional study involving several geographic regions and public and private universities would mitigate the effects of any characteristics or qualities unique to this transfer student population.
Further, though nearly all the data pointed to movement in the expected (positive) direction, effects sizes on the variables showed a range, from 6 effect sizes >.2; 5 effect sizes >.5, and two effect sizes greater than .8. Also, there were only two results that were significant at the <.05 level, and no correction for multiple comparisons was made. Therefore, these quantitative findings, though promising, need further replication with a large group sample. On the other hand, it should be noted that the N = 1 data and the qualitative data are quite important to consider, as the goal of the pedagogical intervention was not just pre and post shifts in the aggregate, but individual changes for each student based on their unique control profile and goals. From this perspective the results were quite promising, as noted in the results section.
One final caution is that this was an educational class, not group therapy (even though it was taught by a professor with experience as a clinician). Sensitivity to issues of "confidentiality" and level of self-exploration appropriate in an education setting need to be considered. There were no concerns or problems resulting from this class, but this is at least an issue to be aware of. Twelve of the 13 students wrote highly positive comments about the personal focus of the class. One student clearly had a negative experience, and that should be taken as a caution to potential adverse effects and explore how they might be best addressed. There may be potential psychological discomforts when students reflect on their thoughts or choices they have made in the past. If a student feels like he or she is not making progress towards his or her goal, he/ she may become distressed. In this class, the instructor noted this possibility at the start of the class, provided information on the university resources for "counseling and self-exploration"; monitored each student's progress on a weekly basis, including their journals turned in at weeks 3 and 7, monitored their classroom participation, had office hours which he encouraged each to utilize. Yet, he "missed" the concerned student, and nothing was said by the student until the feedback at the end of the class.
Final Comment
While Control Therapy has been shown to be of value to individuals in psychotherapy, this is the first indication that the principles and practice of Control Therapy can have broader usage-i.e., in an educational setting. The Dalai Lama (2017) has recently called for an expansion of education-"my wish is that one day formal education will pay attention to the education for the heart, teaching love, compassion, justice, forgiveness, mindfulness, tolerance and peace." He suggests that this expansion be applicable to allthose who believe in God, those who don't. The principles and practice of Control Therapy involve a framework which is sensitive to a continuum of beliefs of self-agency, and other/ Other agency (cf. Smith, 1983 on self and other power). Students in this class were Buddhist, Christian, existential/atheist, and agnostic. They were able to find ways to match their control strategies to their beliefs. Further, the SCI was developed to be culturally sensitive, and it has been translated into Korean (Park & Sung, 2008); Portuguese (Bogiazian, 2015); Chinese, (Liu & Chang, 2103;Lee, Chan, Kwok, & Hsu, 2005 ;Chia, Cheng, & Chuang, 1998;Zili & Taisheng, 2015); Spanish, (Santibañez, Galego, & Iraurgi, in press); and (versions are under development in German, French, Italian, Hebrew and Farsi-see footnote 10, references).
The importance of individuals finding ways to learn the skills of self-regulation, and to find ways to gain and re-gain a positive sense of control in their lives are critically important in all cultures, even if there are differences in emphasis on modes and agency of control (e.g., Weisz et al., 1984, in Japan;Shapiro, 1989, 1990Lee et al., 2005 in Hong Kong and Australia;Soh, Surgenor, Touyz, & Walter, 2007, North European Australian and Chinese Singaporean women).
Finally, as has been noted, this was a preliminary study. In scientific investigation there are four stages of research development. The first stage asks, is there something promising here to investigate. The last stage is a placebo controlled double blind study (including multiple institutions; multiple cultural contexts/countries; different types and levels of learners. Our intent in this study is to offer a promising "seedling" paper upon which others could then build. This class shows the potential for students to be able to learn the skills of gaining awareness through self-reflection on their control profile, learning to monitor their own behavior, setting self-directed goals, and learning to match appropriate control-enhancing techniques to help them achieve their goals (e.g., assertive/change mode of control and yielding/accepting mode of control strategies. Thus, the teaching the principles and practice of Control Therapy in an educational setting can have a positive impact on student's lives-personally and interpersonally. It seems promising to continue this research, by adding appropriate control groups, and to expand and evaluate this teaching to other "grade levels" and to test its efficacy in educational settings in other cultures. | 2019-07-21T08:06:53.748Z | 2017-01-01T00:00:00.000 | {
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267156068 | pes2o/s2orc | v3-fos-license | Analysis of Regional Financial Independence and Effectiveness in North Buton District
This research aims to determine and analyze the regional financial independence and effectiveness of North Buton Regency. This research was quantitative
INTRODUCTION
To increase fixed assets, regional governments set aside money in the APBD capital expenditure budgets.The distribution of capital expenditures is determined by the infrastructure and facility requirements specific to the region, both for public facilities and for the efficient execution of governmental duties.Therefore, local governments should alter the makeup of their spending in an attempt to raise the caliber of public services.Thus far, routine spending has accounted for the majority of regional spending, despite it being substantially less productive (Yovita 2011).
Low capital expenditure can affect the performance of various government agencies.Capital expenditure is an important factor in improving the economy, so it is necessary to intervene in government services to cover the low level of budget disbursement.Budget absorption in 2016 was still less than 90%, indicating issues with capital spending.
The toughest challenge in infrastructure development is the very high need for infrastructure throughout Indonesia.Meanwhile, the government has a relatively limited budget in the APBN (State Revenue and Expenditure Budget).The APBN budget for infrastructure development is still regarded as inadequate, despite the government having raised capital spending and infrastructure development budget.As a result, the Regional Government ought to be permitted to spend its APBD for capital expenditures rather than for hiring staff and other regular expenses.Apart from this, the involvement of BUMN (State-Owned Enterprises) and the private sector in collaborating with the government in providing infrastructure needs to be expanded and improved.
The importance of observing what proportion of teachers' salaries are in Employee Expenditures is because so far many parties have highlighted and criticized the amount of Employee Expenditures which are considered too large in the APBD.Many parties said that this resulted in reduced allocations for Capital Expenditures, which were seen as having a more significant influence on the fulfillment of public services to the community (Director General of Financial Balance 2012).Seeing that there is a lack of attention to capital expenditure in the APBD in Indonesian provincial governments, regional governments should be able to allocate their APBD for capital expenditure and not use it up for personnel and routine expenditure.
Since the implementation of regional autonomy, it has provided regional governments with the opportunity to further develop regional potential.To develop regional potential, regional governments need to increase the capital expenditure budget.The sources of funds used to finance capital expenditure consist of Regional Original Income (PAD), General Allocation Funds (DAU), Special Allocation Funds (DAK), and Sharing Funds.Results (DBH).
As a result, the Regional Government ought to be permitted to spend its APBD for capital expenditures rather than for hiring staff and other regular expenses.On the other hand, poor APBD management can hamper local government performance in improving regional development and people's welfare.The problem that arises is when local governments are faced with a small amount of regional spending but have to cover large needs.Meanwhile, at the same time, the local government lacks creativity in managing the APBD, so that the government at the level above (provincial or central government) is not optimal in managing the APBD (Suara Merdeka 2012).
One of the focuses of President Jokowi's current administration is infrastructure development in addition to Human Resources (HR) development.One of the policies in promoting infrastructure development in the regions is through allocating an infrastructure budget of 25% of general transfer funds which include the General Allocation Fund (DAU) and Profit Sharing Funds (DBH).This policy aims to ensure that regional spending is not only for spending on apparatus but rather on spending that is indicated for public services (Sofi, 2020).
Finance Minister Sri Mulyani noted that around 13.4 percent of APBD funds were used for official travel.Then around 17.5 percent for office services.Meanwhile, the amount of employee spending reached around 36 percent.The Minister of Finance concluded that around 70 percent of APBD spending was only used to deal with regional officials.According to him, this is ironic because the community only gets the remaining 30 percent or one third.He said that this portion of the budget must be changed so that people can feel the benefits.This demonstrates that capital spending, which ought to have a significant impact on regional development, only receives a small percentage of it.(Thomas, 2019) North Buton Regency is a regional government that implements regional autonomy well because of the support from resource factors that can move the wheels of government to achieve goals.One of the factors is the financial factor, the financial factor is the main factor which is the financial source for the administration of regional government.Law Number 23 of 2014 states that the sources of income include, among other things, original revenue from the region, balancing funds, and other sources of valid income.
Table 1.Sources of Income for North Buton Regency 2016-2020 (in Rupiah) Source: Processed Data 2023 Salihi 248 Based on the table above, the author describes that the largest contribution comes from central government transfer funds in the form of balancing funds which include general allocation funds and special allocation funds with the highest contribution occurring in 2016 amounting to IDR 595,322,355,796.Meanwhile, original regional income, which is a reflection of the implementation of autonomy and fiscal decentralization, has a low contribution with the highest value in 2017 amounting to 27,660,740,065.
The Regional Revenue and Expenditure Budget (APBD) is still used more for personnel expenditure than capital expenditure."Low capital expenditure absorption has the potential to cause public losses.Because capital expenditure is usually used to build public facilities and facilities, an integrated budgetary control mechanism must be implemented.This is considered important so that integration from upstream to downstream occurs, and budget absorption can be maximized (Kppod, 2019).The multiplier effect is a broad influence, which is caused by one activity and then influences other activities (Rochimah, 2021).Indonesian Center of Reform on Economics (CORE) economist Yusuf Rendy said that capital expenditure essentially functions as expenditure that can provide a multiplier effect as well as a government step in increasing productive assets.Based on the phenomenon in the explanation above, research on Regional Financial Independence and Effectiveness was used as a research title, then, North Buton district became the chosen research location.The researcher hopes that by carrying out the research, the research results can provide information, evaluation and improvements related to regional financial independence and effectiveness.
Formulation of Problem
North Buton Regency is a district where the amount of the APBD continues to fluctuate, therefore the formulation in this research is what is the financial independence and effectiveness of the North Buton Regency area?
Research Purposes
This research aims to determine and analyze the regional financial independence and effectiveness of North Buton Regency.
Benefits of Research
This research can be used as a source of reference for future researchers, especially regarding the independence and effectiveness of regional finances, and used as an illustration and reference for regional governments to allocate regional income sources more productively.
LITERATURE REVIEW
According to Halim (2008: 101), budgetary expenses for the purchase of fixed assets and other assets that yield benefits over multiple accounting periods are referred to as capital expenditures.This understanding is based on the definition of capital expenditures found in Government Accounting Standards Law No. 71 of 2010.
Meanwhile, according to Mardiasmo (2004: 187), a collection of direct expenses used to fund investment activities is known as a capital expenditure (increasing assets).Halim (2008: 101) According to Darise (2008: 135), Original Regional Income is income obtained by the region which is collected based on regional regulations.Based on these two definitions, Original Regional Income (PAD) is defined as one of the revenue streams that a region receives and that comes from the potential of each region, which the region can explore and use on its own.In the Explanation of Law Number 33 of 2004, it is explained: Original Regional Income (PAD) is defined as regional income that comes from regional taxes, levies, the outcomes of independent regional wealth management, and other permissible regional original income.Its goal is to give regions the latitude they need to look for funding when implementing regional autonomy to fulfill the decentralization principle.
Original regional income is all regional revenue originating from original regional economic sources consisting of: 1. Regional Taxes, namely regional government revenues originating from regional tax collections.Types of regional income are detailed according to income objects by the law on regional taxes and regional levies.Regional taxes consist of provincial government regional taxes and district/city government regional taxes.Provincial government regional taxes are taxes managed by the provincial government, for example, motor vehicle tax, motor vehicle title transfer fees, and underground water tax.Meanwhile, district/city government regional taxes include hotel tax, restaurant tax, advertising tax and parking tax. 2. Regional levies, namely regional government revenues originating from regional levies.The types of regional levies are detailed according to income objects by the law on regional taxes and regional levies, for example, health service levies, water levies, weighbridge levies and others.3. Separated Regional Wealth Management Results, namely regional revenues originating from the results of separate regional wealth management.4. Other Legitimate Regional Original Income, namely revenue from PAD which is not within the PAD classification mentioned previously.
Fund for General Allocation
Fund for General Allocation (DAU) is a source of regional income that is part of the Balancing Fund and is one of the factors that influences the amount of Capital Expenditure allocated in a region.General Allocation Funds are transfer funds from the central government to regional governments whose use is left entirely to the regions.
In Law number 33 of 2004, it is explained that the General Allocation Fund is funds sourced from the State Revenue and Expenditure Budget which are allocated to equalize financial capacity between regions in the context of implementing decentralization and regional autonomy.According to Kuncoro (2014: 63), to bridge the gap between their capacity and fiscal needs, all districts and cities receive the General Allocation Fund, a block grant that is distributed using a formula based on certain principles that generally suggest that impoverished and underdeveloped regions should receive more than rich areas.According to Mardiasmo (2004: 144), the General Allocation Fund is intended to maintain financial equality and balance between the center and the regions, so that in distributing the General Allocation Fund it is necessary to pay attention to regional potential, financing needs to support government administration in the regions and the availability of the APBN.The General Allocation Fund functions as a fiscal equalization factor.Factors that influence the amount of General Allocation Funds for each region are the fiscal gap and regional potential (fiscal capacity).The principle of allocation of General Allocation Funds is that regions with large fiscal potential but small needs will receive relatively small General Allocation Funds.On the other hand, if a region's potential is small while its needs are large, then the region will receive a relatively large General Allocation Fund allocation.
Special Allocation Fund
Capital Expenditures are also influenced by Special Allocation Funds.Special Allocation Funds are transfer funds from the central government to regional governments other than the General Allocation Funds.The legal basis governing General Allocation Funds is Law Number 33 of 2004 concerning Financial Balancing between the Central Government and Regional Governments, Government Regulation number 55 of 2005 Budget revenues which are allocated to certain regions to help fund special activities which are regional affairs and national priorities.Mardiasmo (2004: 144) explains that Special Allocation Funds are funds allocated to help finance certain needs, namely national programs or activity programs that are not available in other regions.Meanwhile, Law Number 23 of 2014 concerning Regional Government explains that Special Allocation Funds are funds sourced from APBN revenues allocated to certain regions to fund special activities which are government affairs that fall under regional authority.After examining some definitions, it is possible to determine that the term "Special Allocation Funds" refers to transfers of funds from the central government to regional governments that are obtained from the APBN and assigned to specific regions to finance unique initiatives that fall under the purview of the regional authority in terms of infrastructure and facility provision (physical facilities).The Special Allocation Fund allocation is meant to support special activities in specific areas that are regional affairs and, by national priorities, to finance the need for infrastructure and basic community service facilities that are still in need of improvement or to promote the acceleration of regional development.(Nurlan Darise, 2014: 137).
Regional Revenue and Expenditure Budget (APBD)
By Law 33 of 2004, article 1 number 17 "Regional Revenue and Expenditure Budget, hereinafter referred to as APBD, is the annual financial plan of the Regional Government which is discussed and approved jointly by the Regional Government and the Regional People's Representative Council, and stipulated by Regional Regulations".
A regional financial plan with a detailed description, a source of revenue that is a minimum target to cover costs-costs associated with these activities and the existence of costs that constitute the maximum limit of expenditures to be implemented-types of activities and projects that are stated in the form of figures, and the budget period, which is typically 1 (one) year-are all included in Abdul Halim's (2004: 15-16) definition of a regional budget.
Indra Bastian (2006: 189) defines APBD as the materialization of regional government work plans for a year, expressed as monetary units, with a focus on public welfare objectives.
Balancing Fund
Balancing funds are funds sourced from the State Revenue and Expenditure Budget (APBN) which are allocated to regions to finance regional needs.The investment fund groups are divided into types 1.Profit Sharing Funds are funds sourced from the APBN which are allocated to regions as part of tax and non-tax revenue sharing 2. General Allocation Funds are funds sourced from APBN revenues which are allocated to regions in the form of block grants whose use is left entirely to the regional government.3. Special Allocation Funds are funds sourced from the APBN which are allocated to regions for a specific/special purpose.Other Legitimate Regional Income Other legitimate regional income is other revenue that does not come from the classification of Original Regional Income and Balancing Funds as previously explained.Miscellaneous legitimate regional income groups are divided according to the type of income they include 1. Grants originating from the government, other regional governments, domestic private agencies/institutions/organizations, community groups/individuals and non-binding foreign institutions, either in the form of foreign exchange, rupiah or goods and/or services, including experts and training that does not need to be repaid.2. Emergency funds from the government to deal with victims/damage caused by natural disasters.3. Tax revenue sharing funds from the provincial government to the district/city government 4. Special adjustment and autonomy funds 5. Financial assistance from the provincial government or other regional governments
METHODOLOGY
Through the use of descriptive analysis, this research was quantitative.Data regarding APBD realization reports specifically for regional revenue components, namely PAD, DAU, and DAK in 2016-2020, which had been collected from the North Buton Regency Regional Financial Agency, were analyzed using a ratio that was calculated by dividing the total of each local revenue component by the total expenditure and multiplying by 100%.This ensured that each component's contribution matched.The analysis process involved gathering data in the form of APBD realization reports for the years 2016-2020 and studying regional revenues under law number 33 of 2004.
Draw conclusions from the data for the APBD Realization Report for the years 2016-2010, and offer advice or comments to the North Buton Regency's Regional Financial Agency on how to manage the APBD going forward.The formula used was as follows: ( Source : Abdul Halim, 2012 : 23) Information : K = Contribution PAD = Original Regional Income DAU = General Allocation Fund DAK = Special Allocation Fund BM = Capital Expenditure Table 1.
International Journal of Advanced Technology and Social Sciences (IJATSS)
Vol .1, No.4, 2023: 245-258 253 RESULTS AND DISCUSSION 1. Regional Original Income 2016-2020 According to Abdul Halim (2007: 94), revenue derived by a region from sources inside its borders that is gathered in line with regional regulations and applicable laws and regulations is known as Regional Original Income, or PAD.The following table shows the North Buton Regency Regional Revenue Agency's Regional Original Income from 2016 to 2020: Table 1.North Buton Regency Original Regional Income from 2016 to 2020 Source: Processed Data The table above shows that PAD for North Buton Regency for the 2016-2020 fiscal year, its achievements exceeded targets in 2016, 2017 and 2019, while in 2018 and 2020 PAD did not reach the targets that had been set.
General Allocation Funds for 2016-2020
General Allocation Funds received from 2016 to 2020 at the North Buton Regency Regional Revenue Agency can be seen in the following table : Table 2. General Allocation Funds for North Buton Regency from 2016 to 2020 Source: Data Processed in 2023 The table above shows that the DAU for North Buton Regency for the 2016-2020 fiscal year achieved targets in 2016, 2018 and 2019, while in 2017 and 2020 it did not reach the targets that had been set.
Special Allocation Funds For 2016-2020
The receipt of special allocation funds from 2016 to 2020 at the North Buton Regency Regional Revenue Agency can be seen in the following table : Table 3.General Allocation Funds for North Buton Regency from 2016 to 2020 Source: Data Processed in 2023 The table above shows that the North Buton Regency DAK for the 2016-2020 fiscal year fluctuated to meet the target in 2018 and 2019, whereas in 2016, 2017 the achievement exceeded the target but in 2020 it did not reach the target 4. Capital Expenditures 2016-2020 Table 4. Budgeted Capital Expenditure from 2016 To 2020 at the North Buton Regency Regional Revenue Agency Can be Seen in the Following Table Source: Data Processed in 2023 The table above shows that North Buton Regency Capital Expenditure for the 2016-2020 fiscal year fluctuated above the target in 2016, 2017, 2018 and 2019, while in 2020 North Buton Regency Capital Expenditure did not exceed the target.5. Contribution of Original Regional Income to Capital Expenditure Allocation Table 5. Contribution of Original Regional Income to the North Buton Regency Capital Expenditure Allocation for the 2016-2020 Fiscal Year Source: Data Processed in 2023 In the table above, it can be seen that the contribution of Original Regional Income to the North Buton Regency Capital Expenditure allocation for the 2016-2020 Fiscal Year varies between 5.34% to 14.62%.The largest contribution occurred in 2020, namely 14.62% and the lowest was in 2016, amounting to 5.34%.With an average contribution of 9.98%, it proves that Regional Original Income contributes in an ineffective category to the allocation of Capital Expenditures in North Buton Regency.6.General Allocation Fund contribution to Capital Expenditure allocation Table 6.Contribution of the General Allocation Fund (DAU) to the North Buton Regency Capital Expenditure Allocation for the 2016-2020 Fiscal Year Source: Data Processed 2023 In the table above, it can be seen that the contribution of the General Allocation Fund to the Capital Expenditure allocation for North Buton Regency for the 2016-2020 budget year always increases between 162.96% to 311.83%.The largest contribution occurred in 2020, namely 311.83% and the lowest occurred in 2016, namely 162.96%.With an average contribution of 237.39%, it proves that the General Allocation Fund contributes in a very effective category to the allocation of Capital Expenditures in North Buton Regency.
states that what is included in Capital Expenditures are: 1) Land Capital Expenditures; 2) Shopping for Equipment and Machinery; 3) Building and Building Capital Expenditures; 4) Capital Expenditure on Roads, Irrigation and Networks; 5) Other Fixed Asset Expenditures; and 6) Spending on Other Assets.Government Accounting Standards (SAP) which are regulated in Government Regulation Number 71 of 2010 is an amendment to Government Regulation Number 24 of 2005.Locally-generated revenue concerning Balancing Funds and Minister of Finance Regulation Number 145/PMK.07/2013concerning Budget Allocations Transfer to Region.According to Government Regulation Number 55 of 2005 concerning Balancing Funds, it is explained that Special Allocation Funds are funds sourced from State Revenue and Expenditure | 2024-01-24T18:17:53.907Z | 2023-12-28T00:00:00.000 | {
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196523002 | pes2o/s2orc | v3-fos-license | Fast Track Colorectal Surgery for Deep Endometriosis: A Prospective Randomized Trial
Background and study aims: Application of fast track protocols in laparoscopic colorectal surgery has been assessed in oncological cases with contrasting results. This study was to assess the feasibility and advantages in a group of young women suffering from bowel endometriosis. Patients and methods: Over one year, 227 women were recruited for this prospective randomized study on fast track protocol for laparoscopic surgery for bowel endometriosis. Patients were allocated to a perioperative fast-track or conventional care in a 1:3 ratio and clinical outcomes and costs were evaluated. Results: Clinical outcomes and re-admissions within thirty days were homogenous between the two groups. Direct and indirect costs were significantly lower in the fast track group (p < 0.5). Discussion: A fast track protocol for laparoscopic surgery for bowel endometriosis can be applied in referral centers providing a direct impact on clinical management and a definite economic advantage.
gery) [1]. Endometriosis is a benign disease that occurs in women of reproductive age and may affect seriously the quality of life and fertility [2]. The severity of the disease ranges widely between asymptomatic ovarian cyst to severe infiltrating endometriosis [2]. Severe endometriosis (stage IV of ASRM classification, reference [2] occurs in less than 10% of cases and the incidence of bowel endometriosis in such cases [3] is as high as 50% with colorectal localization accounting for more than 90% of cases [4]. There is a general consensus on the opportunity of a multidisciplinary surgical approach when severe deep endometriosis is diagnosed in order to reduce the chronic pelvic pain that is associated to the disease [5,6] and improve the fertility rate [7]. We decided to set up a fast-track protocol study mainly because it is a homogeneous population of patients of young women that may present a quicker recover after surgery than older patients with colorectal cancer.
Patients and Methods
This prospective randomized study (clinical trial registration: UMIN-000014199) was carried out at Sacro Cuore Don Calabria General Hospital of Negrar (Italy) between January and December 2013. A prospective recruitment of 227 consecutive women undergoing elective laparoscopic bowel resection for deep infiltrating intestinal endometriosis was carried out ( Figure 1). Patients were randomly assigned to a perioperative fast-track (group A, n = 62) or conventional (group B, n
Introduction
Many studies on fast track colorectal cancer surgery have been published in the past ten years with contrasting results mainly because of different populations and surgical approach (laparoscopic/open sur-= 165) care in a 1 to 3 ratio. Surgeons and anesthetists were blind on the group assigned. Preoperative polyethylene glycol administration for bowel preparation was used only in the Group B while a low-residue diet in Group A, in both cases without preoperative antibiotic prophylaxis. Fast-track program was based also on prompt removal of nasogastric tube after surgery, early postoperative oral fluid intake, resumption of oral feeding (solid/semi-liquid) within 24 hours after surgery, no antibiotic therapy after surgery, getting the patient out of bed and walking on day 1, and discharge from hospital as soon as the bowel function was restored. The average cost of routine care for laparoscopic colorectal surgery is 6,141 Euros per patient while it is slightly increased by ileostomy (6,716 Euros) but definitely more costly in case of complications such as hemorrhage or pyrexia (10,768 Euros). The application of a fast-track program allows a reduction of costs both in cases with an uneventful postoperative course and in cases of complications as reported in Figure 2B (p < 0.5). Furthermore, the hospital stay affects the average costs not only directly but also on the possibility to admit another patient (implementation of cost-effectiveness and reduction of waiting list).
The postoperative hospital stay was significantly shorter in group A (Figure 2A) with 52% of patients discharged on day 3 (median in group B was 7 days). No significant difference was found in re-admissions within thirty days (17.7% vs. 15.8%, p > 0.5). osis (DIE) with bowel envolvement results in complex procedure associated with postoperative complications such as anastomotic leakage, rectovaginal fistula, bleeding and abdominal abscess.
The management of this benign disease that occurs in women of reproducing age, seriously affecting the quality of life, should be executed in dedicated centers and also specialized minimally invasive approach.
Surgical mamagement is the primary treatment for symptomatic bowel endometriosis [8] and long term clinical outcomes are satisfactory when rectosigmoid obstruction due to severe bowel endometriosis occurs [9].
In recent years, there also are developed early rehabilitation programs in order to reduce postoperative pain and perioperative stress to lead to enhanced recovery after surgery.
Since Kehlet Introduced this concept in early 1990's, many studies have come out and several randomized trials and meta-analyzes have shown that the use of fast-track protocols are useful for early recovery of patients after colorectal resection for oncological disease. [10][11][12].
Both the laparoscopic approach fast track programs may Enhance recovery after surgery like several cohort series, meta-analyzes and prospective studies suggest [13][14][15].
Conversely literature is poor in the field of fast track programs applied to the surgical treatment of endometriosis. Ours would be the first prospective randomized study unicentric reported.
Kondo, et al. recently published a retrospective study of 161 patients with deep endometriosis undergoing fast track surgery reporting a shorter length of hospital stay and a lower readmission rate in treated patients.
In Our study we confirm that the application of a fasttrack protocol for elective colorectal surgery in young women with deep infiltrating endometriosis decreases not only the length of hospital stay (52% of patient in the fast track group was discharged on day 3, while the median in the control group was 7 days), but also the hospitalization costs decrease without increasing postoperative morbidity. The readmission within 30 days was similar in both groups, suggesting that this event is not directly related to the perioperative protocol strategy.
This study was registered at the Local Ethics Committee and on the UMIN-Clinical Trial Registry website with registration number UMIN-000014199. | 2019-07-15T22:29:28.357Z | 2019-06-01T00:00:00.000 | {
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264174424 | pes2o/s2orc | v3-fos-license | Numeracy Numeracy
Abstract How supportive of quantitative literacy (QL) are the Common Core State Standards in Mathematics (CCSSM)? The answer is tentative and conditional. There are some QL-supportive features including a strong probability and statistics strand in grade 6 through high school; a measurements and data strand in K-5; ratio and proportional reasoning standards in grades 6 and 7; and a comprehensive and coherent approach to algebraic reasoning and logical argument. However, the standards are weak in supporting reasoning and interpretation, and there are indications that the applications in CCSSM – mostly unspecified – will not include many QL contextual situations. Early indicators of assessment items follow a similar path. Except for statistics, most of the high school standards are aimed at development of algebra and precalculus topics, and there will likely be little room for more sophisticated applications of the QL-friendly mathematics of grades 6-8. The experience with CCSSM is limited at this point, leaving several crucial results uncertain, including assessments, emphases on statistics, and kinds of modeling and other applications.
Introduction
Will the implementation of the Common Core State Standards in Mathematics (CCSSM)1 change education for quantitative literacy (QL), and, if so, how?My answer to this question has several conditions and unknowns, but, in sum, at this time, I conclude that there will be minimal effect.This does not distinguish CCSSM from other K-12 mathematics standards -articulated or not -that I have known over the years.
In "Two Mathematics: Ever the Twain Shall Meet?" (Madison 2004), I echoed Alan Schoenfeld's "Reflections on and Impoverished Education" (Schoenfeld 2001).Similar to Alan's, my education in mathematics was totally devoid of the kinds of problem situations I have learned are central in QL, or, for that matter, any real applications and the whole of probability and statistics.My "Two Mathematics" article in Peer Review focused on the two different mathematics of U.S. society, namely school mathematics and QL mathematics, and the divide has existed since pre-colonial days.In several ways, CCSSM (2010) is more supportive of QL than was my mathematics education or some of the state standards being replaced, in part because awareness and importance of QL is worlds above what it was when I was in school or when many of the state standards were developed.The increasing need for QL, however, has outstripped those increases, at least cancelling the relative gain.
Just as the jury is still out on the general success of CCSSM (not our main goal here), it is too soon to know its potential to support QL.As noted above, there are unknowns.Some of them are: • How will CCSSM be implemented in the steady state, i.e. after the assessments are given and validated?Will the goals of more coherence and more depth be achieved?
• What kind of assessments will be used?Will these become the de facto standards, and if so, will the tests be worth teaching to?
• What kinds of "real world" or "real life" problems will be solved?In numerous places in CCSSM (2010) there are unspecified applications.Will modeling situations be substantial, be realistic, and include QLfriendly applications?
• Will the probability and statistics strand, one that is critical for QL, be a substantial component, or will it be relegated to the "cover if time permits" status?
• Is it realistic to believe that all students can achieve competence in the understandings and skills in CCSSM by grade 11?
• Will the political objections to CCSSM that have surfaced undermine its success?
In what follows, I will elaborate on some of these items, mainly in relation to QL but occasionally on the general effect of CCSSM.First, in the interest of full disclosure, I should elaborate that my experience with CCSSM has been fairly extensive.
The Author and CCSSM
Approximately five years ago, October 2009, my involvement in CCSSM began.I was one of approximately 25 people convened in Washington DC to evaluate CCSSM at that point and advise on its continued development under the auspices of the Conference Board of the Mathematical Sciences (CBMS) and the American Council on Education (ACE).Subsequently I was part of a three-person team (along with Jason Zimba and Pat Thompson) to write for CCSSM on quantitative reasoning.And, later, I was one of three members, chaired by Alan Tucker of State University of New York at Stony Brook, of the Mathematical Association of America (MAA) to evaluate CCSSM for MAA.Later still, I was the higher education mathematics faculty member from Arkansas who advised one of the assessment consortiums, Partnership for Assessment of Readiness for College and Careers (PARCC).Finally, I wrote the first draft of the standards progression (something like a learning progression) for modeling in CCSSM high school2 .
Aside from the progressions draft, I can detect none of my fingerprints on CCSSM, so pride of ownership is not at issue.For the past five years, as a part of an NSF-funded mathematics and physics partnership,3 I have led several professional development workshops every summer for middle and high school mathematics and science teachers with CCSSM as a guiding framework.The shifting and new responsibilities for teachers brought on by the implementation of CCSSM have been very apparent, and many weaknesses in teachers' preparations to teach using CCSSM have become evident to me.Not that these issues are new; rather, they temper my optimism that CCSSM will significantly change the outcome of school mathematics.The most-popular workshops we conducted for teachers in the partnership were the ones on QL.Teachers were hungry for everyday, contemporary applications to use in their classrooms, and the QL workshops provided that because we used the Casebook of Media Articles (Madison et al. 2012) that we use in our college course in quantitative reasoning.No such applications are specified in CCSSM.
In October 2009, about the only parts of CCSSM that were available were the eight Standards for Mathematical Practice, which had been distilled from the five strands of mathematical proficiency from Adding It Up (Kilpatrick et al. 2001) and the process standards from the NCTM Standards for School Mathematics (NCTM 2000).The distillation and explanation producing the eight practice standards in the form, "Mathematically proficient students do …," was very effective and impressive as a beginning.The practice standards set a high bar.I recall Denny Gulick, University of Maryland, remarking at the CBMS-ACE forum in 2009 that he would be delighted if all faculty colleagues understood and performed as the practice standards indicated.As would be expected, adding detailed content standards to these elegant practice standards was difficult and often troublesome.Many constituencies with varying interests had to be satisfied, so no single perspective prevailed.In 2009 there were some beginnings of example problems that explained the standards and set goals for understanding.Evidently, development of these problem examples did not continue as they are not part of CCSSM at present.Undoubtedly they will be part of the assessments in the guise of sample items that have begun to unfold.One of the dangers of sample items or example problems is that they become the standards.
What Do I Mean by QL?
What I mean by QL will help explain my opinions on how CCSSM does support QL and how it could be more supportive.Over the years there have been several published meanings or frameworks for QL, and CCSSM is more supportive of some than others.For example, the MAA report on quantitative reasoning (MAA 1994) gives a goal for QL that is reasonably well supported by CCSSM, namely applying simple mathematical methods to the solution of real-world problems.This conception of QL is similar to the two key characteristics of a numerate person as stated in the Cockroft (1982) report.The two characteristics were the ability to use mathematics in everyday life and to understand and appreciate information presented in mathematical terms.According to Maguire and O'Donoghue (2002), the Cockroft report's conception of numeracy (called QL in the U.S.) ushered in the mathematical phase of numeracy that gave way to the integrative phase around 2000.Yet, numeracy surveys and literacy testing in the U.S. and Europe have been and are still dominated by this mathematical context.As Lynn Steen and I (2008) wrote: Further arguments for focusing mathematics education in a cross-disciplinary and functional direction emerged in a U.S. report on "what work requires of schools" that stressed practical competencies (in, e.g., resources, information, systems, and technology) built on a broad foundation that included basic skills, decision making, and problem solving (SCANS 1991).The emphasis on numeracy as a functional skill-now giving rise to the term "functional mathematics" (Murnane and Levy 1996;Forman and Steen 1999)-dominates QL assessments and has influenced many state mathematics standards.
It is likely that this view of QL as functional mathematics influenced CCSSM if consideration of supporting QL was ever an issue.A framework for QL by Dossey (1997) moved more toward the integrative version of numeracy by having chance, data interpretation, and measurement as three of the six major aspects.Gal (1997: 41) went further, including affective aspects in his conception of numeracy tasks that "require adults to integrate seamlessly both numeracy and literacy skills."CCSSM would be more supportive of Dossey's version than Gal's, however. Finally, Wilkins (2000) gives a framework for QL that contains affective and motivational aspects of QL, e.g., recognition of societal impact of mathematics, understanding the nature and historical development of mathematics, and having a positive disposition toward mathematics.CCSSM, not surprisingly, is silent on these affective aspects.
My meaning for QL is based on the six core competencies for QL as developed in the AAC&U VALUE rubric for QL as modified by Boersma, et al. (2011).The six core competencies are interpretation, representation, calculation, analysis/synthesis, assumption, and communication.Most, if not all QL situations can be resolved by applying the six competencies.In sum, I find CCSSM supportive of the calculation competency, somewhat supportive of the representation competency (via modeling) and the analysis/synthesis competency, and not very supportive of interpretation and communication competencies.The support for assumptions is unclear.
Overview of my Conclusions
Here I list my conclusions on how supportive CCSSM is of QL.I will elaborate on some reasons in the remainder of this perspective.
Strengths of CCSSM in support of QL
• The practice standards are elegant, challenging and very supportive of QL.
• The mathematics and statistics content is sufficient for QL, especially the strands (called domains in CCSSM) on measurement and data, ratios and proportional relationships, and statistics and probability.
• The development of algebraic thinking and logical reasoning from Kindergarten through grade 11 is coherent and systematic.
Support for QL that is yet undetermined
• The assessments are yet to be administered and validated.
• The applications and mathematical models in CCSSM are mostly unspecified, leading possibly to applications within the assessments becoming dominant in the classrooms.
• The pressure to "cover" the standards by grade 11 may reduce the attention to substantial and challenging applications.
Some evident weaknesses in CCSSM's support of QL
• Beyond the practice standards, the emphasis on interpretation and conceptualization is weak, as is the emphasis on communication and reflection on results of computations.
• There are very few suggestions of applications or mathematical models that deal with critical citizenship issues such as political arguments, government economics, and health risks.
• The language used in CCSSM does not encourage conceptualization and interpretation, especially in regard to quantitative reasoning and conceptualization of functions.
Are Goals of "All Students" and "College and Career Readiness" Realistic?
On a panel that I chaired on CCSSM and college placement examinations at the Joint Mathematics Meetings in 2013, Zalman Usiskin, looked to history to make a point about CCSSM.Usiskin (2013: 17) noted that … in 1892, Charles Eliot, the President of Harvard, chaired a committee which came to be called the Committee of Ten, whose purpose was to standardize the high school curriculum in the United States for students intending to go to college.The Committee of Ten ( 1893) recommended that all students follow a college-preparatory curriculum, that "every subject which is taught at all in a secondary school should be taught in the same way and to the same extent to every pupil so long as he pursues it, no matter what the probable destination of the pupil may be, or at what point his education is to cease.…"The report recommended that all students take one year of algebra and one year of
The Practice Standards and QL
As I have written elsewhere (Madison 2014), the first four practice standards are central to QL: making sense of problems; modeling with mathematics or statistics; reasoning quantitatively; and drawing, supporting and communicating conclusions.Critiquing the reasoning of others is often the entry point into a QL situation.Less central to QL are practice standards 5 -8: use appropriate tools strategically, attend to precision, look for and make use of structure, and look for and make use of regularity in repeated reasoning.Tools for QL include calculators or spreadsheets and quantitative benchmarks for detecting reasonableness of answer.There is attention to precision but most numerical precision focuses on the precision needed, realistic or possible, in resolving the QL situation.Precise definitions and correct units are important when available in a context or by assumption.The final two standards -looking for and using structure and looking for and expressing regularity -are more applicable to mathematics development.
Better than the Standards Being Replaced?
Are the CCSSM standards better or more supportive of QL than the ones they are replacing?This question is difficult to answer as the standards being replaced vary from state to state.I can say with confidence that the Common Core standards are stronger and better aligned with QL than were the Arkansas Frameworks as they eventually became practiced.I do not have direct experience with standards from other states.One has to wait to see how CCSSM is implemented and assessed.If the assessments are "tests worth teaching to" and, as is likely, the assessments become the standards, then we will have moved forward.
So far, what I have seen of the sample items released by the two assessment consortia, PARCC4 and the Smarter Balanced Assessment Consortium,5 the assessments will not be very supportive of QL.As an example, the PARCC sample assessments in grades 6-high school had 192 items, and only 18 of these were on probability, data analysis and statistics.Since that statistics strand is the most QL-friendly of all the strands, having more items from that strand would be better for QL.
CCSSM Mathematics Content and QL
There are standards at every grade level that are very supportive of QL.Aside from the fundamentals of arithmetic, the parts of CCSSM that are omnipresent in QL are the Measurement and Data strand in K-5, the Statistics and Probability strand in grades 6-high school, and the Ratios and Proportional Reasoning strand in grades 6-7.The latter strand has only six standards, with two of these having four problem types.These standards are important in QL.For example, one of them reads "Use proportional relationships to solve multistep ratio and percent problems.Examples: simple interest, tax, markups and markdowns, gratuities and commissions, fees, percent increase and decrease, percent error."One aspect of CCSSM that could very well be very supportive of QL is the development of algebraic thinking from K on.This development would be more supportive were it rooted in quantitative reasoning as I will note below.Nevertheless, this broader view of algebraic thinking in CCSSM -as well as the broader view of logical reasoning -could produce significantly stronger QL reasoning among high school graduates.How well that works depends heavily on implementation and emphases.
Applications and CCSSM
The extent and kinds of applications will be critical in determining how CCSSM supports QL or how the impoverishment that Alan Schoenfeld and I experienced in our mathematics education is remedied.Some applied mathematicians have criticized CCSSM because of the lack of applications.Specifically, I recall that Alan Tucker was open in this kind of criticism of CCSSM when we were evaluating CCSSM for the MAA.As noted above, the kinds of "real world" problems that are to be solved by students using various content designations in CCSSM are not specified.There are a few examples that help clarify what is meant by various standards.One of these, on exponential functions, is fairly supportive of QL: "… identify percent rate of change of functions such as y = (1.02)^t,y = (0.97)^t, … , and classify them as representing growth or decay." One difficulty of inserting QL-friendly applications in K-12 is that many are sophisticated uses of elementary mathematics.6This is difficult because the students may not be knowledgeable about the contexts of the sophisticated uses such as economics, political science, and personal finance.This is especially so in grades 6 and 7 where the standards on ratios and proportional reasoning occur.It is seen as impractical by teachers because they have more complex mathematics to teach and the sophisticated contexts are unlikely to occur on assessments.
Modeling, CCSSM, and QL
Modeling, once viewed as a likely strand, is not a separate strand in CCSSM, and modeling is very much a part of QL.Representing with mathematics or statistics models is a first step after interpreting in resolving many QL situations.Instead of a separate strand, various standards in the existing strands are starred as an indication that they are a modeling standard.As stated in CCSSM, Modeling is best interpreted not as a collection of isolated topics but rather in relation to other standards.Making mathematical models is a Standard of Mathematical Practice and specific modeling standards appear throughout the high school standards indicated by a star symbol (*).
From the progression draft7 : … About one in four of the standards in Number and Quantity, Algebra, Functions, and Geometry have a star, but the entire conceptual category of Statistics and Probability has a star.In statistics, students use statistical and probability models-whose data and variables are often embodied in graphs, tables, and diagrams-to understand reality.Statistical problem solving is an investigative process designed to understand variability and uncertainty in real life situations.Students formulate a question (anticipating variability), collect data (acknowledging variability), analyze data (accounting for variability), and interpret results (allowing for variability).
Readiness for College Mathematics
Readiness for college mathematics has different meanings.The most common meaning is readiness for success in a degree credit-bearing college mathematics course, often college algebra.That appears to be the meaning of college readiness assumed in CCSSM.Since college algebra courses generally are not supportive of QL (See, for examples (Gaze 2014) and (Gillman 2010)), CCSSM's targeting college algebra could be the major reason for not being supportive of QL.Not all the CCSSM standards are proposed as necessary for career and college readiness.Standards that are not proposed as necessary are marked with a (+) indicating that these are for preparation for more-advanced courses such as calculus, advanced statistics or discrete mathematics.There are various measures used for determining readiness for college mathematics.The ACT assessment has one (Allen and Sconing 2005), namely an ACT mathematics score of at least 23.One of the stated intents of the PARCC assessment of CCSSM was to have higher education institutions recognize the assessment results as indicating readiness for college mathematics.Whether or not that happens, or becomes operational, is to be determined.It certainly should be an indicator, but likely will not be definitive.Indeed, if the CCSSM summative assessment is first administered in 2014-15, then it will be a few years before the results can be evaluated as to whether they measure readiness for college mathematics.Use as an indicator of college readiness was also the same intent of a predecessor of CCSSM, the Algebra II end-of-course examination, the development of which was led by Achieve,8 which was also the leader of PARRC.The PARCC CCSSM assessment is still unknown, but the Algebra II end-of-course examination was not friendly to QL.In any event, mathematical sciences faculty are likely to continue placement testing to determine where entering students begin in mathematics.Such tests contain little information about QL.Consequently, no assessments of QL are likely very soon for college admissions or college readiness.
Algebra, Quantitative Reasoning and Functions
The definitions and assumptions in CCSSM tend to be directed toward numbers and procedures rather than conceptualization and interpretation.For example, CCSSM defines quantity as a number with a unit.This definition moves right past the conceptualization of a quantity and avoids student participation in the dialectic among object, attribute and quantification as advocated by Thompson (1993).In fact, Smith and Thompson (2007), arguing for developing algebraic reasoning by way of quantitative reasoning (as opposed to a generalization of arithmetic), state the following: For too many students and teachers, mathematics bears little useful relationship to their world.It is first a world of numbers and numerical procedures (arithmetic), and later a world of symbols and symbolic procedures (algebra).What is often missing is any linkage between numbers and symbols and the situations, problems, and ideas that they help us think about.
In a similar vein, Thompson and Carlson (in press) note that CCSSM does not promote student thinking about variation and covariation as the historical and cognitive roots of the concept of functions in mathematics.As they state: … the words "covary" and "variation" do not appear in CCSSM's 93 pages.The word "variation" appears just four times -three times in the context of statistics and once about variation in assumptions.The word "vary" also occurs only four times -once about opportunities, twice about changing assumptions, and once in the context of statistical variation.
Both these examples are at the core of potential strengths of CCSSM -successful development of algebraic thinking and correct meanings by students for the fundamental notion of function.Consequently this goes beyond a weak support for conceptualization and interpretation to a way of developing major domains of CCSSM.Since CCSSM should be a formative document, these fundamental issues should be monitored, studied and researched carefully.
Final Thoughts
How supportive of QL will CCSSM be?Will the implementation of CCSSM change education for QL? Probably not, because, as with previous standards, education for QL is not a primary aim -college and career readiness are.Since much of school mathematics now is mired in recall and apply, significant change would require a strong position by CCSSM.That does not seem to be present, and the absence of specific applications means that assessments will likely determine what kinds of applications and models are emphasized, a crucial circumstance.There are QL-supportive features of CCSSM -e.g. the ratios and proportionalreasoning standards, the probability and statistics standards, and the aim of coherence of the development of algebraic thinking and logical reasoning, but assessments are likely to become the de facto standards.There are signs already that the assessments of CCSSM will not be QL-supportive-that, rather, they will focus more on college readiness where QL and statistical analysis are, at this time, not major issues, whereas college algebra is.Complete implementation is still unknown, and implementation issues have generated substantial political opposition.If colleges and universities make QL a more-substantive issue in admission and in graduation, then college readiness, which is dominant in CCSSM, will bend CCSSM toward the Committee of Ten's goal of sending graduates -high school or college -"into the world with fullest equipment for citizenship" (Mackenzie 1894: 149).
Madison: Quantitative Literacy and Common Core Published by Scholar Commons, 2015 geometry, and that is how those courses became standard in the U.S. curriculum in grades 9 and 10.The Common Core committee, charged with coming up a curriculum appropriate for both college and the world of work, thought the same way as the Committee of Ten, and they created a framework for a secondary school curriculum in which appropriateness of mathematics for later college study was viewed as the best preparation for all students, whether college-bound or not."Usiskin's point was that for a long time colleges have had difficulty dealing with the diverse spectrum of high school graduates and that CCSSM is not likely to change that.It is indeed an ambitious challenge to prepare all students for either college or work.That is not happening now, and CCSSM is not likely to make it so.Along those lines, at the 2009 ACE forum, I remarked that it seemed that an additional c-word should have been added to the Common Core State Standards for College and Career Readiness, namely that readiness for citizenship should have been added.Had that been the case, CCSSM would have had a broader but more QL-friendly goal.In 1892, the Committee of Ten recognized the need for citizenship readiness(Mackenzie 1894: 149): … only 3 per cent. of our high school pupils enter our colleges.It follows, therefore, that the best possible provision for secondary education, particularly in our high schools should be made, if we would send into the world with fullest equipment for citizenship the 97 per cent of high school pupils who do not enter college. | 2017-09-07T13:46:56.569Z | 2015-01-01T00:00:00.000 | {
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265018956 | pes2o/s2orc | v3-fos-license | DialogBench: Evaluating LLMs as Human-like Dialogue Systems
Large language models (LLMs) have achieved remarkable breakthroughs in new dialogue capabilities by leveraging instruction tuning,which refreshes human impressions of dialogue systems. The long-standing goal of dialogue systems is to be human-like enough to establish long-term connections with users. Therefore, there has been an urgent need to evaluate LLMs as human-like dialogue systems. In this paper, we propose DialogBench, a dialogue evaluation benchmark that contains 12 dialogue tasks to probe the capabilities of LLMs as human-like dialogue systems should have. Specifically, we prompt GPT-4 to generate evaluation instances for each task. We first design the basic prompt based on widely used design principles and further mitigate the existing biases to generate higher-quality evaluation instances. Our extensive tests on English and Chinese DialogBench of 26 LLMs show that instruction tuning improves the human likeness of LLMs to a certain extent, but most LLMs still have much room for improvement as human-like dialogue systems. Interestingly, results also show that the positioning of assistant AI can make instruction tuning weaken the human emotional perception of LLMs and their mastery of information about human daily life.
Introduction
Large language models (LLMs) (Bai et al., 2022;Du et al., 2022;Sun et al., 2023;OpenAI, 2023) have achieved remarkable breakthroughs by leveraging instruction tuning (Wei et al., 2021), especially unlocking new dialogue capabilities.Such new dialogue capabilities empower humans to naturally interact with LLMs, which has refreshed human's impression of dialogue systems.The long-standing goal of dialogue systems requires LLMs to be sufficiently human-like to establish long-term connections with users by satisfying the need for communication, affection and social belonging.Specifically, human-likeness generally covers the following fine-grained capabilities: correctly understanding the dialogue context, making reasonable use of relevant knowledge, detecting the user's emotions and personality when necessary, and finally generating friendly and reasonable responses that are coherent and consistent with the dialogue context (Huang et al., 2020).However, the heightened human likeness could not correspond to improved scores on existing LLM benchmarks.
Existing LLM benchmarks are mostly oriented to evaluate the LLMs' abilities for task completion as assistant AI, such as human-knowledge mastery (Zhao et al., 2023a;Zeng, 2023;Huang et al., 2023;Cobbe et al., 2021) or instruction following (Mishra et al., 2022;Zheng et al., 2023).However, these benchmarks do not focus on whether LLMs as dialogue systems are sufficiently humanlike to establish long-term connections with users.Therefore, an in-depth evaluation benchmark of those abilities related to human likeness is essential for identifying the strengths and limitations of LLMs as multi-turn dialogue systems.
The most ideal approach is to collect corresponding high-quality dialogues from real humans.However, most real-human dialogues, whether from social networks or open datasets, are likely to have been leaked during the pre-training of LLMs.To prevent the issue of "data leakage", the evaluation benchmark must contain new evaluation instances and be updated frequently.Due to the difficulty of human writing, it is necessary to construct new human-human dialogues as evaluation instances automatically.Inspired by Møller et al. (2023) and Whitehouse et al. (2023), we explore the use of GPT-4 as a surrogate for humans to generate massive evaluation instances.
In this paper, we propose a novel Dialogue Evaluation Benchmark with GPT-4 as Data Generator, DialogBench for short.Since dialogues generated without restrictions may not involve commonsense use or emotional expression, we generate corresponding evaluation instances for different finegrained capabilities.To evaluate comprehensive abilities, we select 12 dialogue tasks.Each task requires LLMs to possess at least one ability to perform it well.For each task, we prompt GPT-4 to generate evaluation instances.Specifically, we first design the basic prompt based on widely-used design principles and further mitigate the existing biases to generate most of the available evaluation instances.Afterward, we filter out detrimental evaluation instances via a filter mechanism.Consequently, we construct English and Chinese dialogue evaluation benchmarks towards human likeness.
We conduct a comprehensive evaluation of 26 LLMs using DialogBench, including pre-trained and supervised instruction-tuning models.Experimental results reveal that instruction tuning can improve the human likeness of LLMs.For supervised instruction-tuning models, top-tier models can handle a wide array of dialogue tasks, indicating the potential for developing LLMs into humanlike dialogue systems.However, we observe significant performance gaps between top-tier models and other LLMs, which suggests that their performance lags considerably.In addition, LLMs generally perform better at correctly understanding context but are relatively poor at perceiving emotions and personality.Current LLMs also do not understand much about daily human life.This underscores the necessity for more efforts to enhance the abilities related to the human likeness of most LLMs.
Our contributions are summarized as follows: (1) We present DialogBench, a comprehensive benchmark to standardize the evaluation of LLMs as human-like dialogue systems.(2) We perform a thorough evaluation of 26 different LLMs using Di-alogBench, uncovering a significant performance evaluation under diverse dialogue tasks.It illuminates the top-tier LLM in human likeness and highlights dimensions for improvement.
Related Work
Evaluation of LLMs.To better understand LLM's strengths and limitations, many benchmarks are proposed to evaluate broad capabilities.These benchmarks mainly evaluate the LLMs' ability to complete tasks as an assistant AI and can be divided into the following categories (Zhao et al., 2023a).Comprehensive-evaluation bench-marks (Liang et al., 2022;Srivastava et al., 2023;Li et al., 2023a;Choi et al., 2023) are applied to holistically evaluate LLMs on multiple NLP tasks.Human-centric benchmarks (Zeng, 2023;Zhong et al., 2023;Huang et al., 2023;Xu et al., 2023b;Clark et al.) primarily focus on evaluation in human-centric scenarios by collecting qualification exams.In addition, special-ability benchmarks (Ahn et al., 2022;Liu et al., 2023;Li et al., 2023b;Babe et al., 2023;Chalamalasetti et al., 2023) place more emphasis on advanced abilities.Despite the emergence of various benchmarks, no benchmark comprehensively evaluates LLMs as human-like dialogue systems.
Dialogue Benchmarks.There are several benchmarks for evaluating dialogue capabilities (Reddy et al., 2019;Mehri et al., 2020;Gupta et al., 2022).These benchmarks can be used to evaluate language models that have been fine-tuned on the corresponding training sets but cannot directly evaluate instruction-following LLMs.In addition, these previous benchmarks may have been leaked during the pre-training of LLMs.In contrast, Dialog-Bench contains new evaluation instances with natural language, which can be directly used to evaluate instruction-following LLMs and avoid data leakage.Zheng et al. (2023) evaluates LLMs' multiturn instruction-following abilities, which focuses on assessing its alignment with human preference, rather than LLMs as human-like dialogue systems.Recent researchers (Zhao et al., 2023b;Wang et al., 2023b;Rao et al., 2023;Ji et al., 2023;Wang et al., 2023a) also focus on human-like characters of GPT-4 or ChatGPT.However, our work holistically evaluates capabilities related to human likeness.
LLMs for Data Generation.Many recent researches (Whitehouse et al., 2023;Yu et al., 2023;Tang et al., 2023;Xu et al., 2023a;Whitehouse et al., 2023) also leverage GPT-4 for data generation, mainly using several training instances as fewshot examples to prompt the generation of more training instances.In contrast, our work leverages GPT-4 to generate new evaluation instances for constructing benchmarks without few-shot examples.
DialogBench
In this section, our goal is to generate evaluation instances using GPT-4.To this end, in section 3.1, we describe the selection of dialogue tasks.In section 3.2, we describe how to determine the ques- tion type of evaluation instances, like generation questions or multi-choice questions, to effectively reflect the quality of LLMs as human-like dialogue systems.In section 3.3, we design the basic prompt as the input of GPT-4.In section 3.4, we describe the biases of the basic prompt and the corresponding solutions, along with introducing a filter mechanism to pick out high-quality data.The overall architecture of DialogBench construction is shown in Figure 1.
Task Selection
To confirm what capabilities LLMs need to have to be like a human, we refer to the main dimensions that are concerned when evaluating human likeness of open-domain dialogue systems, including coherence, consistency, diversity, and fluency (Mehri and Eskenazi, 2020).Considering that LLMs have made great progress in diversity and fluency, along with having more requirements in correctness and safety (Yuan et al., 2023;Cheng et al., 2023), we refine the evaluation dimensions, including coherence, consistency, correctness, and safety.Consequently, we apply each evaluation dimension as a guide and select tasks that focus on the corresponding evaluation dimension.Accordingly, those abilities can be reflected by the quality of task completion.Specifically, we elaborately tease out 12 dialogue tasks.The detailed selection process and task definitions are presented in Appendix A. The overall selection results are shown in Figure 2.
Question Setting
The selected tasks not only include understanding tasks but also generation tasks, and their corresponding evaluation metrics are different.To unify evaluation, we follow most existing benchmarks (Li et al., 2023a;Hendrycks et al., 2021;Huang et al., 2023) to adopt multi-choice questions and use accuracy as the evaluation metric.Consequently, an evaluation instance requires LLMs to select the correct answer from candidate options based on the given multi-turn dialogue context for the given test question relevant to the specific task.The question templates are shown in Figure 4.
Prompt Formatting
A well-designed prompt helps generate highquality evaluation instances.We create prompts according to the prompt design proposed by Zhao et al. (2023a), which summarizes four key ingredients of prompts and several basic design principles.Specifically, we take slot filling as an example to describe the prompt creation.We first clarify the core content based on these key ingredients and then integrate them into an effective prompt based on the design principles.The detailed creation is described in Appendix B. The final prompt is the exact string that concatenates each content of the four ingredients, shown in Figure 3.
Quality Control
We observe several biases and low-quality instances in generated evaluation instances.Next, we present the corresponding solutions for mitigating biases and filter mechanisms.The optimized prompts for all tasks are shown in Table 9-20.
Basic Prompt
Goal Description [Background Introduction]We are currently testing the annotation capabilities of data annotators for slot filling in multi-turn dialogues.
[Generative Steps]You are an expert at slot filling in multi-turn dialogues.Now act as the test creator.Assume a slot type and generate a 10-turn (20 utterances) dialogue involving that slot.Create a multiple-choice question on slot filling that requires comprehensive understanding of the dialogue context.
Input Data
[Multi-turn dialogues]Multi-turn dialogues are chat records between speakers engaging in continuous interactions using natural language.
[Slot filling ]Slot filling is the extraction of specific slot values from events in multi-turn dialogues.
] [Difficult Setting]The candidate options should be derived from the dialogue, making the incorrect options more confusing.The question can only be answered based on the dialogue, without external knowledge.All candidate options need be similar in length and content.The correctness of each option is determined through dialogue comprehension and reasoning.
Bias Mitigation
The dialogue should be relevant to [DOMAIN].
Before setting question, you need to randomly set the personalities of both speakers, and you have a certain probability of setting the personalities of the speakers to be unfriendly in order to generate the unfriendly, quarreling, offensive, negative, specifying, ironic and weird content.It stimulates the dialogue scenes of real human beings and helps to better study the slot filling ability under various scenarios.
A multi-turn dialogue generated by Speaker1 and Speaker2
A multi-choice question for a certain task Information used to answer the question, such as knowledge or personality The inner ring is the proportion of speaker1's style, while the outer ring is the proportion of the corresponding speaker2's style given speaker1's style.
Bias Mitigation.For domain bias, we first count the number of each domain covered by all evaluation instances.Specifically, we employ GPT-4 to detect the domain that the given dialogue context is about.Their statistics are shown in Figure 6, which show that GPT-4 tends to generate several common domains, leading to a long-tail distribution.This may cause two issues: (1) The imbalanced numbers in each domain will cause the overall results to be biased; (2) The results in domains with insufficient data are not accurate enough.Thus, it is necessary to balance the amount of instances in each domain and ensure that each domain has enough instances.By observing the domains involved in human dialogues, we manually designate 20 domains, mainly involved in two major categories: daily life and professional knowledge.Specifically, we externally introduce the domain into the "input data" of the basic prompt, shown in the right of Figure 3.The domain information is shown in Appendix C.
For style bias, we observe that the generated dialogues have almost no unfriendly dialogue style.We roughly divide dialogue styles into friendly, neutral, and unfriendly, and further calculate the proportion of each style on both speakers of all dialogues.The results are shown in Figure 5(a), which shows that GPT-4 hardly generates unfriendly dialogues via the basic prompt.However, there are quite a few unfriendly dialogues in the real human world.Unfriendly communication would greatly increase the difficulty of interaction, and evaluating LLMs in unfriendly scenarios can reflect the true level of LLMs as human-like dialogue systems.Therefore, we induce GPT-4 to generate a certain proportion of unfriendly dialogues by optimizing the basic prompt.Since the dialogue style is related to the personalities of both speakers, we require For position bias of correct answers, GPT-4 does not guarantee that the correct answers in all generated evaluation questions are evenly distributed among the candidate options.Furthermore, we observe that several LLMs have their selection preferences, shown in Figure 7. Specifically, we calculate the accuracy of these LLMs when placing the correct answers in different positions on the whole evaluation set.Therefore, the accuracy of LLMs may be inaccurate when we apply the evaluation instances that GPT-4 generates without correction.To mitigate position bias, we assign the position of the correct answer among candidate options randomly (Zheng et al., 2023).It can be effective at a large scale with the correct expectations.
Data Filter.The generated evaluation set inevitably contains low-quality instances.Inspired by Zhou et al. (2022), we propose to adopt GPT-4 to filter out low-quality instances.We prompt GPT-4 to check whether the multiple-choice questions are correct.The prompt is displayed in (2) Supervised instruction-tuning LLMs: which mostly release from the academia and companies.Except for GPT-4 and ChatGPT, the remaining are open-sourced LLMs.In addition, we test the human level in these dialogue tasks.Specifically, we randomly choose 50 evaluation instances for each task and then employ 3 experts to do these questions.Finally, a question is considered correct if at least 2 experts answer it correctly.These results can reveal not only the quality of DialogBench but also the human level of this benchmark.
Evaluation Method.For the above LLMs, we use accuracy as the metric and adopt different evaluation methods.(1) Pre-trained LLMs: each option content is independently scored by concatenating it with the instruction along with the given dialogue and question as a prompt and computing the probability of "option content".Specifically, we calculate the perplexity of each option content and then choose the label corresponding to the option content with the lowest perplexity as the predicted answer.This evaluation method is consistent with the training method of pre-trained LLMs (i.e., next token prediction), stimulating the optimal performance of LLMs.
(2) Supervised instruction-tuning LLMs: We regard the given dialogue as the history of chatting between the user and the LLM.In the current interaction turn, we concatenate the instruction, along with the question and all options to form an exact string as the user's question to the LLM, and then the LLM gives the option label.In implementation, we allow LLMs to output at most 256 tokens, and then extract the outputted label as the predicted answer.
Implementation Details.We further describe the parameter settings for GPT-4 when generating data and LLMs to be evaluated.When using GPT-4 to generate evaluation instances, we set temperature to 1, presence_penalty to 0.6, frequency_penalty to 0, and other parameters to default for the API parameters of GPT-4.When evaluating LLMs, we set the temperature to 0, the presence_penalty to 0.6, and the frequency_penalty to 0 for Chat-GPT and GPT-4.Besides, the temperature is set to 0, max_new_tokens is set to 256, and other parameters to default for other open-source models.Furthermore, the versions of ChatGPT and GPT- 4 we use in our work are gpt-3.5-turbo-0613and gpt-4-0314.We implement our code using Pytorch 2 and Huggingface 3 and experiment on A100 80GB GPUs, spending an average of 20 minutes to 2 hours on each task while inferring with open-source models.We also show the evaluation prompts in Appendix D.2.
Main Results
Overall and task-specific scores on English and Chinese DialogBench are reported in Table 2 and 3.The overall score of all LLMs on English Dialog-Bench is slightly better than the score on Chinese DialogBench.Additionally, the overall performance of all LLMs on each task generally has the same trend on English and Chinese DialogBench.
We further observe that: (1) For correctness, most pre-trained LLMs can perform well on slot filling (SF) but are relatively poor on the other 3 tasks.(2) For personalization consistency, pretrained LLMs as a whole have good performances in emotion perception (ED), whereas poor performance in personality following (PRG).For semantic consistency, the decent performance on dialogue summarization (DS) indicates that pretrained LLMs perform well in maintaining semantic alignment.However, it is still relatively difficult in scenarios that require one-step reasoning, as shown by the performance on dialogue NLI.(3) For coherence, the average performance of LLMs on dialogue infilling (DI) and multi-turn response generation (MRG) is relatively similar, and there is still much room for improvement.(4) For offensive detection (OD), most pre-trained LLMs can empower a certain capability of offensive detection.Overall, current pre-trained LLMs perform relatively well on correctness-related tasks and have greater room for improvement on tasks related to coherence and safety.For consistency-related tasks, pre-trained LLMs must be continuously optimized to possess corresponding high-order capabilities.Supervised Instruction-tuning LLMs.We begin by observing that GPT-4 presents the best performance, which represents the strongest capabilities as a human-like dialogue system.Additionally, the instruction-tuning LLMs achieve higher scores than the corresponding pre-trained LLMs on most dialogue tasks (e.g., Baichuan2-13B), suggesting that instruction tuning is an efficient means for improving the capabilities that LLMs as human-like dialogue systems should have.
We further observe that: (1) For correctness, most LLMs perform relatively well on all 4 tasks, indicating that these LLMs have decent abilities to generate correct dialogues.(2) For personalization consistency, most LLMs perform unsatisfactorily.Interestingly, most LLMs achieve inferior scores on emotion classification than the corresponding pre-trained LLMs, such as QWen-7B.It might be because the positioning of assistant AI enables instruction tuning to focus on the ability to complete tasks, abandoning the ability to perceive emotions.
(3) For coherence and safety, although instruction tuning has enhanced the LLMs' abilities, there is still much room for improvement.Overall, there is the same trend on different evaluation dimensions for supervised instruction-tuning LLMs as pre-trained LLMs.Due to space limitations, a more detailed experimental analysis is in Appendix E.
Further Discussion
We probe LLMs' performance for different domains and validate the effectiveness of adjusting dialogue style and introducing filtering mechanisms.
Performance on Different Domains.We calculate the average accuracy of all supervised instruction-tuning LLMs on each domain, as shown in Figure 8.The detailed results are displayed in Table 8.We observe that the average performance in daily life is overall lower than that in professional knowledge (e.g., 52.14% vs. 56.07%).We speculate that this is related to the current positioning of supervised instruction-tuning as assistant AI.Assistant AI needs to follow instructions to complete various knowledge-based tasks, which particularly requires LLMs to master a variety of professional knowledge.Correspondingly, information relevant to the daily life of humans might be underestimated when fine-tuning LLMs.This suggests that improving the human-likeness of LLMs as dialogue systems requires introducing more daily dialogues into supervised fine-tuning.
Ablation Study.We perform the following ablation tests to validate the effect of each component: (1) Remove the description of mitigating the style bias in the prompt (-Styles); (2) Remove the filter mechanism (-Filter).We use GPT-4 to conduct this experiment.The results are shown in Table 4.We observe that: (1) The accuracy improves to varying degrees without mitigating the style bias, which validates that unfriendly communication would greatly increase the difficulty of interaction.(2) The accuracy has dropped to varying degrees, indicating that the filtered instances are indeed incorrect and LLMs cannot answer.
Conclusion
We present DialogBench, a systematically designed dialogue benchmark for evaluating LLMs as human-like dialogue systems.DialogBench includes 12 dialogue tasks to probe the capabilities related to human likeness for comprehensive evaluation.For each task, we prompt GPT-4 to generate evaluation instances.Specifically, we design the basic prompt based on widely-used design principles and further eliminate existing biases to generate higher-quality instances.An extensive study of 26 LLMs, including pre-trained and supervised instruction-tuning, is conducted in Chinese and English DialogBench.We unveil that instruction finetuning can improve the human likeness of LLMs to a certain extent.However, there is still a long way to go for most LLMs as human-like dialogue systems.In addition, LLMs are generally better at understanding context, but relatively poor at perceiving emotions and personality.We expect Di-alogBench to serve as a cornerstone for future study to develop better human-like LLMs.
Limitations
Multilingual Benchmark Expansions.Dialog-Bench can only be used to evaluate English and Chinese LLMs, and cannot evaluate LLMs in other languages.However, our proposed evaluation framework is applicable to all LLM evaluations, which only need to use the top-tier LLM of the corresponding language as a data generator to construct evaluation instances for quickly building a testbed.
Additional Dimensions and Dialogue Tasks.
Human-like dialogue systems require a variety of fine-grained capabilities to ensure long-term connections with users.Although we conduct extensive literature references to select comprehensive dimensions and dialogue tasks, we fully acknowledge that some other dimensions and dialogue tasks were not included in our benchmark.In addition, we employ GPT-4 as a data generator, which sets restrictions on the selection of dimensions and dialogue tasks.some researchers (Chang et al., 2023;Bubeck et al., 2023) have highlighted clear limitations of GPT-4, including limited reasoning, output length limit, and toxic content generation.Therefore, we pay more attention to dimensions and dialogue tasks that GPT-4 experts in.
Technical limitations.Due to limited computational and financial resources, we only include pretrained LLMs with no more than 70B and supervised instruction-tuning LLMs with no more than 20B in DialogBench's first edition of evaluation.
Although recent research suggests that when LLMs expand beyond a certain threshold, they may begin to exhibit emerging capabilities (Wei et al., 2022a), we were unable to test all very large language models.We welcome future researchers to study our benchmarks and evaluate LLMs as human-like dialogue systems.
Reproducibility of Closed Access Models.Some of the LLMs (e.g., ChatGPT and GPT-4) being evaluated are only accessible through a programming interface that essentially adds a black box on top of a black box.The mechanisms behind these interfaces may change at any time, so the evaluation results from different periods may vary arbitrarily.
Ethics Statement
Since GPT-4 is trained on online data, GPT-4 may encode biases that perpetuate stereotypes, discrimination, or marginalization of specific languages or communities.This results in DialogBench potentially generating toxic and harmful instances Furthermore, we induce GPT-4 to generate a certain proportion of unfriendly dialogues for evaluating LLMs in unfriendly scenarios, which can reflect the true level of LLMs as human-like dialogue systems.Accordingly, this might lead to some unkind and harmful instances.In addition, we employ three experts to manually do these evaluation questions.We pay 0.2 to each expert for each instance.
A.1 Selection Process
We apply evaluation dimensions, including coherence, correctness, consistency and safety as guides and elaborately select tasks that focus on the corresponding evaluation dimension.Accordingly, those abilities can be reflected by the quality of task completion.The detailed selection process is provided as follows: • For coherence, We elect two tasks that are often focused on in the dialogue field, dialogue infilling (Xue et al., 2022) and multi-turn dialog generation (Li et al., 2017).
• For correctness, we follow Zhao et al.Conversely, the open-scenario correctness provides a testbed for probing the knowledge encoded by LLMs.We mainly select the ability to use commonsense correctly which is necessary for human-like dialogue systems, i.e., commonsense-aware response generation (Zhou et al., 2021).
• For consistency, it mainly falls into two dimensions, including personalization consistency and semantic consistency (Huang et al., 2020).For personalization consistency, we focus on capabilities necessary for real-human interactions, containing emotional perception, personality following, and relationship maintaining between speakers.As a result, we prioritize emotion detection • For safety, some researchers (Chang et al., 2023;Bubeck et al., 2023) have highlighted clear limitations of GPT-4, including toxic content generation.Therefore, we currently prioritize an important task that GPT-4 experts in, i.e., offensive detection (Dinan et al., 2019).
Consequently, we tease out 12 dialogue tasks.The overall selection results are shown in Figure 2. Please see Appendix A.2 for detailed task definitions.
A.2 Task definitions
The detailed task definitions are shown in Table 5.
B Prompt Formatting
Firstly, we clarify the core content of our prompt according to four key ingredients.The key ingredients depict the functionality of a prompt for eliciting the abilities of GPT-4 to complete the goal, including goal description, input data, contextual information, and prompt style.
• Goal description.The goal description is typically a specific instruction that GPT-4 is expected to follow.For a given dialogue task, we design the following information in natural language to describe the goal, including the background introduction and the generative step of the evaluation questions.By providing a well-clarified goal description, GPT-4 can more effectively understand the goal and generate the desired output.
• Input data.The input data provides the necessary information to guide the output generation that meets the requirements.The requirements primarily involve the difficulty of the evaluation instance.Inspired by human-level qualification exams, we heuristically set up the construction techniques for candidate options, formatted by the exact string, to control the difficulty.The clear and detailed input data allows GPT-4 to produce more controllable evaluation instances.
• Contextual information.In addition, contextual information is also essential to make prompts clear.In our creation, we find that
Intent Classification
Intent classification is a task of identifying which action the user wishes to take based on the dialogue context (Louvan and Magnini, 2020).
Slot Filling
Slot filling is a task that maps the input slot key to the corresponding slot value based on the given dialogue context (Chen et al., 2017).
Emotion Detection
Emotion detection is a task of classifying the emotion of a speaker on a specific event in a dialogue (Acheampong et al., 2020).
Personality-grounded Response Generation
Personality-grounded response generation is a task that generates an appropriate response that is consistent with the personality characteristics of the dialogue context (Ma et al., 2020).
Multi-turn Response Generation
Multi-turn response generation is a task of generating a coherent response given a dialogue context (Li et al., 2017).
Dialogue Summarization
Dialogue summarization is the process of extracting, summarizing, or refining key information from a multi-turn dialogue, turning it into a summary paragraph that can be used to present the main points of that multi-turn dialogue(Feng et al.).
Commonsense-aware Response Generation
Commonsense-aware response generation is a task of generating an appropriate response incorporating correct commonsense knowledge (Zhou et al., 2021).
Dialogue Infilling
Dialogue infilling is a task of infilling the missing utterance of the given dialogue that is consistent with the preceding and subsequent context (Xue et al., 2022).
Offensive Detection
Offensive detection is a task that detects whether utterances contain uncivil, discriminatory, or aggressive content in the given dialogue (Dinan et al., 2019).
Dialogue Natural Language Inference
Dialogue natural language inference is a task of inferring the semantic relationship between a certain part of a dialogue and a given hypothesis, including entailment, contradiction, and neutral (Welleck et al., 2019).
Relation Classification
Relations classification is a task of inferring the interlocutor's interpersonal relationship from the information implied in the dialogue (Jia et al., 2021).
Table 5: The definitions of all selected dialogue tasks.
it is necessary to provide some contextual information for explaining specific concepts appearing in the designed prompt.Therefore, we introduce the definition of multi-turn dialogue and the description of the dialogue task specifically to better depict our goal.
• Prompt style.A suitable prompt style can decompose the difficult task into several detailed sub-tasks to help GPT-4 accomplish the goal step by step.Inspired by this, we introduce the chain-of-thought (CoT) technique (Wei et al., 2022b), which guides GPT-4 to generate evaluation instances step by step according to the order of the dialogue context, the task question, the candidate options, the problemsolving analysis, and the answer.
When constructing each content of the four key ingredients, We mainly refer to the following design principles: (i) expressing the goal clearly, (ii) decomposing into easy, detailed sub-tasks, and (iii) utilizing a model-friendly format.These design principles help create prompts that are clearer and easier to understand.The final prompt is the exact string that concatenates each content of the four ingredients, as shown in Figure 3.In addition, the prompts of data generation for all tasks are listed in Table 9-20.
C Domain Bias
The detailed descriptions of each domain are shown in Table 8.The selected domain involves two categories: daily life and professional knowledge.Daily life mainly covers gourmet cooking, travel, household chores, film, neighborhood, workplace, music, shopping, games, and sports; while professional knowledge covers history, philosophy, sociology, psychology, economics, geography, physics, biology, computer science, and medicine.The detailed descriptions we give are the specific topics that are typically talked about in each domain.
D.1 LLMs to evaluate
Table 6 shows the details of pre-trained or supervised instruction-tuning LLMs for evaluation.
D.2 Evaluation Prompt Setup
We evaluate LLMs in answer-only and zero-shot settings.Prompts used for two types of LLMs are shown in Figure 9 and Figure 10 respectively.Similar to the evaluation method, we use different instructions to induce LLMs to generate answers.
E Main Results
A central objective for our evaluation is to achieve a common and unified understanding of the corresponding capabilities of LLMs as human-like dialogue systems.We first evaluate the pre-trained LLMs using DialogBench to provide a baseline of LLMs' capabilities as human-like dialogue systems.Further, we evaluate the supervised instruction-tuning LLMs and analyze the impact of instruction fine-tuning on LLMs as human-like dialogue systems.
E.1 Pretrained LLMs
Overall and task-specific scores in Chinese and English DialogBench are reported at the top of Table 3 and 2 respectively.The overall score of all LLMs on English DialogBench is slightly better than the score on Chinese DialogBench.Additionally, the overall performance of all LLMs on each task generally has the same trend on Chinese and English DialogBench.Next, we mainly conduct analysis based on the results on Chinese DialogBench.We first give an overall analysis and further highlight findings at the task level from evaluation dimension perspectives.
Overall Analysis.On this challenging benchmark, surprisingly we discover that some pretrained LLMs have pretty good performances.Specifically, Baichuan2-13B presents the best performance, scoring an overall accuracy of 54.43% on DialogBench.Qwen-7B and InternLM-7B follow closely behind with overall accuracy scores of 53.63% and 53.37% respectively.For other pre-trained LLMs, despite their relatively poorer performance, most of them can score above 43%.
It suggests that the model scale is monotonically correlated with the model accuracy win rate within a model family.
Dimension-specific Analysis.For the 4 tasks in the correctness dimension, the average accuracy scores of all pre-trained LLMs are 72.63% for slot filling (SF), 61.61% for knowledge-grounded response generation (KRG), 54.55% for intent classi- Assistant: Well, I can't help it.Work leave is at that time.
User: Well, You are rich.I'm just going to a small mountain village in Arizona at the end of this month.
Assistant: Are there any attractions nearby?
User: Not really.But the only good thing is that I can experience the pristine environment and the fresh air.
Assistant: That's good too.I hope you're having fun.
User: Thank you.I can only imagine how comfortable you'll be in Hawaii during the high season.
Assistant: Ha ha.Yeah, I hope it's not too hot.
User: Don't forget your sunscreen!Heat and sun exposure, careful to become cooked meat!Assistant: Don't worry, I will take care of the sun protection.
A
Figure 10: An evaluation prompt for supervised instruction-tuning LLMs is an exact string, i.e., "Based on the content of the above dialogue, only output the option letter corresponding to the correct answer in the candidate options according to the test question.[TestQuestion]{test_question}[Options]{option_str}".We regard the given dialogue as the history of chatting that has occurred between the user and the LLM.In the current interaction turn, this evaluation prompt is regarded as the user's question to the LLM, and then the LLM gives the option label.We take slot filling as an example.The purple text is the content generated by LLMs.
fication (IC), and 51.81% for commonsense-aware response generation (CRG).Accordingly, the best scores are 81.84%,67.93%, 61.79%, and 60.57% respectively.As these suggest, most pre-trained LLMs can perform well on slot filling but have relatively poor performance on the other 3 tasks about correctness.Furthermore, we observe that the margin varies across different tasks: the largest margin is on knowledge-grounded response generation (KRG) where LLaMA2-70B achieves an accuracy of 67.93% compared to second place from Baichuan2-13B at 63.18%, whereas the smallest margin is for slot filing (SF), i.e., 81.84% for LLaMA2-70B vs. 80.91% for QWen-7B.In general, the margins between the various pre-trained LLMs are not significant, which indicates that these pre-trained LLMs have modestly different performances in correctness-related abilities.
For the 5 tasks in the consistency dimension, we divide these tasks into two groups for analysis, including personalization consistency and semantic consistency.For the 3 tasks about personalization consistency, the average accuracy scores of all LLMs are 55.09% for emotion detection (ED), 29.97% for relation classification (RC), and 26.46% for personality-grounded response generation (PRG).The best scores are 69.93%,56.89%, and 28.90% respectively.These show that pretrained LLMs as a whole have good performances in emotion perception, whereas the performance on personality following is unsatisfactory.We speculate that the pre-trained LLMs have not seen many instances related to personality following.The finding about personality is consistent with (Safdari et al., 2023).For the 2 tasks about semantic consistency, the average accuracy scores of all LLMs are 59.45% for dialogue summarization (DS) and 36.49% for dialogue NLI (NLI).The best scores are 69.52%, and 45.11% respectively.The decent performance on dialogue summarization indicates that pre-trained LLMs perform well in maintaining semantic alignment, however, it is still relatively difficult to maintain semantic consistency that requires one-step reasoning, as shown by the performance on dialogue NLI.Overall, we see significant heterogeneity across the results on consistencyrelated tasks, which may be because each task requires different levels of abilities.
For the 2 tasks in the coherence dimension, the average accuracy scores of all LLMs are 44.01%for dialogue infilling (DI) and 47.22% for multiturn response generation (MRG), along with the best scores of 47.56% and 54.58% respectively.It indicates that the average performance of LLMs on both tasks is relatively similar, and there is still much room for improvement in maintaining dialogue coherency.For offensive detection (OD) in the safety dimension, the average accuracy of all LLMs is 53.44% and the best performance is an accuracy of 62.57%, which shows that most pre-trained LLMs have scores around 50% and empower a certain capability of offensive detection.
Overall, current pre-trained LLMs perform relatively well on correctness-related tasks and have greater room for improvement on tasks related to coherence and safety.For consistency-related tasks, it is necessary for pre-trained LLMs to continue to be optimized to possess corresponding high-order capabilities.
E.2 Supervised Instruction-tuning LLMs
The overall and task-specific scores in Chinese and English DialogBench are reported at the bottom of Table 3 and 2. We also conduct an analysis based on the results on Chinese DialogBench, along with giving an overall analysis and task-level analysis respectively.Additionally, we analyze the performance changes of pre-trained LLMs and instruction-tuning LLMs in the same model family on different tasks.
Overall Analysis.As shown in Table 3, the results show that the overall performances of different models are different, and the performance of the same LLM on different dialogue tasks also varies widely.We further observe that: (1) GPT-4 presents the best performance on overall accuracy with 81.54%, which basically represents the best performance that existing supervised instructiontuning LLMs can achieve.This excellent score also indicates that GPT-4 has strong capabilities as a human-like dialogue system.In addition, ChatGPT achieves an overall scores of 70.42%, ranking second.(2)They are closely followed by Baichuan2-13B-Chat with 66.44%, and InterLM-Chat-7B with 64.40%.The performance gap between GPT-4 and the best open-source LLMs (81.54% vs. 66.44%)shows that there is still much room for improvement in the capabilities that LLMs should have as human-like dialogue systems.Compared with ChatGPT, Baichuan2-13B-Chat have achieved better performances on 3 out of 12 tasks, which indicates that Baichuan2-13B-Chat currently has pretty good capabilities related to human likeness.(3) The instruction-tuning LLMs achieve higher scores than the corresponding pre-trained LLMs on most dialogue tasks (e.g., QWen-7B vs. QWen-7B-Chat, Baichuan2-13B-Base vs. Baichuan2-13B-Chat), which suggests that instruction tuning is an efficient and effective means for improving the capabilities that LLMs should have as human-like dialogue systems.
Dimension-specific Analysis.For the 4 tasks in the correctness dimension, GPT-4 and ChatGPT achieve scores of over 81.46% and 76.69% on all tasks, including slot filling (SF), intent classification (IC), knowledge-grounded response generation (KRG), and commonsense-aware response generation (CRG), which demonstrates that it is not unachievable currently to improve the correctnessrelated capabilities of LLMs.Most other LLMs (e.g., Qwen-7B, ChatGLM, Baichuan variants, In-ternLM) also have impressive results on these tasks, with the average accuracy remaining around 73.13%, 70.19%, 74.07%, and 66.31% respectively.This shows that most supervised instructiontuning LLMs can understand the intent and slot in the dialogue context, along with selecting appro-priate knowledge or commonsense for generating responses with reasonable accuracy.In addition, the supervised instruction-tuning LLMs achieve higher scores than the pre-trained LLMs in the same model family (e.g., QWen, Baichuan2, and In-ternLM) on almost all corresponding tasks, which indicates that instruction finetuning benefits LLMs improving those capabilities related to correctness.However, there is no such improvement in slot filling (SF), probably because this task is simple enough and the pre-trained LLMs already have quite good capabilities.
For the 5 tasks in the consistency dimension, we also analyze personalization and semantic perspectives respectively.For the 3 tasks about personalization consistency, GPT-4 only achieves 61.90% and 72.83% in emotion detection (ED) and personalitygrounded response generation (PRG), and Chat-GPT obtains relatively inferior scores correspondingly.Most other LLMs also perform unsatisfactorily on these two tasks, with the average scores of the remaining LLMs around 39.19% and 34.96%.We speculate that the current positioning of LLMs is assistant AI, which would weaken the LLMs' abilities of emotional perception and personality following.Relatively speaking, LLMs perform relatively better on relation classification (RC), with an average score of all LLMs except GPT-4 and ChatGPT around 40.93%.But overall, all LLMs still have much room for improvement in tasks related to personalization consistency.Interestingly, most supervised instruction-tuning LLMs achieve inferior scores on emotion classification than the pre-trained LLMs in the same model family, such as QWen-7B, Baichuan2-13B and InternLM-7B.It might be due to the fact that the positioning of assistant AI enables instruction tuning to focus on the ability to complete tasks, abandoning the ability to perceive emotions.For the 2 tasks about semantic consistency, there is the same conclusion that LLMs perform well on dialogue summarization (DS), e.g., GPT-4 with a score of 90.22%, but perform relatively poorly on dialogue NLI (NLI) that requires one-step reasoning (e.g., GPT-4 with a score of 75.39%).In addition, we observe that instruction tuning can also generally improve consistency-related capabilities.
For the 2 tasks in the coherence dimension, GPT-4 achieves scores of 79.22% and 77.75% on dialogue infilling (DI) and multi-turn response generation (MRG) respectively.Accordingly, Chat-GPT achieves scores of 65.53% and 70.72% respectively.The other LLMs have relatively inferior performances, with the average accuracy of 48.20% and 50.35%.Furthermore, instruction tuning also improves coherence-related capabilities compared with the pre-trained LLMs and the supervised instruction-tuning LLMs in the same model family.These indicate that although instruction tuning has enhanced the LLMs' ability to generate coherent responses to a certain extent, there is still much room for improvement.For offensive detection (OD) in the safety dimension, GPT-4 and ChatGPT obtain scores of 83.15% and 61.50% respectively, which suggest that there is still room for research on this task.In addition, Some top LLMs (e.g., Baichuan2-13B and QWen-7B) achieve higher scores via instruction tuning, however, this improvement does not appear on other LLMs.
Overall, there is the same trend of performances on different evaluation dimensions for supervised instruction-tuning LLMs as pre-trained LLMs.The difference is that supervised instruction-tuning LLMs generally have stronger performances than the corresponding pre-trained LLMs.
Prompt for Intent Classification
We are currently testing the annotation capabilities of data annotators for intent classification in multi-turn dialogues.Multi-turn dialogue refers to a chat record generated by two speakers engaging in continuous interactions using natural language.Intent classification refers to determining a specific view, plan, or action to be taken by a speaker from the information reflected in the multi-turn dialogue.
You are an expert at intent classification in multi-turn dialogues, and now please act as the test creator.Firstly, please generate a two-party dialogue with at least 10 turns(10 turns=20 utterances), followed by a multi-choice question of intent understanding that requires a comprehensive understanding of the context to answer.The dialogue content should be relevant to {Domain}.
Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings and helps to better study the intent understanding ability under the various scenarios.
When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The last turn of the dialogue is not the end of the dialogue session.The multi-choice question you generated should be as difficult as possible, requiring a comprehensive understanding of the whole dialogue rather than a certain turn to answer.Meanwhile, the information involved in candidate options should be from the dialogue, which makes the wrong options even more confusing.The question can be answered only based on the dialogue without external knowledge.Please especially note that the correct answer should not be a snippet of the dialogue, which makes the question too simple.All candidate options need to be similar in terms of length and content to make it more difficult to distinguish between correct and wrong candidates.All candidate options can be determined their correctness after comprehensive understanding and extensive reasoning on the dialogue.
Prompt for Emotion Detection
We are testing data annotators on their annotation capabilities for emotion classification multi-turn dialogues.A multi-turn dialogue is a chat transcript produced by multiple turns of sustained interaction between two speakers using natural language.Emotion detection refers to classifying the emotion of a speaker on a specific event from the information reflected in the multi-turn dialogue.
You are an expert at emotion classification in multi-turn dialogues, and now please act as the test creator.You begin by assuming the emotions of both sides of the dialogue, with emotions limited to eight types: disgust, fear, disappointment, neutrality, anger, sadness, joy, and surprise.Then please generate a two-party dialogue with at least 10 turns(10 turns=20 utterances) reflecting the emotions of both sides, followed by a multi-choice question of emotion classification that requires a comprehensive understanding of the context to answer.The dialogue content should be relevant to {Domain}.
Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings and helps to better study the intent understanding ability under the various scenarios.
When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The content of the last turn of the sub-dialogue is not a closing statement.Your questions must be as difficult as possible, and the dialogue content does not literally reflect any candidate options directly, but by analyzing the content or the tone of voice in the dialogue, etc., you can clearly determine which candidate emotions are correct.
Prompt for Commonsense-aware Response Generation
We are currently testing the annotation capabilities of data annotators for response selection in multi-turn commonsense-aware dialogues.Multi-turn dialogue refers to a chat record generated by two speakers engaging in continuous interactions using natural language.Commonsense, namely generic knowledge, refers to the basic knowledge that a mentally and physically grown-up adult should possess to live in society, including survival skills (self-care ability), basic labor skills, common knowledge in natural science, humanities and social science, etc.The commonsense-aware response selection in multi-turn dialogue refers to selecting the response by annotators according to the context as the response to the utterance by speaker1 in the last turn, which uses correct commonsenses.
You are an expert at commonsense-aware response selection in multi-turn dialogues, and now please act as the test creator.Firstly, please generate a two-party dialogue with at least 10 turns(10 turns = 20 utterances) and a piece of commonsense, followed by a multi-choice question based on this common sense.The dialogue content should be relevant to {Domain}.
Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings, and helps to better study the response selection ability in multi-turn commonsense-aware dialogues under the various scenarios.
When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The last turn of the dialogue is not the end of the dialogue session.The last turn of the two-party dialogue you generate contains only the speaker1's utterance, not the speaker2's response.The multi-choice question you generated should be as difficult as possible.The content of the last turn of dialogue must be related to the whole dialogue, not only to the utterances of the most recent turns.All candidates must include the commonsense.At the same time, all of the candidates seem to be plausible responses, but only one commonsense they contain is correct.This commonsense is what a sane adult living in society can judge between right and wrong.None of the candidate options can be literally similar or overlap with the generated commonsense.
Prompt for Knowledge-grounded Response Generation
We are currently testing the annotation capabilities of data annotators for response selection in multi-turn knowledge-grounded dialogues.Multi-turn dialogue refers to a chat record generated by two speakers engaging in continuous interactions using natural language.The knowledge-grounded response selection means that according to the speaker1's response in the last turn of the multi-turn dialogue, the annotators must select the most correct and appropriate response from the candidates, considering the context of the multi-turn dialogue and the given background knowledge.You are an expert at knowledge-grounded response selection in multi-turn dialogues, and now please act as the test creator.You first need to generate a paragraph of background knowledge of more than 1000 words, then generate more than 10 turns(10 turns=20 utterances) of two-party dialogue on the topic of background knowledge.Furthermore, you should write a multi-choice question that requires you to select the correct knowledge from the background knowledge to answer.The dialogue content and background knowledge are required to be relevant to {Domain}.
Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings and helps to better study the response selection ability in multi-turn knowledge-grounded dialogues under various scenarios.
When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The last turn of the dialogue is not the end of the dialogue session.The last turn of the two-party dialogue you generate contains only the utterance of speaker1, not the response of speaker2.The response in the last turn can only be generated based on the dialogue and background knowledge, with no additional external knowledge required.The multi-choice question you generated should be as difficult as possible.All candidates must be connected to the dialogue, and the knowledge used must come from background knowledge.However, none of the candidate options can literally resemble or overlap the background knowledge.In particular, the wrong option cannot be a negative expression of a piece of background knowledge.All candidate options require comprehensive understanding and extended reasoning dialogue, and background knowledge to determine the plausibility of the options.In addition, all candidate options must be similar enough in terms of length and content, making it more difficult to distinguish between right and wrong candidate options.
Prompt for Dialogue Natural Language Inference
We are currently testing the annotation capabilities of data annotators for dialogue natural language inference in multi-turn dialogues.Multi-turn dialogue refers to a chat record generated by two speakers engaging in continuous interactions using natural language.Natural language inference refers to giving an utterance of a speaker in a multi-turn dialogue as a premise and giving a hypothesis at the same time, and semantic analysis is carried out to determine the semantic relationship between the premise and the hypothesis, including entailment, contradiction, and neutral.You are an expert at natural language inference in multi-turn dialogues, and now please act as the test creator.You first need to assume a semantic relationship of {Relation}.
Then, you should generate a two-party dialogue with at least 10 turns(10 turns = 20 utterances) and specify an utterance of a speaker at a turn as the premise.At the same time, you should generate a hypothesis that fits the specified semantic relationship.Finally, write a multi-choice question of dialogue natural language inference that requires a comprehensive understanding of the context of multi-turn dialogue.The dialogue content should be relevant to Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings and helps to better study the dialogue natural language inference ability under various scenarios.
When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The last turn of the dialogue is not the end of the dialogue session.The multi-choice question you generated should be as difficult as possible.When the semantic relation belongs to entailment, the hypothesis can be fully inferred from the premise.When the semantic relation belongs to contradiction, the premise can fully infer the negation of the hypothesis.When the semantic relation belongs to neutral, the premise neither entails nor contradicts the hypothesis, and the semantic relation between the premise and hypothesis belongs to other cases except entailment and contradiction.
Table 13: The prompt for data generation of dialogue natural language inference by GPT-4.
Prompt for Offensive Detection
We are currently testing the annotation capabilities of data annotators for offensive detection in multi-turn dialogues.Multi-turn dialogue refers to a chat record generated by two speakers engaging in continuous interactions using natural language.Offensive detection refers to judging whether utterances of the speaker contain uncivil, discriminatory, or aggressive content in a multi-turn dialogue.
You are an expert at offensive detection in multi-turn dialogues, and now please act as the test creator.You first limit the dialogue generated to {contain or not contain} offensive remarks.Then, you should generate a two-party dialogue with at least 10 turns(10 turns = 20 utterances) that meets the above requirements.Finally, you need to generate a multi-choice question about offensive detection in the multi-turn dialogue.The dialogue content should be relevant to {Domain}.
Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings and helps to better study the offensive detection ability under various scenarios.
When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The last turn of the dialogue is not the end of the dialogue session.The multi-choice question you generated should be as difficult as possible.The content of the dialogue may contain some offensive statements.If the dialogue you generate contains offensive statements, one turn will be offensive at most, helping make your question difficult.The offensive statement you generate cannot be literally offensive in order to increase the difficulty of the test.
Prompt for Personality-grounded Response Generation
We are testing data annotators on their annotation capabilities for response selection in multi-turn personality-grounded dialogues.A multi-turn dialogue is a chat record generated by multiple turns of continuous interaction between two speakers using natural language.The persona-grounded response selection refers to the fact that for the last turn of the multi-turn persona-grounded dialogues, the annotator needs to combine the multi-turn dialogues context and the given speaker2's personality to select the most appropriate response from the candidate responses that reflects the given persona-grounded dialogues.
You are an expert at persona-grounded response selection in multi-turn dialogues, and now please act as the test creator.You first need to generate a detailed personality of speaker2, followed by a two-person dialogue with more than 10 turns, where the last turn of the two-person multi-turn dialogue contains only speaker1's words and not speaker2's response.Then a response selection multi-choice question in multi-turn persona-grounded dialogues is also generated, where each candidate option is a candidate response from speaker2 in response to the dialogues above.The dialogue is required to be related to {Domain}.
Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings and helps to better study the intent understanding ability under the various scenarios.When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The content of the last turn of the sub-dialogue is not a closing statement.The questions you come up with must be as difficult as possible, and the content of the last turn's dialogue must be coherent with the entire dialogue, not just the sentences from the most recent turn.At the same time, the words of speaker1 in the final turn must be able to detect the personality of the other speaker in a targeted way.For this reason, the content of the last turn cannot be the closing sentence of the entire dialogue.None of the candidate options can literally resemble or overlap with that personality.In addition, the candidate choices are all coherent and reasonable responses to the above part of the dialogue, and they differ only in personality, which can be clearly distinguished after a comprehensive understanding of the entire dialogue and the personality.The choice of which option is the correct answer is also based only on the degree of personality.
Prompt for Dialogue Infilling
We are currently testing the annotation capabilities of data annotators for dialogue infilling in multi-turn dialogues.Multi-turn dialogue refers to a chat record generated by two speakers engaging in continuous interactions using natural language.Dialogue infilling means, in the last turn of a multi-turn dialogue, the utterance spoken by speaker1 is unknown, and the response spoken by speaker2 is known.Then, the annotator needs to predict what utterance spoken by the speaker1 should be in the last turn in consideration of the context of the multi-turn dialogue and the response spoken by the speaker2.You are an expert at dialogue infilling in multi-turn dialogues, and now please act as the test creator.Firstly, please generate a two-party dialogue with at least 10 turns(10 turns = 20 utterances).Then, you generate 4 candidate utterances spoken by the speaker1 based on the dialogue content.Finally, randomly select one of the candidate utterances of speaker1 to generate a response from speaker2 who can answer the utterance.The dialogue content should be relevant to {Domain}.Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It stimulates the dialogue scenes of real human beings, and helps to better study the dialogue-infilling ability under the various scenarios.When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The last turn of the dialogue is not the end of the dialogue session.The multi-choice question you generated should be as difficult as possible.The utterance that you generated spoken by speaker1 must be related to the whole dialogue so that the annotators must rely more on the dialogue context to make decisions.In addition, each candidate of the multi-choice question is as similar as possible, but there are good and bad differences between the options, and these differences can be distinguished by a comprehensive understanding of the dialogue above and the response spoken by speaker2 in the last turn.Finally, there should be no literal similarity or overlap between speaker1's utterance and speaker2's responses that you generate, lest the annotator filter the answers directly by literal similarity.The format of the question is as follows: {"id": "xx", "task": "Dialogue Infilling", "domain": "{Domain}", "speaker1 personality": "xx", "speaker2 personality": "xx", "dialogue": [{"speaker1": "xx", "speaker2": "xx"}, {"speaker1": "xx", "speaker2": "xx"}, {"speaker1": "xx", "speaker2": "xx"}, ..., {"speaker1": "xx", "speaker2": "xx"}], "option": "xx", "analysis": "xx", "label": "xx", "response": "xx"}.Where "id" denotes a randomly generated id, "speaker1 personality" denotes the character personality of speaker1, "speaker2 personality" denotes the character personality of speaker2, "dialogue" denotes more than 10 turns of two-person English dialogue where the two speakers are represented by speaker1 and speaker2 in each turn of interaction, it does not include the perosn1's utterance to be completed and the following speaker2's response."option" denotes a dictionary that contains 4 candidate options, with capital letter sequence identifiers as the key.Each of these options is a possible candidate for speaker1's utterance."analysis" denotes the reason for choosing that correct option."label" denotes the correct option represented by a capital letter.Note that the generated question should be in JSON format.Now, please set the question.
Prompt for Relation Classification
We are currently testing the annotation capabilities of data annotators for relation classification in multi-turn dialogues.Multi-turn dialogue refers to a chat record generated by two speakers engaging in continuous interactions using natural language.Relation classification means predicting the relationship type between the two dialogue speakers based on the context of the multi-turn dialogue.
You are an expert at relation classification in multi-turn dialogues, and now please act as the test creator.Firstly, you should assume the relationship between the two sides of the dialogue is {Relation}.Then, you generate a two-party dialogue with at least 10 turns(10 turns = 20 utterances) that fits the relationship.It is followed by a multi-choice question of relation classification that requires a comprehensive understanding of the context to answer.The dialogue content should be relevant to {Domain}.
Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings, and helps to better study the relation classification ability under the various scenarios.When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The last turn of the dialogue is not the end of the dialogue session.The multi-choice question you generated should be as difficult as possible.The relationship between two speakers cannot be directly reflected in the dialogue content, but the relationship can be clearly judged by understanding the dialogue content and the implicit information contained therein, such as the dialogue tone, attitude, scene, and other rich information.Candidates need to be as similar as possible, which is more confusing, but with a deep understanding of the context of the dialogue, the annotators can pick the right answer without debate.
Prompt for Multi-turn Response Generation
We are currently testing the annotation capabilities of data annotators for response selection in multi-turn dialogues.Multi-turn dialogue refers to a chat record generated by two speakers engaging in continuous interactions using natural language.Response selection refers to the process in which the annotators select the correct response from the candidates as the most coherent and reasonable response in the dialogue after comprehensively understanding the content of the multi-turn dialogue.
You are an expert at response selection in multi-turn dialogues, and now please act as the test creator.You first need to generate more than 10 turns(10 turns=20 utterances) of two-party dialogue.Then, you need to generate a multi-turn dialogue response selection question that requires a comprehensive understanding of the dialogue context.The dialogue content is required to be relevant to {Domain}.Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings and helps to better study the response selection ability in multi-turn dialogues under various scenarios.
When you generate the dialogue, please avoid using interrogative sentences in the dialogue.The last turn of the dialogue is not the end of the dialogue session.Each candidate of the question matches the personality of speaker2.When analyzing which choice is the correct answer, do not mention the speaker2's personality.In addition, each candidate of the question is only good or bad in terms of coherence.That is, the most coherent response is an effective continuation of the conversation above, which is an orderly chain of events under a common theme with the topic discussed above.The wrong choice may refer to a topic far removed from the one discussed in the conversation.These differences can be easily distinguished after a comprehensive understanding of the entire dialogue so that the annotator can pick the correct choice without dispute.Finally, in order to ensure fairness, the generated dialogue and options do not involve external specific knowledge as much as possible.If external specific knowledge must be involved, try to be common sense as much as possible, and do not involve unfamiliar concepts or entities.
Prompt for Slot Filling
We are currently testing the annotation capabilities of data annotators for multi-turn dialogue slot filling.Multi-turn dialogue refers to chat records generated by continuous interaction between two speakers using natural language.Slot filling refers to extracting the corresponding value for a specific slot (such as time, location, name, etc.) from the events reflected in multi-turn dialogues.
You are an expert in multi-turn dialogue slot filling tasks, and now you are asked to be the question setter.First, you assume the slot of interest, then generate a more than 10-turn dialogue between two people that involves the slot multiple times.After that, create a multi-turn dialogue slot filling multi-choice question that requires a comprehensive understanding of the dialogue context to answer.The dialogue content must be related to {Domain}.
Before setting the questions, you need to randomly set the personalities of the two speakers.There is a certain probability that you will set the speaker's personality to be unfriendly, generating unfriendly, sarcastic, offensive, argumentative, sophistical, weird, or negative content.This simulates real human dialogue scenarios and helps to better study slot filling capabilities in rich scenarios.
Try not to use questions in the generated multi-turn dialogue.The last turn of dialogue should not be a closing statement.Your question must be as difficult as possible, requiring a comprehensive understanding of the entire dialogue, rather than focusing on a single turn or sentence.At the same time, the information involved in all candidate options must come from the dialogue, and all candidate options are seemingly correct values for the given slot.So, you need to make sure that these candidate values appear when generating multi-turn dialogues.The question must be answerable based on the multi-turn dialogue and does not require external knowledge.In particular, all candidate options should be similar enough in length and content to make it more difficult to distinguish between correct and incorrect options.All candidate options need to comprehensively understand and extend the reasoning of the multi-turn dialogue content to judge the correctness of the options.
Prompt for Dialogue Summarization
We are testing data annotators on their ability to annotate multi-turn dialogue summarizations.A multi-turn dialogue is a chat transcript produced by multiple turns of continuous interaction between two speakers using natural language.Dialogue summarization is the process of extracting, summarizing, or refining key information from a multi-turn dialogue, turning it into a short summary paragraph that can be used to present the main points or big ideas of that multi-turn dialogue.
You are an expert at dialogue summarization in multi-turn dialogues, and now please act as the test creator.Firstly, you should generate a two-party dialogue with at least 10 turns(10 turns = 20 utterances).Then, you should create a dialogue summarization multiple-choice question that requires a comprehensive understanding of the context of the multi-turn dialogue.The dialogue content should be relevant to {Domain}.
Before setting the question, you should randomly set the personality of both speakers, and you have a certain probability of setting the personality of the speakers to be unfriendly so as to generate unfriendly, ironic, offensive, quarreling, specifying, weird, and negative content.It simulates the dialogue scenes of real human beings and helps to better study the intent understanding ability under the various scenarios.
The question you come up with must be as difficult as possible, but there are clear differences in strengths and weaknesses between each of the candidate options for multi-choice questions in terms of (1) inconsistencies between the information in the summarization and in the given dialogue, (2) the presence of information in the summarization that is not present in the given dialogue, and (3) the loss of important information in the given dialogue in the summarization, which can be clearly differentiated by a synthesized comprehension of the entire dialogue content.Candidate options only generate summarizations of the dialogue content, and it is not necessary to refer to the character names and personality information of the two speakers when judging which option to choose as the correct answer, but only from the degree of comprehensiveness of the summarization in presenting the main content of the dialogue.
Prompt for Data Filter
As a data quality inspector, you should conduct a comprehensive quality assessment of the following multi-choice question related to {task}.Your assessment needs to take into account both the correctness of the test question and the answer.Specifically, question correctness refers to whether the question is clear and relevant to the given dialogue and external knowledge if exists.Answer correctness refers to whether the content corresponding to the given label can correctly answer the given question.Next, please assess whether the given multi-choice question is correct.Do not analyze and directly give "correct" or "incorrect" as the output.
Figure 3 :
Figure 3: The basic prompt on the left and the external description for mitigating bias on the right.We take slot filling as an example.DOMAIN is the placeholder of the given domain.[DOMAIN_PROMPT] and [STYLE_PROMPT] are the positions of the corresponding description to add.
Figure 4 :Figure 5 :
Figure 4: The template for multi-choice questions in DialogBench.The red text is the explanation.
Figure 6 :Figure 7 :
Figure 6: The proportion of generated evaluation instances based on the basic prompt in each domain.
Figure 8 :
Figure 8: The average accuracy on all supervised instruction-tuning LLMs for each domain.
(2023a) and mainly examine the correctness of two aspects, including closed-scenario and openscenario correctness.The closed-scenario correctness requires LLMs to generate the output only based on the given dialogue context or background knowledge.To this end, we select representative slot filling (Chen et al., 2017), intent classification (Louvan and Magnini, 2020), along with knowledge-grounded response generation (Santhanam et al., 2021).
(
Acheampong et al., 2020), relation classification(Jia et al., 2021) and personality-grounded response generation(Ma et al., 2020) respectively.Semantic consistency refers to the actual semantic content contained in the dialogue context can entail the semantic content understood by humans, facilitating consistent response generation.Thus, we select the corresponding tasks, dialogue summarization (Feng et al.) and dialogue NLI(Welleck et al., 2019).
Figure 9 :
Figure9: An evaluation prompt for pre-trained LLMs is an exact string that concatenates all contents by "Read the following dialogue content, generate the correct answer according to the given question[Dialogue]{dialogue_content}[Test Question]{test_question}[Answer]{answer_content}".We take slot filling task as an example.The purple text is the answer content that LLMs need to calculate probability, which is selected from the Options and calculated one by one.
Table 21 .
We further retain only those evaluation instances that
Table 2 :
Accuracy on Engilsh DialogBench.Bold and underlined indicate the best results and second-best results.
Table 3 :
Accuracy on Chinese DialogBench.Bold and underlined indicate the best results and second-best results.
Knowledge-grounded response generation is a task of generating an informative response based on both dialogue context and the given external knowledge(Santhanam et al., 2021).
Table 6 :
The details of pre-trained or supervised instruction-tuning models LLMs for evaluation.
Table 7 :
Accuracy of supervised instruction-tuning LLMs in Chinese DialogBench for all 20 domains.All domains are mainly divided into two categories, including daily life and professional knowledge.Bold and underlined indicate the best results and the second-best results respectively except for GPT-4 and ChatGPT.
Table 9 :
The prompt for data generation of intent classification by
Table 10 :
The prompt for data generation of emotion detection by
Table 11 :
The prompt for data generation of commonsense-aware response generation by
Table 12 :
The prompt for data generation of knowledge-grounded response generation by
Table 14 :
The prompt for data generation of offensive detection by
Table 15 :
The prompt for data generation of Personality-grounded Dialogue Generation by
Table 16 :
The prompt for data generation of dialogue infilling by
Table 17 :
The prompt for data generation of relation classification by
Table 18 :
The prompt for data generation of multi-turn response generation by
Table 19 :
The prompt for data generation of slot filling by
Table 20 :
The prompt for data generation of dialogue summarization by
Table 21 :
The prompt for data filtering. | 2023-11-06T06:41:04.523Z | 2023-11-03T00:00:00.000 | {
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258855454 | pes2o/s2orc | v3-fos-license | A Retrospective Study on Vitamin D Status and Its Association With Cardiometabolic Risk Factors Among Children With Chronic Kidney Disease at King Abdulaziz University Hospital
Background: Vitamin D deficiency is a significant global health issue. It is prevalent in chronic kidney disease (CKD) patients, which is an important cause of death among children. Many studies have found a link between low vitamin D status in CKD patients and cardiovascular disease (CVD) risk factors. However, there are no data on this relationship in children with CKD in Saudi Arabia. Aims: We aimed to demonstrate this association among children with CKD admitted to the King Abdulaziz University Hospital (KAUH) in Jeddah, Saudi Arabia. Materials and methods: Data were collected between June and August 2020 from a convenience sample of pediatric patients. Results: In total, 153 pediatric patients with CKD stages 2-5 were admitted to the KAUH between 2010 and 2019, and 67.3% had CKD stage 5. Approximately 4.6% and 10.5% of the participants were overweight or obese, respectively. Patients who fell into the lower 25-hydroxyvitamin D (25[OH]D) tertile were older, had higher body mass index (BMI) values, and had higher blood pressure than those in the upper two tertiles; however, these differences were not statistically significant. There was a significant inverse association of 25(OH)D levels with BMI, blood pressure, and serum creatinine levels. Conclusions: The results of this retrospective study suggest that patients with CKD and lower vitamin D levels have a higher BMI and blood pressure and are therefore at higher risk of developing CVD. Future prospective studies with a larger sample size are needed to confirm these findings. Randomized clinical trials are also needed to investigate the effect of sufficient vitamin D status on reducing CVD in patients with CKD.
Introduction
Nearly one billion people around the world suffer from vitamin D deficiency [1]. Sun exposure causes the skin to synthesize vitamin D in the form of cholecalciferol, which then goes through two hydroxylation reactions as it reaches the bloodstream. The liver performs the first hydroxylation process, turning it into 25-hydroxyvitamin D (25[OH]D), while the second process takes place in the kidney's proximal tubules, where it is transformed into 1,25-dihydroxyvitamin D (1,25(OH)2D) [2], which is the active, or hormone, form of vitamin D and is responsible for its biological functions [3].
Globally, renal dysfunction, often known as chronic kidney disease (CKD), is a significant public health issue with high rates of morbidity and mortality [4]. The most common causes in pediatric patients are congenital kidney and urinary tract abnormalities, followed by glomerulonephritis and inherited nephropathies [5]. Vitamin D deficiency, described as a 25[OH]D level <20 ng/mL, occurs frequently in people with CKD and increases the risk of morbidity and mortality [6]. Patients with CKD are exposed to age-related and other risk factors for 25(OH)D deficiency, including female sex, adiposity, proteinuria, low physical activity, peritoneal dialysis, diabetes mellitus, and reduced vitamin D receptor (VDR) [7].
Cardiometabolic disorders are a major contributor to cardiovascular disease (CVD), which is one of the leading causes of death for people with CKD [7]. The interaction between cardiovascular and metabolic risk factors, such as the association of CKD with obesity, hypertension, dyslipidemia, and insulin resistance, presents difficulty in terms of risk classification. Consequently, optimizing metabolic health is essential for managing children with CKD [8]. Vitamin D plays an important role in cardiovascular health [9], for example, in blood pressure regulation and heart, smooth muscle, and endothelial cell functions. Additionally, it has been discovered that there is a link between CVD and vitamin D in CKD patients.
Previous studies have found that low vitamin D levels in people with CKD are reliable indicators of disease development and mortality [10]. In a randomized clinical trial, high-dose cholecalciferol (2000-8000 IU/day) administration in children with CKD showed favorable effects on cardiovascular and endothelial parameters [11]. Another study in the US found that approximately 30% of deaths in patients with CKD were secondary to cardiovascular causes. Moreover, increased left ventricular mass and diastolic dysfunction have been linked to vitamin D deficiency [12]. Other studies have shown that children with CKD have a high prevalence of CVD risk factors that persist even after renal transplantation [13].
Therefore, understanding the effects of vitamin D deficiency on CVD development in pediatric age groups is of great interest. However, in Saudi Arabia, there are no data on the relationship between cardiometabolic risk factors and low vitamin D levels in children with CKD. Identifying cardiometabolic risk factors among CKD children with severe vitamin D deficiency would provide a basis for future research on the advantages of vitamin D supplementation and improving vitamin D status in lowering their cardiovascular risk. Thus, this study aimed to determine the association between cardiovascular and metabolic risk factors and low vitamin D levels in children with CKD in Jeddah, Saudi Arabia.
Study design and setting
This retrospective record review study was carried out between June and August 2020 at the Pediatric Nephrology Center of Excellence (PNCE) at King Abdulaziz University Hospital (KAUH), a tertiary center in Jeddah, Saudi Arabia. Ethical approval was obtained from the institutional review board of KAUH . This study was performed according to the ethical standards of the institutional and/or national research committee, the 1964 Helsinki Declaration, its later amendments, or comparable ethical standards. Informed consent was obtained from the parents or guardians of the children.
Study population
All pediatric patients of both sexes (aged 1-18 years) with CKD stage based on an estimated glomerular filtration rate (eGFR) <90 mL/min/1.73 m 2 between 2010 and 2019 were included in the study. Children with congenital heart disease, diabetes, thyroid malfunction, renal cancer, those who underwent a renal transplant, and those with missing data on 25(OH)D levels were excluded.
Data collection instruments
Records were reviewed from the Phoenix system at KAUH using a data collection sheet via Google Forms. The data sheet included information on the patient's demographics (date of birth, sex, and nationality), socioeconomic data (birth order, monthly income, and parents' education), and family history of renal disease acquired by contacting a family member who knew the most about the child. Using the recorded height and weight, the body mass index (BMI) was calculated by dividing the weight in kilograms by the square of the height in meters. Heart rate, systolic blood pressure (SBP), and diastolic blood pressure (DBP) were also noted. Laboratory readings (eGFR mL/min/1.73 m 2 , serum 25(OH)D in nmol/L, serum creatinine in µmol/L, hemoglobin in g/dL, the parathyroid hormone in pmol/L, serum calcium in mmol/L, serum phosphate in mmol/L, albumin in g/L, and magnesium in mmol/L levels) were also recorded.
Risk factors were defined as follows
SBP and DBP above the 95th percentile for the child's age and sex were considered to indicate high blood pressure. Obesity was defined as BMI ≥95th percentile for the child's age and sex based on the Centers for Disease Control and Prevention's growth charts, hypoalbuminemia as a plasma albumin level <30 g/L, hyperparathyroidism as a serum parathyroid hormone level >7 pmol/L, anemia as a hemoglobin level <10 g/dL, hyperphosphatemia as plasma inorganic phosphate >1.6 mmol/L, and hypercalcemia as plasma total calcium >2.52 mmol/L. The corrected calcium level (mmol/L) was calculated using the adjusted Payne formula [14]: serum calcium (mmol/L) + 0.02 * (40 -serum albumin [g/L]).
Data analysis
Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) version 21 (SPSS, version 23, IBM Corp., Chicago, IL, USA). The normality of the data was examined using the Shapiro-Wilk test. The terms "median" and "interquartile range (IQR)" were used to describe non-normally distributed data. For categorical variables, numbers and percentages were used, whereas the mean ± standard deviation (SD) was computed for continuous normally distributed variables. Tertiles of 25(OH)D were evaluated, and variables in the lower tertile were compared with those in the upper two. The differences between continuous and categorical variables were assessed using the Student's t-test and the Chi-square test, respectively. The independent relationship between 25(OH)D level and various cardiometabolic risk factors was investigated using multiple linear regression analysis, which was adjusted for age, sex, nationality, eGFR, monthly income, mother's education, parathyroid hormone level, heart rate, corrected calcium level, and BMI. The threshold for significance was set at P<0.05.
Results
In total, 153 pediatric patients from 1 to 18 years of age with CKD stages 2-5 (62% males) were admitted to KAUH between 2010 and 2019. More than two-thirds (67.3%) of the recruited children had CKD stage 5. The sociodemographic and clinical features of the patients are shown in Table 1. Approximately 95% of the patients were on vitamin D3 supplements; however, 44% had 25(OH)D levels of ≤50 nmol/L and 96% had high parathyroid hormone levels. Finally, 4.6% and 10.5% of the participants were overweight or obese, respectively. The study cohort's initial characteristics are shown in Table 2 for reference. The study cohort's mean 25(OH)D level (SD) was 65 (42) nmol/L. Table 3 shows the different variables studied according to the tertiles of serum 25(OH)D levels. Patients in the lower 25(OH)D tertile were older and had higher BMI and blood pressure (both SBP and DBP) than those in the upper two tertiles; however, these differences lacked statistical significance. The relationships between the various examined variables and serum 25(OH)D levels are shown in Table 4. BMI, DBP, and serum creatinine levels showed statistically significant inverse correlations with 25(OH)D levels. In contrast, significantly higher 25(OH)D levels were positively associated with corrected calcium levels, albumin levels, and eGFR. Table 5 shows the independent predictors of the studied cardiometabolic risk factors (adjusting for age, sex, nationality, eGFR, monthly income, mother's education, parathyroid hormone level, heart rate, corrected calcium level, and BMI). Among the different cardiometabolic risk factors studied, 25(OH)D level was the only significant predictor of BMI and blood pressure (both SBP and DBP); BMI was reduced by 0.024 kg/m 2 for every 1 nmol/L increase in 25(OH)D, while SBP decreased by 0.155 mmHg and DBP decreased by 0.130 mmHg.
Discussion
According to our findings, patients in the lower 25(OH)D tertile were older, had a higher BMI, and had higher blood pressure (both SBP and DBP) than those in the upper two tertiles; these differences were not statistically significant; however, according to regression analysis, the 25(OH)D level was found to be a significant predictor of BMI and blood pressure (both SBP and DBP).
These results are consistent with those of previous studies [14]. Furthermore, other studies have reported inconsistencies in the association between vitamin D levels and metabolic syndromes. However, they found that low vitamin D levels were associated with a higher risk of developing type 2 diabetes, as vitamin D helps to improve sensitivity to insulin [15][16][17]. Additionally, several studies have discovered that higher 25(OH)D levels are associated with a reduced risk of cardiovascular illnesses [18]. The differences between these findings could be due to different study types: studies that found positive correlations were observational studies, in which the patients were observed and outcomes measured to achieve more accuracy. Another reason could be that the patient's data were poorly documented in the hospital's system, resulting in false statistics.
Vitamin D and obesity
Our findings support other research findings, indicating that obese children and adolescents are more likely to be vitamin D deficient than their non-obese counterparts [19][20][21][22]. To explain this, several theories have been put forth on the links between vitamin D deficiency and obesity. First, because of societal stigma, obese people typically avoid going out in the daytime, hardly engage in any outdoor activities, and/or dress in clothing that covers most of their bodies, restricting sun exposure and cutaneous vitamin D production [23]. Alternatively, the increased local utilization of 25(OH)D could be explained by the presence of the enzyme 1-hydroxylase, which activates vitamin D in the adipose cells of obese people [23]. According to one study [24], regardless of the quantity of cutaneous vitamin D precursor present, the increase in serum 25(OH)D concentration was lower in obese persons after exposure to sunlight than in other individuals.
However, in line with earlier findings, evidence of a link between low vitamin D levels and obesity remains unknown. The majority of these results depend on variations in the VDR gene, which has been linked to obesity features in some studies. Several other genes have also been studied for obesity-related features. However, these results have been inconsistent [21,22].
Vitamin D and blood pressure
Vitamin D deficiency activates the renin-angiotensin-aldosterone system (RAAS), which increases blood pressure and affects the cardiovascular system. However, randomized clinical trials and meta-analyses have shown conflicting results on the effects of vitamin D supplementation on lowering blood pressure [25,26]. On the other hand, a systematic review and meta-analysis concluded that vitamin D supplementation in patients with vitamin D deficiency who are older than 50 years or obese lowers SBP, while it lowers SBP and DBP in patients with vitamin D deficiency and hypertension [26]. Large, randomized trials with a focus on patients with severe vitamin D deficiency and hypertension are necessary before vitamin D may be recommended for the prevention or treatment of hypertension.
Limitations
This study has several limitations. The significance level of the outcomes could not be attained in our research because of the presumably small sample size. The lack of information on vitamin D dosages is a significant issue because the recommended doses of vitamin D3 may differ substantially. Additionally, dietary data and lipid profiles were unavailable. Therefore, the association between these factors should be investigated in future studies.
Future research should also assess how vitamin D specifically affects the diagnosis, treatment, and prognosis of cardiovascular diseases in children with CKD. Certain issues, such as the cutoff value that would indicate risk and the boundary value that would indicate the necessity for action, still need to be resolved. Additional research is needed to show vitamin D levels as a biomarker for pediatric cardiovascular disorders.
Conclusions
The results of this retrospective study suggest that children with CKD and lower vitamin D levels have a higher BMI and blood pressure and are therefore at higher risk of developing CVD. Future prospective studies with larger sample sizes are needed to confirm these findings. Additionally, randomized clinical trials are required to determine the effects of sufficient vitamin D status on reducing CVD in patients with CKD.
Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. The research ethics committee (REC) issued approval Reference No 366-20. The REC recommended granting permission of approval to conduct the project. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. | 2023-05-24T15:11:52.070Z | 2023-05-01T00:00:00.000 | {
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13575130 | pes2o/s2orc | v3-fos-license | On Some Modifications of the Fueter Operator
We present some classes of functions that are defined on the quaternions as solutions for a linear operator that resembles the Cauchy-Riemann conditions. Unlike the Fueter regular functions; in this case the identity function is a solution and solutions are closed under the quaternionic product.
I. INTRODUCTION
"Quaternionic analysis" refers to many different types of extensions of the Cauchy-Riemann equations on the quaternion field. Fueter's approach; the socalled regular functions, is a rich theory that contains quaternionic versions of Cauchy's theorem and Cauchy's integral formula. Probably the best modern reference for the regular functions, in their general form, is Sudbery. The elegance of this generalization is perhaps clouded by the lack of some algebraic properties of the Complex Analysis: Regular functions cannot be multiplied or composed to obtain new regular functions and the identity function is not even regular. In recent years S.L Eriksson and H. Leutwiler have introduced a modified Dirac equation (related to the Cauchy-Riemann-Fueter condition for regularity), which gives rise to the Hyperholomorphic functions, which has also shown to be of great richness. It is interesting to note that Hyperholomorphic functions contains the identity function as well as its powers. Following that spirit this work is an attempt to study some other modifications of the Fueter operator whose solutions are algebraic closed (under the quaternionic product) and thus complex-like. What is complex-like in the quaternionic field is per se a broad question. Another way to reformulate it could be the following: In how many ways the quaternions contains the complex plane? The answer seems to depend on which special property of the complex numbers one is interested in. As we want a system of solutions for some Cauchy-Riemann equations that: are algebraic closed as quaternions and that contains the identity, we start by adding a rather strict condition: the functions evaluated at some point will not change the direction of the imaginary part of the quaternion. The study of such functions was first done by Rinehart and Cullen. Informally one obtains a process that turns the complex function z n onto the quaternion function p n .
We consider the set of commutative function such that they are analytic when restricted to some complex plane into the quaternions and it was introduced by S. De Leo and P. Rotelli. This is our starting point for the first of three classes of functions. The second class appears naturally when one evaluates the Fueter operator on the power of quaternions and appeared first on C.A Deavours article on the quaternion calculus. The third class, the most basic, are actually the Cullen functions. Deavours deduced that the Cullen functions would be solutions for the operator that defines the second class. We show that this result is incomplete as there exists solutions for this class that are essentially different from the Analytic Intrinsic functions studied by Cullen. Our main tool is the observation that the Fueter operator is invariant under some coordinate system related to the spherical coordinates. We show how our solutions classes are related to the Fueter's regularity. The bridge, in this case, is first based on a observation by Rinehart, that gives sufficient and necessary conditions for a complex function to be regular when turned into a quaternionic function by the Cullen method. These complex functions satisfy a non-standard version of the Cauchy-Riemann conditions.
II. DEFINITIONS
Let p be a quaternion, we write p in the canonical coordinates as: Let f be a quaternionic map that commutes with its own argument in a open set ω: They are naturally related to complex maps. As we want to emphasize this we call these maps for Complex Extrinsic (CE).
where ι := xi + yj + zk By fixing ι ∈ S 2 we associate to ω a open subsetω in the upper complex plane, in the following way: where r := x 2 + y 2 + z 2 In the same way we associate a CE map to a family of complex maps, parametrized by S 2 and defined onω ι : In the rest of this work we will use the standard spherical coordinates: ι = (cos α sin β, sin α sin β, cos β) We shall denote by ι α , ι β the derivative of ι respect to α and β as a variable. We say f ip is a complex component of the CE quaternionic map f . If f is such that it only has one component we say f is complex instrinsic (CI).
We add now differential conditions on our CE functions to relate them to complex analytic functions. All the Classes we are going to define are understood to be C 1 .
We say a CE map f is of Class I if and only if it satisfies the the following Cauchy-Riemann equation: A CE function is called of Class II if and only if: A function of Class I that is also CI, that is, with only one complex component is called of Class III.
Finally, a CE function is called regular if and only if: The operator first defined in (11) is known as the Fueter (or sometimes Cauchy-Fueter or even Cauchy-Riemann-Fueter) operator. It has a left and right version: We first rewrite ∂f ∂ lp as: Now let φ(t, x, y, z) = (t, r cos α sin β, r sin α sin β, r cos β) the parametrization. It is tempting to write then the following operator: (16) And we inquire how this modified operator is related to the original Fueter operator. It is somehow surprising that they represent exactly the same operator: Proof This is a mere use of the chain rule. We start writing (17) explicitly: Finally, the validity of the proposition depends of the fact that:
III. SOME PROPERTIES ON CLASSES I TO III
Given two regular functions f, g (not necesarilly CE) in some open set ω in general it is not true that f g or gf will be regular. This is an important difference between the Fueter regularity and the classical analyticity, and makes the generation of regular functions more difficult. Note that that in the Fueter regularity the identity function f (p) = p is not regular. Classes I to III, on the other side, behave nicely with respect to the quaternionic product and contain the identity function.
Proposition 3 Let f ,g be both of Class I,II or III then f g = gf and f + g are of respective Class I,II or III. If there exists the algebraic inverse f −1 then it is also of respective Class I, II or III.
Proof This is a straight-forward calculation and will be omitted.
Proposition 4 Class I ⊃ Class II ⊃ Class III
Proof We first observe that the Class III is by definition contained in the Class I. Let f = u(p) + ιv(p) be of Class II, if we write f = u + ιv this is equivalent as to say that u and w := v r satisfy the following set of equations in (t, x, y, z) coordinates: Multiplying (27) by x, (28) by y and (29) by z and summing turns these three equations in only one. So we obtain only two equations, namely: Turning back these two equations in coordinates t, r, ι is exactly (9). So f is of Class I. Now let f be of Class III. Let (α, β) be the parametrization of S 2 , in spherical coordinates. We observe that if f is of Class III this then: Applying the Fueter operator to f in (t, r, ι) = (t, r, α, β) coordinates is: We observe that A Class III functions is by definition of Class I, the first two summands in (32) are precisely the Class I condition and so they vanish, this together with the previous calculation shows that: and so f is of Class II. By a straight-forward calculation is obtained a simple formula for the jacobian determinant of a Class I to III function at a given point.
Proposition 5 Let f be a function of Class II, the determinant of the jacobian is given by the following formula: where: Proof Because f is assumed of Class II then for the parametrization of the sphere f satisfies: We observe that and ( ∂p ∂β By the assumption: r = 0 and we rewrite (38) as: We substitute f = u + ιv in (41) and obtain: Since ι 2 = −1 then ∂ι ∂α ι + ι ∂ι ∂α = 0, and the same holds if we replace α by β. As these two imaginary vectors anticommute their quaternionic product is exactly their cross product. The anticommutativity still holds if we replace any of these vectors by their inverses. We continue this calculation expressing these vectors in terms of the parametrization: We substitute this expressions in (43): Which finally implies: Consider the function f = xr −1 ι is a Class I function that is not Class II. We observe also that the following function: is a Class II function that is not of Class III. The expression for this solution in (t, x, y, z) coordinates is: From which we construct two more solutions: ̺(x, y, z) := arctan( y z ) + ι arctanh( x As expected,ρ denotes the quaternionic conjugate of the function ρ. We appreciate chirality by the following observation, which holds also for ̺,σ: This observation can be generalized: Proposition 7 Let f be a left-Class II function such that then the conjugate of f will be a right-Class II.
Proof We consider which can be rewritten as: On the other side, a Class III function is central: Proposition 8 Let f be a Class II function. Then if and only if f is of Class III Proof Suppose as the dot product of ι −1 α ι and ι −1 β ι is zero we conclude that ∂v ∂α = ∂v ∂β = 0 (67) and from here So f is of Class III. The reciprocal is immediate.
V. REGULAR IC FUNCTIONS
We now look for sufficient and necessary conditions that a CI function has to meet in order to be regular, in other words, that f satisfies: Because the function is CI, it only has one complex component, so: Which occurs if and only if As before, let us callf the (unique) complex component of the quaternionic function f . It is clear thatf has to satisfy the non-analytic condition: We summarize all of this in the following Proposition 9 (Rinehart conditions) Let f be a CI and regular function andf its complex component, then.
Let f be a Class III quaternionic function andf its complex component, then Proof The first affirmation is immediate. For the second affirmation is just enough to note that Class III functions are in particular Class II. As a consequence we can provide a functional that turns analytical functions onto complex functions that satisfy (72) and therefore will result into regular functions when transformed in quaternionic functions.
Proposition 10 Let z = x + iy and f = u + iv be a analytical function, defined on the upper complex plane. Let f ′ (z) be its total derivative evaluated at a point z. Define a functional L as: Then L(f ) will satisfy (72)
VI. THE IMAGINARY DERIVATIVE
We now investigate the generation of regular functions taking as seed Class II functions. We present what we shall call the imaginary derivative: Proposition 11 Let f be a (left)-Class II function, then Proof This is immediate after we remember a Class II function is Class I, so: We now use the fact that for Class II functions the Fueter operator (and the imaginary derivative) result in a scalar functions, let f be again a (left)-Class II then After reordering we obtain So the chiral difference of a Class II function is regular.
VII. LAURENT SERIES
Let f be a Class I function. We denote by ι(α 0 , β 0 ) a fixed point in S 2 . Let c be a point in this upper complex plane. c can be written as c 1 + c 2 ι(α 0 , β 0 ) where c 1 , c 2 are real numbers. We consider the annulus As f is of Class I then its restriction to this annulus is a complex holomorfic function. Lets denote f (t, r, α 0 , β 0 ) this restriction. In this annulus we represent this complex function by its Laurent series: Now let V (α, β) denote a connected open set in S 2 .
We consider an open set W defined in the quaternionic space as the cartesian product of these two open sets : We observe that the function defined as: and a n (α, β) is of Class II for all n.
We can now provide a functional that will turn a left-Class II function into a right-Class II function defined on a open set W.
Proposition 13 (The Mirror operator) Let f be a left-Class II function defined in a open set W as previously described, then Proof Assume f is left regular. We write f with its Laurent series. | 2014-10-01T00:00:00.000Z | 2004-12-06T00:00:00.000 | {
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220383906 | pes2o/s2orc | v3-fos-license | The Impact of Product and Labour Market Reform on Growth: Evidence for OECD Countries Based on Local Projections
This paper examines the impact of labour and product market reforms on economic growth in 25 OECD countries between 1985 and 2013, and tests whether this impact is conditioned by the fiscal policy stance, i.e. whether there are fiscal expansions or adjustments. Our local projection results suggest that controlling for endogeneity of reforms and the stance of fiscal policy is crucial. To control for endogeneity, we use the Augmented Inverse Probability Weighted estimator. Our results suggest that product market reforms mostly cause slight negative growth, except when implemented during periods of neutral fiscal policy. Labour market reforms hurt growth under tight and neutral fiscal policy but are conducive to economic growth if introduced during periods of expansionary fiscal policy. “In every press conference since I became ECB President, I have ended the introductory statement with a call to accelerate structural reforms in Europe. The same message was also conveyed repeatedly by my predecessors, in three quarters of all press conferences since the introduction of the euro. The term “structural reforms” is actually mentioned in approximately one third of all speeches by various members of the ECB Executive Board. By comparison, it features in only about 2% of speeches by governors of the Federal Reserve” (Draghi, 2015).
"In every press conference since I became ECB President, I have ended the introductory statement with a call to accelerate structural reforms in Europe. The same message was also conveyed repeatedly by my predecessors, in three quarters of all press conferences since the introduction of the euro. The term "structural reforms" is actually mentioned in approximately one third of all speeches by various members of the ECB Executive Board. By comparison, it features in only about 2% of speeches by governors of the Federal Reserve" (Draghi, 2015).
Introduction
Productivity growth is the most important component of economic growth. Regulation is widely believed to play a role in explaining cross-country productivity differences, as regulation limits the competitive pressures that challenge firms to thrive (Nicoletti and Scarpetta, 2003;Aghion and Griffith, 2005;Cette et al., 2016). Structural reforms are therefore often called for as illustrated by the quote from former ECB President Mario Draghi. Given the centrality of labour and product markets to the functioning of the economy, most research focuses on the output effects of reforms in these fields. 1 As pointed out by , these reforms broadly involve deregulating retail trade, professional services and certain segments of network industries, primarily by reducing barriers to entry; easing hiring and dismissal regulations for regular workers; and increasing the ability of and incentives for the non-employed to find jobs. 2 Product market reform may enhance productivity growth. For instance, Arnold et al. (2016) report that banking, telecommunications, insurance and transport reforms all had significant positive effects on the productivity of manufacturing firms in India. One mechanism through which reform may affect productivity is by enhancing firm dynamics, i.e., the process of entry, thrive, and exit from the market (de Haan and Parlevliet, 2018). Firm entry and exit (business churning) is often regarded as key to economic growth, as Schumpeterian creative destruction facilitates the resource shifts from less productive firms to more productive ones, fostering innovation and adoption of new technology (ECB, 2018). Business churning is affected by country-specific conditions influencing the incentives for firms to invest in new technology or adapt existing technologies to maintain their competitive edge. But competitive pressure may also spur productivity growth through other channels. For instance, the entry of new competitors may directly encourage productivity growth in incumbent firms (Aghion et al., 2004), while more competition in the markets for intermediate goods allows firms to boost productivity through cheaper inputs (Bourlès et al., 2013). Several studies have examined the impact of product market reform on economic growth, reporting mixed results (see Parlevliet et al., 2018 for a review of the literature).
Most research on labour market reform focuses on its effect on unemployment (see Brancaccio et al., 2018 for a discussion of the literature). However, this type of reform may 1 Note, however, that several studies (to be discussed below) do not find strong evidence that structural reform enhances growth. For instance, estimate panel VARs for 20 OECD countries over the period and report that labour and product markets deregulation involves potential short-run costs materialized by higher unemployment and lower output. 2 It is not clear whether labour and product market reforms are substitutes or complements. Following the theoretical work of Blanchard and Giavazzi (2003), who demonstrated a degree of substitutability between product and labour market regulations in a general equilibrium setting, several studies have investigated this relationship empirically with different outcomes (see Parlevliet et al., 2018). The relationship between both types of reform may also be different in the short and the long run. For instance, find complementarity in the short run, but substitutability in the longer run. also affect economic growth. As pointed out by Brancaccio et al. (2018), several models suggest that a high degree of labour market rigidity causes wage stickiness which would be an impediment to the spontaneous balance of demand and supply of labour (Blanchard and Giavazzi, 2003). Removal of these rigidities would move the economy towards the production frontier and thereby promote economic growth. For example, in the Solow (1956) growth model, labour market rigidities can be argued to lead to a high capital labour ratio, which results in low levels of savings and capital accumulation and thus low growth. Furthermore, a rigid labour market implies that the economy produces below its potential and slows convergence of the economy toward its steady state (Alonso et al., 2004). Most empirical studies, however, do not provide strong support for the growth-enhancing effects of labour market reform. For instance, Campos and Nugent (2018), who use the change in their index of the rigidity of employment laws as proxy for labour market reform in a panel of more than 140 countries over the 1960-2004 period, report that changes in rigidity do not systematically affect economic growth. Likewise, Brancaccio et al. (2018), who consider the effects of a change in an employment protection index for a sample of 23 countries and 24 years, find no statistically significant positive impact of labour market reform on GDP growth.
We examine the impact of labour market and product market reforms on economic growth in 25 OECD countries. In addition, we test whether this impact is conditioned by the fiscal policy stance. We employ the local projections (LP) approach (Jordà, 2005). LP is a flexible alternative to vector autoregression models since it does not impose dynamic restrictions. Furthermore, it is better suited to estimating non-linear or state-dependent impacts, like, in our case, the stance of fiscal policy. In estimating our models, we follow Teulings and Zubanov (2014) and include the leads of the reform dummies. This approach alleviates the bias caused by overlapping forecast horizons. When calculating the forecast horizon, outcomes for observations prior to a treatment by construction overlap with the treatment ahead in time, but this is not registered in the data for the affected observation when using a standard LP setup.
The effects of structural reforms on growth are important to study on its own. However, an important related issue is whether reforms are better at delivering if implemented in combination with specific types of fiscal policy. Ignoring this conditionality, may explain why the results of previous studies on the growth-enhancing effect of structural reform differ. We therefore condition on fiscal policy. Bordon et al. (2018) report that the impact of product market reforms on employment when initiated with non-restrictive fiscal policy is positive and significant in the medium term. However, under a restrictive fiscal policy stance the impact of product market reforms on employment is negative and statistically significant five years after the reform has been launched. Furthermore, the likelihood that a structural reform occurs may depend on the presence of fiscal adjustments of expansions (Mirau et al., 2007). This is important, as we endogenize the occurrence of labour and product market reforms.
There are some papers closely related to our work. Bordon et al. (2018) investigate the impact of structural reforms on employment using OECD labour marker reform indicators and the local projection (LP) approach, while controlling for endogeneity. However, unlike Bordon et al. (2018), we examine the impact of reforms on economic growth. Most importantly, instead of using the OECD reform indicators, we use the reform indicators of . The most important advantage of this database is that it identifies the exact timing of major legislative and regulatory actions by advanced economies since the early 1970s in key labour and product market policy areas. Furthermore, it captures reforms in areas for which OECD indicators exist but do not cover all relevant policy dimensions .
Another paper that is strongly related to our work is Duval and Fuceri (2018) who also use local projections and the same database as the current paper. These authors examine the effects of labour and product market reforms on output, employment and productivity, and analyse how these vary with prevailing macroeconomic conditions and policies. Product market reforms are found to raise productivity and output, but gains materialise only slowly. The impact of labour market reforms is primarily on employment. The authors also find that the economy's response to reforms significantly improves when they are accompanied by fiscal or monetary stimulus. There are three main difference with our work. First, instead of using fiscal policy shocks identified as the forecast error of government expenditure, we identify the stance of fiscal policy based on the presence of fiscal adjustments and fiscal expansions following the approach suggested by Wiese et al. (2018). In our view, fiscal adjustments or expansions better capture the stance of fiscal policy; it builds upon the literature initiated by Alesina and Perotti (1995); see also Alesina et al. (2019). Second, we control for the endogeneity of reforms using the Augmented Inverse Probability Weighted (AIPW) estimator proposed by Jordà and Taylor (2015), following Bordon et al. (2018). Third, we include treatment leads to alleviate the bias from overlapping forecast horizons as proposed by Teulings and Zubanov (2014).
Our findings indicate that controlling for endogeneity of reforms and the stance of fiscal policy is crucial. Our results suggest that product market reforms mostly cause slight negative growth. Labour market reforms hurt growth under restrictive and neutral fiscal policy but are conducive to economic growth if introduced during periods of expansionary 3 fiscal policy.
The remainder of the paper is organized as follows. Section 2 discusses the data used. Section 3 outlines our methodology, while section 4 presents our main findings. Section 5 offers a robustness analysis, while section 6 concludes.
Structural reform
Most previous research on the impact of structural reform uses OECD regulation indicators (see, for instance, Bouis et al., 2012;Faccini, 2014;and Bordon et al., 2018). These range from 0 to 6 to capture the restrictiveness of regulation in labour and product markets. A reform is then identified as a fall of the index. In our research, we instead use the narrative reform database of . Drawing on , we may summarize the methodology used to construct this database as follows. In a first step, all legislative and regulatory actions related to product and labour market regulation mentioned in the OECD Economic Survey are identified for the 26 countries over the entire sample. Next, out of the more than 1000 actions, major reforms are identified based on three criteria: (1) the OECD Economic Survey uses strong normative language to define the action, suggestive of an important measure; (2) the policy action is mentioned repeatedly across different editions of the OECD Economic Survey for the country considered; (3) the OECD indicator of product and labour market regulation displays a very large change. Reform variables are two dummies (labour and product market reforms) equal to one if there is a major reform identified by . There are 248 product market reforms and 79 labour market reforms in our sample which runs from 1985 to 2013 (see Table 1).
Fiscal policy stance
Following the approach suggested by Wiese et al. (2018), we apply the Bai and Perron (B&P) (1998,2003) approach to the cyclically adjusted primary budget balance (CAPB) as share of GDP to identify fiscal adjustments and fiscal expansions. This approach is more objective than those used in the literature on fiscal adjustments so far, because it takes the variability of the budget balance within countries into account. Adjustments are generally defined in the literature as a discretionary (i.e. cyclically adjusted) and significant decline in the general government's budget balance. Significant in this case does not refer to statistical significance, but rather whether the change in the cyclically adjusted (primary) budget balance exceeds some (subjectively selected) threshold. So, these filters are based on a 'one-size-fits-all' principle and they therefore do not take into account that the budgetary processes in some countries may lead to a much more volatile budget balance than those in other countries. A filter that does not take volatility into account is prone to identify fiscal adjustments and expansions that are the result of the budgetary institutions in place (or other factors driving fiscal policy volatility), rather than deliberate attempts of politicians to improve the budget balance or to make fiscal policy expansionary. Our approach to identify the beginning of a period with tight or expansionary fiscal is based on the identification of statistically significant changes in the Data Generating Process of the CAPB. 4 Perron (1998, 2003) developed a general method for this purpose. Consider a model with m possible structural breaks: Where yt is the dependent variable, in our case, the cyclically adjusted primary budget balance in each individual country separately, δ j is a vector of estimated constants, i.e., the mean of the m + 1 different segments of the time series y t , and u t is the error term. The B&P filter generates the segmented route through the series that yields the lowest Sum of Squared Residuals (SSR) up to a maximum number of breaks. The maximum number of breaks is restricted by the trimming parameter h, which specifies a minimum number of observations that have to occur between consecutive breaks. We have set h=0.15 which means that 15% of the within country time series has to pass between breaks. 5 The process underlying the algorithm is straightforward. First, it searches for all possible sets of breaks up to a maximum restricted by the trimming parameter h, and determines the set that minimizes the SSR for each number of breaks. Then, F-tests determine whether the improved fit produced by allowing additional breaks is sufficiently large compared to what can be expected randomly on the basis of the asymptotic distribution derived in Bai and Perron (1998). We used the test procedure recommended by Bai and Perron (2003) to select the optimal number and timing of breaks. That is, dependent on properties of the individual time series, we chose the appropriate filter specification and test. Generally, though, the error distribution is allowed to differ across segments. 6 Autocorrelation and potential heteroskedasticity is modelled non-parametrically by running the filter using a heteroskedasticity and autocorrelation-consistent estimate of the variance-covariance matrix. The B&P method identifies the break date (fiscal adjustment or fiscal expansion initiation) as the first year after an upward or downward structural break in CAPB. Therefore, we take a one-year lag to identify the start of the fiscal adjustment or expansion. This method will identify the beginning but not the end of periods with tight or expansionary fiscal policy. In line with Wiese et al. (2018), we define the periods such that tight fiscal policy after an upward structural break continues as long as the change in the CAPB is positive, and expansionary fiscal policy after a downward structural break continues as long as the change in the CAPB is negative. Observation that are not classified as either expansionary or tight fiscal policy are labelled as observations with neutral fiscal policy.
Data for the cyclically adjusted budget balance (CAPB) come from the OECD and begin in 1985 for some countries, but later for several other. 7 Due to the limited availability of CAPB data we lose observations when we partition the data on fiscal policy stance (see Figure 1).
As Figure 1 shows, product market reforms seem unrelated to fiscal policy. However, labour market reforms happen more frequently during periods of tight fiscal policy than during periods with expansionary fiscal policy, but this is also caused by the fact the we observe fewer fiscal expansions than fiscal adjustments.
Figure 1. Fiscal policy stance and market reforms
Note: The figure displays the distribution of major product and/or labour market reforms during loose (expansionary), neutral and tight (restrictive) fiscal policy. In total there are 140 observations with tight fiscal policy, 112 with loose fiscal policy and 545 with neutral fiscal policy. There are 24 observations where a product and labour market reform is introduced simultaneously.
Dependent and other variables
Most other data come from the Penn World Tables (PWT) version 9.0 (Feenstra et al., 2015). The dependent variable is cumulative real GDP growth per capita projected stepwise forward in time, so 0 to 1, 0 to 2 etc. until 0 to 5 years. The cumulative growth rates are calculated based on real growth rates (log differences of real GDP in PPP 2011 US$, divided by populations size, both from PWT 9.0).
The political variables considered in the AIPW estimator are own updates of variables used in Wiese et al. (2018). They are explained in more detail in section 3. Table A2 in Appendix 2 provides a description of all variables used and their sources.
Estimation methods
The basic regression model that we estimate is: log %,'() − log %,' = % + 0 2,%,' + 3 ∆ %,'89 + : Where h=1…,5 is the forecast horizon, and log %,'() − log %,' denotes the cumulative growth rates of real GDP over the forecast horizon. % denotes country fixed-effects and %,2,' are the reform dummies (where r denotes the product and labour markets). Notice that both reform indicators are always included simultaneously in the regressions. In all the OLS LP regressions (and in the AIPW LP outcome regression, see below) we include an AR(4) term for the growth rate between t-1 and t. 8 %' is a vector of additional control variables. %' contains the output gap calculated using the Hodrick-Prescott filter using high smoothing (l=100) as recommended in Jordà and Taylor (2015). Our results are robust to using OECD data on the output gap which is calculated using a production function approach; the correlation coefficient between the OECD output gap and our own is 0.845. %' also includes the change in physical capital (gross investments relative to GDP) and the percentage change from year to year in the human capital index from PWT 9.0.
Treatment leads and lags are also included as suggested by Teulings and Zubanov (2014). The leads are included to avoid the bias that results from overlapping forecast horizons. 9 The bias is in that sense deterministic as it is a consequence of calculating the projections themselves, under the hypothesis that reforms do have an effect on economic growth. We also include treatment lags in our models. But contrary to the leads, it is an empirical issue how long the effect of reforms persists in the data. Again, we use Akaike's information criterion to determine the lag length which consistently tells us to use 5 lags of the treatment variable.
The major drawback of equation (2) is that it ignores that structural reforms may be introduced in countries/years where the expected benefits of reform are higher than in countries/years were no reforms are introduced. In other words, failing to account for this can lead to selection bias. Therefore, we proceed with a quasi-experimental method, namely the Augmented Inverse Probability Weighted estimator proposed by Jordà and Taylor (2015). In the first step, we estimate logit models to estimate the likelihood that a structural reform occurs. This latent variable framework captures the idea that reforms are introduced in periods where the expected benefits of reforms are large. In the second step, we use local projections specified as equation 2, but weighing observations inversely according to the predicted probabilities from the logit model. Specifically, observations in which a reform took place are assigned a weight by the inverse of the probability score, whereas the observations where no reform took place receive a weight of the inverse of one minus the probability score. This means that treated observations with a low probability score receive a higher weight in the regression along with control observations with a high probability score. This places more weight on observations that are comparable and hence reduces selection bias. The augmented 8 Our choice of information criteria is based on the fact that the Akaike criterion is least likely to choose an autoregressive process of too low order; see Lutkepohl and Kratzig (2004). 9 The bias increases with the forecast horizon, see Teulings and Zubanov (2014). The leads of the treatment dummies ensure that it is registered in the data if the outcome for a specific observation is affected by a treatment ahead in time. This most often is the case for control observations, i.e. country-year pairs where no reform took place. However, in the IMF narrative reform data, reforms at times occur repeatedly within our forecast horizon of 5 years. In that case the Teulings and Zubanov (2014) approach also registers that the outcome of a treated observation may be affected by later treatments, which otherwise would have meant an upward bias in the effect of reforms. This is especially the case for product market reforms which happens frequently in the data (see Table 1).
weighting adds an adjustment factor to the treatment effect when the estimated probability scores are close to zero or one. The method is said to be double robust as it only requires one of the following two conditions to hold: The conditional mean model is correctly specified or the probability score model is correctly specified. Weighting can be interpreted as removing the correlation between the covariates and the reform indicator, and regression removes the direct effect of the covariates (see Imbens and Wooldridge, 2009 for more details). We report the Average Treatment Effect (ATE), which is calculated as the average difference between treated and non-treated (control) observations based on the weighted OLS regression line for both groups. Table 2 shows the LP estimates of our basic model (eq. 2). So, here we do not control for endogeneity and do not condition on the stance of fiscal policy. Figure 2 shows the corresponding impulse response functions for product and labour market reforms, based on the estimates shown in Table 2. Table 2. The dark grey shaded areas display the 90% error bands, the light grey shaded areas display the 95% error bands. Year t=1 is the first year after a reform took place at t=0.
Local Projection results
It is quite remarkable that in this very simple setup, product and labour market reforms do not affect output growth. The estimated coefficient on the output gap coefficient is negative (as expected). This means that on the upturn of the business cycle growth in the future is predicted to be significantly negative and vice versa. It controls for a reversion to the mean effect. 25 Notes: The table shows the local projection estimates of labour and product market reforms on cumulative economic growth, unconditional of the fiscal policy stance. The models are based on equation 2, and include treatment leads equal to the forecast horizon, five lags of the treatment variable, and country fixed-effects. The number of treatment lags was determined by Akaike's information criterion. Standard errors clustered at the country level are shown in parentheses: *** p<0.01, ** p<0.05, * p<0.1.
The sum of the coefficients on the lagged dependent variable in column (1) is larger than one, which may imply a non-stationary growth process. However, panel stationarity tests reject non-stationarity (results available on request). In columns (2)-(5) of Table 2, the sum is much larger than one, but that is not a surprise since we estimate cumulative growth rates. The in-significant physical and human capital elasticities are perhaps not such a surprise in a sample of OECD countries; see Mankiw et al. (1992) for a similar finding.
Next, we included the stance of fiscal policy. Table 3 presents the estimation results and Figure 3 shows the corresponding impulse response functions. Surprisingly, the main takeaway from Table 3 is that product and labour market reforms only have an effect during periods of tight fiscal policy. Specifically, for product market reforms the accumulative effect on growth after 5 years is almost 2% of additional GDP. For labour market reforms the effect is significant and positive, after 5 years 3.7% of additional GDP is estimated. Table 3 with the solid black lines. The impulse responses are partitioned on fiscal policy stance, the panels on the left display the impulse responses under tight fiscal policy, the panels in the middle under neutral fiscal policy and the panels on the right under loose fiscal policy. The dark grey shaded areas display the 90% error bands, the light grey shaded areas display the 95% error bands. Year t=1 is the first year after a reform took place at t=0. Yes Notes: The table shows the local projection estimates of labour and product market reforms on cumulative economic growth, conditional of fiscal policy stance. The models are based on equation (2), but also include treatment leads equal to the forecast horizon, five lags of the treatment variable, and country fixed-effects. The number of treatment lags was determined by Akaike's information criterion. Standard errors clustered at the country level are shown in parentheses: *** p<0.01, ** p<0.05, * p<0.1.
Quasi-experimental results
In an ideal RCT (Randomized Controlled Trial) setting where treatments are assigned randomly, we would expect the probability density function for each control variable included in equation (2) to be the same for each sub-population of treated and control units.
The overlap of the densities should be close to perfect. For example, the distribution of the deviation between actual GDP and potential GDP (the output gap) should be similar for the subpopulation where a major product market reform takes place and the subpopulation of all other (control) observations. A simple way to check whether this condition holds is to do a test of equality of means between the subsamples. This is done in Table 4 below. As evident, especially the balance of the output gap between treated and control observations is a cause of concern. This is an indication that we cannot assume that treatments are assigned randomly as is done in the simple LP analysis above. Specifically, the balance test in Table 4 indicates that the output gap on average is negative (implying that the economy is running below it potential) for treated observations compared to control observations. This suggests that labour and product market reforms cannot be viewed as exogenous events.
Notice that this balance condition is also behind the implicit assumption that we can estimate the simple LPs presented above by restricting the coefficients of the controls in equation 2 to be the same for the treatment and the control groups. The AIPW estimates below relaxes this assumption, as a regression is specified separately for both the treatment group and the control group (Imbens and Wooldride, 2009;Jordà and Taylor, 2015). The difference in the predicted outcomes of log %,'() − log %,' between each regression for the treatment and the control group then serves to calculate the (weighted) ATEs. Observations 691 Note: Standard errors of a two-sided t-test are reported in parentheses: *** p<0.01, ** p<0.05, * p<0.1 When policy interventions like labour and product market reforms are driven by endogenous responses to control variables (as shown in Table 4), the observed treatment and control units can be viewed as being oversampled from the part of the distribution in which the propensity score of treatment reaches high values. The simple LP projections presented above are based on the sampled distribution and will therefore be biased. Too much weight is given to treated observations with a high probability of treatment and too little weight is given to control observations with a high probability of treatment. Inverse weighting using propensity scores shift the probability mass away from the oversampled region of the distribution towards the under-sampled region. This shift rebalances the sample such that we can view the re-weighted sample as reconstructing the true distribution of outcomes under treated and control observations. In other words, we can view the rebalancing as if we had observed a random sample for each group, unaffected by endogenous responses to control variables. Thus, the regression for both the control group and the treatment group are less susceptible to bias and their difference can be used to calculate an unbiased estimated of the ATE of reforms on economic growth (see Imbens andWooldridge, 2009 andJordà andTaylor, 2015 for more details). 10 To estimate the propensity scores, ideally any predictor of treatment should be included regardless of whether that variable is included in the model specified in eq. (2). Therefore, we follow Jordà and Taylor (2015) and estimate a saturated propensity score model. As predictors of the likelihood of structural reform ("the treatment in t+1"), we use the following variables. First, the output gap and the lagged growth rates as these variables capture the idea that reforms are more likely to occur after times of economic crisis (Drazen and Grilli, 1993). In line with this argument, we also include the unemployment rate and the inflation rate, as these variables may also signify difficult economic times. We also add the fiscal policy stance to take into account that reforms may be more likely during different types of fiscal policy regimes. We add political variables to account for the fact that reforms are more viable under certain political circumstances. Specifically, we add: (1) A variable counting the number of years a government has held office to capture the idea that reforms become less likely the longer a government holds office. (2) An election variable reflecting that an executive or legislative election took place to capture the idea that reforms typically are more likely after a new government takes office (Haggard and Webb, 1993). (3) A variable measuring government ideology to capture the idea that the political colour of a government matters in terms of the policies it implements (Hibbs, 1977). (4) A variable measuring political fractionalisation to capture the idea that more politically fragmented governments may find it harder to implement economic reforms (Alesina and Drazen, 1991). We also control for the possibility that labour and product market reforms may be related (Fiori et al., 2012) by including labour market reforms as predictor of product market reforms in t+1, and vice versa for labour market reforms. In the logit model for labour market reforms, we also add institutional variables capturing the strictness of hiring and firing conditions for workers on temporary or regular contracts. This takes a level effect into account, as countries with very flexible hiring and firing conditions are typically less likely to reform the labour market (Turrini et al., 2015). We also include variables for duration dependence (Carter and Signorino, 2010). Specifically, we add a variable that counts the number of years since the last reforms, plus its squared and cubed term. F-tests show that the duration dependence variables are jointly significant and therefore should be in the model. Table A1 in Appendix 2 provides a description of the variables used.
An important thing to note here is that although relatively few variables are highly significant, the model has a high predictive ability: the 'area under the ROC curve' is above or close to 0.8 in all the reported logit models. 11 In all specifications shown in Table 5, the area under the ROC curve is statistically significantly different from 0.5. 12 Figures 4-6 provide smooth kernel density estimates of the distribution of the propensity scores for treatment and control units to check for overlap. The plotted densities are based on models 1-3 in Table 5, respectively. In the ideal RCT setting, the overlap between the distribution of propensity scores for treated and control units would be near identical. Although the logit models used to estimate the propensity scores all have high predictive ability, Figures 4-6 make clear that we have considerable overlap between the distributions for treated and control units. This indicates that we have a satisfactory logit model that can be used to identify the ATEs properly using our quasi-experimental estimation strategy. Figures 5 and 6 also make clear that some treated units have a propensity score that is very close to 0. In practice, this means that these observations get very high weights when weighing inversely with the propensity score. Although the AIPW estimator adds an adjustment factor to the treatment effect when the estimated probability scores are close to 0 for treated observations (and close to 1 for control observations) this is not enough to stabilise the estimator in our setting. Therefore, we truncate the propensity scores for labour market reforms and joint reforms (see the notes to Figures 5 and 6), following Imbens (2004) and Cole and Hernan (2008).
Figure 4. Overlap of propensity scores for product market reforms
11 ROC stands for Receiver Operating Characteristics. It is also referred to as the Correct Classification Frontier. If the model had no predictive ability, the area under the ROC curve would be 0.5. A perfect classification ability would correspond to an area under the ROC curve equal to 1. The area under the ROC curve has an approximate normal distribution in large samples. 12 In line with Jordà and Taylor (2015), we include country-dummies in the estimations. If we estimate the models in Table 5 without fixed-effects the predictive ability declines, but the area under the ROC curve is still statistically different from 0.5. We also estimated the model without country fixed effects, but the model with country fixed effects turned out to be superior in predicting treatment. So, we proceed with the FE specification for the propensity scores in the AIPW estimates regardless of the incidental parameter problem in the logit model.
Figure 5. Overlap of propensity scores for labour market reforms
Note: In the AIPW estimates of labour market reforms below we truncate propensity scores at 0.1 for p-scores lower than 0.1 due to many observations with a very low p-score. Otherwise the estimator becomes unstable (1 divided with a very small number will give a very large weight to treated observation with a low predicted p-score). To keep symmetry, we also truncate at high propensity scores, so above 0.9, but this has no consequences as can be seen in Figure 4.
Figure 6. Overlap of propensity scores for the interaction of labour and product market reforms
Note: In the AIPW estimates of the interaction of labour and product market reforms bellow we truncate propensity scores at 0.05 for p-scores lower than 0.05 due to observations with a very low p-score. Otherwise the estimator becomes unstable (1 divided with a very small number will give a very large weight to treated observation with a low predicted p-score). To keep symmetry, we also truncate at high propensity scores, so above 0.95, but this has no consequences as can be seen in Figure 5. Note: The table reports point estimates of a logit specification to predict the probability of treatment in t+1. In model 3 treatment is defined as observations in which both a product market reform and a labour market reform occurred simultaneously, there are 24 treatments in that case. As a consequence, we can only use the 13 countries in which reforms occurred at least once simultaneously to estimate the model, due to the inclusion of fixed effects. Standard errors clustered at the country level are shown in parentheses: *** p<0.01, ** p<0.05, * p<0.1.
Tables 6 and 7 present the results using the quasi-experimental doubly-robust Augmented Inverse Probability Weighted (AIPW) estimator proposed by Jordà and Taylor (2015). Table 6 shows the estimation results if we do not condition on fiscal policy, while Figure 7 offers the corresponding impulse response functions. If we do not condition on the fiscal policy stance at the time of the reform, the effects of reform on GDP growth are small. Only labour market reforms affect economic growth: after 3 years, economic growth has declined by 0.3%.
However, if we split the sample and estimate ATEs for each type of reform during different types of fiscal stances a more fine-grained pattern emerges (Table 7 and Figure 8). Product market reforms mostly cause slight negative growth, except for a very small positive effect during periods of neutral fiscal policy. Labour market reforms hurt growth if introduced during periods of tight fiscal and neutral fiscal policy, but they are conducive to economic growth if introduced during periods of loose fiscal policy. Yes Notes: The table shows the ATE responses of the AIPW local projection estimates of labour and product market reforms on cumulative economic growth, unconditional of fiscal policy stance. Compared to Table 2, the number of observations drop due to unavailability of some of the reform predictor variables. The models are based on equation 2, but also include treatment leads equal to the forecast horizon, five lags of the treatment variable, and country fixed-effects. The number of treatment lags was determined by Akaike's information criterion. Standard errors clustered at the country level are shown in parentheses: *** p<0.01, ** p<0.05, * p<0.1.
Figure 7. ATE responses of AIPW local projection estimates of the effect of product and labour market reforms on cumulative economic growth in t+1-5
Notes: The figure plots the ATE responses of product (left panel) and labour market (right panel) reforms on cumulative economic growth from Table 6 with the solid black lines. The dark grey shaded areas display the 90% error bands, the light grey shaded areas display the 95% error bands. Year t=1 is the first year after a reform took place at t=0. Yes Notes: The table shows the ATE responses of the AIPW estimates of labour and product market reforms on cumulative economic growth, conditional of fiscal policy stance. The models are based on equation 2, but also include treatment leads equal to the forecast horizon, five lags of the treatment variable, and country fixedeffects. The number of treatment lags was determined by Akaike's information criterion. Standard errors clustered at the country level are shown in parentheses: *** p<0.01, ** p<0.05, * p<0.1.
Figure 8. ATE responses of AIPW local projection estimates of the effect of product and labour market reforms on cumulative economic growth in t+1-5, partitioned by fiscal policy stance
Notes: The figure plots the ATE responses of product market (panel above) and labour market (panel below) reforms on cumulative economic growth from Table 7 with the solid black lines. The impulse responses are partitioned by fiscal policy stance, the left panels display the ATE responses under tight fiscal policy, the middle panels under neutral fiscal policy and the left panels under loose fiscal policy. The dark grey shaded areas display the 90% error bands, the light grey shaded areas display the 95% error bands. Year t=1 is the first year after a reform took place at t=0.
As shown, our findings change drastically when we control for the fact that the assignment of treatment is non-random. Unconditional of fiscal policy, product market reforms have no statistically significant effect on economic growth, while the unconditional effect of labour market reforms is significantly negative throughout the evaluation period.
When conditioning on fiscal policy a more fine-grained pattern emerges. Contrary to the simple LP results where treatment selection is ignored, we now find mostly negative effects of both product and labour market reforms during fiscal adjustments. However, labour market reforms have a positive effect on growth if implemented during a fiscal expansion, while their effect on growth is again negative if implemented when fiscal policy is neutral. Product market reforms have little to no effect if fiscal policy is neutral or loose.
Robustness analysis
As a first robustness test, we analyse the joint effect labour and product market reforms. In practice, that amounts to analysing whether reforms work better or worse when implemented as broad reform packages, i.e. simultaneous reforms in both the product and labour market.
Unfortunately, we only have 25 observations in which major reforms occur in both the product and labour market simultaneously. Therefore, it is not possible to conduct this analysis while conditioning on fiscal policy. There are simply too few treated observations in each cell for the types of fiscal policy.
The results reported in Table 8 and Figure 9 suggest that the initial effect of joint labour and product market reforms is negative in the short term but in the medium term the effect becomes positive. This conclusion follows from the fact that in the short run the effect of economy wide reforms is negative and just falls short of 10% significance after one year. After 2 years the effect becomes positive and after five years GDP have grown by 3%. The effect after 5 years is almost significant at the 5% level. The table shows the ATE responses of the AIPW estimates of labour and product market reforms occurring simultaneously on cumulative economic growth, unconditional of fiscal policy stance. The models are based on equation 2, but also include treatment leads equal to the forecast horizon, five lags of the treatment variable, country fixed-effects, and the indicators of labour and product market reforms. The number of treatment lags was determined by Akaike's information criterion. Standard errors clustered at the country level are shown in parentheses: *** p<0.01, ** p<0.05, * p<0.1. Table 8 with the solid black lines. The dark grey shaded areas display the 90% error bands, the light grey shaded areas display the 95% error bands. Year t=1 is the first year after a reform took place at t=0. Additionally, we check if our main AIPW findings are sensitive to the way we identify the fiscal policy stance. As an alternative to the method based on structural break tests, we apply threshold criteria as usual in the literature on fiscal adjustments (cf. Alesina and Perotti, 1995;see Wiese et al., 2018 for an overview of thresholds used in the literature). Specifically, we define the start of a fiscal adjustment as a positive change in the CAPB larger than 1.5 percent of GDP; the adjustment continues as long as the change in CAPB is positive. A negative change in CAPB smaller than -1.5 percent of GDP indicates the start of a fiscal expansion, which continues as long as the change in the CAPB is negative. That way, we identify 152 periods with fiscal adjustments, 128 periods with fiscal expansions and 517 periods with neutral fiscal policy; see Figure A3 in the online Appendix 1 for the distribution of reforms over these types of fiscal policy stances. Yes Notes: The table shows the ATE responses of the AIPW estimates of labour and product market reforms on cumulative economic growth, conditional on the fiscal policy stance determined using a threshold approach. The models are based on equation 2, but also include treatment leads equal to the forecast horizon, five lags of the treatment variable, and country fixed-effects. The number of treatment lags was determined by Akaike's information criterion. Standard errors clustered at the country level are shown in parentheses: *** p<0.01, ** p<0.05, * p<0.1.
Figure 10. ATE responses of AIPW local projection estimates of the effect of product and labour market reforms on cumulative economic growth in t+1-5, partitioned by alternative fiscal policy stance
Notes: The figure plots the ATE responses of product market (panel above) and labour market (panel below) reforms on cumulative economic growth from Table 9 with the solid black lines. The impulse responses are partitioned by the fiscal policy stance determined using a threshold approach. The left panels display the ATE responses under tight fiscal policy, the middle panels under neutral fiscal policy and the left panels under loose fiscal policy. The dark grey shaded areas display the 90% error bands, the light grey shaded areas display the 95% error bands. Year t=1 is the first year after a reform took place at t=0. Figure 10 suggests that our conclusion that labour market reforms enhance economic growth under loose fiscal policy also holds under the alternative definitions of fiscal adjustments and expansions. Under tight and neutral fiscal policy, labour market reforms have a negative effect on growth; under tight fiscal policy this negative effect is significant only in the first years after the reform, while under neutral fiscal policy it becomes significant after some years. In line with our previous findings, product market reforms generally have a negative or non-significant effect on economic growth.
Finally, a cause of concern about our estimates may be the Nickell (1981) bias. Specifically, we estimate a dynamic panel model with fixed-effects. As Nickell (1981) shows, the demeaning process creates a correlation between regressor and error which creates a bias in the estimated coefficient of the lagged dependent variable. If the independent variables of interest are correlated with the lagged dependent variable their coefficients may be biased as well. This is particular a problem in a large N, small T context. We have small N and relatively large T. The bias can be gauged in the following way.
If the AR(1) coefficient 3 on ∆ %,' is positive (as in our case), the bias is invariably negative, so that the persistence of the 3 coefficient on ∆ %,' will be underestimated. For reasonably large values of T, the limit of 3 on ∆ %,' as N → ∞ will be approximately −(1 + 3 )/(T − 1). In our case 3 = 0.67, the bias will be about -0.062, or less than 1/10 of the true value. This is even assuming that N tends to infinity, which is far from the case in our application. Furthermore, the correlation between the labour and product market indicators and ∆ %,' is low and negative. The correlation coefficient for product (labour) market reforms and lagged GDP growth is -0.04 (-0.08). Because of this negative correlation, the Nickell bias also leads to an underestimation of the impulse responses of reforms on growth. This, in combination with the relative low size of the biased AR(1) term and the large T relative to N leads us to conclude that the Nickell bias in our case is negligible. 13
Conclusion
Our findings indicate that controlling for endogeneity of reforms and the stance of fiscal policy is crucial. Our results suggest that product market reforms mostly cause slight negative growth. Labour market reforms hurt growth under tight and neutral fiscal policy but are conducive to economic growth if introduced during periods of expansionary fiscal policy. Additionally, we show that product and labour market reforms are substitutes in the short run, but complements in the medium run.
One important topic for future research is to analyze the election effects of reforms. Recently, Alesina et al. (2020) found that liberalizing reforms are costly to incumbents when implemented close to elections. They also find that the electoral effects depend on the state of the economy at the time of reform: reforms are penalized during contractions; liberalizing reforms undertaken in expansions are often rewarded. Our results suggest that in analysing the electoral consequences of reform, it is important to distinguish between labour and product market reforms, as they may affect economic growth differently, and to take the fiscal policy stance into account as well, since expansionary fiscal policy may alleviate the negative short-run growth effects of reform. 13 GGM estimation is not suited in cases of large T and small N. Rather a method based on recursive substitutions could be used. But as noted in Teulings and Zubanov (2014), a disadvantage of such an approach is a sizeable efficiency loss. | 2020-11-14T08:18:38.455Z | 2022-01-06T00:00:00.000 | {
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55852255 | pes2o/s2orc | v3-fos-license | A UAV-BASED LOW-COST STEREO CAMERA SYSTEM FOR ARCHAEOLOGICAL SURVEYS-EXPERIENCES FROM DOLICHE (TURKEY)
The use of Unmanned Aerial Vehicles (UAVs) for surv eying archaeological sites is becoming more and mor e c mmon due to their advantages in rapidity of data acquisition, cost-ef fici ncy and flexibility. One possible usage is the documentation and visualization of historic geo-structures and -objects using UAV-a ttached digital small frame cameras. These monoscop ic cameras offer the possibility to obtain close-range aerial photograph s, but – under the condition that an accurate nadir -waypoint flight is not possible due to choppy or windy weather conditions – at the same time implicate the problem that two single aer ial images not always meet the required overlap to use them for 3D photogramme tric purposes. In this paper, we present an attempt to replace the monoscopic camera with a calibrated low-cost stereo camera tha t takes two pictures from a slightly different angl e at the same time. Our results show that such a geometrically predefined stereo im age pair can be used for photogrammetric purposes e .g. the creation of digital terrain models (DTMs) and orthophotos or the 3D ext raction of single geo-objects. Because of the limite d g ometric photobase of the applied stereo camera and the resulting base-he ight ratio the accuracy of the DTM however directly depends on the UAV flight altitude.
INTRODUCTION
Archaeology offers a wide field of possible applications for Unmanned Aerial Vehicles (UAVs).Because of their small weight and compact dimensions and the possibility to collect data without having ground based physical access to areas of interest, they are very well suited for the usage at archaeological excavation sites.Traditionally the documentation in Archaeology is based on accurate mapping by hand drawings or by using terrestrial survey instruments like theodolites or laser scanner.Both methods are extremely time-consuming and costintensive, so consequently one important methodological issue is the documentation of historic geo-structures and -objects by close-range aerial photography.This can be achieved by using UAV-attached digital small frame cameras.The common UAV remote sensing system set-up is usually driven by the idea of high flexibility, efficiency and economical aspects and even more important: to overcome the limitation of the restricted payload by an optimal sensor set up.Consequently lightweighted monoscopic consumer digital cameras (which are often not calibrated) are being used for image acquisition and geo-object mapping.In order to be able to use such individual images for 3D photogrammetric purposes, two corresponding images require an overlap of at least 60% (Kraus, 2007).Especially if the excavation site is located at an unsheltered and exposed spot, unstable and windy weather conditions can make an accurate UAV-waypoint nadir-flight impossible.Even images that were taken at the same position in a very short interval of time may show significant displacements of the camera perspective (Lo Brutto, Borruso and D'Argenio 2012).This may result in the difficulty to achieve the necessary level of geometric orientation and overlap between single images.In contrast to this conventional practice, our approach is to replace the monoscopic camera by a calibrated stereoscopic camera, commonly referred to as '3D stereo camera'.The main objective of this study is to evaluate to what extent the combination of a quadrocopter and such low-cost digital stereo camera is able to produce stereoscopic evaluable sets of aerial photographs for the (3D-) documentation of archaeological geostructures.
SYSTEM SET UP
The archaeological excavation site in Doliche that served as a testing area for our field study is located in South East Turkey (Province of Gaziantep) and covers approximately 10.000 m 2 at the summit region of Mt.Dülük Baba Tepesi.For our purpose of taking aerial images of this site we chose a small, flexible UAV that is easy to handle and transport.Equipped with a small digital stereo camera, aerial images with high resolution can be obtained from various altitudes and analysed later on.
UAV
Unmanned Aerial Vehicles (UAVs), also called Unmanned Aerial Systems (UASs) or drones are basically aircrafts without a pilot onboard.They may however be radio controlled by a pilot on the ground or fly autonomously along a previously programmed route.There are a variety of UAVs differing in size, shape, weight, possible time of flight, load capacity and fields of application.The wide range of their potential photogrammetric applications has been verified in different studies.In particular, it has been shown that UAV-platforms with attached digital cameras are very well suited for aerial surveys of archaeological sites (e.g.Eisenbeiss et al, 2005).The UAV we used for our study needed to be light-weight, relatively small and easy to transport, capable of carrying the stereo camera, which weights about 250g, mounted to a servo-driven bracket.For these reasons we chose a self-built Quadro XL (MikroKopter, 2012a) equipped with a modified landing skid and a servo-driven camera bracket (Figure 1).The copter is propelled by four rotors.It has a diameter of 60cm and a load capacity of up to 1kg.It was radio controlled from the ground using a Spektrum MX-20 RC.
Stereoscopic Camera
The camera applied in our ongoing study is the FinePix Real 3D W3, manufactured by Fuji Company (Fujifilm, 2013).It was launched on the market in 2010 as a low-cost digital stereo camera for amateur photographers.It represents a small-format and light-weight stereoscopic digital camera which is equipped with two parallel lenses able to acquire two images simultaneously with a defined stereo-overlap of at least 90% at a digital resolution of 10MP.The images data can be stored as a single MPO (Multi Picture Object) file or as two different JPEG files.The stereo-concept of the Finepix 3D W3 equates to the principle of stereoscopic viewing with human eyes.As a consequence, a geo-object is imaged from two different perspectives resulting in two corresponding but slightly different photos: One point on the ground is projected under a certain convergence angle, latter one depending on the base line between the lenses, the flight altitude and the height of the objects.The differences between several angles result in different distances of the points on each image.Instead of measuring the Z-value of the points, the disparity of the distances of the points' projections, the stereoscopic parallax, is measured and hence creates the depth perception (figure 2).
FIELD WORK
The flight campaign was carried out in autumn 2012 in South East Turkey, close to the ancient village of Doliche (Gaziantep Province) where an archaeological site on the summit of Mt.Dülük Baba Tepesi is been excavated and documented by a Turkish-German archaeologist team since 2001 (Winter and Blömer, 2006).In ancient periods, the Doliche region has been famous as the homeland of the roman god Iupiter Dolichenus, who especially gained popularity in the Roman Empire during the first three centuries AD.It has been verified that the main sanctuary of Iupiter Dolichenus was located on top of Dülük Baba Tepesi (Blömer and Winter, 2006).
In the past years this site has served as a location for some interdisciplinary archaeological work focussing also on geodata processing, image acquisition, GIS-technologies and the interpretation of close range high resolution aerial images acquired by a balloon-mounted digital remote sensing camera system (Lasar, 2008;Krüger, 2009;Prinz, Lasar and Krüger, 2010).In Turkey, the recording and acquisition of aerial images is restricted by military law and it is difficult to obtain permissions for surveying flights with planes.Alternate ways of gathering aerial images, as with a UAV, are much more practicable.
UAV flights were carried out on seven days between 11 th and 22 nd September 2012.Prior to the flight missions full ground control points (including X, Y and Z-values) were put down and picked up with a GPS total station so that the corresponding aerial images could be oriented using the control points' coordinates later on.In total more than 460 control points were distributed over the area.Due to the choppy wind conditions on the mountain Dülük Baba Tepesi (1.211m a.s.l.) flights were mainly performed during the calmer morning hours.One flight could last up to twenty minutes before the copter's batteries had to be changed.The attached stereo camera was oriented towards the ground and hold in a nadir-position by the servo-driven camera bracket.Most of the camera's settings were adjusted to the settings as they had been defined when the camera was calibrated in the lab.After the start of the rotors, a stereo image was taken every 4.6 seconds and saved as an MPO-file that was later unpacked to individual sets of two corresponding JPEG-files.Pictures that were taken during take-off or landing procedures were excluded from further analysis.After all 425 appropriate stereo image pairs remained.
IMAGE PROCESSING AND DATA ANALYSIS
The 3D-image analysis was performed using ERDAS LPS/Stereo Analyst (ERDAS Imagine, 2012).Since the inner orientation of the camera was known due to the calibration report we used the ground control points' coordinates to perform an aerial triangulation in order to determine the exterior orientation parameters of the images and to eliminate radial distortions.This procedure included an automated extraction of a digital terrain model (DTM).Exploiting this model, further quantitative analysis is possible, including manual or automated measuring and mapping of unknown point coordinates, heights, distances, areas and volumes (Aber, 2010).Furthermore we used the DTM as input data for the orthorectification of the images and thus were able to create geometrically corrected orthophotos (Figure 3).Such images may now be used for different documentation purposes, e.g. as geobase maps in a GIS environment, combined with corresponding thematic map layers such as strata findings or other relevant archaeological information.Lasar (2008) has previously shown that such close range orthoimages yield additional crucial information, especially if used as 'time slots' within time-spatial considerations of the ongoing excavation progress (monitoring).A commonly used way to reveal a stereoscopic impression and to view the excavation site as a three dimensional scene is to display the stereo image pair as one anaglyph 3D image by combining the corresponding two images in a shifted cyan/red mode (Figure 4).By using cyan/red glasses, it is now possible to view the excavation site as a three dimensional scene and to carry out dimensional measurements without any further image processing.We also successfully used ERDAS Stereo Analyst to perform a three-dimensional extraction of single geo-objects like ashlar rocks out of the anaglyph 3D images (Figure 5).
DISCUSSION
The analysis has shown that stereo image pairs obtained by a calibrated UAV-based low-cost stereo camera system can be used to create digital terrain models and orthophotos that may serve for a variety of further archaeological purposes.Even the 3D extraction of single geo-objects can be realized with the obtained images.As a consequence of the limited photobase of the stereo camera, the resulting base-height-ratio and the increasing ground sample distance with increasing flight altitude, however, one has to state that the accuracy of the outcomes directly depends on the UAV flight altitude: Differences in height can be much better perceived if the flight altitude is low.Small-scaled images are therefore much more vulnerable for errors in Z-values.We observed that the lower a stereo image flight campaign was conducted, the better the three-dimensional quality/resolution of the calculated surface models becomes.One traditional way of archaeological documentation is to draw the position of geo-objects at the excavation site by hand.To assure the geometric accuracy of these drawings, points are continuously picked up with a total station.Even though this is a very time-consuming technique it is reasonable to assume that it is also very accurate.We used these drawings as a reference and overlaid our orthophotos with them using ArcMap 10.1 (ESRI, 2012).It becomes obvious that orthophotos derived of stereo image pairs that were taken at a low flight altitude have a smaller offset than orthophotos that derived of stereo image pairs that were taken at a higher altitude (Figures 6 and 7).We therefore can conclude that the quality of our orthophoto (which depends on the quality of the DTM) declines with the shooting distance.This means the lower the flight altitude is, the better the accuracy of the surface model and the orthophoto becomes.One has to obtain a reasonable compromise between the desired accuracy and the area that can be covered by one stereo image pair.This can only be decided upon the concrete use case.Further research will have to show to what extent the quality of the DTMs may be improved by adding more ground control points.As Eisenbeiss, 2009 has observed, not all commercially available software packages for image processing can be used in all possible photogrammetric applications of UAVs, since most of them are designed for standard aerial images.Some studies have explored the possibilities of alternate, free and low cost software that for example make use of structure-from-motion techniques (Neitzel and Klonowski, 2011).Further research will have to evaluate the possibilities of these computer vision techniques to perform the image processing with image pairs obtained from a UAV-based stereo camera.Possible applications for the technique of using a UAV-based low-cost stereo camera system may however go beyond archaeology.One imaginable example is the digital representation of city structures and the visualization of planned architectural changes where the relation of different buildings is much more important than their accurate position.Another use case could be the usage of stereo image pairs for the creation and digital representation of time series to monitor ongoing social-spatial processes for geographical analysis, e.g. in remote villages, where up-to-date aerial images with high resolution are not available.Here again, the exact positions of geo-objects are less important than the observation of their general structures and their change over time.Thus, our approach can serve as a fast, cost-effective and flexible way for the production of geometrically predefined stereo image pairs even under challenging weather conditions.
CONCLUSION
Our study has shown that a UAV-based low-cost stereo camera system is able to produce stereoscopic evaluable sets of aerial photographs that can be used for the documentation of archaeological geo-structures.If the inner orientation of the camera is known and the outer orientation can be determined by ground control points, a digital terrain model (DTM) and orthophotos can be generated out of stereo image pairs.The 3D extraction of single geo-objects is possible as well.We therefore conclude that the combination of an Unmanned Aerial Vehicle and a stereo camera bears a high potential for the geocoded documentation of archaeological surface structures and geoobjects during ongoing excavation and survey stages.It can indeed serve as a promising alternative to conventional monoscopic systems, since the major problem of latter imaging set up is the (scale depending!)large amount of resulting single images and the laborious calculation of their individual orientation.Even though due to the limited geometric photobase of the stereo camera lower flight altitudes result in better three-dimensional qualities, the main advantage of our approach is the possibility to produce collections of geometrically predefined stereo image pairs instantaneously.
Figure 1 .
Figure 1.MikroKopter (Quadro XL) with the attached Fuji FinePix Real 3D W3 stereo camera during a flight mission at the survey site Doliche (photo by R. Dylka, 2012)
Figure 3 .
Figure 3. Orthophoto mosaic (rotated) of ca.70 m 2 of the excavation site in Doliche derived from a digital terrain model that was created out of a stereo image pair
Figure 6 .
Figure 6.Detail of an orthophoto with overlaid drawings.Flight altitude was approx.12m; the photo reveals an offset of up to 20cm.
Figure 7 .
Figure 7. Detail of an orthophoto with overlaid drawings.Flight altitude was approx.6m.The DTM's and thus the orthophoto's accuracy rise considerably with lower flight altitude (note the two green and blue ground control points).
Table 1 .
Specifications of the Fujfilm Finepix Real 3D W3 stereo camera
Table 2 .
Settings of the Fujifilm Finepix Real 3D W3 stereo camera | 2018-12-11T10:06:11.177Z | 2013-08-16T00:00:00.000 | {
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46262311 | pes2o/s2orc | v3-fos-license | Activation of CaMKIIδC Is a Common Intermediate of Diverse Death Stimuli-induced Heart Muscle Cell Apoptosis*
Ca2+-calmodulin-dependent protein kinase II (CaMKII) is expressed in many mammalian cells, with the δ isoform predominantly expressed in cardiomyocytes. Previous studies have shown that inhibition of CaMKII protects cardiomyocytes against β1-adrenergic receptor-mediated apoptosis. However, it is unclear whether activation of CaMKII is sufficient to cause cardiomyocyte apoptosis and whether CaMKII signaling is important in heart muscle cell apoptosis mediated by other stimuli. Here, we specifically enhanced or suppressed CaMKII activity using adenoviral gene transfer of constitutively active (CA-CaMKIIδC) or dominant negative (DN-CaMKIIδC) mutants of CaMKIIδC in cultured adult rat cardiomyocytes. Expression of CA-CaMKIIδC promoted cardiomyocyte apoptosis that was associated with increased mitochondrial cytochrome c release and attenuated by co-expression of Bcl-XL. Importantly, isoform-specific suppression of CaMKIIδC with the DN-CaMKIIδC mutant similar to nonselective CaMKII inhibition by the pharmacological inhibitors (KN-93 or AIP) not only prevented CA-CaMKIIδC-mediated apoptosis but also protected cells from multiple death-inducing stimuli. Thus, activation of CaMKIIδC constitutes a common intermediate by which various death-inducing stimuli trigger cardiomyocyte apoptosis via the primary mitochondrial death pathway.
CaMKII family encoded by four different genes (␣, , ␦, and ␥). The ␦ isoform of CaMKII family is predominantly expressed in the heart of many mammalian species, including humans (2)(3)(4)(5). Two primary splicing variants of the ␦ isoform, CaMKII ␦B and CaMKII ␦C , have been cloned from rat heart (2). Compared with CaMKII ␦C , CaMKII ␦B has an additional 11-amino acid sequence that contains a nuclear targeting signal (6). Thus, CaMKII ␦B and CaMKII ␦C localize to the nuclear and the cytosolic compartments, respectively, in cardiac myocytes (6).
Over the past decade, a number of studies have been focused on the role of CaMKII (without reference to specific isoforms) in regulating cardiac excitation-contraction coupling. Most if not all of the characterized CaMKII target proteins in the heart are involved in the regulation of intracellular Ca 2ϩ and excitation-contraction coupling. These include sarcoplasmic reticulum (SR) Ca 2ϩ release channels known as the ryanodine receptor (RyR2) (7), SR Ca 2ϩ pump and its regulator phospholamban (PLB) (8,9), and sarcolemmal L-type Ca 2ϩ channels (10 -13). Phosphorylation of these substrates plays an essential role in regulating diverse and important cardiac functions such as the well established frequency-dependent accelerations in contractile relaxation and Ca 2ϩ transient decay (14,15) and cardiac pacemaker activity (16). However, CaMKII activation might be abnormally enhanced under certain pathological conditions that disrupt intracellular Ca 2ϩ mobilization, such as cardiac ischemia, acidosis, myocardial infarction, and excessive -adrenergic receptor (AR) stimulation. Because of its positive feedback biochemical nature (i.e. autophosphorylation) (1), the exaggerated CaMKII activation or expression has been implicated in the pathogenesis of arrhythmia (17,18), cardiac hypertrophy (19 -22), and cardiomyopathy (5,(22)(23)(24).
It has been demonstrated that CaMKII is persistently activated during sustained AR stimulation in cardiac myocytes both in cell culture and in vivo (25)(26)(27) and that this activation is part of the signaling pathway relaying  1 AR-evoked apoptotic signals in the heart (25). In this model, inhibition of CaMKII prevents  1 AR-induced cardiac myocyte apoptosis, whereas overexpression of CaMKII ␦C enhances the  1 AR pro-apoptotic effect (25). It is also noteworthy that the cytosolic isoform, CaMKII ␦C , is selectively up-regulated in a rabbit heart failure model (24), whereas there is little or no change in the expression or activity of the nuclear isoform (CaMKII ␦B ). Despite these studies implicating CaMKII ␦C in the  1 AR-induced cell death, it is currently unclear whether activation of the cytosolic isoform CaMKII ␦C is sufficient to cause heart muscle cell apoptosis and whether other death-inducing stimuli require CaMKII activity to cause cell death. Because apoptosis is a key cause factor of chronic heart failure, the identification of fundamental molecular players involved in heart muscle cell loss has became, in the past decade, an important research focus in the field of cardiovascular biology and medicine.
The goals of the present study were (a) to determine whether activation of the cardiac cytosolic isoform, CaMKII ␦C , is sufficient to trigger cardiac myocyte apoptosis and, if so, (b) to determine whether CaMKII ␦C is a necessary intermediate in the death signaling caused by stimuli other than chronic stimulation of  1 ARs. To address these questions, we used cultured adult rat cardiac myocytes in conjunction with adenoviral gene transfer of constitutively active (CA-CaMKII ␦C ) or dominant negative (DN-CaMKII ␦C ) mutants of CaMKII-␦C to elevate or suppress CaMKII ␦C activity, respectively. We demonstrate that enhanced activation of CaMKII ␦C is sufficient to trigger robust cardiac myocyte apoptosis via activating the mitochondrial apoptotic pathway and that inhibition of CaMKII protects cardiomyocytes not only from  1 AR-induced apoptosis, as previously reported (25), but from a number of other death-inducing stimuli as well. These findings demonstrate that activation of CaMKII ␦C is an important common intermediate in the death signaling pathway of many different stimuli that induce apoptosis in heart muscle cells.
EXPERIMENTAL PROCEDURES
Construction of Viral Vectors-HA-tagged constitutively active CaMKII␦ C (CA-CaMKII) was generated by replacing the residue Thr 287 with Asp (T287D) using the transformer site directed mutagenesis kit (Clontech), whereas dominant negative CaMKII␦ C (DN-CaMKII) was generated by replacing the residue Lys 43 with Ala (K43A), as described previously (27). Adenovirus expressing WT-CAMKII ␦C (25) was kindly provided by Dr. Joan Heller Brown at the Department of Pharmacology, University of California San Diego. The generation and amplification of adenoviruses harboring the target gene were performed in HEK293 cells (27). Recombinant replication-adenovirus expressing Bcl-X L was obtained from the University of Pittsburgh NHLBI, National Institutes of Health Pre-clinical Vector Core.
Cell Culture and Adenoviral Gene Transfer-Single cardiac myocytes were isolated from the hearts of 2-3-month-old Sprague-Dawley rats using a standard enzymatic technique, then cultured, and infected with adenoviral vectors at a multiplicity of infection (m.o.i.) indicated, as described previously (27,28). Briefly, myocytes were plated at a density of 0.5 to 1ϫ 10 4 /cm 2 on coverslips or in dishes precoated with 10 g/ml laminin. The culture medium was M199 (Sigma) plus 5 mmol/ liter creatine, 2 mmol/liter L-carnitine, 5 mmol/liter taurine, 0.1% insulin-transferrin-selenium-X, 1% penicillin and streptomycin, and 25 mmol/liter HEPES, pH 7.4, at 37°C. Adenovirusmediated gene transfer was implemented by adding adenoviral vectors encoding rat WT-CaMKII ␦C , DN-CaMKII ␦C , CA-CaMK ␦C , Bcl-X L , or -gal into the culture dish. The experiments were done with cells cultured 24 Cell Apoptosis-Cell apoptosis was detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay as previously described (25). The percentage TUNEL-positive cells was determined by randomly counting 500 -800 cardiac myocytes over 20 randomly chosen fields in each culture dish. DNA fragmentation was also visualized by DNA laddering assay, as previously described (25).
Western Blotting-CaMKII-dependent phosphorylation of PLB at Thr 17 (PLB-Thr 17 ) was detected by Western blot using a site-specific antibody (Badrilla, West Yorkville, UK) (15,27). To quantify the expression of WT and mutant CaMKII ␦C , cell lysate (30 -50 g of protein) were loaded in a Ca 2ϩ -free loading buffer containing 20 mM EDTA and immunoblotted using anti-HA (1:1000, Covance) or anti-CaMKII antibody (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Following incubation with a peroxidase-conjugated antibody, the films were exposed to the chemiluminescence (ECL; Amersham Biosciences) reaction and quantified with a video documentation system (Bio-Rad). The actin amount used as protein loading control was detected with an anti-actin antibody (Santa Cruz).
Statistical Analysis-The results are presented as the means Ϯ S.E. Statistical significance was determined by oneway analysis of variance or unpaired Student's t test when appropriate. A p value of Ͻ0.05 was considered to be statistically significant.
Manipulation of CaMKII ␦C Activity in Cultured Adult Rat
Cardiomyocytes-Previous studies have shown that CaMKII activation has been implicated in  1 AR-induced apoptosis of adult mouse cardiomyocytes (25) and Ca 2ϩ influx-mediated apoptosis of feline cardiomyocytes (31). Here, we sought to determine whether there is a causal relation between the activation of CaMKII ␦C and heart muscle cell apoptosis. We specifically enhanced or inhibited CaMKII ␦C activity by adenoviral gene transfer of a constitutively active (CA-CaMKII ␦C ) or a dominant negative CaMKII ␦C mutant (DN-CaMKII ␦C ), respectively. The expression of adenovirally delivered HA-tagged WT-, CA-, and DN-CaMKII ␦C was determined by Western blotting with either an anti-HA or an anti-CaMKII antibody in cultured adult rat cardiomyocytes 24 h after infection (Fig. 1A). The autonomous activity of CaMKII, i.e. Ca 2ϩ -calmodulin-independent kinase activity, assayed by 32 P incorporation into a substrate peptide, was elevated by 5.8-fold in cardiomyocytes infected with Adv-CA-CaMKII ␦C , whereas it was suppressed by 70% in cardiomyocytes infected with Adv-DN-CaMKII ␦C (Fig. 1B). To further evaluate the functional consequence of manipulating CaMKII activity, we examined CaMKII-mediated PLB-Thr 17 phosphorylation. Consistent with the kinase activity profile, the expression of CA-CaMKII ␦C and DN-CaMKII ␦C caused a significant increase and decrease, respectively, in the phosphorylation of PLB-Thr 17 (32). These results indicate that we can specifically enhance or suppress CaMKII ␦C activity using adenoviral gene transfer techniques in cultured intact adult rat cardiac myocytes.
Increased CaMKII ␦C Activity Is Sufficient to Cause Heart Muscle Cell Apoptosis-We next explored the potential effect of CaMKII ␦C signaling on myocyte viability. Enforced expression of CA-CaMKII ␦C alone caused increased cardiomyocyte apoptosis, documented by increased TUNEL staining-positive cells (Fig. 2, A and B) and DNA fragmentation revealed by DNA laddering assay, whereas overexpression of WT-CaMKII ␦C did not induce apoptosis in these cells (Fig. 2, A and C), consistent with the kinase activity profile under the same experimental conditions (Fig. 1B). CA-CaMKII ␦C -mediated myocyte apoptosis occurred within 24 h after adenoviral gene transfer and increased in a time-dependent manner (Fig. 2B). Inhibition of CaMKII activity with the peptide inhibitor, AIP (5 M), or coexpression of the DN-CaMKII ␦C mutant effectively suppressed CA-CaMKII ␦C -induced apoptosis (Fig. 2B). Furthermore, CA-CaMKII ␦C protein abundance and the kinase activity were closely correlated with the amount of Adv-CA-CaMKII ␦C virus delivered to the cultured cells (Fig. 3, A and B), with the severity of myocyte apoptosis correlating with the level of CaMKII activity (Fig. 3, C and D).
Involvement of Mitochondrial Death Machinery in Cardiomyocyte Apoptosis Induced by CaMKII ␦C Activation-To investigate whether the mitochondrial death pathway is involved in CaMKII ␦C -mediated heart muscle cell apoptosis, cytochrome c release into the cytosol was monitored in subcellular fractions by Western blotting. We found that cytochrome c was markedly increased in the cytosolic fraction but decreased in mitochondrial fraction in cardiomyocytes expressing CA-CaMKII ␦C , but not in those expressing DN-CaMKII ␦C or WT-CaMKII ␦C (Fig. 4, A and B), suggesting that enhanced CaMKII ␦C activity leads to cytochrome c release from mitochondria into the cytoplasm. The involvement of mitochondrial death signaling was further substantiated by the fact that overexpression of the anti-apoptotic Bcl-2 family member, Bcl-X L , suppressed CA-CaMKII ␦C -mediated cardiac myocyte apoptosis (Fig. 4, C and D).
Activation of Endogenous CaMKII by Various Stimuli That Trigger Myocyte Apoptosis-We next explored the possibility that other cell death-inducing stimuli, such as increased intracellular Ca 2ϩ concentration, acidosis, and oxidative stress, activate endogenous CaMKII to induce myocyte cell death. Indeed, CaMKII-dependent phosphorylation of PLB, the key physiological regulator of SR Ca 2ϩ -ATPase whose phosphorylation at PLB-Thr 17 serves as an intracellular marker of increased CaMKII activity, was augmented by 2-3-fold in myocytes (Fig. 5), demonstrating that endogenous CaMKII activity was elevated in response to those treatments.
The Ca 2ϩ -elevating agents markedly promoted myocyte apoptosis as assessed by increased TUNEL staining (Fig. 6A). On average, the percentage of apoptotic cells was increased ϳ3.5-fold. Similarly, intracellular acidosis or oxidative stress (H 2 O 2 , 12 M) markedly augmented the percentage of TUNELpositive cells (Fig. 6, B and C). Inhibition of CaMKII with KN-93 (2 M) or AIP (5 M) effectively protected cells not only from the Ca 2ϩelevating agents (Fig. 6A) (Figs. 7). Thus, increased CaMKII activity is associated with the administration of a number of different cell death-inducing stimuli. Expression of DN-CaMKII ␦C is able to inhibit the kinase activity and the associated cell death as well, indicating that activation of CaMKII ␦C is a common intermediate in the signaling pathways triggered by multiple stimuli that induce myocyte apoptotic death.
DISCUSSION
In the present study, there are two novel findings. First, isoformspecific activation of CaMKII ␦C by expression of a constitutively active mutant is sufficient to trigger myocyte apoptosis. Second, inhibition of CaMKII effectively protects myocytes not only from adenoviral gene transfer of CA-CaMKII ␦ but also from that caused by increased intracellular Ca 2ϩ , intracellular acidosis, and oxidative stress. These results support the conclusion that enhanced activation of CaMKII ␦C in cardiac myocytes is a common intermediate in the death signaling pathways initiated by diverse cell death-inducing stimuli.
Activation of Cardiac CaMKII ␦C Functions as a Common Intermediate Converging a Multitude of Cardiac Apoptotic
Signaling Pathways-As is the case for most cells, upstream apoptotic signaling of cardiac myocytes can be classified into two general pathways: the death-ligand receptor-mediated pathway involving activation of caspase-8 and downstream executioner caspases and the mitochondrial or intrinsic pathway, the terminal steps of which involve the release of cytochrome c, recruitment of apoptotic protease activating factor-1 (Apaf-1), and the activation of caspase-9 and downstream executioner caspases (33,34). In contrast to the extrinsic pathway that transduces death signaling from a specialized set of death receptors, the intrinsic pathway integrates a broader spectrum of extracellular and intracellular stresses that activate signals converging on the mitochondria, leading to the release of a number of apoptogenic proteins that either directly or indirectly activate caspases. The present study has shown that activation of the cardiac cytosolic CaMKII isoform, CaMKII ␦C , is a common intermediate that integrates various apoptotic stimuli and relays the apoptotic signals to the mitochondrial death machinery, as evi-denced by the robust increase in mitochondrial cytochrome c release and the protective effect of Bcl-X L , an important anti-apoptotic member of Bcl-2 family (35). Altogether, the present findings have identified a potentially crucial molecular intermediate involved in heart muscle cell loss and may shed new light on our understanding of the pathogenesis of heart failure and lead to a potentially important therapeutic approach for reducing myocyte loss, thus preventing or retarding the progression of heart failure caused by various etiologies.
Intracellular Distribution Dictates CaMKII Isoform-specific Functions-Recent work has demonstrated that stimulation of G protein-coupled receptors such as ET-1 leads to cardiac myocyte hypertrophy via a signaling pathway sequentially involving G q , phosphatidylinositol 1,4,5-trisphosphatemediated nuclear Ca 2ϩ release, activation of nuclear CaMKII (CaMKII ␦B ), HDAC5 phosphorylation, and activation of MEF2dependent transcription (35). Remarkably, this Ca 2ϩ -dependent, nuclear CaMKII-mediated hypertrophy signaling pathway cannot be activated by the global Ca 2ϩ transients that cause myocyte contraction (36). Thus, it is reasonable to assume that the distinct intracellular localization of cardiac CaMKII ␦B and CaMKII ␦C may enable myocytes to distinguish simultaneous local and global Ca 2ϩ signals and exhibit different functional roles. This assumption is supported by studies in CaMKII transgenic mouse models. Specifically, overexpression of the cytoplasmic CaMKII ␦C isoform leads to hyperphosphorylation of substrates involved in cardiac excitation-contraction coupling, resulting in SR Ca 2ϩ leak and cardiomyopathy (37,38), whereas overexpression of the nuclear CaMKII ␦B isoform leads to cardiac hypertrophic gene expression profile (21). These findings indicate that the intracellular distribution of the kinase dictates its accessibility and sensitivity to physiological and pathological stimuli.
Our previous studies have shown that overexpression of CaMKII ␦C but not CaMKII ␦B enhances  1 AR-mediated cardiac myocyte apoptosis (25). The present study has further demonstrated that increased activation of CaMKII ␦C is sufficient to trigger cardiac myocyte apoptosis, whereas isoform-specific inhibition of CaMKII ␦C is able to significantly protect cardiomyocytes against multiple death-inducing stimuli-mediated death. These findings provide an explanation for the observation of more severe heart failure and premature death phenotype in transgenic mice overexpressing cardiac CaMKII ␦C (37, 38) compared with those overexpressing cardiac CaMKII ␦B (21). Future investigation is merited to better appreciate the distinct functional roles of these cardiac CaMKII isoforms and their regulation under a variety of physiological or pathological circumstances.
Potential Implications of CaMKII Deregulation in the Pathogenesis of Heart Failure-Multiple lines of evidence suggest that deregulation of CaMKII acts as an important pathogenic factor for heart failure, although this kinase plays a pivotal role in normal cardiac excitation-contraction coupling and pacemaker activity (10 -16). First, previous studies have demonstrated that sustained  1 AR stimulation, a characteristic of the failing heart, causes cardiomyocyte apoptosis via activation of CaMKII signaling, independently of the classic cAMP/cAMP-dependent protein kinase pathway (25). Second, CaMKII expression is increased in the failing hearts of humans and animals (5,(22)(23)(24). Third, transgenic overexpression of the cytosolic isoform CaMKII ␦C induces severe heart failure (37), which is associated with enhanced SR Ca 2ϩ leak, reduced SR Ca 2ϩ content and enhanced fractional SR Ca 2ϩ release (37). Moreover,  1 AR-induced fetal gene expression, a hallmark of cardiac hypertrophy, is mediated by a CaMKII-dependent mechanism in cultured rat neonatal cardiac myocytes (39). In contrast, inhibition of CaMKII activity prevents cardiac arrhythmias and suppresses after depolarizations, a crucial mechanism responsible for heart failure-associated arrhythmias (17,18). Finally, CaMKII inhibition in vivo improves cardiac functional performance and reduces catecholamine-and myocardial infarc-tion-induced maladaptive cardiac remodeling (26). These studies have demonstrated that deregulation of CaMKII signaling is essentially involved in many aspects of cardiac hypertrophy and heart failure, marking CaMKII as a promising therapeutic target for the treatment of heart failure. | 2018-04-03T06:18:20.225Z | 2007-04-06T00:00:00.000 | {
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119295386 | pes2o/s2orc | v3-fos-license | Forms and currents on the analytification of an algebraic variety (after Chambert-Loir and Ducros)
Chambert-Loir and Ducros have recently introduced real differential forms and currents on Berkovich spaces. In these notes, we survey this new theory and we will compare it with tropical algebraic geometry.
Introduction
Antoine Chambert-Loir and Antoine Ducros have recently written the preprint "Formes différentielles réelles et courants sur les espaces de Berkovich" (see [CD12]). This opens the door for applying methods from differential geometry also at nonarchimedean places. We may think of possible applications for Arakelov theory or for non-archimedean dynamics. In the Arakelov theory developed by Gillet and Soulé [GS90], contributions of the p-adic places are described in terms of algebraic intersection theory on regular models over the valuation ring. The existence of such models usually requires the existence of resolution of singularities which is not known in general. Another disadvantage is that canonical metrics of line bundles on abelian varieties with bad reduction can not be described in terms of models. In the case of curves, there is an analytic description of Arakelov theory also at finite places due to Chinburgh-Rumely [CR93], Thuillier [Th05] and Zhang [Zh93]. Now the paper of Chambert-Loir and Ducros provides us with an analytic formalism including (p, q)-forms, currents and differential operators d ′ , d ′′ such that the crucial Poincaré-Lelong equation holds. This makes hope that we get also an analytic description of the p-adic contributions in Arakelov theory. In Amaury Thuillier's thesis [Th05], he has given a non-archimedean potential theory on curves. For the case of the projective line, we refer to the book of Baker and Rumely [BR10] with various applications to non-archimedean dynamics. Again, we may hope to use the paper of Chambert-Loir and Ducros to give generalizations to higher dimensions.
The purpose of the present paper is to summarize the preprint [CD12] and to compare it with tropical algebraic geometry. We will assume that K is an algebraically closed field endowed with a (non-trivial) complete non-archimedean absolute value | |. Let v := − log | | be the corresponding valuation and let Γ := v(K × ) be the value group. Note that the residue fieldK is also algebraically closed. For the sake of simplicity, we will restrict mostly to the case of an algebraic variety X over K. In this case, there is quite an easy description of the associated analytic space X an and so we require less knowledge about the theory of Berkovich analytic spaces than in [CD12]. The main idea is quite simple: Suppose that X is an ndimensional closed subvariety of the split multiplicative torus T = G r m . Then there is a tropicalization map trop : T an → R r . Roughly speaking, the map is given by applying the valuation v to the coordinates of the points. Tropical geometry says that the tropical variety Trop(X) := trop(X an ) is a weighted polyhedral complex of pure dimension n satisfying a certain balancing condition. The thesis of Lagerberg [La12] gives a formalism of (p, q)-superforms on R r together with differential operators d ′ , d ′′ similar to ∂,∂ in complex analytic geometry. Using the tropicalization map, we have a pull-back of these forms and differential operators to X an . In general, we may cover an arbitrary algebraic variety X of pure dimension n by very affine open charts U which means that U has a closed immersion to G r m and we may apply the above to define (p, q)-forms and currents on X an . Chambert-Loir and Ducros prove that there is an integration of compactly supported (n, n)-forms on X an with the formula of Stokes and the Poincaré-Lelong formula. The main result of the paper [CD12] is that the non-archimedean Monge-Ampère measures, which were introduced by Chambert-Loir [Ch06] directly as Radon measures on X an , may be written as an n-fold wedge product of first Chern currents. We will focus in this paper on the basics and so we will omit a description of this important result here.
Terminology
In A ⊂ B, A may be equal to B. The complement of A in B is denoted by B \ A as we reserve − for algebraic purposes. The zero is included in N and in R + .
All occurring rings and algebras are with 1. If A is such a ring, then the group of multiplicative units is denoted by A × . A variety over a field is an irreducible separated reduced scheme of finite type. We denote by F an algebraic closure of the field F .
The terminology from convex geometry is introduced in §2 and §3. Note that polytopes and polyhedra are assume to be convex.
Superforms and supercurrents on R r
In this section, we recall the construction of superforms and supercurrents introduced by Lagerberg (see [La12], §2). They are real analogues of complex (p, q)-forms or currents on C r . So let us first recall briefly the definitions in complex analytic geometry. On C r , we have the holomorphic coordinates z 1 , . . . , z r . A (p, q)-form α is given by α = where I (resp. J) ranges over all subsets of {1, . . . , r} of cardinality p (resp. q) and where the α IJ are smooth functions. Here, use the convenient notation dz I := dz i1 ∧ · · · ∧ dz ip and dz J := dz j1 ∧ · · · ∧ dz jq for the elements i 1 < · · · < i p of I and j 1 < · · · < j q of J. We have linear differential operators d ′ , d ′′ and d = d ′ + d ′′ on differential forms which are determined by the rules for smooth complex functions f on C r . Very often, these differential operators are denoted by ∂ := d ′ and∂ := d ′′ . A current is a continuous linear functional on the space of differential forms on C r . Continuity is with respect to uniform convergence of finitely many derivatives on compact subsets. Differential forms may be viewed as currents using integration and the differential operators d, d ′ , d ′′ extend to currents. For details, we refer to [De12], Chapter I, or to [GH78]. The goal of this section is to give a real analogue in the following setting: Let N be a free abelian group of rank r with dual abelian group M := Hom(N, Z). For convenience, we choose a basis e 1 , . . . , e r of N leading to coordinates x 1 , . . . , x r on N R . Our constructions will depend only on the underlying real affine structure and the integration at the end will depend on the underlying integral R-affine structure, but not on the choice of the coordinates. Here, an integral R-affine space is a real affine space whose underlying real vector space has an integral structure, i.e. it comes with a complete lattice. The definition of the integrals in [CD12] does use calibrations which makes the integrals in some sense unnatural. In the case of an underlying canonical integral structure (which is the case for tropicalizations), there is a canonical calibration (as in [CD12], §3.5) and both definitions of the integrals are the same.
2.1 Let A k (U, R) be the space of smooth real differential forms of degree k on an open subset U of N R , then a superform of bidegree (p, q) on U is an element of Formally, such a superform α may be written as where I (resp. J) consists of i 1 < · · · < i p (resp. j 1 < · · · < j q ), α IJ ∈ C ∞ (U ) and The wedge product is defined in the usual way on the space of superforms A(U ) := p,q≤n A p,q (U ) which means that d ′ x i and d ′ x j anticommute. There is a canonical C ∞ (U )-linear isomorphism J p,q : A p,q (U ) → A q,p (U ) obtained by switching factors in the tensor product. The inverse of J p,q is J q,p . We call α ∈ A p,p (U ) symmetric if J p,p α = (−1) p α.
2.2 There is a differential operator d ′ : A p,q (U ) → A p+1,q (U ) given by This does not depend on the choice of coordinates as is an intrinsic characterization using the classical differential d on the space A p (U, R) of real smooth p-forms. Similarly, we define a differential operator d ′′ : By linearity, we extend these differential operators to A(U ). Moreover, we set d := d ′ + d ′′ .
2.3
If N ′ is a free abelian group of rank r ′ and if F : N ′ R → N R is an affine map with F (V ) ⊂ U for an open subset V of N ′ R , then we have a well-defined pullback F * : A p,q (U ) → A p,q (V ) given as usual. The affine pull-back commutes with the differential operators d, d ′ and d ′′ . The pull-back is defined more generally for smooth maps, but then it does not necessarily commute with d, d ′ and d ′′ .
2.4 Let A c (U ) denote the space of superforms on U with compact support in U . Recall that r is the rank of M . For α ∈ A c (U ), we define . . , r} and the usual integration of r-forms with respect to the orientation induced by the choice of coordinates on the right hand side. If F is an affine map as in 2.3 and if r = r ′ , then we have the transformation formula (see [La12], equation (2.3)). We conclude that the definition of the integral depends only on the underlying integral R-affine structure of N R . The sign (−1) [CD12] for more details about positive forms).
2.5 Now let σ be a polyhedron of dimension n in N R . By definition, σ is the intersection of finitely many halfspaces A polytope is a bounded polyhedron. We say that σ is an integral Gaffine polyhedron for a subgroup G of R if we may choose all u i ∈ M and all c i ∈ G.
In this case, we have a canonical integral R-affine structure on the affine space A σ generated by σ. If L σ is the underlying real vector space of A σ , then this integral structure is given by the lattice N σ := L σ ∩ N . Using 2.3 and the above, we get a well-defined integral σ α for any α ∈ A n,n c (U ), where U is an open neighbourhood of σ.
Using the basis e 1 , . . . , e r of N and assuming α ∈ A r,r c (U ), the contraction α; e 1 , . . . , e r {r+1,...,2r} is a (r, 0)-superform which may be viewed as a classical r-form on U . Then it is immediately clear from the definitions that we have Next, we are looking for an analogue of Stokes' theorem for superforms.
2.7
Let H be an integral R-affine halfspace in N R . This means that H = {ω ∈ N R | u, ω ≤ c} for some u ∈ M and c ∈ R. Using a translation, we may assume that c = 0 and hence the boundary ∂H is a linear subspace of N R . Let [ω ∂H,H ] be the generator of N/(N ∩ ∂H) ∼ = Z which points outwards, i.e. there is u ∂H,H ∈ M such that u ∂H,H (H) ≤ 0 and u ∂H,H (ω ∂H,H ) = 1. We choose a representative ω ∂H,H ∈ N and we note also that u ∂H,H is uniquely determined by the above properties.
2.8 Let U be an open subset of N R and let σ be an r-dimensional integral Raffine polyhedron contained in U . For any closed face ρ of codimension 1, let ω ρ,σ := ω ∂H,H using 2.7 for the affine hyperplane ∂H generated by ρ and the corresponding halfspace containing σ. We note that ω ρ,σ ∈ N is determined up to addition with elements in N ρ = N ∩ L ρ , where L ρ is the linear hyperplane parallel to ρ. For η ∈ A r−1,r c (U ), we have introduced the contraction η; ω ρ,σ {2r−1} as an element of A r−1,r−1 c (U ) which is obtained by inserting the vector ω ρ,σ for the (2r−1)-th argument of the corresponding multilinear function (see 2.6). Note that the restriction of this contraction to ρ does not depend on the choice of the representative ω ρ,σ . Then we define where ρ ranges over all closed faces of σ of codimension 1. On the right, we use the integrals of (r − 1, r − 1)-superforms from 2.4. For η ∈ A r,r−1 c (U ), we define similarly 1 Note that the integrals do depend only on the integral R-affine structure of N R but do not depend on the choice of the orientation of N R . If σ is an integral R-affine polyhedron of any dimension n and if η ∈ A n−1,n c (U ) for an open subset U of N R containing σ, then we define ∂σ η by applying the above to the affine space A σ generated by σ and to the pull-back of η to A σ . We give now a concrete description of ∂σ η in terms of integrals over classical (n − 1)-forms. For every closed face ρ of σ, let N σ = L σ ∩ N be the canonical integral structure on the affine space generated by σ. If e ρ 1 , . . . , e ρ n−1 is a basis of N ρ , then ω ρ,σ , e ρ 1 , . . . , e ρ n−1 is a basis of N σ . We note that the contraction η; ω ρ,σ , e ρ 1 , . . . , e ρ n−1 {n,...,2n−1} may be viewed as a classical (n − 1)-form on U and hence we get Proof: This is just a reformulation of [La12], Proposition 2.3, in the case of a polyhedron using the formalism introduced above. In the quoted result, the boundary was assumed to be smooth, but as the classical Stokes' formula holds also for polyhedra (see [Wa83], 4.7), this applies here as well.
Proposition 2.10 (Green's formula) We consider an n-dimensional integral Raffine polyhedron σ contained in the open subset U of N R . Assume that α ∈ A p,p (U ) and β ∈ A q,q (U ) are symmetric with p + q = n − 1 and that the intersection of the supports of α and β is compact. Then we have Proof: This follows from Stokes' formula as in [CD12], Lemma 1.3.8.
2.11
A supercurrent on U is a continuous linear functional on A p,q c (U ) where the latter is a locally convex vector space in a similar way as in the classical case. We denote the space of such supercurrents by D p,q (U ). As usual , we define the linear differential operators d, d ′ and d ′′ on D(U ) := p,q D p,q (U ) by using (−1) p+q+1 times the dual of the corresponding differential operator on A p,q c (U ). The sign is chosen in such a way that the canonical embedding A p,q (U ) → D r−p,r−q (U ) is compatible with the operators d, d ′ and d ′′ . Here, α ∈ A p,q (U ) is mapped to [α] ∈ D r−p,r−q (U ) given by [α](β) = N R α ∧ β for any β ∈ A r−p,r−q c (U ).
Superforms on polyhedral complexes
We keep the notions from the previous section and we will extend them to the setting of polyhedral complexes. We will introduce tropical cycles and we will characterize them as closed currents of integrations over weighted integral R-affine polyhedral complexes.
3.1 A polyhedral complex C in N R is a finite set of polyhedra with the following two properties: Every polyhedron in C has all its closed faces in C . If ∆, σ ∈ C , then ∆ ∩ σ is a closed face of ∆ and σ. Note here that the empty set and also σ are allowed as closed faces of a polyhedron σ (see [Gu12], Appendix A, for details).
A polyhedral complex C is called integral G-affine for a subgroup G of R if every polyhedron of C is integral G-affine. The support |C | of C is the union of all polyhedra in C . The polyhedral complex C is called pure dimensional of dimension n if every maximal polyhedron in C has dimension n. We will often use the notation C k := {σ ∈ C | dim(σ) = k} for k ∈ N.
3.2 Let C be a polyhedral complex in N R . A superform on C is the restriction of a superform on (an open subset of) N R to |C |. This means that two superforms agree if their restrictions to any polyhedron of |C | agree. Let A(C ) be the space of superforms on C . It is an alternating algebra with respect to the induced wedge product. We have also differential operators d, d ′ and d ′′ on A(C ) given by restriction of the corresponding operators on A(N R ). Let A p,q (C ) be the space of (p, q)-superforms on C . The support of α ∈ A(C ) is the complement of {ω ∈ |C | | α vanishes identically in a neighbourhood of ω} in |C |. We denote by A p,q c (C ) the subspace of A p,q (C ) of superforms of compact support. Let N ′ be a free abelian group of rank r ′ and let F : N ′ R → N R be an affine map. Suppose that C ′ is a polyhedral complex of N ′ R with F (|C ′ |) ⊂ |C |, then the pull-back in 2.3 induces a pull-back F * : A p,q (C ) → A p,q (C ′ ).
3.3 A polyhedral complex D subdivides the polyhedral complex C if they have the same support and if every polyhedron ∆ of D is contained in a polyhedron of C . In this case, we say that D is a subdivision of C . All our constructions here will be compatible with subdivisions. This is no problem for the definition of superforms on C as they depend only on the support |C |.
A weight on a pure dimensional polyhedral complex C is a function m which assigns to every maximal polyhedron σ ∈ C a number m σ ∈ Z. Then we get a canonical weight on every subdivision of C . For a weighted polyhedral complex (C , m), only the polyhedra ∆ ∈ C which are contained in a maximal dimensional σ ∈ C with m σ = 0 are of interest. They form a subcomplex D of C and we define the support of (C , m) as the support of D. The polyhedra of C \ D will usually be neglected.
3.4 Let (C , m) be a weighted integral R-affine polyhedral complex of pure dimension n. For α ∈ A n,n c (C ), we set where we use integration from 2.4 on the right. We define integrals over the bound- where we use the boundary integrals from 2.8 on the right. Note that the boundary ∂C may be defined as the subcomplex consisting of the polyhedra of dimension at most n − 1, but there is no canonical weight on ∂C . Indeed, the boundary integral ∂(C ,m) β depends on the relative situation ∂C ⊂ C because of the weight m σ and the contraction with respect to the vectors ω ρ,σ used in the definitions. This is similar to the situation in real analysis where boundary integrals depend on the relative orientation. These classical boundary integrals do depend only on the restriction of the differential form to the boundary which is clearly wrong for our boundary integrals. However, it is still true that ∂(C ,m) β = 0 if the support of β is disjoint from ∂C .
Proposition 3.5 (Stokes' formula) Let (C , m) be a weighted integral R-affine polyhedral complex of pure dimension n. For any η ′ ∈ A n−1,n c (C ) and any η ′′ ∈ A n,n−1 c (C ), we have Proof: This follows immediately from Stokes' formula for polyhedra given in Proposition 2.9.
3.7 A weighted integral R-affine polyhedral complex (C , m) of pure dimension n is called a tropical cycle if its weight m satisfies the following balancing condition: Here, N ρ is the canonical lattice contained in the affine space generated by ρ and ω ρ,σ ∈ N σ is the lattice vector pointing outwards of σ (see 2.8). Tropical cycles are the basic objects in tropical geometry.
Proposition 3.8 Let (C , m) be a weighted integral R-affine polyhedral complex of pure dimension n on N R . Then the following conditions are equivalent: Proof: Let α ∈ A n−1,n c (N R ). By Stokes' formula in Proposition 3.5, we have where ρ (resp. σ) ranges over all elements of C of dimension n − 1 (resp. n).
Suppose now that σ⊃ρ m σ ω ρ,σ ∈ N ρ for some (n − 1)-dimensional ρ ∈ C . Recall that we may view α as a multilinear map N 2n−1 R → C ∞ (N R ) which is alternating in the first n − 1 arguments and also alternating in the last n arguments. But an alternating n-linear map on a vector space of dimension n − 1 is zero and hence the restriction of α; σ⊃ρ m σ ω ρ,σ {2n−1} to ρ is zero. Then the above display proves (a) ⇒ (b).
Conversely, if σ⊃ρ m σ ω ρ,σ ∈ N ρ for some (n − 1)-dimensional ρ ∈ C , then there is an α ∈ A n−1,n c (N R ) such that the restriction of α; σ⊃ρ m σ ω ρ,σ {2n−1} to ρ is non-zero. We may also assume that the support of α is disjoint from all other (n − 1)-dimensional polyhedra of C . Then the above display proves (b) ⇒ (a). The equivalence of (a) and (c) is shown similarly.
3.9 Now let F : N ′ R → N R be an affine map whose underlying linear map is integral, i.e. induced by a homomorphism N ′ → N . We will define the push-forward of a weighted integral R-affine polyhedral complex (C ′ , m) of pure dimension n on N ′ R . For details, we refer to [AR10], §7. After a subdivision of C ′ , we may assume that is a polyhedral complex in N R . We define the multiplicity of an n-dimensional Endowed with these multiplicities, we get a weighted integral R-affine polyhedral is also a tropical cycle. It might happen that F * (C ′ , m) is empty, then we get the tropical zero cycle.
Proposition 3.10 (projection formula) Using the assumptions above and α ∈ A n,n Proof: Let σ ′ be an n-dimensional polyhedron of C ′ . Then σ := F (σ ′ ) is an integral R-affine polyhedron in N R . We assume for the moment that σ is also ndimensional. As above, we consider the lattice N σ := N ∩ L σ in N R , where L σ is the linear space which is a translate of the affine space generated by σ. Let A be the matrix of the homomorphism F : N σ ′ → N σ with respect to integral bases. Then we have | det(A)| = [N σ ′ : N σ ] and hence the transformation formula (1) shows If dim(σ) < n, then both sides are zero and hence formula (2) is true in any case. Using the weighted sum over all σ ′ , the claim follows immediately from (2).
Moment maps and tropical charts
A complex manifold is locally defined using analytic charts ϕ : U → C r . The charts help to transport the analysis from C r to M . The idea in the non-archimedean setting is similar replacing the above charts by algebraic moment maps ϕ : U → G r m to multiplicative tori and the corresponding tropicalizations ϕ trop : U → R r . The restriction of ϕ an to the preimage of an open analytic subset will be called a tropical chart. In this section, K is an algebraically closed field endowed with a complete nontrivial non-archimedean absolute value | |. Note that the residue fieldK is also algebraically closed. Let v := − log | | be the associated valuation and let Γ := v(K × ) be the value group. We will study analytifications, tropicalizations and moment maps of the algebraic variety X over K. This will be used in the next section to define (p, q)-forms on X an .
4.1
We recall first the construction of the analytification of X. Let U = Spec(A) be an open affine subset of X, then U an is the set of multiplicative seminorms on A extending the given absolute value | | on K. This set is endowed with the topology generated by the functions U an → R, p → p(a) with a ranging over A. By glueing, we get a topological space X an which is connected locally compact and Hausdorff. We can endow it with a sheaf of analytic functions leading to a Berkovich analytic space over K which we call the analytification of X. For a morphism ϕ : Y → X of algebraic varieties over K, we get an analytic morphism ϕ an : Y an → X an induced by composing the multiplicative semiorms with ϕ ♯ on suitable affine open subsets. We refer to [Be90] for details, or to [BPS11], §1.2, for a neat description of the analytification.
4.2
We will define some local invariants in x ∈ X an . On an open affine neighbourhood U = Spec(A), the point x is given by a multiplicative seminorm p on A and we often write |f (x)| := p(f ) for f ∈ A. Dividing out the prime ideal I := {f ∈ A | p(f ) = 0}, we get a multiplicative norm on the integral domain B := A/I which extends to an absolute value | | x on the quotient field of B. The completion of this field is denoted by H (x). It does not depend on the choice of U and it may be also constructed analytically. The absolute value of H (x) is denoted by | | as it extends the given absolute value on K. Note that the completed residue field H (x) of x remains the same if we replace the ambient variety X by the Zariski closure of x in X.
Let s(x) be the transcendence degree of the residue field of H (x) overK. The quotient of the value group of H (x) by Γ is a finitely generated abelian group and we denote its Q-rank by t(x). Finally, we set d( Example 4.3 Let T = G r m be the split multiplicative torus of rank r with coordinates z 1 , . . . , z r . Then a point x of T an could be visualized by the coordinates z 1 (x), . . . , z r (x) ∈ H (x) and the multiplicative seminorm corresponding to x is given by |f (x)| = |f (z 1 (x), . . . , z r (x))| for every Laurent polynomial f on T . Conversely, every field extension L/K with an absolute value extending the given absolute value on K and every (β 1 , . . . , β r ) ∈ (L × ) r give rise to a point x ∈ T an by |f (x)| := |f (β 1 , . . . , β r )|. Note that L and (β 1 , . . . , β r ) are not uniquely determined by x.
In particular, we get an inclusion of T (K) into T an . For every x ∈ T (K), we have d(x) = 0. However, there can be also other points with d(x) = 0. If T = G 1 m , then precisely the points of type 1 (i.e. the K-rational points) and the points of type 4 satisfy d(x) = 0 (see [Be90], 1.4.4).
Returning to the case T = G r m , there are some distinguished points of T an which behave completely different than K-rational points. For positive real numbers s 1 , . . . , s r , we define the associated weighted Gauss norm on K[T ] by It follows from the Gauss Lemma that the weighted Gauss norm is a multiplicative seminorm giving rise to a point η s ∈ T an . The set S(T an ) := {η s | s 1 > 0, . . . , s r > 0} is called the skeleton of T an . Every point η s ∈ S(T an ) satisfies d(η s ) = r (see [Du12], (0.12) and (0.13)).
4.4
Let T := G r m be a split multiplicative torus over K with coordinates z 1 , . . . , z r . Then we have the tropicalization map It is immediate from the definitions that the map trop is continuous and proper. To get a coordinate free approach, we could use the character group M and its dual N . Then trop is a map from T an to N R . We refer to [Gu12] for details about tropical geometry.
Remark 4.5 Note that we have a natural section R r → T an of the tropicalization map. It is given by mapping the point ω ∈ R r to the weighted Gauss norm η s associated to s := (e −ω1 , . . . , e −ωr ). It follows from [Be90], Example 5.2.12, that this section is a homeomorphism of R r onto a closed subset of T an which is the skeleton S(T an ) introduced in 4.3. In this way, we may view the tropicalization map as a map from T an onto S(X an ). Then it is shown in [Be90], §6.3, that the tropicalization map is a strong deformation retraction of T an onto the skeleton S(T an ). This point of view is used very rarely in our paper.
4.6
For a closed subvariety Y of T of dimension n, the tropical variety associated to X is defined by Trop(Y ) := trop(Y an ). The Bieri-Groves theorem says that Trop(Y ) is a finite union of n-dimensional integral Γ-affine polyhedra in R r . It is shown in tropical geometry that Trop(Y ) is an integral Γ-affine polyhedral complex. The polyhedral structure is only determined up to subdivision which does not matter for our constructions. We will see below that the tropical variety is endowed with a positive canonical weight m satisfying the balancing condition from 3.7. We get a tropical cycle of pure dimension n which we also denote by Trop(Y ) forgetting the weight m in the notation.
4.7
The tropical weight m on an n-dimensional polyhedron σ of Trop(Y ) is defined in the following way. By density of the value group Γ in R, there is ω ∈ Γ r ∩relint(σ). We choose t ∈ G r m (K) with trop(t) = ω. Then the closure of t −1 Y in (G r m ) K • is a flat variety over K • whose special fibre is called the initial degeneration in ω (Y ) of Y at ω. Note that in ω (Y ) is a closed subscheme of (G r m )K . Let m W be the multiplicity of an irreducible component W of in ω (Y ). Then the tropical weight m σ is defined by m σ := W m W , where W ranges over all irreducible components of in ω (Y ). One can show that the definition is independent of the choices of ω and t. It is a non-trivial fact from tropical geometry that (Trop(Y ), m) is a tropical cycle (see [Gu12], §13, for details).
4.8
For an open subset U of the algebraic variety X, a moment map is a morphism ϕ : U → T to a split multiplicative torus T := G r m over K. The tropicalization of ϕ is Obviously, this is a continuous map with respect to the topology on the analytification U an . Note that our moment maps are algebraic which differs from the moment maps in [CD12] which are defined analytically. We say that the moment map ϕ ′ : U ′ → T ′ of the open subset U ′ of X refines the moment map ϕ : U → T if U ′ ⊂ U and if there is an affine homomorphism ψ : T ′ → T of the multiplicative tori such that ϕ = ψ • ϕ ′ on U ′ . Here, an affine homomorphism means a group homomorphism composed with a (multiplicative) translation on T . This group homomorphism induces a homomorphism M → M ′ of character lattices. Its dual is the linear part of an integral affine map Trop(ψ) : ) i∈I is a moment map which refines every ϕ i . Moreover, it follows easily from the universal property of the product that every moment map ϕ ′ : U ′ → T ′ which refines every ϕ i refines also ϕ. Proof: Let ω ∈ ϕ trop (U an ). We note that ϕ −1 trop (ω) is a Laurent domain in U an and hence it has the same dimension as U . We conclude that ϕ −1 trop (ω) is not contained in the analytification of the lower dimensional Zariski-closed subset U \U ′ and hence ω ∈ ϕ trop ((U ′ ) an ). is a tropical cycle on R r . If ϕ is generically finite, then this tropical cycle is of pure dimension dim(X) and the support is equal to ϕ trop (U an ) (see Lemma 4.9).
The following result is called the Sturmfels-Tevelev multiplicity formula. It was proved by Sturmfels and Tevelev [ST08] in the case of a trivial valuation and later generalized by Baker, Payne and Rabinoff [BPR11] for every valued field.
Proposition 4.11 Let ϕ ′ : U ′ → T ′ be a moment map of the non-empty open subset U ′ of X which refines the moment map ϕ : U → T , i.e. there is an affine homomorphism ψ : in the sense of tropical cycles (see 3.9).
Proof: In fact, the Sturmfels-Tevelev multiplicity formula is the special case where X = U ′ is a closed subvariety of T ′ (see [Gu12], Theorem 13,17, for a proof in our setting deducing it from the original sources). In the general case, we conclude that Since U ′ is dense in U , the claim follows.
4.12
We will show that every open affine subset U of X has a canonical moment map. We note that the abelian group M U := O(U ) × /K × is free of finite rank (see [Sa66], Lemme 1). Here, we use that K is algebraically closed (or at least that X is geometrically reduced). We choose representatives ϕ 1 , . . . , ϕ r in O(U ) × of a basis. This leads to a moment map ϕ U : U → T U = Spec(K[M U ]). By construction, ϕ U refines every other moment map on U . Note that this moment map ϕ U is canonical up to (multiplicative) translation by an element of T U (K). Let f : X ′ → X be a morphism of algebraic varieties over K and let U ′ is an open subset of X ′ with f (U ′ ) ⊂ U . Then f ♯ induces a homomorphism M U → M U ′ of lattices. We get a canonical affine homomorphism ψ U,U ′ : T U ′ → T U of the canonical tori with ψ U,U ′ • ϕ U ′ = ϕ U • f . This will be applied very often in the case where U ′ is an open subset of U in X ′ = X and f = id. Then we get a canonical affine homomorphism ψ U,U ′ : T U ′ → T U .
4.13
Recall that an open subset U of X is called very affine if U has a closed embedding into a multiplicative torus. Clearly, the following conditions are equivalent for an open affine subset U of X: On a very affine open subset, we will almost always use the canonical moment map ϕ U : U → T U which is a closed embedding by the above. To simplify the notation, we will set Trop(U ) for the tropical variety of U in T U . It is a tropical cycle in (N U ) R , where N U is the dual abelian group of M U . The tropicalization map will be denoted by trop U := (ϕ U ) trop : U an → (N U ) R . Recall that ϕ U is only determined up to translation by an element of T U (K) and hence trop U and Trop(U ) are only canonical up to an affine translation. This ambiguity is no problem as our constructions will be compatible with affine translations.
The following result of Ducros relates the local invariant d(x) from 4.2 with tropical dimensions. Proof: We choose rational functions f 1 , . . . , f s on X with |f 1 (x)| = · · · = |f s (x)| = 1 such that the reductions f 1 , . . . , f s form a transcendence basis of the residue field extension of H (x)/K. There are rational functions g 1 , . . . , g t which are regular at x such that |g 1 (x)|, . . . , |g t (x)| form a basis of (|H (x) × |/|K × |) ⊗ Z Q. By definition, we have d(x) = s + t. By (0.12) in [Du12], f 1 (x), . . . , f s (x), g 1 (x), . . . , g t (x) reduce to a transcendence basis of the graded residue field extensions of H (x)/K in the sense of Temkin. There is a very affine open neighbourhood U of x in X such that f 1 , . . . , f s , g 1 , . . . , g t are invertible on U . Let ϕ 1 , . . . , ϕ r ∈ O(U ) × be the coordinates of the canonical moment map ϕ U : U → T U = G r m . Then the graded reductions of ϕ 1 , . . . , ϕ r generate a graded subfield of the graded residue field extension of H (x)/K. By construction, this graded subfield has transcendence degree d(x) over the graded residue field of K. By [Du12], Theorem 3.2, Trop U (V ) is a finite union of integral Γ-affine polytopes for every compact neighbourhood V of x in U an which is strict in the sense of [Be93]. For any open neighbourhood W of x in U an , Theorem 3.3 in [Du12] shows that there is a compact strict neighbourhood V of x in W such that trop U (V ) is a finite union of d(x)-dimensional polytopes. We say that the tropical chart (V ′ , ϕ U ′ ) is a tropical subchart of (V, ϕ U ) if V ′ ⊂ V and U ′ ⊂ U . We note that the definition of tropical chart here is different from the tropical charts in [CD12], §3.1, which consist of an analytic morphism to a split torus and a finite union of polytopes containing the tropicalization. Proof: To prove (a), we may assume that X = Spec(A) is a very affine scheme. A basis of X an is formed by subsets of the form V := {x ∈ X | s 1 < |f 1 (x)| < r 1 , . . . , s k < |f k (x)| < r k } with all f a ∈ A and real numbers s a < r a . Using the ultrametric triangle inequality as applied to f a + π for a non-zero π ∈ K of small absolute value if f a (x) = 0, it is easy to see that we may choose the basis in such a way that 0 < s a for all a = 1, . . . k. Note that V is contained in the analytification of the very affine open subset U := {x ∈ X | f 1 (x) = 0, . . . , f k (x) = 0} of X. It is obvious that (V, ϕ U ) is a tropical chart proving (a).
To prove (b), let us consider the moment map Since X is separated, it is easy to see that Φ is a closed embedding and hence U ∩ U ′ is very affine. By definition of a tropical chart, Ω := trop U (V ) (resp. is an open subset of Trop(U ) (resp. Trop(U ′ )). Note that is an open subset of Φ trop ((U ∩ U ′ ) an ). An easy diagram chase yields Φ −1 trop (Ω ′′ ) = V ∩ V ′ . Since ϕ U∩U ′ refines the moment map Φ, we deduce that (V ∩ V ′ , ϕ U∩U ′ ) is a tropical chart. This proves (b). Remark 4.17 In [CD12], everything is defined for an arbitrary analytic space. In Section 7, we will compare their analytic constructions with our algebraic approach.
Differential forms on algebraic varieties
On a complex analytic manifold M , we use open analytic charts ϕ : U → C r to define (p, q)-forms on U by pull-back. The idea in the non-archimedean setting is similar replacing the above charts by tropical charts (V, ϕ U ) from the previous section in order to pull-back Lagerberg's superforms to U an .
In this section, K is an algebraically closed field endowed with a complete nontrivial non-archimedean absolute value | |. Let v := − log | | be the associated valuation and let Γ := v(K × ) be the value group. The theory could be done for arbitrary fields (see [CD12]), but it is no serious restriction to assume that K is algebraically closed as the theory is stable under base extension and in the classical setting, the analysis is also done over C. We will introduce (p, q)-forms on the analytification X an of a n-dimensional algebraic variety X over K. Suppose that we have another tropical chart (V ′ , ϕ U ′ ). Then (V ∩ V ′ , ϕ U∩U ′ ) is a tropical chart (see Proposition 4.16) and we get a canonical affine homomorphism ψ U,U∩U ′ : T U∩U ′ → T U of the underlying tori with ϕ U = ψ U,U∩U ′ • ϕ U∩U ′ on U ∩ U ′ (see 4.12). The associated affine map Trop(ψ U,U∩U ′ ) : (N U∩U ′ ) R → (N U ) R maps the tropical variety Trop(U ∩ U ′ ) onto Trop(U ) (use Lemma 4.9). Then we define the restriction of the superform α ∈ A p,q (trop U (V )) to a superform α| V ∩V ′ on trop U∩U ′ (V ∩ V ′ ) by using the pull-back to trop U∩U ′ (V ∩ V ′ ) with respect to Trop(ψ U,U∩U ′ ). This plays a crucial role in the following definition: Definition 5.2 A differential form α of bidegree (p, q) on an open subset V of X an is given by a covering (V i ) i∈I of V by tropical charts (V i , ϕ Ui ) of X an and superforms α i ∈ A p,q (trop Ui (V i )) such that α i | Vi∩Vj = α j | Vi∩Vj for every i, j ∈ I. If α ′ is another differential form of bidegree (p, q) on V given by α ′ j ∈ A p,q (trop U ′ j (V ′ j )) with respect to the tropical charts (V ′ j , ϕ U ′ j ) j∈J covering V , then we consider α and α ′ as the same differential forms if and only if α i | Vi∩V ′ j = α ′ j | Vi∩V ′ j for every i ∈ I and j ∈ J. We denote the space of (p, q)-differential forms on V by A p,q (V ). As usual, we define the space of differential forms on V by A(V ) := p,q A p,q (V ). The subspace of differential forms of degree k ∈ N is denoted by A k (V ) := p+q=k A p,q (V ).
5.3
It is obvious from the definitions that the differential forms form a sheaf on X an . Using the corresponding constructions for superforms on tropical cycles, it is immediate to define the wedge product and differential operators d, d ′ , d ′′ on differential forms on V . By 4.6, we have A p,q (V ) = {0} if max(p, q) > dim(X).
For a morphism ϕ : X ′ → X and open subsets V (resp. V ′ ) of X an (resp. (X ′ ) an ) with ϕ(V ′ ) ⊂ V , we get a pull-back ϕ * : A p,q (V ) → A p,q (V ′ ) defined in the following way: Suppose that α ∈ A p,q (V ) is given by the covering (V i ) i∈I and the superforms α i ∈ A p,q (trop Ui (V i )) as above. Then there is a covering for the corresponding very affine open subsets. Then ϕ * (α) is the differential form on V ′ given by the covering (V ′ j ) j∈J and the superforms ϕ * (α i(j) ) ∈ A p,q (Trop(U ′ j )). We leave the details to the reader. This construction is functorial as usual.
Remark 5.4 We obtain the same sheaf of differential forms on X an as in [CD12], §3. In the latter reference, all analytic moment maps were used to define differential forms on X an and so it is clear that our differential forms here are also differential forms in the sense of [CD12]. To see the converse, we argue as follows: By Proposition 4.16, tropical charts (V, ϕ U ) form a basis in X an . It follows from Proposition 7.2 that an analytic moment map ϕ : V → (G r m ) an may be locally in x ∈ V approximated by an algebraic moment map ϕ ′ : Here, U ′ is a suitable very affine open subset of U with x ∈ (U ′ ) an . It follows from [CD12], Lemma 3.1.10, that we may use algebraic moment maps to define differential forms in the sense of [CD12]. Using that ϕ U ′ factorizes through ϕ ′ (see 4.12), we get the claim.
Definition 5.5 Let α be a differential form on an open subset V of X an . The support of α is the complement in V of the set of points x of V which have an open neighbourhood V x such that α| Vx = 0. Let A p,q c (V ) be the space of differential forms of bidegree (p, q) with compact support in V .
Proposition 5.6 Let (V, ϕ U ) be a tropical chart of X an and let α ∈ A p,q (V ) be given by α U ∈ A p,q (trop U (V )). Then α = 0 in A p,q (V ) if and only if α U = 0 in A p,q (trop U (V )).
5.8
In analogy with differential geometry on manifolds, we set C ∞ (V ) := A 0,0 (V ) for any open subset V of X an and a smooth function on V is just a differential form of bidegree (0, 0). Since tropicalization maps are continuous, it is clear that a smooth function is a continuous function on V . By the Stone-Weierstrass theorem, the space C ∞ c (V ) of smooth functions with compact support in V is a dense subalgebra of C c (V ) (see [CD12], Proposition 3.3.5).
Definition 5.9 Let (V i ) i∈I be an open covering of an open subset V of X an . A smooth partition of unity on V with compact supports subordinated to the covering (V i ) i∈I is a family (φ j ) j∈J of non-negative smooth functions with compact support on V with the following properties: (i) The family (supp(φ j )) j∈J is locally finite on V .
(ii) We have j∈J φ j ≡ 1 on V .
(iii) For every j ∈ J, there is i(j) ∈ I such that supp(φ j ) ⊂ V i(j) .
Proposition 5.10 Let (V i ) i∈I be an open covering of an open subset V of X an . Then there is a smooth partition of unity (φ j ) j∈J on V with compact supports subordinated to the covering (V i ) i∈I .
Proof: It is enough to show that for every x ∈ V , there is a non-negative smooth function φ with compact support in V and with φ(x) > 0. Since X an is a locally compact Hausdorff space which is also σ-compact, the open subset V is paracompact and hence standard arguments from differential geometry yield the existence of the desired partition of unity (see [Wa83], Theorem 1.11).
To prove the crucial claim at the beginning of the proof, we may assume that V is coming from a tropical chart (V, ϕ U ) (see So far, we have seen properties of differential forms which are completely similar to the archimedean case. The next result of Chambert-Loir and Ducros ([CD12], Lemme 3.2.5) shows that the support of a differential form of degree at least one is disjoint from X(K).
Lemma 5.11 Let W be an open subset of X an . We consider α ∈ A p,q (W ) and x ∈ W with d(x) < max(p, q). Then x ∈ supp(α).
Proof: Using Proposition 4.16 and shrinking the open neighbourhood W of x, we may assume that W is a tropical chart (W, ϕ U ) on which α is given by the superform α U ∈ A p,q (trop U (W )). By Proposition 4.14, there is a very affine open subset U x of U and a compact neighbourhood V x of x in (U x ) an ∩ W such that trop Ux (V x ) is of dimension d(x). By Proposition 4.16, there is a tropical chart (V ′ , ϕ U ′ ) with x ∈ V ′ ⊂ V x and U ′ ⊂ U x . By 4.12, there is an affine homomorphism ψ : T U ′ → T U such that ϕ U = ϕ U ′ •ψ. Using the same factorization for the tropicalizations, we see that the restriction of α to V ′ is given by Trop(ψ) * (α U ) ∈ A p,q (trop U ′ (V ′ )). The inclusion U x ⊂ U yields that trop Ux factorizes through trop U (use 4.12). Since V ′ ⊂ V x , we get dim(trop U (V ′ )) ≤ dim(trop Ux (V ′ )) ≤ d(x) < max(p, q). As trop U (V ′ ) = Trop(ψ)(trop U ′ (V ′ )), we conclude that Trop(ψ) * (α U ) = 0. This proves α = 0.
Corollary 5.12 Let W be an open subset of X an and let U be a Zariski open subset of X. If α ∈ A p,q (W ) with dim(X \ U ) < max(p, q), then supp(α) ⊂ W ∩ U an .
Proposition 5.13 Let α ∈ A p,q c (X an ) be a differential form with max(p, q) = dim(X). Then there is a very affine open subset U of X such that supp(α) ⊂ U an and such that α is given on U an by a superform α U ∈ A p,q c (Trop(U )).
Proof: By assumption, the support of α is a compact subset of X an . We conclude that there are finitely many tropical charts (V i , ϕ Ui ) i=1,...,s covering supp(α) such that α is given on V i by the superform α i ∈ A p,q (trop Ui (V i )). Recall that Ω i with respect to (ϕ U ) trop is equal to V . We conclude that (V, ϕ U ) is a tropical chart of X an . Note that α is given on U an ∩ V i by α ′ i := Trop(ψ i ) * (α i ) ∈ A p,q (Ω ′ i ). By Proposition 5.6, α ′ i agrees with α ′ j on Ω ′ i ∩Ω ′ j for every i, j ∈ {1, . . . , s} and hence they define a superform α U ∈ A p,q (Ω). By construction, α U gives the differential form α on V . It follows from Remark 5.7 that α U has compact support in Ω. Since α has compact support in V , we conclude that α U is a superform on Trop(U ) which defines α on U an . 5.14 Let α ∈ A n,n c (W ) for an open subset W of X an , where n := dim(X). Obviously, we may view α as an (n, n)-form on X an with compact support. We call a very affine open subset U as in Proposition 5.13 a very affine chart of integration for α. Then α is given by a superform α U ∈ A n,n c (Trop(U )). We define the integral of α over W by Here, we view Trop(U ) as a tropical cycle (see 4.6) and we integrate as in 3.4.
Proposition 5.16 Let λ, ρ ∈ R and let α, β ∈ A n,n c (W ). Then we have Proof: By Lemma 5.15, we may choose a simultaneous very affine chart of integration for both α and β. Then the claim follows by the corresponding property of the integration of superforms.
We have also Stokes' theorem for differential forms on the open subset W of X an . Note that W has trivial boundary in the algebraic situation [Be90, Theorem 3.4.1] and hence the boundary does not occur as in the version [CD12, Theorem 3.12.1] for analytic spaces.
Proof: By Proposition 5.13, there is a very affine open subset U of X such that supp(α) ⊂ U an and such that α is given on U an by a superform α U ∈ A 2n−1 c (Trop(U )). Then U is a very affine chart of integration for d ′ α and d ′′ α and the claim follows from Proposition 3.5 and Proposition 3.8.
Remark 5.18 Integration of differential forms on complex manifolds is defined by using a partition of unity with compact supports subordinated to a covering by holomorphic charts. Surprisingly, this was not necessary in our non-archimedean algebraic setting as we have defined integration by using a single suitable tropical chart. In fact, the use of a smooth partition of unity (φ j ) j∈J with compact supports subordinate to an open covering of W by tropical charts (V i , ϕ Ui ) i∈I would not work here directly. To illustrate this, suppose that α ∈ A n,n c (W ) is given on V i by α i ∈ A n,n (trop Ui (V i )). If the functions φ j are of the form )), then we could set W α = j∈J Trop(U i(j) ) f j α i(j) . However, the functions φ j could not be expected to have this form and so this approach fails.
Chambert-Loir and Ducros define integration more generally for differential forms on paracompact good analytic spaces (see [CD12], §3.8). The idea is to use a covering by the interiors of affinoid subdomains. Then there is a smooth partition of unity with supports subordinated to this covering which reduces the problem to defining integration over an affinoid subdomain. But in the affinoid case, one can find a single tropical chart of integration similarly as in Proposition 5.13. It follows from Remark 7.6 and Proposition 7.11 that both definitions give the same integral on the analytification of an algebraic variety.
Currents on algebraic varieties
In this section, K is an algebraically closed field endowed with a non-trivial nonarchimedean complete absolute value | |. We consider an open subset W of X an for an algebraic variety X over K of dimension n. Similarly as in the complex case, we will first define a topology on A p,q c (W ) and then we will define currents as continuous linear functionals on this space. We will see that the Poincaré-Lelong equation holds for a rational function.
6.1 Let (V i , ϕ Ui ) i∈I be finitely many tropical charts contained in W and let ∆ i be a polytope contained in the open subset Ω i := trop Ui (V i ) of Trop(U i ). We consider the space A p,q (V i , U i , ∆ i : i ∈ I) of (p, q)-forms α on W with support in C := i∈I trop −1 Ui (∆ i ) such that α is given on V i by a superform α i ∈ A p,q (Ω i ) for every i ∈ I. Since the tropicalization map is proper (see 4.4), the set C is compact. Similarly as in the complex case, we endow A p,q (V i , U i , ∆ i : i ∈ I) with the structure of a locally convex space such that a sequence α k converges to α if and only if all derivatives of the superforms α k,i converge uniformly to the derivatives of the superform α i on ∆ i . Here, α k,i (resp. α i ) is the superform on Ω i which defines α k (resp. α) on V i and we mean more precisely the derivatives of the coefficients of α k,i | ∆i (resp. α i | ∆i ). It follows easily from Proposition 4.16 that A p,q c (W ) is the union of all spaces A p,q (V i , U i , ∆ i : i ∈ I) with (V i , U i , ∆ i : i ∈ I) ranging over all possibilities as above.
6.2 A current on an open subset W of X an is a linear functional T on A p,q c (W ) such that the restriction of T to all subspaces A p,q (V i , U i , ∆ i : i ∈ I) is continuous. The space of currents is a C ∞ (W )-module denoted by D p,q (W ). As usual (cf. 2.11), we define the differential operators d ′ , d ′′ and d := d ′ + d ′′ on the total space of currents D(W ) := p,q D p,q (W ). Using partitions of unity from Proposition 5.10, it is easy to show that the currents form a sheaf on X an .
Generalizations to analytic spaces
The final section shows how our notions fit with the paper [CD12]. While we restricted to the algebraic case, the paper of Chambert-Loir and Ducros works for arbitrary analytic spaces. We assume that the reader is familiar with the theory of analytic spaces as given in [Be93] or [Te10]. For simplicity, we assume again that K is algebraically closed, endowed with a non-trivial non-archimedean complete absolute value | | with corresponding valuation v := − log | | and that all occurring analytic spaces are strict in the sense of [Be93]. This situation can always be obtained by base change without changing the theory of differential forms and currents. As usual, we use the value group Γ := v(K × ).
7.1 Let Z be a compact analytic space over K. An analytic moment map on Z is an analytic morphism ϕ : Z → T an for a split torus T = G r m over K as before. Let M be the character group of T , then we have T = Spec(K[M ]). The map ϕ trop := trop • ϕ : Z → N R is called the tropicalization map of ϕ and we may use the coordinates on T to identify N R with R r .
The next result shows that for the construction of differential forms in the algebraic case, we may restrict our attention to algebraic moment maps.
Proof: We may assume that X = Spec(A). Similary as in the proof of Proposition 4.16, there is a neighbourhood V ′ := {x ∈ X | s 1 ≤ |f 1 (x)| ≤ r 1 , . . . , s k ≤ |f k (x)| ≤ r k } of x in W with all f a ∈ A and real numbers 0 < s a < r a . We may assume that f 1 , . . . , f k form an affine coordinate system y 1 , . . . , y k on X. Using coordinates on T = G r m , the moment map ϕ is given by analytic functions ϕ 1 , . . . , ϕ r on W which restrict to strictly convergent Laurent series in y 1 , . . . , y k on V ′ . Cutting the Laurent series in sufficiently high positive and negative degree, we get Laurent polynomials p 1 , . . . , p r with |p a | = |ϕ a | on V ′ for a = 1, . . . , r. By Proposition 4.16, there is a very affine open subset U of X such that U an contains x and such that p 1 , . . . p r define an algebraic moment map ϕ ′ : U → T with ϕ trop = ϕ ′ trop on V ′ . Choosing a neighbourhood V of x in U an ∩ V ′ , we get the claim.
We have the following generalization of the Bieri-Groves theorem. Working with analytic spaces, the boundary ∂Z of Z becomes an issue.
Theorem 7.3 (Berkovich, Ducros) If Z is a compact analytic space over K of dimension n and if ϕ : Z → T an is an analytic moment map, then ϕ trop (Z) is a finite union of integral Γ-affine polytopes of dimension at most n. Moreover, ϕ trop (∂Z) is contained in a finite union of integral Γ-affine polytopes of dimension ≤ n − 1. If Z is affinoid, then ϕ trop (∂Z) is equal to a finite union of such polytopes.
Proof: The first claim is due to Berkovich and the remaining claims are due to Ducros (see [Du12], Theorem 3.2).
7.4
We consider now a compact analytic space Z over K of pure dimension n. Theorem 7.3 shows that the tropical variety ϕ trop (Z) is the support of an integral Γ-affine polytopal complex in N R . Our next goal is to endow this complex with canonical tropical multiplicities. This will lead to the definition of a weighted polytopal complex (ϕ trop ) * (cyc(Z)) which is canonical up to subdivision.
If dim(ϕ trop (Z)) < n, then we set (ϕ trop ) * (cyc(Z)) = 0 meaning that we choose all tropical weights equal to zero. It remains to consider the case dim(ϕ trop (Z)) = n. We choose a generic surjective homomorphism q : T → T ′ onto a split multiplicative torus T ′ = Spec(K[M ′ ]) of rank n = dim(Z). Generic means that the corresponding linear map F := Trop(q) is injective on every polytope contained in ϕ trop (Z). By Theorem 7.3, there is an integral Γ-affine polytopal complex C in N R with |C | = ϕ trop (Z) such that F (τ ) is disjoint from (q • ϕ) trop (∂Z) for every n-dimensional face σ of C and τ := relint(σ). By passing to a subdivision, we may assume that F * (C ) is a polyhedral complex in N ′ R as in 3.9, where N ′ is the dual of M ′ as usual. We identify F (τ ) ⊂ N ′ R ∼ = R n with an open subset of the skeleton S((T ′ ) an ) as in Remark 4.5. Then it is clear that q • ϕ restricts to a map (q • ϕ) −1 (τ ) → F (τ ) which agrees with F • ϕ trop using the identification S((T ′ ) an ) = N ′ R . It is shown in [CD12], §2.4, that this restriction of q • ϕ is a finite flat and surjective morphism which means that every point p of F (τ ) has a neighbourhood W ′ in (T ′ ) an such that (q • ϕ) −1 (W ′ ) → W ′ has these properties. Using that F −1 (F (τ )) = τ ′ τ ′ , where τ ′ is ranging over all open faces of C with F (τ ′ ) = F (τ ), we get We conclude that the map ϕ −1 trop (τ ) ∩ (q • ϕ) −1 (F (τ )) → F (τ ) is finite, flat and surjective. Again, this has to be understood in some open neighbourhoods. Since τ is connected, the corresponding degree depends only on τ and not on the choice of p. We denote this degree by [ϕ −1 trop (τ ) : F (τ )]. Recall that N σ is the canonical lattice in the affine space generated by σ. Then the character lattice M ′ of T ′ is of finite index in M σ = Hom(N σ , Z).
Definition 7.5 Using the notation from above, the tropical multiplicity m σ along σ is defined by m σ := [ϕ −1 trop (τ ) : F (τ )] · [M σ : M ′ ] −1 . Furthermore, (ϕ trop ) * (cyc(Z)) is the weighted polyhedral complex C endowed with these tropical multiplicities. The weights might be rational numbers, at least we have no argument that they are integers in the analytic case.
Remark 7.6 It is not so easy to show that the tropical multiplicity is well-defined, i.e. independent of the choice of q. Chambert-Loir and Ducros do not use tropical multiplicities, but the latter are equivalent to the canonical calibration introduced in [CD12], §3.5. To summarize this construction, let e 1 , . . . , e n (resp. f 1 , . . . , f n ) be a basis of M ′ (resp. M σ ). Then the canonical calibration of σ is defined as together with the orientation induced by the pull-back of e 1 , . . . , e n with respect to the linear isomorphism F | (Nσ) R . The canonical calibration is equal to the calibration m σ f 1 ∧ · · · ∧ f n together with the orientation induced by f 1 , . . . , f n . Since the canonical calibration does not depend on the choice of q up to refinement ([CD12], §3.5), the same is true for the tropical multiplicities.
Remark 7.7 One can define the irreducible components of an analytic space (see [Con99]). A compact analytic space Z has finitely many irreducible components Z i . Then we define the cycle cyc(Z) associated to Z as a positive formal Z-linear combination of the irreducible components Z i by restriction to affinoid subdomains and then by glueing (see [Gu98], §2). One can show that the weighted n-dimensional polyhedral complex (ϕ trop ) * (cyc(Z)) depends only on cyc(Z) and this dependence is linear. We leave the details to the reader.
The next result shows that the Sturmfels-Tevelev multiplicity formula holds for analytic spaces.
Proof: The corresponding statement for canonical calibrations is shown in [CD12], Lemma 3.5.2, and hence the claim follows from Remark 7.6. Proposition 7.9 Let Z be a compact analytic space over K of pure dimension n and let C be the same integral Γ-affine polytopal complex with support ϕ trop (Z) as in 7.4. Then for every (n − 1)-dimensional polyhedron ρ of C not contained in ϕ trop (∂Z), the balancing condition σ∈Cn, σ⊃ρ m σ ω ρ,σ ∈ N ρ from 3.7 holds in ρ.
Proof: Chambert-Loir and Ducros prove in [CD12], Theorem 3.6.1, that ρ is harmonious in C which is a condition for the canonical calibration equivalent to the balancing condition by Remark 7.6.
7.10
In an algebraic setting, our goal is to compare the tropical multiplicities introduced in 4.7 with the ones from Definition 7.5. Let us consider an algebraic variety X over K of dimension n and an algebraic moment map ϕ : X → T = Spec(K[M ]) ∼ = G r m over K. Note that ϕ trop (X an ) = Trop(ϕ(X)). We endow ϕ trop (X an ) with the tropical multiplicities m alg σ of the tropical cycle Trop(ϕ * (X)) := deg(ϕ)Trop(ϕ(X)) of N R .
The analytification X an is not compact (unless n = 0), but as ∂X = ∅, we can define tropical multiplicities in the same analytic manner as in Definition 7.5. This means that we choose a generic projection q : T → T ′ = Spec(K[M ′ ]) onto a torus T ′ of rank n and an integral Γ-affine polyhedral complex C with support equal to ϕ trop (X an ) such that F * (C ) is a polyhedral complex on N ′ R for the associated linear map F : N R → N ′ R . For every σ ∈ C n and τ := relint(σ), we define as in Definition 7.5. Since the tropical multiplicities m alg and m an are compatible with subdivision, we may assume that the underlying integral Γ-affine polyhedral complex C is the same in both definitions. Now we are ready to compare these two tropical multiplicities.
Proposition 7.11 Let ϕ : X → T be an algebraic moment map and n := dim(X).
Using the notations from above, we have m an σ = m alg σ for every σ ∈ C n .
Proof: The following argument is quite close to the proof of the Sturmfels-Tevelev formula given by Baker, Payne and Rabinoff (see [BPR11], Theorem 8.2). We may assume that ϕ is generically finite, otherwise ϕ trop (X an ) has dimension < n and all tropical multiplicities are zero. Let Y be the closure of ϕ(X) in T and let q : T → T ′ be a generic homomorphism onto a split torus T ′ = Spec(K[M ′ ]) of rank n with associated linear map F : There is an open dense subset U of Y such that ϕ is finite over U . Since the tropical multiplicities m an and m alg are compatible with subdivision of the polyhedral complex C , we may assume that Trop(Y \ U ) is contained in the support of C n−1 . Let Y ′ be the closure of q(Y ) in T ′ and let ω ∈ τ ∩ N Γ . We consider the affinoid subdomains U ω := trop −1 (ω) in T an and U ′ ω ′ := trop −1 (ω ′ ) in (T ′ ) an . By finiteness of ϕ over U , the set X ω := (ϕ an ) −1 (U ω ) = ϕ −1 trop (ω) is an affinoid subdomain of X an and ϕ restricts to a finite morphism X ω → Y ω := Y an ∩ U ω . Let X ω , Y ω , U ω , U ′ ω ′ be the canonical formal affine K • -models of X ω , Y ω , U ω , U ′ ω ′ associated to the algebra of power bounded elements in the corresponding affinoid algebra. Moreover, let Y ω be the closure of Y ω in U ω . Then we have canonical morphisms of admissible formal affine schemes over K • in the sense of Bosch, Lütkebohmert and Raynaud (see [BL93], §1). We claim that all these morphisms are finite and surjective. Obviously, the generic fibres of the first and second morphism are finite and surjective. To see that the generic fibre of the third morphism is finite, we note first that F −1 (ω ′ ) ∩ Trop(Y ) is finite by construction of q and hence q −1 (U ′ ω ′ ) ∩ Y an is in the relative interior of an affinoid subdomain of T an which is contained in q −1 (U ′ ω ′ ). We conclude that q −1 (U ′ ω ′ ) ∩ Y an → U ′ ω ′ is a proper map (see the proof of Theorem 4.31 in [BPR11] for more details about the argument). Since q −1 (U ′ ω ′ ) ∩ Y an is the disjoint union of the finitely many affinoids U ρ ∩ Y an , ρ ∈ F −1 (ω ′ ) ∩ Trop(Y ), we conclude that q induces a proper morphism Y ω → U ′ ω ′ of affinoids. By Kiehl's direct image theorem ([BGR84], Theorem 9.6.3/1), this morphism is finite and hence also surjective using dimensionality arguments. We conclude that all three morphisms in (4) are surjective and finiteness follows from [BPR11], Proposition 3.13.
The degree [X ω : U ′ ω ′ ] of X ω over the affinoid torus U ′ ω ′ is well-defined as U ′ ω ′ is irreducible (see [BPR11], Section 3, for a discussion of degrees). Since the degree does not change by passing to an affinoid subdomain of U ′ ω ′ (see [BPR11], Proposition 3.30), we get where B ranges over all irreducible components of (X ω ) s . We conclude from (5) and (6) where C ranges over all irreducible components of (Y ω ) s and B ranges over all irreducible components of (X ω ) s mapping onto C. Since the special fibre of Y ω is isomorphic to the initial degeneration in ω (Y ), all irreducible components C are isomorphic to the torus Spec(K[M σ ]) (see [BPR11], Theorem 4.29) proving Since X ω is the preimage of the affinoid subdomain Y ω of T an , we deduce from [BPR11], Proposition 3.30, that X ω is of degree deg(ϕ) over Y ω and hence the projection formula again shows the equality deg(ϕ)cyc((Y ω ) s ) = (ι • ϕ) * (cyc((X ω ) s ) of cycles in (U ω ) s . Inserting (10) in (9) by using that the special fibre of X ω is reduced, we get m an σ = deg(ϕ) where m(C, (Y ω ) s ) is the multiplicity of the irreducible component C in the special fibre of Y ω . By definition, the right hand side is equal to m alg σ which proves the claim.
Remark 7.12 Note that in the algebraic case, Proposition 7.11 yields that the tropical multiplicities in Definition 7.5 are well-defined integers, i.e. independent of the choice of the generic projection q. Moreover, the argument of Chambert-Loir and Ducros for Proposition 7.9 gives a new proof for the classical balancing condition for tropical varieties which is based mainly on degree considerations. | 2013-04-10T13:33:39.000Z | 2013-03-29T00:00:00.000 | {
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207814947 | pes2o/s2orc | v3-fos-license | DESIGN, DEVELOPMENT, AND EVALUATION OF AN ION-ACTIVATED OPHTHALMIC IN SITU GEL OF MOXIFLOXACIN HYDROCHLORIDE
Objective: The objective of this study was to develop an in situ ophthalmic gel of an anti-infective drug, moxifloxacin (MOX) hydrochloride (HCL), for sustained ocular delivery for the treatment of bacterial infections of the eye. Results: Optimized formulation batch F7 (0.12% gelrite and 0.6% sodium alginate) was liquid before addition of simulated tear fluid (STF) and underwent rapid gelation on addition of STF and had given 84.05% cumulative drug release; the formulation was found to be clear, having good in situ gelling capacity, good antibacterial efficacy, having drug content 99.75%; optimized formulation was sterile and showed sustained drug release over 8 h period as compared to marketed eye drop. Conclusions: From the above results, we can concluded that 32 full factorial design and statistical models can be successfully used to optimize the formulations, and it was concluded that the trial batch F7 (0.12% gelrite and 0.6% sodium alginate) is the best formula (percentage cumulative drug release over 84.05%) and it is possible to formulate in situ ophthalmic gels of MOX HCL using gelrite in combination with sodium alginate for the treatment of various bacterial infections of the eyes.
INTRODUCTION
Ophthalmic drug delivery is the most challenging and interesting area for upcoming pharmacists and formulation chemist due to unique anatomy and physiology of the eye. Eye drops that are conventional ophthalmic delivery systems often result in poor bioavailability and therapeutic response because high tear fluid turnover and dynamics cause rapid precorneal elimination of the drug. A high frequency of eye drop instillation is the main cause of patient non-compliance. Addition of excess drug in the formulation is an attempt to overcome bioavailability problem which is potentially dangerous if the drug solution drained from the eye is systemically absorbed from the nasolacrimal duct. Various other ophthalmic vehicles such as ointments, suspension, inserts, and aqueous gels have been developed to enhance the ophthalmic bioavailability. This ocular drug delivery system, however, has not been used frequently due to some disadvantages such as blurred vision from ointments or low patient compliance from inserts.
Above-mentioned problems can be overcome by the use of in situ gelling systems, a liquid dosage form suitable to be administered by instillation into the eye, which on exposure to physiological conditions, changes to the gel phase thus increase the precorneal residence time of the delivery system and enhance the ocular bioavailability. It comprises the ease of eye drop instillation and patient compliance as well as sustained release property that is described to intensify ocular bioavailability. The concept of in situ forming gels came into existence in the early 80s.
Depending on the method employed to cause sol to gel phase transition on the ocular surface, the following three types of systems have been recognized: • pH-triggered -the polymers used in this system are pseudolatexes -carbomer (carbopol), cellulose acetate phthalate latex. • Temperature dependent -poloxamers (pluronic and tetronics), cellulose derivatives (methylcellulose and hydroxypropylmethylcellulose), xyloglucan. • Ion-activated induced -alginates and gelrite (gellan gum) [1].
Moxifloxacin (MOX) hydrochloride (HCL) is a fourth-generation fluoroquinolone broad-spectrum antibacterial, Biopharmaceutical Classification System Class-I drug, having low ocular bioavailability and therapeutic response due to high tear fluid turn over and rapid precorneal elimination of ocular dosage form. Hence, the rationale is to increase the bioavailability and patient compliance of MOX by formulating ophthalmic in situ gel which gives better residence time using a combination of polymers (as a release retardant) and make it more effective.
The aim of present work is to design, develop, and evaluate in situ ophthalmic gel of an anti-infective drug (MOX HCL 0.5% w/v) for sustained ocular delivery using a combination of gelrite as gelling agent and sodium alginate as viscosifying agent which is used for the treatment of various infective diseases of the eye, to get better patient compliance by increasing residence time and bioavailability.
The formulation would be useful to treat external infections of the eye such as acute and subacute conjunctivitis, bacterial keratitis, bacterial endophthalmitis, and keratoconjunctivitis.
Materials
MOX HCL was purchased from Yarrow Chem Products Pvt., Ltd., Mumbai, Gelrite was provided by Yarrow Chem Products Pvt., Ltd., Mumbai.
Kondalkar and Dev
Sodium alginate was purchased from Thomas Baker (Chemicals) Pvt. Ltd., Mumbai, and all other ingredients were of analytical grade.
Selection of drug and polymers
MOX is a fourth-generation fluoroquinolone with expanded activity against Gram-positive, Gram-negative bacteria as well as atypical pathogens. MOX is the hydrochloride salt of a fluoroquinolone antibacterial antibiotic. In common with quinolone antibiotics, the bactericidal action of MOX results from inhibition of the topoisomerase II (DNA gyrase) and topoisomerase IV required for bacterial DNA replication, transcription, repair, and recombination. Topoisomerase IV is the primary activity inhibited for many Gram-positive bacteria whereas DNA gyrase is the primary quinolone target in many Gram-negative microbes [2].
Determination of absorbance maxima of MOX by ultraviolet (UV) spectrophotometer
A solution of MOX containing the concentration 10 μg/ml was prepared in simulated tear fluid (STF) pH 7.4 and UV spectrum was taken using Shimadzu (UV-1800) double beam spectrophotometer. The solution was scanned in the range of 200-400 nm (Fig. 1).
Identification of MOX
Identification of MOX was carried out by Fourier-transform infrared (FTIR) spectrophotometer (Fig. 2).
Interaction studies [3] The proper design and formulation of a dosage form require consideration of the physical, chemical, and biological characteristics of all drug substances and excipients to be used in the formulation of the product. The drug and excipients must be compatible with one another to produce a stable, efficacious, and safe product. The interaction study of prepared in situ gel formulations was carried out using IR spectroscopy following potassium bromide (KBr) dispersion method. The spectrum of a dried mixture of drug and KBr was then run followed by drug with excipients in the wavelength region between 4000 and 400 cm −1 (Fig. 4).
The drug-polymer compatibility was confirmed by differential scanning calorimetric (DSC). Thermal characterization of pure drug and polymer mixture was performed with a calorimeter, which was carried out by heating drug and the physical mixture of the drug with polymers
Kondalkar and Dev
separately from 20°C to 300°C at the heating rate of 10°C/min in a nitrogen environment (Fig. 5).
Preparation of in situ gelling system
Factorial design [4,5] A 3 2 randomized full factorial design was used in the present study. In this design, 2 factors were evaluated; each at 3 level and experimental trials were performed for all 9 possible combinations. The concentration of gelrite (X1) and concentration of sodium alginate (X2) was chosen as an independent variable in 32 full factorial designs, while percent cumulative drug release was taken as dependent variable (Table 1a-c and Fig. 13).
The formulation layout for the factorial design batches (F1-F9) is shown in Tables 2 and 3. Procedure A 3 2 factorial design was used for formulation design, gellan gum (gelrite) and sodium alginate were chosen as an independent factor. Their effect on dependent factors such as drug release and viscosity was observed. An aqueous solution of a varying concentration of gelrite and sodium alginate was prepared and evaluated for gelling capacity and viscosity to identify the compositions suitable for as an in situ gelling system. Polymer solution was prepared by dissolving required quantity of sodium chloride in deionized water followed by dispersing gelrite and sodium alginate
Kondalkar and Dev
in above solution and heat up to 90°C for 20 min followed by cooling to room temperature, drug solution was prepared by dissolving MOX in mixture of propylene glycol and deionized water (8:100), drug solution was mixed with polymer solution using a magnetic stirrer under constant stirring until a uniform solution was obtained. The pH of the formulation was then set to 4.4 using 0.1 N HCL. The prepared in situ gels were filled in glass vials closed with rubber closures and sealed with aluminum caps and sterilized by autoclave at 121°C 15 psi for 20 mi [4,5].
Evaluation of prepared in situ gelling system
Interaction studies [6] IR spectra were taken using FTIR spectrophotometer (Jasco 4100). The pellets of drug and KBr were prepared by compressing the powders (Ratio of drug to KBr 1:100) at 20 psi on KBr press, and the spectra were scanned in the wave number range of 4000-400 cm −1 FTIR study was carried on pure drug, physical mixture of drug and polymers, formulations to confirm the compatibility of drug with other excipients used in the preparation of in situ gels (Fig. 6).
Visual appearance and clarity [7] Visual appearance and clarity were checked under fluorescent light against a white and black background for the presence of any particulate matter (Table 4).
pH [8] The pH of the prepared in situ gelling system after addition of all the ingredients was measured using pH meter (Table 4).
In vitro gelation [8]
The gelling capacity of formulations was evaluated to identify the formulations suitable for use as in situ gelling systems. Gelling capacity was determined by mixing the formulation with STF in the proportion 25:7 and examined visually.
Rheological studies [8] The viscosity of the instilled formulation is an important factor in determining residence time of drug in the eye. The prepared solutions were allowed to gel in the STF, and then the viscosity determination was carried out using Brookfield viscometer DV-II+PRO model, angular velocity range from 1 to 100 rpm. Viscosity of the formulations increased with increase in polymer concentration. The hierarchy of shear rate was reversed and an average of three readings was used to calculate viscosity (Tables 5 and 6 and Figs. 8 and 9).
Sterility testing [8,9] Sterility testing is intended for detecting that the presence of a viable form of microorganisms and was performed for aerobic and anaerobic bacteria and fungi using fluid thioglycolate medium and soybean casein digest medium, respectively, as per the Indian Pharmacopoeia (Table 7).
In vitro release studies [8,10] In vitro drug release from the formulations was studied by the diffusion cell. Here, the pH of the lacrimal fluid and the blinking rate of the eye were taken into consideration and were simulated. The procedure for standard calibration is the same as mentioned under drug content determination.
Comparative evaluation of marketed product with prepared in situ gels
In vitro release studies of the marketed formulation were carried out using bi-chambered donor receiver compartment model (Franz diffusion cell) using cellophane membrane soaked overnight in the receptor medium (STF pH 7.4). The diffusion medium was 12 ml of STF stirred at 50 rpm at 37±0.5°C. One end of the diffusion tube was covered by a cellophane membrane. The 1 ml formulation was spread on the cellophane membrane, and membrane was placed such that it just touches the diffusion medium STF present in the From diffusion medium and analysed by a UV spectrophotometer at 288.5 nm using STF as blank (Fig. 11).
Kondalkar and Dev
Pharmacokinetic release studies [10] All the optimized formulations were subjected to study the release kinetics, and the best fit kinetic model was determined for the optimized formulations (Table 9).
Antimicrobial efficacy studies [11] The antimicrobial efficacy studies were carried out to ascertain the biological activity of the optimized formulations. Staphylococcus aureus and Escherichia coli were used as the test organisms. Antimicrobial efficiency was determined by agar diffusion test employing cup-plate method. Sterile solutions of MOX (standard solution) and the developed formulations were diluted at different concentration (test solutions) these solutions were poured into cups bored into sterile nutrient agar previously seeded with test organisms (E. coli and S. aureus), after allowing diffusion of the solutions for 2 h, the agar plates were incubated at 37°C for 24 h. The zone of inhibition (ZOI) measured around each cup and was compared with that of control. The entire operation except the incubation was carried out in a laminar air flow unit. Both positive and negative controls were maintained during the study (Table 10 and Fig. 12).
Accelerated stability studies [7,9] Stability is defined as the extent, to which a product retains within specified limits and throughout its period of storage and uses, i.e., shelf life. Stability studies were carried out on optimized formulations according to the International Conference on Harmonization (ICH) guidelines.
A sufficient quantity of formulations in previously sterilized vials was stored in desiccators containing a saturated solution of sodium chloride, which gives a relative humidity of 75±5%. The desiccators were placed in a hot air oven maintained at a temperature 40±5°C and room temperature. Samples were withdrawn at 7 days interval for 42 days. Percent drug remaining was calculated and plotted against time in days (Table 10 and Figs. 14 and 15).
Solubility study
The solubility of MOX was found to be dependent on pH. MOX was soluble in a cosolvent mixture of propylene glycol and water, glycerine and water 2.4 g of MOX soluble in 100 ml water.
Melting point determination
The melting point of MOX was found to be approximately 238-242°C.
Where "-"sign indicate the no growth. SD: Standard deviation
FT-IR spectroscopy study
FTIR study was carried on the pure drug, a physical mixture of drug and polymers, formulations to confirm the compatibility of the drug with other excipients used in the preparation of in situ gels.
DSC
Thermal characterization of pure drug and the physical mixture was performed with a calorimeter. The sample was placed in sealed aluminum pans. The samples were scanned at 20°C/min from 20°C to 300°C.
EVALUATION OF PREPARED IN SITU GELLING SYSTEM
Interaction studies
FTIR spectral analysis
The prepared in situ gelling systems were evaluated for interaction studies to ensure that there is no interaction occurred in between drug and polymers. For confirmation of stability of the drug in the prepared formulations, the IR spectra were taken and compared with that of pure drug. The result of these studies revealed that there were no definite changes obtained in the bands of the drug with respect to pure drug (Fig. 6).
DSC analysis of MOX
Evaluation of visual appearance, clarity, pH, and drug content All the prepared in situ gelling systems were evaluated for preliminary steps such as visual appearance, clarity, pH, and drug content. These formulations were translucent and clear. The pH of the formulations was found to be 4.4, and drug content was in between 73% and100% (Table 4).
Rheological studies
For the development of optimum in situ gelling system, two major prerequisites viscosity and gelling capacity should be taken in consideration, since the ocular shear rate is very high ranging from 0.03/S during inter-blinking periods to 4250-28500/S during blinking, viscoelastic fluid with a viscosity that is high under low shear rate condition and low under high shear rate condition, which is called pseudo-plastic fluid, is often preferred, so dynamic viscosity of formulations was measured as the change of shear rate before and after gelation (Tables 5 and 6 and Figs. 8 and 9).
Sterility testing
All the prepared in situ gelling systems were evaluated for the sterility. After 7 days of incubation, the results showed no microbial growth in all formulations ( Table 7). Estimation of MOX by spectrophotometric method A simple spectrophotometric method for estimation of MOX was developed in STF, which exhibited λ max at 288.5 nm. Results are shown in Table 8 and Fig. 10.
In vitro release studies
The in vitro release of MOX from the prepared formulations was studied through cellophane membrane using diffusion cell. The release studies of prepared in situ gelling systems were carried out up to 8 h (Fig. 11).
Antimicrobial efficacy studies
The optimized in situ gelling formulations showed antimicrobial activity when tested microbiologically by the cup-plate technique. Clear zones of inhibition were obtained in all the formulations. The diameter of ZOI produced by formulations against all test microorganisms is given in Table 10 and Fig. 12.
Accelerated stability studies
According to the ICH guideline, the accelerated stability studies were carried for prepared in situ gelling systems. All the formulations were analyzed for visual appearance, clarity, pH, and drug remaining. 6 weeks of stability studies reveal that there was no change in visual appearance and clarity. All the formulations showed slight changes in pH, but it was in acceptable limits (±0.5). Study of percentage drug remaining in all formulations reveals that there were no definite changes observed to justify for drug degradation (Table 11 and Figs. 14 and 15).
DISCUSSION
Optimized formulations F6 (0.1% gelrite and 1.0% sodium alginate), F7 (0.12% gelrite and 0.6% sodium alginate), and F8 (0.12% gelrite and 0.8% sodium alginate) and were liquid before instillation in to eye and underwent rapid gelation on instillation in to eye and had given 85.55%, 84.05%, and 81.21% percentage cumulative drug release, respectively, the formulations were found to be clear, having good in situ gelling capacity, and having drug content 81-100%, optimized formulations were sterile and showed sustained drug release over 8 h period as compared to marketed eye drop, release kinetic study showed that the formulation followed Higuchi model diffusion controlled and non-Fickian release mechanism, the optimized formulations were having good antibacterial efficacy. As per the ICH guidelines, the stability study of formulations was carried out results showed that formulations were stable (translucent and clear) at room temperature as well as at 40°C.
Hence from the above results, we can conclude that F7 (0.12% gelrite and 0.6% sodium alginate) is the best formula (percentage cumulative drug release over 84.05%) and it is possible to formulate in situ ophthalmic gels of MOX using gelrite with sodium alginate in combination for the treatment of various bacterial infections.
CONCLUSION
The present work was carried out to develop ion activated in situ gel of MOX, a broad spectrum antibacterial agent used in the treatment of ocular infections, was successfully formulated as in situ gel-forming eye drops using gelrite as a gelling agent in combination with sodium alginate as a viscosity enhancing agent. Thus, the developed formulation is a viable alternative to conventional eye drops of its ability to sustain drug release. Furthermore, important is the ease of administration afforded and decreased frequency of administration resulting in better patient acceptance.
AUTHERS' CONTRIBUTIONS
Mr. Asish Dev conceived of the presented idea. I developed the theory and performed the computations. Mr. Asish Dev verified the analytical methods. All authors discussed the results and contributed to the final manuscript.
CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest. | 2019-10-24T09:22:54.804Z | 2019-02-13T00:00:00.000 | {
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239012660 | pes2o/s2orc | v3-fos-license | Assessing the Potential for Sustainable Aquaculture Development in Cambodia
Inland capture fisheries are central to livelihoods and food security in Cambodia, but are under threat from growing anthropogenic pressures. Policy discourse in Cambodia increasingly frames aquaculture as a viable alternative to capture fisheries, and seeks to promote its development. This paper presents results from the first comprehensive survey of Cambodia's aquaculture value chain. The study combines qualitative (46 Key Informant Interviews) and quantitative surveys (1,204 farmers and 191 other aquaculture value chain actors) to investigate potential for aquaculture in Cambodia to grow, support livelihoods, and contribute to food security. We found the following: (i) The fish farm sector in Cambodia is comprised mainly of small family farms raising carnivorous fish species or pangasius, using direct inputs of “trash fish” harvested from the wild; (ii) Most fish seed and pelleted feed are imported, and domestic producers of these inputs struggle to compete; (iii) Fish farmed in Cambodia is mostly sold live. Farm fish are more expensive than the main species harvested from inland capture fisheries, and struggle to compete with imported farmed fish; (iv) Capture fisheries employ many times more people than aquaculture; (v) Space for aquaculture is limited because few locations have both perennial access to water and protection from flooding. These findings raise questions about the potential of Cambodia's aquaculture sector, as currently organized, to contribute significantly to employment, food and nutrition security, and rural economic development. We propose actions to improve the sector's sustainability and contribute to desirable development outcomes.
INTRODUCTION
Cambodia has one of the world's most productive inland fisheries (Baran, 2005), based around the ecosystem of the Tonle Sap Lake. Inland fisheries have been central to livelihoods and food and nutrition security in Cambodia for centuries (Cooke, 2011;Sithirith, 2011), and continue to be so today (Hartje et al., 2018;Freed et al., 2020).
Aquaculture has only relatively recently become the focus of sustained interest from research and development institutions in Cambodia. This interest aligns with predicted, and increasingly realized, declines in inland capture fisheries production. For example, a combination of drought and water impoundment by upstream dams caused reported fish catch from the Tonle Sap to contract 23% in 2020, prompting fears of imminent fisheries collapse (MRC, 2020). Such a collapse would threaten the livelihoods and food security of millions of Cambodians (IFReDI, 2013).
Aquaculture is increasingly framed in Cambodian development policy discourse as having an important role to play in meeting demand for fish and providing rural employment, but has yet to contribute significantly to these outcomes. Development projects supporting Cambodia's aquaculture sector have faced constraints to sustainable impact, and high rates of non-adoption (JICA, 2015;Richardson and Suvedi, 2018).
The relationship between capture fisheries and aquaculture is rarely discussed explicitly when aquaculture development strategies are designed or evaluated, beyond the stylized "facts" of fisheries decline and aquaculture's growth potential (Bush and Hirsch, 2005;Tezzo et al., 2020). Moreover, in common with many other countries in the Global South, little rigorous research has been conducted on aquaculture value chains in Cambodia (Bush et al., 2019). Knowledge gaps and pending development investments in Cambodia's aquaculture sector make it important to understand the characteristics of its aquaculture value chain, and to identify practical strategies that could contribute to sustainable and equitable development outcomes.
We follow the call by Tezzo et al. (2020) for a food systems approach to aquaculture development, that views aquaculture's potential to contribute to food and nutrition security in relation to capture fisheries. We combine qualitative and quantitative analysis to answer four sets of questions: 1. What is the structure of Cambodia's aquaculture value chain, and the conduct and performance of the actors in it? 2. What technical (micro) and structural (meso/macro) challenges does aquaculture in Cambodia face? 3. What is the likely future role of aquaculture in Cambodia's food system, vis-à-vis capture fisheries and with respect to food and nutrition security? 4. What options exist for supporting sustainable and equitable forms of aquaculture that contribute to livelihoods in on-and off-farm segments of the value chain?
Context synthesizes literature on fisheries and aquaculture and associated policy discourses in Cambodia. Methods sets out the methodology and analytical framework. In Results, we answer question 1 by analyzing the structure and performance of key segments of the aquaculture value chain (farms, fish seed and feed supply, fish processing and marketing). The discussion section addresses questions 2-4, with respect to prospects for aquaculture development, and implications for the design of policy and interventions. The final section concludes.
CONTEXT
Cambodia's inland fisheries are the fifth largest in the world (Fisheries Administration, 2017). Most of the fish harvested originate from the flood pulse-driven ecosystem of the Tonle Sap, Southeast Asia's largest lake. During peak monsoon season from June to October water flows upstream from the Mekong River, filling the lake, flooding surrounding forest and rice fields, and expanding the Lake's surface area from 0.25 million hectares to 1-1.3 million hectares. From November-February, water from the lake flows back toward the Mekong, causing the water level to fall (van Zalinge et al., 2003). Half of rural Cambodian households fish, on either a regular or occasional basis (Nasielski et al., 2016) catching >200 kg of fish per year on average, of which more than half is sold (Mousset et al., 2016). In addition, many households migrate to the Tonle Sap river during peak fishing season to purchase fish for transformation into fermented fish paste (LeGrand et al., 2020).
Consequently, fish and other aquatic animals are the main animal source foods eaten in Cambodia (Olney et al., 2009;IFReDI, 2013), accounting for three-quarters of animal protein intake, second only to rice in terms of total food intake, and rich in micronutrients. Fisheries are particularly important for the food and nutrition security of poorer households with insufficient resources to invest in farming or aquaculture (Roos et al., 2007a;Chhoun et al., 2009;Nam and Touch, 2011;Hartje and Grote, 2016). This includes the 19% of rural families that are landless, and the 40% of farm households that own <0.5 ha of land (World Bank, 2006).
Cambodia's inland fisheries face a variety of threats. Hydropower development in the Mekong catchment is expected to reduce fisheries production by up to 42% (ICEM, 2010;IFReDI, 2013). Hydropower dams and smaller-scale infrastructure such as roads, canals, and irrigation dams reduce seasonal water flows including the flood pulse that feeds the Tonle Sap ecosystem, disrupting aquatic habitat connectivity, and blocking movements of sediment and fish migrations (Baran et al., 2007;ICEM, 2010;Basist and Williams, 2020). Agricultural intensification is driving use of chemical inputs, irrigation (Mukherji et al., 2009), and aquatic habitat conversion (Mahood et al., 2020). Destructive fishing practices are widespread (Chan et al., 2020).
These threats are compounded by more frequent droughts linked to climate change. Severe droughts in 2016, 2019, and 2020 caused large catch declines, altered fish species assemblages, and drove shifts in fish size composition toward smaller fish (Ngor et al., 2018;Chan et al., 2020;FISHSTAT, 2020). Higher temperatures and changes in rainfall and river flows are predicted to affect water quality, particularly impacting migratory fish species (ICEM, 2013).
As a result, Cambodia's capture fisheries are predicted to be unable to meet future demand, with negative implications for human nutrition (IFReDI, 2013;Golden et al., 2019). Fish supply is predicted to fall by 34,000-182,000 tons (6 to 34%) depending on the number of dams built on the Mekong mainstream, equivalent to a drop in fish consumption from 63 kg/person/year in 2011 to between 41 and 29 kg/person/year (IFReDI, 2013).
Despite vast differences in the current scale of the inland capture fisheries and aquaculture sectors in Cambodia in terms of quantities of food produced and numbers of people engaged, there is a common tendency to depict capture fisheries as "traditional" and locked in a trajectory of terminal decline, in contrast to aquaculture which is framed as a "modern" sector on the rise (Bush, 2008;Friend et al., 2009;Arthur and Friend, 2011;Tezzo et al., 2020). This framing has the effect of making aquaculture appear as a logical and inevitable substitute for capture fisheries.
Development aid worth more than USD 59 million was granted to Cambodia's aquaculture sector between 2016 and 2019; more than three times the total invested in the sector from 2000 to 2015 1 . Capture fisheries also remain a primary recipient of donor investment in Cambodia, with EUR 87 million allocated from an EU-Cap Fish Capture project, but only a fraction of these resources are used to support active fisheries management, while a significant share is dedicated to upgrading fish processing, and providing alternative livelihoods to fisheries dependent households.
Although policy discourse invokes a promising future for Cambodian aquaculture, implementing projects have encountered multiple challenges. Ex-post-evaluations report these to include inadequate access to quality inputs (VCA4D, 2017; Richardson and Suvedi, 2018); weak technical skills among farmers (Chin, 2015;Bengtson et al., 2016); and limited access to water (Maredia et al., 2017;Richardson and Suvedi, 2018). As such, these studies ascribe low levels of aquaculture adoption mainly to micro-scale farm level constraints, amenable to technical solutions such as training or input provision.
Meso-and macro-scale economic, political, and environmental dimensions that are more difficult to assess are often overlooked in such evaluations. For example, interactions between supply and demand of Cambodian inland capture fish, imported farmed fish from Vietnam and Thailand, and Cambodian aquaculture products are poorly understood and rarely acknowledged. Discussion of the links between capture fisheries and aquaculture focuses mainly on competition for use of small low-cost fish ("trash fish 2 ") for human consumption or as fish feeds (Nam et al., 2005). The physical suitability of the 1 2016-2019 period: Three large projects: EU-AFD Cap Fish Aquaculture = e30 million granted in 2016 until 2021; Sustainable Aquaculture and Community Fish Refuge Management (SAFR) of the German Ministry for Economic Cooperation and Development operated by GiZ granted in 2019 until 2023 with a budget of e4.5 million; USDA Food for Progress funding CAST project USD17 million in 2018. Total approximate USD 59 million funding of granted during this period. From 2000 to 2015 -we count few significant aquaculture projects: JICA FAIEX phases I andII (2005-2015) for a total of USD 8.7 million and USAID HARVEST program (2010-2016) for a total of USD 56 million but including a support to several sectors (rice, horticulture, fisheries, and aquaculture). We estimate the funds dedicated to aquaculture to around USD 10 million. 2 "trash fish" is a common term used to denote small, low market value fish that are utilized as feed for farmed fish or animals. This can include food grade fish that would otherwise edible to humans.
Cambodian environment for aquaculture, in terms of access to perennial water and flood risk, is often absent from the debate.
We address the micro-scale technical concerns and meso/macro-scale structural ones outlined above using primary data from a comprehensive survey of Cambodia's aquaculture sector. The survey methodology is elaborated below.
METHODS
We surveyed 1,204 farmers and 191 other actors in the aquaculture value chain in nine provinces and Phnom Penh municipality from April to July 2019 (WorldFish, 2019). Provinces were selected purposively based on triangulation between key informant interviews and provincial statistics, and include all the main areas where commercial inland aquaculture production occurs.
Quantitative Sampling Method and Data Collection
We implemented a scoping study in these provinces prior to the survey to estimate the population of fish producers and other aquaculture value chain actors. Subsistence farmers and microscale producers were deemed out of scope, and farms with a pond area <200 m 2 or a cage volume <30 m 3 were excluded. Fish traders and processors for whom products sourced from aquaculture accounted for at least 20% of raw material were considered in scope. This exercise identified a total population of 1,617 farms and 406 other value chain actors eligible for inclusion in the survey.
We initially designed a random sample of 1,200 farmers. During survey rollout we found that some respondents had stopped aquaculture, had been selected incorrectly, or could not be contacted. We therefore switched to an exhaustive sampling strategy for farms. The sample represents more than 85% of all active aquaculture producers in the surveyed provinces that met our selection criteria.
We selected a sub-sample of other value chain actors, with the following criteria, per province: (i) all operational feed mills; (ii) three micro-finance institutions; (iii) three fish feed distributors; (iv) three fish wholesalers; (v) five fish collectors (mobile traders); (vi) 30% of fish processors; (vii) all operating hatcheries (30) and nurseries (9). The final sample consisted of 1,204 farms (including 690 pond farms, 504 cage farms and 10 farm operating both types of system), and 191 other actors: 30 hatcheries, nine nurseries, eight feed mills (all of which, at the time of the survey, produced livestock and/or poultry feed but no aqua feed), 24 feed distributors, 34 fish processors, 69 fish traders, and 17 microfinance institutions. We later excluded from the analysis 96 farms that did not report any production in the 12 months preceding the survey, nine farms that operated both ponds and cages, and three farms that reported the type of farm (pond or cage) inconsistently. The questionnaire covered the calendar year 2018 through May 2019 and included modules on assets, employment, farm management practices, and quantities of fish produced and sold in the 12 months preceding the survey. Data were collected on the farm or at business premises during interviews with farmers and business owners (for other value chain actors) by a survey team composed of five enumerators and a supervisor. All data were collected on digital tablets using the KoBo Toolbox platform.
Typology of Farms and Descriptive Statistics
A data cleaning protocol was used to check for inconsistencies or abnormal responses, to generate a clean dataset (WorldFish, 2019). Excel 2016 and R software were used for data analysis to produce descriptive statistics on farm and business performance. We constructed a simple typology of farms based on the distribution of pond and cage farm sizes within the total population, distinguishing three size-based categories of pond and cage farms ("small, " "medium, " and "large") as follow: • Small pond farms (<500 m 2 ) and small cage farms (<50 m 3 ) • Medium pond farms (500 to 3,000 m 2 ) and medium cage farms (50 to 100 m 3 ) • Large pond farms (>3,000 m 2 ) and large cage farms (>100 m 3 ) The cage farm categories can be interpreted as roughly equivalent to number of cages per farm (1, 2, ≤3) given an average cage volume of ∼50 m 3 , whereas the pond farm categories are constructed to include a roughly equal number of farms in each group. We estimated the amount of trash fish used within our sample based on recall data. For homemade feed with trash fish, we use an average ratio of 20% of trash fish per kilogram of feed to estimate the volume of trash fish used in farms (Nam et al., 2005). Extreme values were excluded from the calculation.
Qualitative Sampling, Data Collection, and Analysis
In addition to the quantitative survey described above, we collected qualitative data to capture contextual information, with an emphasis on private service provision, quality control systems, and relationships governing coordination between actors in the value chain. Key informant interviews were conducted with 46 value chain actors, government representatives, development partners, and fish retailers. The interviews were recorded and translated in English before coding using NVivo 12 software. After coding was completed, the team produced node reports that were further analyzed to identify emerging themes.
The results section below combine both qualitative and quantitative results. We use the quantitative survey results to assess the performance of different segments of the value chain and the qualitative interview results to provide context and add nuance to the interpretation of the results of the quantitative survey. In the results section, all information derived from qualitative interviews is identified as such.
RESULTS
We analyze the following value chain segments in the order indicated: (1) producers (pond farms and cage farms); (2) input supply (hatcheries, nurseries, and feed suppliers); (3) traders and processors of farmed fish.
Producers
Using a simple typology of farms, we evaluate the structure of the farm segment of the value chain, with respect to production systems, farm size distribution, location, and water use. We then evaluate the conduct (feeding practices) and performance (productivity) of pond and cage farms for the three most common fish species cultivated in each.
Farm Structure
Our typology distinguishes three size-based categories of pond and cage farms ("small, " "medium, " and "large"; Table 1). Surveyed fish farms average 2,369 m 2 (ponds) and 114 m 3 (cages) in size, respectively. This is small compared to other countries in the region. For example, in nearby Central Thailand pond farms range from 1 to 100 ha with farms around 2-3 ha most common, and small and medium cage farms are sized 180 and 915 m 3 , respectively (Belton et al., 2009). Thus, what we refer to here as "large farms" are large relative to other farms in Cambodia, but small relative to farms in some other parts of Southeast Asia. Figure 1 illustrates the location of surveyed farms, over-layed with the extent of flooding in 2011, a major flood year. Cage farms are located around the edges of the perennial Tonle Sap waterbody and on major rivers that enter and exit the lake, indicating a requirement for continuous water throughout the year. Pond farms are located mainly in a narrow band close to national roads running around the outer edge of the area exposed to flooding. Half (49%) of pond farms lie within the area that flooded in 2011 and can thus be considered at risk of flooding. Most land outside the Tonle Sap floodplain is drought prone. Pond farms appear to be clustered in locations that combine adequate access to water and transport with protection from heavy flooding. This spatial distribution highlights the limited extent of areas suitable for aquaculture production.
Pond farms are the dominant form of production, supplying around 80% of farmed fish. Production is concentrated among farms at the upper end of the size distribution. Only large farms operate more than two ponds or cages each, on average. Farms in our "large" category each make up around 18% of pond and cage farms, but account for 71 and 67% pond and cage area and 70 and 52% of pond and cage fish production, respectively. Although farms in our "small" category account for one third of pond farms and more than half of cage farms, they amount only 5 and 16% of pond and cage farm area, and supply 6 and 25% of pond and cage farm production, respectively ( Table 1).
Most farms are managed intensively. For example, the average extrapolated yield for pond farms is equivalent to 43.7 t/ha and 35.5 kg/m 3 for cage farms. Smaller farms are most productive. For example, the yield of small cage farms is 52.1 kg/m 3 , as compared to 27.8 kg/m 3 for large cage farms. All cages are located in natural waterbodies. The main sources of water for pond farms are rivers (used by 45% of farms) and rainwater 25%. Only 8% of farms use groundwater irrigation. Larger farms are somewhat more likely to use river water, and small farms are slightly more likely to be rain fed. As a result, many pond farms are vulnerable to drought, climate variability, and competition for surface water. Key informant interviews confirmed that many producers face water shortages in the dry season, and that surface water pollution by industry and agriculture is increasing. Farms in or near waterbodies or within the floodplain can have easier access to surface water and be less dependent on rainfall, but are vulnerable to flooding. Pond farms in such locations require investments in dikes and/or netting to avoid fish escapes.
Farm Conduct and Performance
In this subsection we analyze farm conduct (farming technologies, and farmer behavior regarding input purchases and product sales) and performance (yields). We analyze the most common species produced in pond and cage farms, by farm size category. Surveyed farms produced 12 species in total, but five dominated, accounting for 97% of the volume. 88% of the farms specialize in production of a single species.
The main fish species raised in cages are carnivorous. Striped snakehead (Channa striata) and giant snakehead (Channa micropeltes) are most popular, raised by 56 and 26% of cage farms, respectively. The high value species, Asian red tail catfish (Hemibagrus wyckioides), is produced mainly by large cage farms (5% of cage farms). Pangasius catfish, an omnivorous species, is the main fish cultivated in ponds (53% of pond farms) and is also raised in 10% of cage farms. Giant snakehead is also found in both types of farm, and raised by 35% of pond farms Silver barb is present in 12% of pond farms ( Table 2). Other species, including tilapia, carps, hybrid walking catfish, climbing perch and snakeskin gourami are produced by 8 and 7% of surveyed pond and cage farms, respectively. Spatial distribution of the main cultivated species is uneven, except for pangasius, which is found in every province (Figure 2, map A to F).
Pangasius accounts for 61% of the total quantity of fish produced by farms in our sample, followed by giant snakehead (25%) and striped snakehead (7%). Snakehead and catfish species are farmed much more intensively than silver barb ( Table 1). Reported yields are within the range of previous estimates (WorldFish, 2010), suggesting that productivity has not improved significantly in the past decade. Average yields of pangasius grown in ponds (53 t/ha) are several times lower than in Vietnam (370 t/ha) (Belton et al., 2011).
Most farms purchase fingerlings from nurseries or traders. These businesses often import fish seed from Vietnam informally. As an indicator, 35 and 31% of farmers acknowledged that the giant snakehead and striped snakehead seed they stocked was imported from Vietnam. Among farms growing pangasius and silver barb in ponds, only 13 and 19% respectively reported buying their fingerlings from hatcheries in Cambodia, with 35% of silver barb fingerlings obtained from NGOs. <2% of the cage farms reported stocking wild caught fingerlings. Production cycles are annual, with close to one cycle completed each year. Average farm gate prices vary by species, ranging from USD 3.12/kg for red tailed catfish to USD 1.24/kg for pangasius. Pangasius farmers reported that local prices are influenced strongly by the volume of cheap imported pangasius from Vietnam. Farmers often time their harvest according to the fluctuating price of imported fish. When prices are too low, they extend the harvest over 10-20 days, waiting for better prices. Harvest duration is also influenced by consumer demand for live fish, which is sold daily in limited volumes. Many farms spread their harvest over multiple days due to low demand, thus increasing stress and mortality of fish. Extended harvests can also raise production costs, as fish continue to require feed.
Most farms use "trash fish" in some form, either exclusively, in combination with other feeds, or as an ingredient in farm-made feed ( Table 3). Most trash fish is comprised of small, low cost, food grade fish species, harvested locally from the inland capture fishery. All snakehead farms use trash fish. Most giant snakehead farms use trash fish exclusively (81% of ponds and 61% of cages), as do 42% of striped snakehead cage farms, possibly suggesting that striped snakehead domestication is more advanced than giant snakehead, making the former more readily weaned onto artificial feeds.
None of the farms surveyed use manufactured pelleted feed exclusively. However, its use is common among pond farms, and those growing pangasius. For example, 87% of pangasius pond farms and 73% of pangasius cage farms use pellets. Conversely, trash fish use is somewhat less common among pond farms-−51% of pangasius pond farms use trash fish in some form, as compared to 80% of pangasius cage farms. We hypothesize that for cage farms, proximity to the Tonle Sap Lake and rivers makes trash fish easily accessible. However, snakehead pond farms are also heavy users of trash fish showing that access to trash fish is not necessarily constrained by distance from fishing grounds.
We estimate that farms in our sample used 13,400 tons of trash fish in the survey year. This is equivalent to ∼0.9 kg of fish for every person in Cambodia. Pond farms and cage farms use 58 and 42% of the total quantity of trash fish utilized, respectively. Smaller farms are more likely to utilize trash fishperhaps harvesting it themselves to save costs-and are less likely to purchase manufactured pelleted feeds. Larger farms are more likely to buy pellets-perhaps in part because it is difficult to obtain trash fish in sufficiently large quantities, or expensive to use if not self-harvested. This pattern is consistent across pond and cage farms and could indicate a partial transition toward more sustainable farming practices among larger farms (Figure 3).
Input Supply
This section we analyze the scale of operations and conduct of actors in upstream fish seed and fish feed supply segments of the value chain. We surveyed 30 hatcheries and nine nurseries, representing all hatcheries and the largest and most active nurseries in the survey area.
Fish Seed Supply
The existence of a competitive domestic fish seed production sector is widely regarded as essential for aquaculture development to occur successfully, as the availability of affordable, high quality seed is a key factor conditioning farm performance (Tran et al., 2021).
Surveyed hatcheries produced a total 26 million fingerlings in 2018 (Figure 4), a relatively low total volume of production. For comparison, tilapia hatcheries in Thailand sold around 80 million fry per month in 2008 (Belton, 2012), and Vietnam produced more than 2 billion pangasius fingerlings in 2019 (VASEP, 2020). The number of fingerlings produced per surveyed hatchery in Cambodia ranges from 5,000 individuals to 7 million, with eighteen hatcheries producing fewer than 0.5 million fingerlings each year. The main species produced are tilapia, pangasius and silver barb, accounting for 40, 36 and 10% of fingerlings, respectively. This species composition differs considerably from that reported by surveyed farms.
Fish seed production is highly seasonal as rainfall determines the timing of stocking on farms, peaking in the monsoon months of July and August. Only 45% of hatcheries reported being able to meet demand for seed during the peak stocking period. Key informant interviews explained this result with reference to the short peak in demand requiring production of large quantities of seed within a brief window, and limited production planning and no marketing strategies to reach new markets beyond their established networks.
Many hatcheries and nurseries face severe environmental constraints. Rain is the main source of water for 43% of hatcheries, putting them at risk of water shortages. 43% of hatcheries also reported having been affected by severe drought in the recent past. A female nursery operator in Phnom Penh mentioned: "Another constraint is the irregular rain that makes fish become sick or die." However, 33% of hatcheries are regularly affected by floods, requiring fencing around ponds to avoid brood fish escaping and mixing populations. Half of nurseries also reported being affected by flooding.
Linkages between hatcheries and nurseries are limited. According to key informant interviews, nurseries do not usually contract Cambodian hatcheries as seed suppliers, as the supply of seed from local hatcheries is considered unreliable and expensive. Nurseries buy and sell seed with a short nursing period, averaging 28 days, and therefore operate more as fingerling traders than conventional nurseries. Most of the seed traded by nurseries is imported from Vietnam. The nine surveyed nurseries traded 38 million fingerlings in 2018. This is almost double the amount sold by all 30 hatcheries in our sample, underlining the importance of nurseries selling imported seed in supplying Cambodia's fish seed market. The main species traded by hatcheries and nurseries are pangasius (38% of the volume), walking catfish 3 (38%) and giant snakehead (19%).
Ten nurseries in Cambodia have a license to import fingerlings (VCA4D, 2017). Our sample only included licensed nurseries, but according to our key informant interviews unlicensed nurseries probably contribute a large share of snakehead seed imports, and some unlicensed nurseries sell wild fingerlings on a seasonal basis. A recent government survey suggested that 47% of fingerlings are imported from Vietnam, 40% produced locally and 10% sourced from the wild (CaPFish Baseline Survey, 2020). Our own study indicates that 78% of farms sourced fingerlings from nurseries that mostly sold imported seed. On this basis we estimate that well-over half the seed used in Cambodian aquaculture is imported. As such the sector is currently dependent on imported seed, over which local actors and decision makers have little influence, especially regarding quality control.
Fish seed sold in Cambodia does not meet basic quality standards in terms of minimum size, homogeneity, and pathogen free status. <20% of hatcheries and 12% of nurseries grade fingerlings before sale to ensure uniform size. Only 30% of hatcheries report checking for parasites, and only 43% follow basic hygiene practices such as sterilizing equipment after spawning.
The average size of fingerlings sold is just 3.5 g each. There are no regulations for the fingerling trade, neither for tracing origin, nor specifying quality. Fish seed sales are made through networks of clients with trusted relationships. Fingerings supplied by surveyed nurseries consistently sell for 20-60% <those sold by local hatcheries. For example, pangasius fingerlings available from nurseries, which are presumed to be imported, sell for USD 0.02 per piece, whereas those supplied by local hatcheries cost USD 0.024.
Fish Feed Supply
No fish feed mill was operating in Cambodia at the time of the survey, but one local feed mill began to produce floating pellets in 2020. All floating pelleted fish feed sold in Cambodia at the time of the survey was imported, from Vietnam and Thailand. Six brands of imported floating pelleted fish produced by large feed companies are marketed through a network of retailers. Retailers sell feed mainly during peak farming season from July to December, with average sales of 20 tons each during this period, but with wide variations (1.5 to 91 t). About half (45%) of feed retailers have a contract with a feed manufacturer that specifies a minimum annual sales target.
Feeds for pangasius and walking catfish are the best-selling items and priced from USD 0.61-0.87/kg. This is somewhat higher than in neighboring Vietnam and Thailand, but does not appear excessive, considering the transport and transaction costs associated with importing feeds. In fact, given the relatively small size of the aqua-feed market in Cambodia, the presence of six international brands suggests a convenient market for international suppliers operating on a large-scale in neighboring countries, resulting in a reasonable level of competition. Nevertheless, key informant respondents stated that to produce fish at a price that could compete with imported fish, they would need to buy feed costing around USD 0.50/kg, which is similar to pangasius feed prices in Vietnam. However, this estimation does not take into account the other relative advantages that Vietnamese farms have, including economies of scale.
Post-harvest Value Chain Segments
In this section, we analyze the processing and trading segments of the aquaculture value chain, focusing on product types, quality standards, and prices. Our sample included 69 fish traders (a mix of "collectors"-mobile traders who aggregate fish, and wholesalers who operate from fixed premises in markets), and 34 fish processors (producing dried, smoked and fermented fish products), all using raw material comprised of a minimum of 20% Cambodian farmed fish.
These businesses are small and medium enterprises, trading an average of 169 tons of fish per trader, and purchasing an average of 19 tons of fish per processor annually. They operate with limited assets. For example, only three wholesalers (or 10% of our sample) owned a refrigerator, and no "modern" industrial or semi-industrial processing of farmed fish was observed.
The main farmed fish traded are snakeheads, walking catfish, and pangasius. Traders reported procuring 68% of the farmed fish they traded from Cambodian farms and the rest from imports. Processors obtained 33% of the fish they processed from Cambodian farms, with the rest from the wild or imported.
Much of the farmed fish sold in Cambodia is marketed live, including both locally produced and imported farmed fish. The main farmed species (snakeheads and catfishes) can breathe atmospheric oxygen and can survive for long periods with little or no water making live marketing relatively simple. 60% of the farmed fish traded by surveyed traders was sold live, through outlets including landing sites, wholesale markets and wet markets. Live sales serve as a guarantee of freshness, and live fish attract a higher average sales value than dead fish. The preponderance of live sales means that reported rates of spoilage are low, at just 6%. Only 27% of Cambodian farmed fish was reported to be sold in fresh, dead form. This was mainly used for producing high value processed products such as dry salted giant snakehead, and smoked walking catfish, but these processors also purchase live fish.
The volume of fish imported by Cambodia in 2017 was estimated to be 120,000 tons, based on a review of import licenses (VCA4D, 2017). Another recent survey indicated that 46% of farmed fish traded in Cambodia was imported (CAST, 2020). Most imported fish are farmed pangasius from Vietnam. Fish are usually imported live, regardless of species. Import volumes fluctuate depending on the ability of Vietnamese producers to export to higher value markets (VCA4D, 2017). The price offered by traders to Cambodian fish farmers is benchmarked against the price of imported fish.
Our qualitative interviews suggest that these conditions are challenging for Cambodian fish farmers, as illustrated by a medium scale male farmer from Kampong Cham producing pangasius who stated "the price (of farmed fish) is not stable, it declines when the imported fish from Vietnam enters the market." Similarly, another medium scale pangasius farmer operating in Battambang mentioned: "The problem is that a lot of pangasius remain unsold since there are a lot of imported fish from Vietnam. A lot of imported fish from Vietnam entered (Cambodia) recently, so the local fish prices declined." Common species of small wild fish harvested around the Tonle Sap Lake, such as gourami or medium and small sized cyprinids, tend to be more affordable than farmed fish from any source, costing approximately half as much at between 0.66 and 0.88 USD/kg (Mille et al., 2016), and contributing disproportionately to fulfilling demand for fish by poorer households.
In sum, most farmed fish marketed in Cambodia currently occupies one of two specific market niches-live fish, or high value processed fish-both of which face stiff competition from Vietnamese live fish imports. To date, neither locally produced nor imported farmed fish can compete on price with small wild fish harvested from the Tonle Sap, which continue to be the mainstay of consumption by lower income consumers.
Quality Standards and Product Differentiation
Surveyed producers, traders, and processors did not report adhering to the hygiene procedures promoted by extension services and development projects. Moreover, supermarkets do not impose their own quality standards on suppliers. Qualitative interviews highlighted that although processors are aware of the existence of good practices, they are reluctant to implement them due to the costs involved, and lack of premiums for higher quality products.
The qualitative survey indicated that demand for locally produced fish is high, due to a positive perception among Cambodian consumers about Cambodian products. Cambodianfarmed fish are considered of higher quality than imported fish because of the perception that fewer antibiotics and hormones are used in their production. Consumer ranking of preferences for fish places Cambodian wild fish first, followed by Cambodian farmed fish, with imported farmed fish least preferred.
Several respondents indicated that there is an emerging market for higher quality products (certified safe product, chemical free) in urban areas, as some consumers are becoming more willing to pay higher prices. This was illustrated by a male wholesaler operating in Pursat who stated, "even though we sell our local fish at a higher price, people still buy our fish. Even though imported fish is sold at cheap prices, buyers would not buy it if they knew it was imported." Supermarkets, restaurants, and caterers stressed the need for a quality standard traceability system, as wealthier consumers, particularly in urban areas, are increasingly aware of food safety issues. These respondents felt that the inability to distinguish between local and imported products was causing missed business opportunities for producers to access markets and to gain higher selling prices. A restaurant manager in Siem Reap made mention of quality control regulations: "Sometimes the laws (guidelines and regulations) have already been established, but we do not understand what's in them, so it's more like everything is okay." However, developing a quality standard traceability system faces several constraints. First, it requires a regulatory framework and the institutional capacity to enforce such regulation. In addition, there is limited interest from wet market retailers, who account for most fish sales, in implementing a system of quality standards as the establishment of a will result in premium prices, making fish more difficult to sell. The current perception among most actors is that live fish are an adequate indicator of quality. As described by a female wholesaler in Pursat: "Live fish means that fish is good quality. No one questions about fish quality; as long as fish are alive, fat and available in the market, those fish are good."
Employment Generation and Livelihoods
Current development discourse frames aquaculture as a potentially significant source of jobs and income for rural Cambodians. To gain a sense of the scale of aquaculture's current contributions to rural employment and livelihoods in Cambodia, we estimated levels of family and wage employment generated by businesses included in our survey. Several results stand out.
First, surveyed enterprises currently generate limited employment, totaling 5,052 full time employment equivalents (FTEs), assuming 240 person days of employment per year (Table 4).
Second, businesses in the aquaculture value chain currently generate few opportunities for hired labor, reflecting the microor small-scale of most of the enterprises involved. 85% of FTEs within our sample (4,296 FTEs) are family employment. Only 15% (756 FTEs) are paid work.
Third, family labor FTEs are evenly split between women (50%) and men (50%). Women and men participate in family labor in roughly equal proportions in all segments of the value chain except nurseries, where women are somewhat underrepresented (accounting for 38% for FTEs). Most family labor is deployed on farms, which are the most numerous category of enterprise in the value chain, accounting for 90% of family labor FTEs. Fourth, most casual paid employment is concentrated in the midstream segments of the value chain (trading and processing), which trade and/or process fish from both capture fisheries and aquaculture. As we are unable to differentiate between work related to products from each source, these figures overestimate aquaculture-linked paid employment among surveyed businesses. The limited number of fulltime workers employed by hatcheries, nurseries, and farms reflects the small size of these enterprises, and is indicative of a lack of professionalization and low levels of skilled labor in the sector.
Fifth, men are the main beneficiaries of paid employment in Cambodia's aquaculture value chain. Most paid work is generated on farms, where men account for 94% of hired FTEs. Women dominate fish processing, accounting for 59% of hired FTEs, but their representation as hired workers in trading, hatcheries and nurseries is more limited (≤25% of hired FTEs).
DISCUSSION
In this section we discuss the implications of results presented above with respect to (1) technical and structural challenges for aquaculture development, (2) capture fisheries-aquaculture interactions, and (3) aquaculture's potential to support livelihoods and employment.
Technical and Structural Challenges to Aquaculture Development
Results presented above indicate that systemic transformation would be required to achieve the ambitious policy targets set for Cambodia's aquaculture sector. We identify constraints at the farm (micro) and sector (meso/macro) scale, across technical, institutional, economic, and biophysical dimensions. At the scale of the production unit (farms, hatcheries, processors), our study reconfirms the existence of technical and economic constraints highlighted in previous studies. These include limited access to water on farms, the high price of feed and poor quality of seed, and the basic nature of most farming practices (Bengtson et al., 2016;Maredia et al., 2017;VCA4D, 2017;Richardson and Suvedi, 2018;Joffre et al., 2019). However, unlike previous studies, we also identify a three fundamental sector-wide structural challenges to the growth of Cambodia's aquaculture sector.
First, there is limited regulation and lack of enforcement of the import of fish and fingerlings from neighboring countries. Prices of imported fish reflect their lower production costs, and it is suspected that fish that are unfit for export due to residues, or surplus to demand in major markets find an outlet in Cambodia (VCA4D, 2017).
This issue is currently being addressed by the government with a temporary ban on imported aquaculture fish to support local industry (MAFF, Press release on January 08 2021). However, the legality of this ban remains unknown, as Cambodia is part of the ASEAN community and trade barriers are subject to WTO treaties. Import controls would better be approached from a food safety and biosecurity perspective, to avoid unregulated imports of material of unknown quality. Similar issues arise from the import of large numbers of fingerlings of unknown origin, health status, and quality (Joffre et al., 2019).
Second, there is no regulatory framework to control the quality of domestic farmed fish products. The absence of standards, quality certification, or traceability systems means that producers and processors have little incentive to upgrade their practices. A lack of such indications also makes it difficult to differentiate domestically produced fish from imported fish of unknown quality, limiting the ability of Cambodian producers to expand their domestic market share. Establishing formal standards would also be a prerequisite prior to targeting export markets in the medium-term.
However, the likelihood implementing greater standardization remains questionable in the current context, where standards will increase operating costs, making it more difficult for producers to compete with imported fish on price. Implementation of missing or unenforced regulations would be more effective if based on local capacity and coordinated with existing regulations in the agriculture and livestock sectors. Without adequately enforced regulations in place, there will be few incentives for hatcheries and producers to make the investments in their operations needed to bring about quality improvements.
Third, the study highlights the extent of spatial and temporal constraints on aquaculture development in Cambodia. We find that most pond-based farms, and many hatcheries and nurseries, face annual drought and flood cycles. Although Cambodia is often depicted as a country with abundant water resources, many producers are affected by drought, often limiting production to the rainy season. To be more competitive, the aquaculture production cycles could complement the seasonality of capture fisheries. Currently the highest catch volume occurs at the same time as the end of the aquaculture production cycle from January to April. To avoid such competition and target the harvest during the period of low catch fish catch (June to September), producers, hatcheries, and nurseries will need access to water to start their production cycle earlier in the dry season, when water is often scarce. Groundwater is rarely used as a water source for pond farms and hatcheries, meaning that exploring methods to ensure affordable groundwater access by fish producers could represent a key policy option.
Although hydropower dams currently limit the flood pulse in the Tonle Sap Lake, Cambodia and the Tonle Sap region can still be heavily flood affected (Sabo et al., 2017). In addition to the survey reports of flooding of pond farms, hatcheries, and nurseries, in October 2020, a severe flood event occurred, impacting thousands of households, including numerous fish farms, resulting in thousands of dollars in losses to aquaculture farmers (CAST, 2020). This fact highlights the persistent vulnerability of the sector to flood, and the need for new financial products such as aquaculture insurance as a risk mitigation tool for investors.
Interactions Between Capture Fisheries and Aquaculture
Capture fisheries provide most of the fish supply for rural Cambodians. Although pressures on inland fisheries productivity are increasing, including recurrent weak flood pulses in 2016, 2019, 2020 (Basist and Williams, 2020;MRC, 2020), our study suggests that Cambodia's aquaculture value chain is unlikely to fill the fish supply gap given current standards of conduct and performance. Aquaculture yields are relatively low, producers are dependent on imported inputs, and production systems are based on carnivorous species that are still highly dependent on capture fisheries for their feeding regime.
Thus, the common discourse of a national aquaculture sector replacing capture fisheries to sustain the supply of fish is unlikely to be realized soon. It is more likely that a continued decline in the productivity of the inland capture fishery would reinforce dependence on imported aquaculture products, especially during months when local aquaculture production is limited.
A major shift in the origin of fish in the human diet could have negative consequences for human health. Shifting consumption from diverse fish species sourced from capture fisheries, including many micronutrient-rich small fish species, to few larger less nutrition farmed fish species could negatively affect nutrient intake and dietary quality (Roos et al., 2007b;Lachat et al., 2018;Bernhardt and Connor, 2021). For example, in Bangladesh research has shown that although the growth of aquaculture has mitigated the supply-demand gap caused by the decline in inland captures fisheries, it has not fully compensated for reduced loss of dietary diversity and micronutrient intakes (Belton et al., 2014;Bogard et al., 2017).
The current affordability of farmed fish, relative to capture fish, is also questionable. The average farm gate pangasius price is above USD 1.20/kg, and 41% of the volume produced is sold above USD 2.00/kg, while other farmed fish produced in Cambodia are considerably more expensive than this on average. In contrast, in rural areas, prices of fish common fish species from capture fisheries are often well-below that threshold (Mille et al., 2016), especially during the main harvesting season of most aquaculture production systems. Moreover, recent studies show that a significant share of fishing households near the floodplain do not sell any of their fish catch, relying on this resource for household food security (Freed et al., 2020).
Hence, the affordability of aquaculture fish to low-income consumers could be a barrier to replacing the affordable fish sourced from capture fisheries. In addition, it has been estimated that only 2% of animal source food consumed in Cambodia originates from aquaculture, as compared to 49% sourced from inland capture fisheries (Vilain and Baran, 2016). Although aquaculture's share has likely grown since this estimate was produced, especially given importance of farmed fish, these figures underline the magnitude of the current gap between the two sectors in terms of their contributions to livelihoods and food and nutrition security.
Our data also show that Cambodia's aquaculture sector is still heavily dependent on "trash fish" with an estimated 13,400 tons of trash fish used for feed during the survey year. Considering predicted declines in inland fisheries productivity and increasing competition for fish between aquaculture and human consumption, the reliance of the sector on local trash fish is a distinct limiting factor for future growth. Fisheries decline would increase trash fish prices, and push producers to convert their farms to production of non-carnivorous fish species, or change their feeding regime by using manufactured pellet, combined with domestication of snakehead and giant snakehead. Large farms are already changing their practices and using more pelleted feed compared to small farms. We expect that producers will soon have to shift their feeding regimes, or increase the pace of this transition, similar to the changes observed in the striped snakehead sector in the Mekong Delta where producers shifted toward pelleted feed with the increasing price of trash fish (Sinh et al., 2014).
Competition between human consumption and aquaculture for low-priced fish is likely to intensify, if fish catches decline further. Already in 2020, record low Dai fisheries catches resulted in a 72% increase in the price of fish harvested compared to 2019 (FAO, 2020;MAFF, 2020). Meanwhile, even if public and private sector investments enable a shift to the use of pelleted feed, the transition to use of manufactured pellets is likely to take several years. Therefore, reliance on trash fish to feed aquaculture fish will remain in the near-term. Meanwhile, existing carp, silver barb and tilapia production systems could intensify by using fertilizer and rice bran and feeding regimes based on pond ecology. However, demand for these fish is low at present.
Based on these findings, we conclude that domestic aquaculture could be complementary to capture fisheries, rather than a replacement. Like in Bangladesh, where capture fisheries was also a pillar of the rural population's food security, a growing aquaculture sector might meet part of the growing demand and mitigate the impact of declining capture fisheries (Belton et al., 2014). However, unless the current production costs of aquaculture production systems (reflected by the selling price at the farm gate) can be reduced significantly, farmed fish is likely to remain less affordable than lower value capture species have been in the past.
Until or unless the sector develops further, and input prices are reduced, aquaculture in Cambodia will continue to respond to demand within specific niches, with the growing middle class in urban areas the preferred target. With an urban population representing 23% of the population and a steady annual growth rate above 3% per annum (World Bank, 2020), urbanization is likely to become one of the main drivers for aquaculture sector.
Potential Contributions to Employment and Livelihoods
Our results show that Cambodia's aquaculture sector is comprised of family-farms and businesses, with limited employment generation outside the family. Within our sample, 90% of labor is family based and fewer than 5,000 full-time equivalent jobs were generated.
Based on the 2019 census of the rural population (NIS, 2019) and the estimated number of households engaged in aquaculture, we estimate that <1.5% 4 of rural households are engaged in aquaculture (including small scale low input systems), compared to 10% of the population engaged full time fishing and 35% employed part time fishing (Ahmed et al., 1998).
These findings align with previous studies highlighting the magnitude of difference in employment between capture fisheries and aquaculture. For example, Mousset et al. (2016) showed that for households in rural Cambodia, aquaculture accounts for a labor input of 0.09 FTE per household, compared to 1.12 FTE per household for capture fisheries.
It is estimated that between 2 million and 4 million people are engaged full-time in fisheries, with an official figure of 2.3 million currently used (Fisheries Administration, 2017). Thus, although decline of fisheries might push a fraction of fishers to enter aquaculture, such a transition would be conditional upon prerequisites such as investment capacity, access to land, and skills, and would not compensate fully for lost employment in capture fisheries.
The apparently rather limited employment generation potential of aquaculture in Cambodia at present also reflects a lack of opportunities for skilled labor for farms and hatcheries. Educational programs for aquaculture are currently limited in Cambodia: there is no vocational training curriculums and academic programs in aquaculture are of low quality (Schkeeper, 2019). Expanded vocational training to support the development of a skilled workforce for farms and hatcheries could thus help to improve the employment generation capacity of the sector, as well as its performance.
CONCLUSIONS
This study is the first comprehensive assessment of Cambodia's aquaculture sector. The study has some limitations. Even though our sampling strategy was exhaustive, recently emerging large commercially oriented farms may be underrepresented. Data collection based on recall did not permit precise analysis of production system performance, especially regarding feed use, where the main raw material for feed is trash fish. Further specific on-farm monitoring surveys will be required to analyze the performance of farms under different feeding regimes. Regarding the analysis of the value chain conduct, the linkages and flows of trash fish between capture fisheries and aquaculture should be investigated further and volumes estimated more accurately. Finally, we did not assess financial performance of the different production system and compare the competitiveness gap with similar production systems operating in neighboring countries.
This study highlights that Cambodia's aquaculture production systems remain constrained by technical, institutional, and economic factors that limit their performance and competitiveness. To achieve the transformation of the aquaculture sector, several macro-scale structural interventions are required, among which the development of a regulatory framework for regulating imports of fish, fingerlings and inputs to Cambodia is key. This also encompasses developing value chain standards to differentiate imported and local farmed fish and respond to increasing demand for locally raised fish.
Commercially oriented fish farming will grow only with perennial access to water and if flood risk is mitigated. Considering the effect of hydropower development on flood patterns (Basist and Williams, 2020), the overall hydrology of the country and climate change predictions in the region (ICEM, 2013) that forecast higher temperatures and longer dry seasons, affordable access to groundwater may be required for aquaculture to grow further. In addition, aquaculture cannot be established, nor expand everywhere in the country and investments should be prioritized to target aquaculture clusters in suitable areas, where support systems, producers and concentrations of other value chain actors exist and can be supported.
In terms of aquaculture inputs and farming practices, our analysis shows that feeding regimes are still heavily dependent on trash fish from capture fisheries, which are increasingly scarce. The transition toward the use of formulated feeds will be facilitated by access to good quality pelleted feeds at a competitive prices, and to knowledge to support efficient feeding practices. Our recommendations regarding feed include creating an enabling environment (e.g., a regulatory framework, accurate information on market demand, and market information systems) for feed producers to invest in local production lines. A widespread shift of feeding regime will also require the transformation of support services, with more accessible sources of information to facilitate behavior change among producers toward more sustainable practices using less trash fish. In addition, the ecological intensification of pond systems producing non-carnivorous species through semiintensive feeding regimes based on pond ecology, green water, and rice bran could also support increased production.
Skilled labor is required facilitate a transition to more efficient value chains and production systems. Trained professionals with practical skills will be needed to provide support services to producers and support sectoral development. Donor-funded projects initiated since 2020 are training farmers and private service providers and supporting access to knowledge. However, additional support and more fundamental changes in access to education, knowledge and support services for the sector are required to support its transformation.
It will prove increasingly difficult for inland capture fisheries to meet future demand for fish in Cambodia, given the growing range of anthropogenic pressures they face, and likely increases in demand for fish from an urbanizing population. This dynamic suggests an important role for aquaculture in Cambodia's food system in years to come, but as a complement to capture fisheries, with respect to food and nutrition security, livelihoods, or employment, not as a substitute for them. Cambodia's aquaculture sector currently faces a unique set of structural and technical challenges. Addressing the areas for intervention listed above, while working to minimize any further pressures on inland capture fisheries productivity are both necessary steps to ensuring that this complementary role can be realized.
GENERAL STATEMENT
Prior to the survey all respondents were provided with an oral informed consent statement in Khmer (or English for key informants who were fluent in English and interviewed in English) and asked for their consent before the interview (both qualitative and quantitative survey). In Cambodia there is no established procedure for ethical review process for socioeconomic research. However, the local authorities reviewed the survey questionnaires. No sensitive topics or vulnerable human subjects were involved.
DATA AVAILABILITY STATEMENT
The datasets presented in this article are not readily available because the dataset is under the license of the Commercialization of Aquaculture for Sustainable Trade (CAST) Project. Access to the dataset requires a specific data agreement with the American Soybean Association. Requests to access the datasets should be directed to James Bernhardt, JBernhardt@soy.org.
ETHICS STATEMENT
Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.
AUTHOR CONTRIBUTIONS
OJ, SF, and JB: designed the study. SF performed the quantitative analysis. OJ, SF, and BB: interpreted the results and drafted the article. ST conducted the GIS analysis. All authors contributed to revising and approving the manuscript. | 2021-10-18T13:29:51.968Z | 2021-10-18T00:00:00.000 | {
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10898423 | pes2o/s2orc | v3-fos-license | Changes in the expression of the Alzheimer ' s disease-associated presenilin gene in drosophila heart leads to cardiac dysfunction
Mutations in the presenilin genes cause the majority of early-onset familial Alzheimer’s disease. Recently, presenilin mutations have been identified in patients with dilated cardiomyopathy (DCM), a common cause of heart failure and the most prevalent diagnosis in cardiac transplantation patients. However, the molecular mechanisms, by which presenilin mutations lead to either AD or DCM, are not yet understood. We have employed transgenic Drosophila models and optical coherence tomography imaging technology to analyze cardiac function in live adult Drosophila. Silencing of Drosophila ortholog of presenilins (dPsn) led to significantly reduced heart rate and remarkably age-dependent increase in end-diastolic vertical dimensions. In contrast, overexpression of dPsn increased heart rate. Either overexpression or silencing of dPsn resulted in irregular heartbeat rhythms accompanied by cardiomyofibril defects and mitochondrial impairment. The calcium channel receptor activities in cardiac cells were quantitatively determined via real-time RT-PCR. Silencing of dPsn elevated dIP3R expression, and reduced dSERCA expression; overexprerssion of dPsn led to reduced dRyR expression. Moreover, overexpression of dPsn in wing disc resulted in loss of wing phenotype and reduced expression of wingless. Our data provide novel evidence that changes in presenilin level leads to cardiac dysfunction, owing to aberrant calcium channel receptor activities and disrupted Wnt signaling transduction, indicating a pathogenic role for presenilin mutations in DCM pathogenesis. © 2001 Bentham Science Publishers Ltd. *Address correspondence to these authors at the Genetics and Aging Research Unit, Department of Neurology, Massachusetts General Hospital, 114, 16th Street, Charlestown, MA 02129, USA; Tel: 617 726 6845; Fax: 617 724 1949; tanzi@helix.mgh.harvard.edu; and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Tel: 617 253 8528; Fax: 617 253 9611; jgfuji@mit.edu. NIH Public Access Author Manuscript Curr Alzheimer Res. Author manuscript; available in PMC 2011 October 2. Published in final edited form as: Curr Alzheimer Res. 2011 May 1; 8(3): 313–322. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript
INTRODUCTION
Alzheimer disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia in the elderly, affecting one in 10 individuals over 65 and nearly half over 85 years old (http://www.alz.org/).The presenilin 1 and 2 genes (PSEN1 and PSEN2) encode highly conserved polytopic membrane proteins (PS1 and PS2) that are required for -secretase activity [1].Mutations in presenilins underlie the majority of cases of early-onset familial AD.Most mutations in presenilins increase the relative ratio between the longer (A 42) and shorter (A 40) amyloid peptides (A 42/A 40) [2].A 42 is the main component of amyloid plaques in the brains of AD patients, and the progressive formation of amyloid plaques is regarded as a key neuropathological feature of AD [3].Recently, mutations in presenilins have also been reported in dilated cardiomyopathy (DCM) patients [4,5], indicative of allelic heterogeneity.
*Address correspondence to these authors at the Genetics and Aging Research Unit, Department of Neurology, Massachusetts General Hospital, 114, 16th Street, Charlestown, MA 02129, USA; Tel: 617 726 6845; Fax: 617 724 1949; E-mail: tanzi@helix.mgh.harvard.edu;and Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Tel: 617 253 8528; Fax: 617 253 9611; E-mail: jgfuji@mit.eduDCM is defined clinically by dilation and impaired contraction of one or both ventricles.DCM occurs in all ages, most common between 20-60 years.DCM causes roughly onethird of cases of congestive heart failure and is the most prevalent diagnosis in individuals referred for cardiac transplantation.Half of DCM patients die within 15 years after diagnosis [6].Presenilin mutations were identified in DCM patients in the PSEN1 5' promoter and a large cytoplasmic loop between transmembrane domains six and seven, and PSEN2 NH 2 terminus [4,5].Presenilin mutations have also commonly been found in 20% of severe DCM patients who required heart transplantation due to heart failure [5].The PSEN1 5' promoter region variants in DCM patients led to significant reduction in transcriptional activities and decreased expression of PS1 protein in the myocardium [5].The PSEN2-R62H missense mutation in DCM patients enhanced PS2 degradation, reduced PS2 stability in mouse embryonic fibroblasts, and altered compromised PS2 function in Notch signaling in C.elegans [7].Together, these data suggest that PSEN1 and PSEN2 mutations or variants contribute to DCM pathogenesis through loss of gene function, and implicate novel mechanisms of cardiomyopathy.
Both AD and DCM are associated with high morbidity and mortality, but currently have no cure.The molecular mechanisms by which presenilin mutations lead to DCM pathogenesis are not yet understood.AD brain contains A oligomers, which have been shown to impair neurotransmission [8].Likewise, amyloid oligomers have been found in the failing heart of DCM patients associated with cardiomyocyte apoptosis [5], in cardiomyocytes derived from human heart-failure patients, and in mice model of desminrelated cardiomyopathy [9].Protein misfolding is implicated in the etiology and pathogenesis of both AD and DCM [5].Apolipoprotein E (APOE) is a multifunctional protein that plays a key role in the metabolism of cholesterol and triglycerides; APOE gene 4 variant is not only the largest known genetic risk factor for late-onset AD [10], but also increases both low-density lipoprotein cholesterol level and coronary heart disease risk [11].Moreover, cerebral angiopathy contributes to cognitive decline and dementia in AD patients and mouse models [12].The expression of vascular MEOX2 is low in AD; silencing of MEOX2 in brain capillaries results in aberrant brain angiogenesis, reduced vascular density and faulty clearance of A [13].These indicate the connection between AD and cardiovascular diseases.
PSEN1 and PSEN2 are strongly expressed in the heart and essential for cardiac morphogenesis [14,15].To date, there is no reported information regarding the in vivo functional role of PS1 in adult heart.Mice null for Ps1 are either dead at birth (Ps1 -/-) or embryonically lethal (Ps1 -/-/Ps2 -/+ ), exhibiting multiple developmental defects including a cardiac anomaly [15].The basic mechanisms of heart development and control of cardiac function are highly conserved between human and Drosophila melanogaster, or the fruit fly, which has been successfully used to characterize genes associated with human diseases, including cardiomyopathy [16,17].Moreover, the morphological and rhythm changes can be readily analyzed in the relatively simple organization of fly heart with an emerging medical imaging technology optical coherence tomography (OCT) [16,18,19].OCT enables real-time, in vivo, micron-scale and three-dimensional imaging of biological tissues [20].OCT has been used for a wide range of clinical applications in human, including ophthalmology, endoscopy, cardiovascular imaging [20][21][22], and recently, studying cardiac functions in live Drosophila [23].
The human PSEN1 and PSEN2 are represented by a highly conserved single ortholog, dPsn, in Drosophila [24,25].To better understand the pathogenic role(s) of presenilin mutations in DCM, we have employed transgenic Drosophila models to analyze the effects of overexpression or RNAi silencing of dPsn on cardiac function, assessed by employing the noninvasive OCT technology.In addition, we also examined heart ultrastructure, calcium channel receptor level, wing development and expression of the wingless (wg) protein, the central component of Wnt signaling transduction pathway that regulates cardiac development.
Optical Coherence Tomography (OCT) for Drosophila Cardiac Function Assessment
The OCT system used is modified based on a swept source OCT engine (Thorlabs Inc).
To study the cardiac function, the flies were anesthetized with Fly Nap for 2 minutes and mounted on glass slides before imaging.Two-dimensional cross-sectional images consisting 64 A-lines covering 250 m over the heart chamber were continuously acquired for 5 seconds at ~120 fps.The imaging plane was consistently chosen to be within segments A6-A7 of the Drosophila heart chamber.This OCT system achieved a finer imaging resolution and significantly improved imaging speed compared to previously reported OCT apparatus for Drosophila heart imaging [23].Three to four repeated measurements were acquired for each fly.The same experimental groups were repeatedly measured and the results were included in the final data.Post-processing the image data was performed with custom written Matlab (Mathworks, Inc) codes to obtain the dimensions of the heart chamber for each frame.M-mode image over the center of the chamber was generated together with the dimension over time.Functional parameters, including heart rate (HR, beat per minute, BPM), rhythmicity of the heartbeat, and the endsystolic and end-diastolic vertical dimensions (ESD and EDD, m) and fraction shorting (FS, %), were extracted from the dimension plots.The prevalence of arrhythmia was quantitatively analyzed via indentifying irregular heartbeat rhythm in the dimension plots for the number of arrhythmia flies in each group (%).
Heart Ultrastructure Analysis by Transmission Electron Microscopy
10-15 fly hearts from 30-day-old flies of each genotype group were fixed, embedded, sectioned and examined as modified from previously in a JEOL JEM 1011 transmission electron microscope for ultrastructure analyses [32].In brief, tissues were fixed in glutaraldehyde, stained en bloc, dehydrated through a graded series of ethanol and then embedded in Epon.Thin sections were cut, collected on formvar-coated grids, stained with uranyl acetate and lead citrate and examined in a JEOL JEM 1011 transmission electron microscope at 80 KV.Images were collected using an Advanced Microscopy Techniques digital imaging system.
Immuno-Staining and Western Blot Analyses
The third instar wing discs were fixed, blocked and probed with primary and secondary anti-bodies accordingly.Wings from adult flies were dissected in isopropanol and mounted in Canada Balsam mounting medium.Western Blot analyses were performed and repeated at least twice using protein isolated from 7-day old fly heart and muscle and the intensity of signals was analyzed using image program Quantity One version 4.6.2.In brief, the levels of analyzed proteins were normalized using the levels of dActin for loading differences controls, and quantification in total protein amounts and presented as the percentage of those in the transgenic flies in comparison with that in controls.The following anti-bodies were used: Mouse anti-wg antibody (Developmental Studies Hybridoma Bank, 1:1000); the secondary anti-mouse Alexa 546 (Invitrogen, 1:200); Rabbit-anti-dPsn (Genescript, against CTF, 1:1000); mouse anti-actin (DSHB; 1:1000).
CDNA synthesis and Real-Time RT-PCR
The cDNAs used for real time RT-PCR were synthesized from total RNA isolated from heart and muscle of 7-day old adult Drosophila.The cDNA was synthesized using SU-PERSCRIPT Preamplification System for First-Strand cDNA Synthesis (Invitrogen).Real-time RT-PCR quantification with dIP3R, dRyR, dSERCA or dActin-specific sense and antisense primers was done on an iCycler (BIORAD) using SYBR Green PCR Core Reagents (Qiagen) according to the manufacturer's instructions.House-keeping gene dActin as internal control was co-amplified under the same PCR conditions.All standards and unknown samples were run in triplicates per reaction.The fluorescence intensity was calculated using iCycler software version 3.1.The expressions of dIP 3 R, dRyR and dSERCA were given as relative number of copies (%) of mRNA molecules, as calibrated by coamplification of dActin.dIP 3 R, dRyR and dSERCA levels were shown as Mean ± Standard Deviation (Std) of repeated experiments.p values were calculated by two-tailed Student's t test.p<0.05 is defined statistically significant.
Statistical Analyses of Cardiac Function
The cardiac functions, including HR, ESD and EDD and FS, were compared between 7-day old and 30-day old flies overexpressing or silencing dPsn vs. age-matched controls and between 7-day old and 30-day old flies of each genotype group.We used a SAS GLM program for statistical analyses.Results were shown as Mean ± Standard Error (SE).p<0.05 is defined statistically significant (actual p values are shown).
The 24B-GAL4 line allows targeted expression in mesoderm and all cardiac and muscular cells with a uniform and high level of expression in the most anterior heart cells [33].Flies in which dPsn was either overexpressed or silenced by 24B-GAL4 appeared normal at eclosion.We quantitatively measured cardiac function, including heart rate (HR, beat per minute, BPM), rhythmicity of the heartbeat, the end-systolic and end-diastolic vertical dimensions -ESD and EDD ( m), fraction shorting (FS, %), in adult flies with a customized OCT system.We analyzed 7-day and 30-day old adult male flies overexpressing dPsn (UAS-dPsn; 24B-GAL4) or RNAi silencing dPsn (UAS-dPsn RNAi ; 24B-GAL4) in comparison with that of age-matched male controls (24B-GAL4/+) Fig. (1 & 2) (Table 1).Compared with age-matched controls, at either 7day, 30-day old age group alone, or 7-day and 30-day old combined age group, overexpression of dPsn significantly increased HR; in contrast, silencing of dPsn significantly decreased HR.Remarkably, either overexpression or RNAi silencing of dPsn in heart resulted in very irregular heartbeats: heartbeat rhythm changed rapidly between fast and slow pace within 1-2 seconds, or vice versa.Quantitative analysis of arrhythmia prevalence demonstrated that silencing of dPsn significantly increased the number of flies with arrhythmia at 30-day old flies (57%) or combined age group (54%) compared to that in age-matched control flies.Likewise, overexpression of dPsn resulted in significant increase in the number of flies with arrhythmia at 30-day old flies (46%) compared to 7-day old flies (19%).Control flies aging from 7-day to 30-day old barely changed arrhythmia prevalence (26% to 27%).Overexpression of dPsn decreased ESD at 30-day old flies or combined age group.Flies in which dPsn was silenced showed significantly reduced ESD (30day old flies alone and combined age group) and EDD (7day old flies and combined age group).Compared with 7day old flies, 30-day old flies in which dPsn was silenced exhibited a significant decrease in HR and a remarkable increase in EDD and FS.30-day old of flies in which dPsn was overexpressed or control flies also showed a trend for agedependent decrease in HR and significant increase in EDD compared with that in 7-day old flies (Table 1).To assess the effects of RNAi silencing or overexpression of dPsn in transgenic flies, we carried out Western blot analysis using protein lysates isolated from the heart and muscle of 7-day old flies Fig. (4).Silencing of dPsn led to a 60% decrease in dPsn expression, and overexpression of dPsn led to a 180% increase in dPsn expression as compared to controls Fig. (4).
Calcium Channel Receptor Expression
In cardiac muscle, Ca 2+ influx during depolarization of action potential initiates the sequence of events leading to contraction.Ca 2+ signaling is primarily modulated by the functional properties of Ca 2+ release channels.There are two main types of Ca 2+ release channels, the inositol 1,4,5trisphosphate receptor (IP 3 R) and the ryanodine receptor (RyR) [34].
The removal of cytosolic Ca 2+ allows for relaxation of muscle fibres.The Ca 2+ uptake into sarcoplasmic reticulum (SR) store occurs predominately by the cardiac isoform of the SR Ca 2+ -ATPase pump SERCA2a [34].We have identified that the human IP 3 R, RyR and SERCA2a are represented by highly conserved single orthologs in Drosophila, as dIP 3 R (Itp-r83A), dRyR (Rya-r44F) and dSERCA (Ca-P60A) (www.flybase.org).We quantitatively determined the expression of dIP 3 R, dRyR and dSERCA levels via real-time RT-PCR amplification of cDNA samples from 7-day old flies in which dPsn was overexpressed (UAS-dPsn; 24B-GAL4), or silenced (UAS-dPsn RNAi ; 24B-GAL4) and control (24B-GAL4/+).dIP 3 R, dRyR and dSERCA levels were normalized by co-amplified internal control gene dActin.dIP 3 R expression levels were 1.17 fold higher in flies overexpressing dPsn, and 2.19-fold higher in flies dPsn was silenced as compared to controls
Wing Development and Wingless Expression
The UAS promoter used in the dPsn transgenic models allows for conditional overexpression or silencing of dPsn with specific GAL4 drivers in the wing or other tissues, in addition to heart.Drosophila adult wing and thoracic body wall arise from the larval wing disc.Wing development is regulated by multiple signaling pathways, including Wnt, Notch, epidermal growth factor receptor, hedgehog and decapentaplegic.The wing pattern provides an important tool in isolating and characterizing genes affecting multiple signaling pathways, e.g.Notch and wg [35].To assess the potential role of dPsn in wing development, we ectopically overexpressed or RNAi silenced dPsn in wing disc using two wing-specific GAL4 drivers, Sd-GAL4 and Ptc-GAL4.
Sd-GAL4 drives the overexpression of dPsn in wing disc in the pattern of scalloped (sd) gene, which regulates the expression of a number of targeted genes including wg, the central component of Wnt signaling transduction pathway [28].Flies in which dPsn was silenced by RNAi in the wing by the Sd-GAL4 driver were embryonically lethal.Compared with control flies (Sd-GAL4/+) Fig. (6A), overexpression of dPsn (UAS-dPsn; Sd-GAL4) in wing disc resulted in the complete loss of both wings, which were consistently observed in over 200 flies in repeated experiments Fig. (6D).We next examined the expression of wg protein by immunostaining 30 wing discs dissected from the third instar larvae with anti-wg antibody.wg was expressed as a broad strip in
Fig. (4).
Western Blotting by anti-dPsn detected dPsn -C-terminal fragment (CTF) (25 KDa) in the heart and muscle of controls and transgenic flies in which dPsn was silenced or overexpressed.The dPsn expression level was normalized with respect to dActin (42KDa) loading control.Silencing of dPsn led to a 60% decrease in dPsn expression, and overexpression of dPsn led to a 180% increase in dPsn expression as compared to controls.
DISCUSSION
We have found that either overexpression or RNAi silencing of dPsn in Drosophila leads to significant changes in HR and to an irregular heartbeat rhythm accompanied with cardiomyofibril defects.While either overexpression or silencing of dPsn decreased end-systolic and end-diastolic heart chamber size and increased FS compared to agematched controls, flies in which dPsn was silenced exhibited an age-dependent remarkably increase in EDD and FS.Taken together, these data indicate that overexpression or RNAi silencing of dPsn in heart primarily leads to irregular heartbeat rhythm via regulating the electrical activity that stimulates the myocardium of the heart for contraction.
Initiation of cardiac muscle contraction requires an increase in intracellular calcium from resting concentration.The increase in intracellular calcium then triggers further release of calcium from SR stores via the Ca 2+ release channel receptor RyR.The release of calcium can be further enhanced by activation of the Ca 2+ release channel receptor IP 3 R.This "calcium-induced calcium release" process ensures rapid and significant increases in intracellular calcium, which are essential to contraction [34].SERCA2a transfers Ca 2+ from the cytosol of the cell to the lumen of the SR during cardiac muscle relaxation [34].In cultured fibroblasts from DCM patients with PSEN1-D333G or PSEN2-S130L mutations, histamine-induced resting intracellular Ca 2+ concentrations are elevated [4].In mouse heart muscle, Ps1 and Ps2 physically interact and colocalize with both IP 3 R and the cardiac form of RyR (RyR2) to SR [36,37].In Ps1-and Ps2deficient mouse embryonic fibroblasts, free Ca 2+ concentrations in SR are decreased and IP 3 R levels are increased [38].In neuronal cells, overexpression of AD-associated PSEN1-M146V or PSEN2-N141I mutants directly increases IP 3 R activity [37].Primary hippocampal neurons from Ps1 mutant knock-in mice exhibit greatly increased levels of RyR and enhanced Ca 2+ release [36].Sorcin reduces the open probability of RyR2 [39].PS2, sorcin, and RyR2 interact with each other in mouse heart [36].Ps2-deficient mice develop normally with no evidence of cardiac hypertrophy and fibrosis, but exhibit increased cardiac contractility and potentiated peak Ca 2+ [36].Elevated Ca 2+ attenuates the association of RyR2 with Ps2, and enhances the association of sorcin with Ps2 [36].Moreover, PS1 and SERCA2a physically interact and are co-immunoprecipitated in the myocardium of DCM patients [5].SERCA activity is diminished in fibroblasts null for both Ps1 and Ps2.Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca 2+ by increasing SERCA activity [40].This suggests that the presenilins play an important role in cardiac excitationcontraction coupling and Ca 2+ signaling via the interaction between PS2, RyR2 and sorcin, and regulation of the SERCA pump.Consistent with previous findings, we found that silencing of dPsn in flies elevated dIP 3 R expression, and that overexprerssion of dPsn led to reduced dRyR expression.Silencing of dPsn remarkably reduced dSERCA expression, which together with degenerated myofibrils may lead to insufficient cardiac muscle relaxation to reduce the endsystolic and end-diastolic cardiac chamber size.These data thus provide evidence that presenilins may regulate SR Ca 2+ storage or intracellular Ca 2+ homeostasis in cardiac cells.
We found that 30-day old flies exhibited significantly decreased HR, increased heart chamber size and arrhythmia prevalence compared to 7-day old flies.This is consistent with previous findings indicating a decline in cardiac function with increasing age in human or Drosophila [41].Compared with flies overexpressing dPsn or control flies, flies in which dPsn was silenced exhibited a more remarkably agedependent decrease in HR and increase in EDD and arrhythmia.This indicates that silencing of dPsn more severely exacerbates age-dependent cardiac dysfunction than does overexpression of dPsn or control flies.Evidence has shown that the insulin receptor (IR) and associated pathways have a dramatic and heart-autonomous influence on age-related cardiac performance in flies, suggestive of potentially similar mechanisms in regulating cardiac aging in vertebrates [41].IR belongs to tyrosine kinase receptor family whose activation is essential for insulin signaling in target tissues.IR undergoes PS/ -secretase-dependent processing to produce IR intracellular domain (ICD).PS/ -secretase inactivation resulted in reduced levels of tyrosine autophosphorylation of the IR -subunit upon insulin stimulation [42].Moreover, insulin-like growth factor I receptor (IGF-IR), which shares strong structural homology with IR, was reported to be a substrate for PS/ -secretase activity [43].These data suggest that the PS/ -secretase proteolysis of IR may act as a modulator of the insulin signaling pathway.
The heart forms very early during mammalian and avian embryogenesis and arises from paired mesodermal regions in the embryo.The regulation of Wnt signal transduction has been implicated as an important event that initiates cardiac development in Drosophila [44].Wnts are a family of secreted signaling proteins including wg.Presenilin has been implicated in regulating Wnt signaling.First, presenilin-mediated -secretase activity is required for the intramembranous cleavage of Notch receptor to release notch intracellular domain (NICD) for the entry to nucleus where it induces the expression of Notch targeted genes [45].Loss-offunction dPsn mutants exhibit early embryo lethality and phenotypes indicative of a general impairment of Notch signaling throughout the development in Drosophila [24].This is particularly interesting in view of the fact that there is inhibitory crosstalk between the Notch and the Wnt signaling pathways (when NICD binds to Wnt) [46].Second, PS1 negatively regulates Wnt signaling by interacting withcatenin to facilitate -catenin turnover in vitro [47][48][49] .cateninsignaling is required for normal cardiac growth and development [50].Moreover, the PSEN1-E318G and PSEN1-D333G mutations found in DCM patients [4,5], are positioned in a large cytoplasmic loop between transmembrane domains six and seven, with which PS1 bindscatenin [51].
We showed that overexpression of dPsn by Sd-GAL4 in wing disc results in severe malformation in wing development accompanied by reduced expression of wg.These data further support the contention that presenilin negatively regulates Wnt signaling.The Notch signaling pathway plays important roles in the patterning and differentiation of the wing veins [35].Overexpression of dPsn by Ptc-GAL4 led to severe loss of intervein cells and formation of extra vein; in contrast, silencing of dPsn resulted in normal intervein cells but loss of acv vein.Given the known role of presenilins in -secretase complex [1], overexpression of dPsn may elevate -secretase activity resulting in enhanced NICD release, thereby potentiating Notch signaling to inhibit intervein cell development but promote vein cell.Conversely, silencing of dPsn may cause wing vein loss due to reduced -secretase activity.Mutations in presenilins alter the proteolytic processing of amyloid precursor protein by aberrant -secretase activities that results in increased neurotoxic A 42 production [8].However, neither DCM-associated PSEN2-R62H nor PSEN2-S130L mutation engender significant effects on A 42 levels or the A 42/40 ratio in vitro [52].In addition, treating WT mice with -secretase inhibitor DAPT did not alter cardiac function, including HR, left ventricle systolic and diastolic pressure [36].These data suggest that the presenilins may regulate cardiac function through other mechanisms in addition to their effect on -secretase activity.Based on the previous and current findings, we propose that mutations in PSEN1 and PSEN2 may alter Wnt signaling viacatenin interaction and NICD inhibitory crosstalk to ultimately cause defects in cardiac function leading to DCM pathogenesis.
In summary, we have demonstrated that either the loss or gain of dPsn levels in heart, results in cardiac dysfunction, owing to aberrant calcium channel receptor activities and disrupted Wnt signaling transduction.Together, these data provide novel in vivo evidence for pathological role(s) of the presenilins in DCM pathogenesis.
Fig. ( 1
Fig. (1).Representative M-mode optical coherence tomography (OCT) imaging of cardiac function in 30-day old adult Drosophila.A: Control showed normal heart rate (HR): 250 beat per minute (BPM) and rhythm; B. Overexpression of dPsn led to increased HR (296BPM) and irregular heartbeats; C: Silencing of dPsn caused reduced HR (167BPM), small heart chamber and irregular heartbeat.
rate.EDD: End-diastolic dimension ESD: End-systolic dimension FS: Fractional shortening *p<0.05,** p<0.01, *** p<0.001, **** p<0.0001: vs. Age-matched controls; # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001: vs. 7-day old age group shows significant increase; shows significant decrease the notum, a thinner strip in the prospective wing margindorsal/ventral (D/V) boundary, and a strip encircling the prospective wing blade in WT Fig. (6B).Consistent with the phenotype observed in adult flies, there was moderately decreased wg expression in flies overexpressing dPsn, especially in the prospective wing blade region Fig. (6E), along with morphological alterations in wing discs.Compared with control flies Fig. (6C), wing discs of flies overexpressing dPsn were thinner, and reduced in size, especially in the dorsal/ventral compartment suggesting disc degeneration Fig. (6F).Together, these data showed that overexpression of dPsn in wing disc by Sd-GAL4 results in severe wing malformation and reduces wg expression.Patched (Ptc) is a segment polarity gene that regulates the development of wing compartments centering at the anterior/posterior border (defined between L3 and L4 vein)[29].Compared with control flies Fig. (6G), overexpression of dPsn driven by Ptc-GAL4 resulted in severe loss of intervein cells narrowing the intervein sector between L3 and L4 vein and forming extra vein cells to fill the intervein region.The intervein sector between L4 and L5 was enlarged; and pcv vein was extended Fig. (6H); in contrast, silencing of dPsn driven by Ptc-GAL4 led to loss of acv vein with normal intervein cells between L3 and L4 vein Fig. (6I).Collectively, these data show that overexpression or silencing of dPsn in wing disc by Ptc-GAL4 alters wing vein formation.
Fig. ( 3 ).
Fig. (3).Transmission electron microscopy of the myofibril.A: Controls showed normal myofibril structure; B: Overexpression of dPsn led to loss of regular structure with vacuoles, a broadening of Z-line and swollen mitochondria in myofibril; C: Silencing of dPsn caused an overall decrease in the density of myofibrils with vacuoles, a breaking Z-line, and severely degenerative mitochondria with impaired structure in myofibril.Magnification: Left: 10,000X, Scale bar: 2 m.Right: 30,000X, Scale bar: 500nm.
Fig ( 6 )
Fig (6).Overexpression or silencing of dPsn in the wing.A. B. C. Control flies had normal wings (A), and wg expression (B) on normal wing disc (C).D. E. F. Overexpression of dPsn in the wing disc by Sd-GAL4 led to complete loss of both wings (D) and moderately decreased wg expression (E) on a thinner and smaller wing disc (F).G.Control fly wings have five longitudinal (L1-5) and two cross-veins: anterior (acv) and posterior (pcv), creating distinct intervein sectors.H. Overexpression of dPsn in the wing by Ptc-GAL4 led to loss of intervein cells between L3 and L4 vein.The intervein sector between L4 and L5 was enlarged and pcv vein was extended.I. silencing of dPsn in the wing by Ptc-GAL4 led to loss of acv vein in the intervein sector between L3 and L4 vein. | 2016-04-23T08:45:58.166Z | 2011-05-01T00:00:00.000 | {
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265384424 | pes2o/s2orc | v3-fos-license | Evaluation of molecular receptors status in breast cancer using an mpMRI-based feature fusion radiomics model: mimicking radiologists’ diagnosis
Objective To investigate the performance of a novel feature fusion radiomics (RFF) model that incorporates features from multiparametric MRIs (mpMRI) in distinguishing different statuses of molecular receptors in breast cancer (BC) preoperatively. Methods 460 patients with 466 pathology-confirmed BCs who underwent breast mpMRI at 1.5T in our center were retrospectively included hormone receptor (HR) positive (HR+) (n=336) and HR negative (HR-) (n=130). The HR- patients were further categorized into human epidermal growth factor receptor 2 (HER-2) enriched BC (HEBC) (n=76) and triple negative BC (TNBC) (n=54). All lesions were divided into a training/validation cohort (n=337) and a test cohort (n=129). Volumes of interest (VOIs) delineation, followed by radiomics feature extraction, was performed on T2WI, DWI600 (b=600 s/mm2), DWI800 (b=800 s/mm2), ADC map, and DCE1-6 (six continuous DCE-MRI) images of each lesion. Simulating a radiologist’s work pattern, 150 classification base models were constructed and analyzed to determine the top four optimum sequences for classifying HR+ vs. HR-, TNBC vs. HEBC, TNBC vs. non-TNBC in a random selected training cohort (n=337). Building upon these findings, the optimal single sequence models (Rss) and combined sequences models (RFF) were developed. The AUC, sensitivity, accuracy and specificity of each model for subtype differentiation were evaluated. The paired samples Wilcoxon signed rank test was used for performance comparison. Results During the three classification tasks, the optimal single sequence for classifying HR+ vs. HR- was DWI600, while the ADC map, derived from DWI800 performed the best in distinguishing TNBC vs. HEBC, as well as identifying TNBC vs. non-TNBC, with corresponding training AUC values of 0.787, 0.788, and 0.809, respectively. Furthermore, the integration of the top four sequences in RFF models yielded improved performance, achieving AUC values of 0.809, 0.805 and 0.847, respectively. Consistent results was observed in both the training/validation and testing cohorts, with AUC values of 0.778, 0.787, 0.818 and 0.726, 0.773, 0.773, respectively (all p < 0.05 except HR+ vs. HR-). Conclusion The RFF model, integrating mpMRI radiomics features, demonstrated promising ability to mimic radiologists’ diagnosis for preoperative identification of molecular receptors of BC.
Introduction
Breast cancer (BC) exhibits significant heterogeneity at both intra-and inter-tumor levels.Different molecular receptor statuses are associated with varying prognoses, treatment responses and survival outcomes (1,2).Profiling of gene expression has identified the four main intrinsic molecular subtypes of BC, including luminal A, luminal B, human epidermal growth factor receptor 2-enriched (HER-2), and triple negative (TN), each of which exhibits distinct molecular receptor statuses and therefore requires tailored therapeutic approach, such as endocrine therapy or neoadjuvant systemic therapy (NST) (3)(4)(5).
Currently, molecular receptor status is mainly determined by gene expression profiling or immunohistochemical (IHC) surrogates from invasive tissue biopsy or surgical specimens in clinical practice.However, due to tumor heterogeneity, a single tissue biopsy is insufficient to capture the global genetic, epigenetic, and/or phenotypic characteristics of a breast tumor, leading to inevitable selection bias (1,2).In addition, as the tumor biology evolves and continuous treatments are administrated, the receptor status and molecular subtypes of BC may change, posing challenges in accurately reflecting the true state of the lesions (5).Therefore, there is a need to develop an effective method for precise assessment of the whole-tumor's histological characteristics, and for spatialtemporal monitoring of the dynamic tumor biological behavior during treatment.MRI-based radiomics, which uses data-mining algorithms or statistical analysis tools on high-throughput imaging features to obtain predictive or prognostic information, has shown promising potentials as an alternative tool for the assessment of BC's molecular receptors status (6)(7)(8).Multiparametric magnetic resonance imaging (mpMRI), which combines morphological (T2 weighted-imaging [T2WI]), functional (diffusion-weighted imaging [DWI]) and kinetic (dynamic contrast-enhanced [DCE]) information, has further demonstrated great promise for preoperative identification of different molecular receptor statuses of BC (8)(9)(10).However, previous investigations mainly selected only one or two single MRI sequence-derived images (e.g., T2WI, DWI-derived apparent diffusion coefficient [ADC] maps, or the early phase of DCE-MRI) for analysis (7,(11)(12)(13)(14), which deviates from the real clinical scenario where radiologists routinely go through all acquired MRI images to make a final diagnosis.Without a comprehensive consideration of the various contributions from different MRI sequences, it may result in subjectivity and an insufficient assessment.
Herein, we hypothesize that a mpMRI-based radiomics method has the potential to provide accurate prediction of molecular subtypes and receptor status of BC.The aim of this study is to develop a novel feature fusion radiomics (R FF ) model that incorporates radiomics features extracted from optimally performed mpMRIs to mimic the routine diagnostic practices of radiologists and preoperatively identify different molecular receptor statuses in BC.
Patient cohort
This study was approved by the Ethics Committee of the Second Affiliated Hospital of South China University of Technology (Guangzhou First People's Hospital) Hospital, with informed consent being waived due to the retrospective nature of this study.A total of 535 patients who underwent breast mpMRI for preoperative assessment at our hospital between January 2017 and April 2022 were included.The inclusion criteria were as follows: (1) histopathological confirmation of BC by surgical resection or needle biopsy; (2) patients who underwent a routine mpMRI including T1WI, T2WI, DWI (with b values of 0 s/mm 2 , 600 s/mm 2 and 800 s/mm 2 ), DWI-derived ADC map and DCE-MRI (with 6 continuous enhancing phases) within one week prior to pathological examinations; (3) no additional therapy prior to MRI.The exclusion criteria were: (1) recurrent BC (n=11); (2) incomplete pathological results, such as those lacking IHC results and Ki-67 scores, or unclear histological types (n=15); (3) cases with Volumes of interest (VOI) that were difficult to delineate due to images artifacts (n=39).( 4) patients with breast implants (n=4).In cases of multicentric or multifocal tumors, only the largest malignant lesion was selected.For bilateral disease, the largest lesions of both breasts were selected according to pathological results.Finally, 460 patients with 466 lesions were enrolled in this study.The lesions were categorized into HR+ (n=336) and HR-groups (n=130), with the HR-group further divided into HEBC (n=76) and TNBC (n=54) subgroups.Based on sample size calculations (15, 16), a required sample size of 210 (42 cases of TNBC and 168 cases of non-TNBC) was sufficient to detect differences between various molecular subtypes of BC with a power of 95%.Appendix 1 showed the detailed information on the sample size calculation process.All lesions were divided randomly into a training/validation cohort (n=337) and a test cohort (n=129) at a ratio of ~3:1, in which a random selected training cohort (n=337) was established to determine the optimal single MR sequence for subsequent experiments, as shown in Figure 1.
The volume of interest delineation
The volume of interest (VOI) was defined on all images that were stored in DICOM format.In order to standardize the extracted image biomarkers from mpMRI, we followed the major procedure outlined by the Image Biomarker Standardization Initiative (IBSI) (17).Before VOI delineation, we used the General registration (elastix) method, available as the "SlicerElastix" plugin in the open-source image analysis platform 3D Slicer (https:// www.slicer.org), to register all sequences' images.This alignment enabled us to better handle morphological variations and structural differences in breast tissue, particularly when aligning the other sequence images with the DCE 2 image.Additionally, we resampled all MRI sequences to a standard resolution of 1.096 x 1.096 x 1.2, ensuring isotropic voxels and reducing variations caused by differences in scanning equipment, protocols, and patient positioning.Furthermore, we normalized the intensity levels of all images to a range of 0-255 to reduce the influence of contrast and brightness variations, which might otherwise affect the quantification of radiomics features (18).
Slice-wise delineation of the VOI was carried out using the ITK-SNAP software (http://www.itksnap.org)on T2W, DWI 600 , DWI 800 , ADC maps, and DCE 1-6 images.The process started Flow chart of the study's population with inclusion and exclusion criteria.BC, breast cancer; HEBC, human epidermal growth factor receptor 2 enriched BC.TNBC, triple-negative breast cancer."n=466" represented the total lesion number.
Radiomics feature extraction and analysis
The radiomics features were extracted from ten VOIs of each lesion using the open-source software toolkit Pyradiomics (19).A total of 109 features were extracted from three categories of features: 1) intensity features (n=19); 2) morphology features (n=15); texture features (n=75).Only the extracted radiomics features with ICC > 0.75 were then fed into 150 classification base models, which were built using 10 classifiers and 15 feature selection methods.Detailed definitions of the above-mentioned features can be found in Pyradiomics documentation and IBSI (17).The full list of radiomics features and the methods employed in this study are summarized in Tables S2, S3, respectively.
Feature fusion radiomics modeling and evaluation
Based on the newly developed mpMRI-based RadioFusionOmics model by our lab, we constructed a feature fusion radiomics (R FF ) model that integrated radiomics information from different MRI sequences to produce more discriminative fused features.A random selected training cohort (n = 337) was used to analyze all radiomics features from each MRI sequence, analogous to a radiologist's initial reviewing of a patient's complete set of MR images.According to the highest cross-validation AUC obtained in the training/validation process, the optimal single sequences that can identify hormone receptor positive (HR+) vs. HR-BC, TNBC vs. HEBC, as well as TNBC vs. non-TNBC were determined and regarded as the single sequence-based radiomics (Rss) model.
Subsequently, the radiomics features from the top four highperforming single sequences were combined to perform multiple sequence feature fusion, similar to a radiologist's final reviewing focusing on specific sequences after a preliminary review.The best combination of sequences (combination of two, three or four sequences, a total of 11 types of combinations) was then identified to develop the R FF models.Utilizing feature-level fusion, the R FF model conducted a feature-wise fusion strategy by finding a transformation to map the feature matrix with a set of MRI sequences (e.g., dimension = 10) to a lower dimensional space (e.g., dimension = 1).By integrating the class structure information (i.e., information on the molecular receptor status of memberships of the training samples) in the calculation of the transformation, the R FF was able to eliminate the between-class correlations and strengthen the within-class correlations during the feature fusion, which can effectively enhance the discriminative power of fused features.Various base models (n=11*150 = 1650) were trained using the fused features and their performances were evaluated and ranked via a stratified ten-fold cross-validation.The optimal base models for Rss and R FF were verified in the training/validation cohort and test cohort.Technical details related to the R FF are shown in Appendix 2. The flow chart of this study was displayed in Figure 2.
Histopathology
All surgical or biopsy specimens were examined by two pathologists (YZ and WD, with 6 and 16 years of experience in the pathological diagnosis of BC, respectively).The following pathological biological markers of BCs were assessed and recorded: tumor maximal diameter, affected side in the breast, number of tumors, histology type, and IHC status of estrogen receptor (ER), progesterone receptor (PR), HER-2, and Ki-67 index.Tumors with ER or PR positive expression (> 10% of tumor nuclei staining) were classified as HR positive (HR+) (20).Positive HER-2 expression was defined as a 3+ IHC score or 2+ accompanied by fluorescence in situ hybridization positive (FISH+) result ( 21).The Ki-67 scores were classified into two groups: < 14% as low Ki-67 level and ≥14% as high Ki-67 level.The molecular subtypes of BCs were classified as follows: luminal A (ER and/or PR positive, HER-2 negative, and Ki-67 < 14%), luminal B (ER and/or PR positive, HER-2 negative, and Ki-67 ≥ 14% or ER and/or PR positive, HER-2 positive, regardless Ki-67 expression), HER-2 enriched (ER and PR negative, HER-2 positive), which was recorded as HEBC, and triple negative cancer (ER, PR and HER-2 negative), named as TNBC.The luminal A and luminal B comprised the HR+ group.The Ki-67 expression was scored as the percentage of positive invasive tumor cells with any nuclear staining, with the mean percentage of positive cells recorded (4).Four cases of different molecular subtypes of breast cancer were presented in the supplementary materials Figures S3-S6.
Statistical analysis
The Chi-square Test and Fisher's Exact Test were used for categorical variables, the One-way ANOVA analysis was used for normally distributed continuous variables, and the Kruskal-Wallis H test was used for non-normally distributed continuous variables to compare demographic and pathological characteristics between different molecular subtypes.The normality of data distribution was evaluated by the Shapiro-Wilk test.The results for normally distributed continuous variables were reported as mean ± SD, while non-normally distributed continuous variables were reported as median (interquartile range, IQR).Categorical variables were presented as numbers and proportions.The performance of each Rss and R FF base models were evaluated via the area under the receiver operative characteristic curve (AUC), sensitivity (SEN), specificity (SPE) and accuracy (ACC) among different subtypes of BC.The performance of the Rss and R FF was compared using the paired samples Wilcoxon signed rank test.Two-sided p < 0.05 was considered statistically significant.All statistical analyses were conducted using the SPSS 25.0 software (IBM SPSS Corporation, USA) and python 3.6.2(Python Software Foundation (USA, https://www.python.org/downloads/).
Demographics data and tumor characteristics
The clinical pathological characteristics of the 460 patients with 466 lesions (6 patients had bilateral lesions) enrolled in the study are presented in Table 1.Among the 466 lesions, 336 lesions (72.1%) were classified as HR+ BCs, with 142 lesions being luminal A and 194 lesions being luminal B. Additionally, 76 lesions (16.3%) were classified as HEBCs, and 54 lesions (11.6%) were classified as TNBCs.The median tumor size of TNBCs (26.0 mm) and HEBC (27.0 mm) was found to be significantly larger than that of HR+ (21.0 mm) (p = 0.000).TNBCs showed a higher prevalence of mass enhancement in DCE MRI (81.5%) and invasive carcinoma (96.2%) compared to HR+ and HEBCs (p < 0.001).TNBCs also had a higher Ki-67 index (> 14%) in comparison with HR+ and HEBCs.Moreover, the age of patients and number of tumors among HR+, HEBC and TNBC groups were significantly different (p < 0.05).Baseline characteristics were not significantly different between both training/validation and test cohorts (Table S4).
Selection of the dominant sequence and development of the Rss model
All the discriminative base models established based on single mpMRI sequence were compared to determine the optimal sequences among HR+ vs. HR-, TNBC vs. HEBC and TNBC vs. non-TNBC.Supplementary Figure S1 demonstrated the discrimination comparison results on ten sequences of the three classification tasks.By analyzing the dominant radiomics features of each sequence, the optimal sequence for discriminating HR+ vs. HR-was DWI 600, the optimal Rss model, namely Rss (DWI 600 ), achieved the highest AUC of 0.787 in the random training cohort (Figure 3), and similar performance in the training/validation cohort (AUC=0.767)and test cohort (AUC=0.768),respectively (Table 2).
The optimal sequence for identifying TNBC and HEBC was DWI-derived ADC map, the best Rss model, recorded as Rss (ADC), yield the highest AUC of 0.788 in the random training cohort (Figure 3), and the best AUC of 0.769 and 0.718 in the training/validation cohort and test cohort, respectively (Table 2).
Regarding TNBC vs. non-TNBC discrimination, the ADC map was also the best sequence, the optimal Rss model (Rss [ADC]) demonstrated the highest AUC of 0.809 in the random training cohort (Figure 3), and the best AUC of 0.784 and 0.735 in the training/validation cohort and test cohort, respectively (Table 2).
R FF model development and evaluation
We selected the top four superior sequences for molecular receptor status classification to build the R FF model.As shown in Figure 3 and Figure S1, the top four superior sequences for HR+ vs. HR-were DWI 600 , DWI 800 , DWI-derived ADC map and DCE 5, with all AUCs > 0.77 in the random training cohort (Figure S1A).Similarly, DWI-derived ADC map, DCE 2 , DCE 3 and DCE 4 were the top four dominant sequences for TNBC vs. HEBC, yielding all AUCs > 0.72 (Figure S1B).While the four most predominant sequences for TNBC vs. non-TNBC were DWI-derived ADC map, DWI 600 , T2WI and DCE 2 , achieving all AUCs greater than 0.73 (Figure S1C).
Subsequently, the performances of each combination of the top two, three or four high-performance mpMRI sequences in random training cohort (a total of 11 types of combinations during each
Top-ranked radiomics features
The top-ranked features associated with the three classification tasks were also sieved by the proposed R FF model and their discriminative capabilities were analyzed.Based on the feature selection procedure of each base model, we counted and ranked the occurrence of each selected feature (only for base models with AUC > 0.6).The fifteen most frequently selected features of the three classification tasks were displayed in Tables S5-S7.Most dominant features were texture features in HR+ vs. HR-(8/15) and TNBC vs. HEBC (8/15), while intensity-based features were the superior discriminative features of TNBC vs. non-TNBC (11/15).The top 5 most frequently selected radiomics features associated with the discrimination of HR+ and HR-included three morphology-based features and two gray level co-occurrence matrix (GLCM) features, while intensity-based features accounted for 80% (4/5) and 100% (5/5), respectively among the top 5 radiomics features of TNBC vs. HEBC and TNBC vs. non-TNBC (Table 3).All the features showed statistically significant differences between HR+ and HR-, TNBC and HEBC, TNBC and non-TNBC with p-values < 0.001.The mean feature values of each group were used as the threshold to identify different molecular receptor statuses.In the task of discriminating TNBC from non-TNBC, the top 5 features outperformed other two tasks, with ~75% of the non-TNBC having larger feature values, while ~65% of the TNBC group had smaller values in all top 5 features (Table 3).
Discussion
Our study aimed to simulate the diagnostic process of radiologists by comprehensively analyzing radiomics features TABLE 3 The top 5 most frequently selected radiomics features of the three classification tasks based on the optimum R FF models.
Classification tasks
Top The 'Mean' shows the mean of the mean radiomics feature values of the two groups in each classification.The letter of '(<Mean | >Mean)' represents the percentage of patients in the two groups with feature value smaller than or larger than the 'Mean' value.Values in bold indicate these features with better discriminative performance.
Initially, the most discriminative MRI sequences (denoted as Rss models) were screened out from the radiomics features, and then the "R FF models" were built by incorporating the top four sequences with high performance on molecular subtype classification.This approach resembles the typical diagnostic process of a radiologist, who first performs a preliminary assessment of all available imaging sequences and then focuses on a subset of sequences with particularly informative features for the final diagnosis.The results showed that the R FF models "DWI 600 +DWI 800 +DCE 5 ", "ADC+DCE 2 +DCE 4 " and "ADC+DWI 6 0 0 +T2WI+DCE 2 " outperformed each Rss model in the classification tasks of HR+ vs. HR-, TNBC vs. HEBC, and TNBC vs. non-TNBC, with all AUC values exceeding 0.7.These findings highlight the effectiveness of fusing multi-sequence MRI radiomics features by the R FF approach to achieve high performance in differentiating different receptor statuses of BCs.Breast cancers exhibit high heterogeneity, leading to distinct therapeutic approaches, such as endocrine therapy for HR+ BCs, targeted therapy with anti-HER-2 monoclonal antibodies for HEBCs, and NST mainly for TNBCs (3).Radiomics, deriving multiple quantitative features from multimodal medical images, may capture spatiotemporal heterogeneity reflected by different molecular receptor statuses before treatment.This improves the discriminative and predictive abilities of medical image in oncology (6,22).Previous studies have applied radiomics preoperatively to assess molecular receptor statuses of BCs and reported preliminary success (7,8,11,12).For instance, Leithner et al. found that radiomic signatures extracted from DCE-MRI via a K-Nearest Neighbors (KNN) classifier were capable of classifying luminal A vs. luminal B, luminal B vs. triple negative, luminal B or HER-2 enriched vs. all other cancers (all ACC >77%) (11).However, most previous studies employed only one or two MRI sequence(s) such as DCE-MRI or DWI-derived ADC maps, without exploring all routine mpMRI sequences, leading to uncertainty regarding which sequences are more important.Our study compared the performances of all ten routine mpMRI sequences, revealing that radiomics signatures from DWI 600 , DWI 800 , DWI-derived ADC map, and DCE 5 sequences exhibited superior discriminative power for HR+ vs. HR-, especially the DWI 600 and DWI 800 sequences.Interestingly, radiomics features from DWI-derived ADC maps contributed more than other sequences for TNBC vs. HEBC and TNBC vs. non-TNBC.
The DWI provides a quantitative ADC parameter that closely reflects the microenvironment of tumor structures such as tumor cellularity, fluid viscosity, the amount of fibrous stroma, and cell membrane permeability, by detecting the Brownian motion of water molecules (23, 24).DWI and ADC maps have been widely used in tumor characterization, particularly in BC.While previous studies have conducted quantitative analyses based on ADC maps to identify different molecular receptor statuses or subtypes of BC, however, the reported results were inconsistent (25-29).For example, Suo et al. found that HER-2 positive subtype exhibited higher mean ADC values than other subtypes of BC with either standard (800 s/mm 2 ) or high (1500 s/mm 2 ) b-values (26).
However, other studies have reported that TNBC had a higher mean ADC value than other subtypes (28,29).These inconsistent findings may be due to the use of different b-values in DWI, different ROI selection strategies (e.g., 2D or 3D ROIs, ROI containing the whole tumor or the lower part of ADC values within the lesion), variations in magnetic field, etc. (27,30,31).Further studies and investigations are warranted, but these trends in ADC values according to clinically relevant subtypes may provide potential imaging biomarkers to aid treatment decisions in BC in the future.The results of our comprehensive analysis revealed that ADC map and DWI sequences played a dominant role in the three classification tasks, suggesting that radiologists should give greater attention to ADC maps and DWI sequences during the clinical interpretation process.
In addition, we found that the DCE 5 sequence, one of the delayed-contrast phases, was more important than other DCE phases in the differentiation between HR+ and HR-BCs.Generally, a time-signal intensity curve on DCE-MRI with a rapid enhancement (corresponding to DCE 2-3 in our study) followed by a washout pattern, is generally indicative of a malignant breast lesion.However, this pattern does not apply to TNBC, which is a common HR-subtype.A previous study showed persistent enhancement pattern on DCE-MRI was significantly associated with TNBC (32).Interestingly, another research showed that a significant proportion (33% [25 of 76]) of familial BCs exhibited a slow or intermediate initial enhancement followed by steady delayed enhancement pattern, which was the general DCE-MRI kinetic feature for benign BC lesions (33).This discrepancy in DCE-MRI enhancement patterns between HR+ and HR-subtypes may be explained by their unique pathohistological features (34, 35).ERnegative BCs are known to have several unique histological features, such as prominent lymphoid stroma, comedo-type necrosis, and central fibrosis (34).TNBC is also highly associated with the presence of a central scar, tumor necrosis, and stromal lymphocytic response (35).These features may result in retaining of contrast agent within the center of lesions and show persistent enhancement, which may be captured as dominant radiomics features from the delayed-contrast phase of DCE-MRI.Our results suggested the potential of the delayed-contrast phase of DCE-MRI in differentiating HR+ and HR-subtypes and in the selection of endocrine therapy candidates.
In this study, we explored the potential of fusing dominant features from mpMRI sequences to improve the accuracy of BC subtype classification.Our hypothesis was that multi-dimensional image information from multiple MRI sequences could be captured and integrated to provide a more comprehensive representation of the breast lesion.Different from previous studies (7,14,36), we investigated all sequences of a routine breast MRI examination and selected the top four high-performance sequences to develop a discriminative model via fusing dominant features of multi-sequences.Incorporating class structure information, the R FF can not only effectively integrate features from different MR sequences, but also ensures that the fused features are more representative and discriminative.The results of our study emphasized the importance of incorporating multiple MRI sequences in the radiomics analysis of breast cancer, as it can lead to improved accuracy in molecular subtype classification.
Our results showed that the top 5 radiomics features that effectively differentiated HR+ and HR-BC were three morphology-based features and two GLCM-based features.This aligns with prior studies, which have shown that molecular subtypes of BC exhibit distinct morphological and textural characteristics on MRI images (11,37).Tumors of the luminal type, for instance, tend to present with irregular shapes and irregular/spiculated contours on MRI due to their slow growth rate and the desmoplastic reaction of the surrounding tissue (10, 33).On the contrary, rapidly growing TNBCs and HEBCs tend to have well-defined, oval/round shapes with smooth outlines (10, 32).According to IBSI, GLCM represents the distribution of intensities of neighboring pixels along image directions and reflects the heterogeneity of image intensity (17).Previous studies have shown that BC subtypes also exhibited distinct ADC values, DWI manifestations and enhancing intensity patterns (11,27,(38)(39)(40).A recent study also reported that non-TNBCs had significantly higher mean/median/5 th percentile washin values compared to TNBCs, indicating that HR+ and HR-lesions have different intensity-derived radiomics features (41).Of note, first order features accounted for 80% (4/5) and 100% (5/5) for classifying TNBC vs. HEBC and TNBC vs. non-TNBC, respectively.The intensity statistical features described intensity distribution within the ROI and also reflected tumor's heterogeneity (11,42).
Limitations
Our study has certain inherent limitations that merit acknowledgment.First, the retrospective design and single-center setting of this study was subjected to selection bias.Conducting a multi-center study was not feasible due to the variations in MRI scan protocols across medical centers, necessitating the inclusion of DCE-MRI with 6 different phases and DWI with b values of 600 and 800 mm 2 /s.Second, a majority of tumors with non-mass enhancement in DCE-MRI were excluded due to challenges in defining the boundaries for VOI delineation, potentially introducing further selection bias.Third, the manual delineation of tumors in this study is time-consuming and prone to subjectivity, and future studies will incorporate semi-or automatic segmentation techniques to enhance objectivity.Fourth, not all radiomics features were analyzed, e.g., gray level dependence matrix (GLDM) being beyond the scope of IBSI was excluded.Fifth, we included a subset of breast cancers that were pathologically confirmed through needle biopsy, which may introduce inherent bias of needle biopsy.Finally, the biological interpretability of the "fused features" used in R FF model was insufficient as a result of implementing the feature fusion strategy, which we will focus on in our future studies.
Conclusion
In conclusion, the R FF model was successfully developed by integrating mpMRI image information to determine different molecular receptors of breast cancer preoperatively.This model, which mimics the diagnostic work pattern of radiologists, outperformed single MR sequence-based radiomics models to distinct molecular receptor status.
FIGURE 2 Flow
FIGURE 2Flow chart of the study.HR, hormone receptor; TNBC, triple-negative breast cancer; HEBC, human epidermal growth factor receptor 2 enriched BC.
classification task) were compared and displayed in Figure S2.Our results illustrated that the model R FF (DWI 600 +DWI 800 +DCE 5 ), R FF (ADC+DCE 2 +DCE 4 ) and R FF (ADC+DWI 600 +T2WI+DCE 2 ) were superior over the other sequences combinations in the random training cohort, yielding the maximal AUC of 0.809, 0.805 and 0.847, respectively.Similar performances were obtained in the training/validation cohort and test cohort, outperforming the Rss model with an AUC of 0.778 and 0.726, 0.787 and 0.773, 0.818 and 0.773, respectively (both p<0.05 except HR+ vs. HR-), as shown in T a b l e 2 .Among R F F ( D W I 6 0 0 + D W I 8 0 0 + D C E 5 ) , R F F (ADC+DCE 2 +DCE 4 ) and R FF (ADC+DWI 600 +T2WI+DCE 2 ), the base model (classifier + feature selection method) were respectively "Logistic Regression + Multi-Cluster Feature Selection" (MCFS), "Logistic Regression + Discriminative Feature Selection" (UDFS) and "Logistic Regression + trace_ratio".The MpMRI-based feature fusion method employed in the task of TNBC vs. non-TNBC achieved the optimal discriminative capability, yielding AUC, ACC, SEN and SPE of 0.818, 0.718, 0.705, 0.721 in the training/ validation cohort and 0.773, 0.767, 0.636, 0.780 in the test cohort, respectively.
TABLE 1
Demographics data and tumor characteristics.P value less than 0.05 was considered statistically significant, presented in bold.HR hormone receptor, HEBC human epidermal growth factor receptor 2 enriched breast cancer, TNBC triple negative breast cancer.
Unless indicated otherwise, data are numbers of cancers, with percentages in parentheses.*Data are median, with interquartile range (IQR) in parentheses.†Other invasive cancers are 1 neuroendocrine carcinoma in HR+ and 1 malignant phyllodes tumor carcinoma in TNBC. a One-way ANOVA analysis.b Kruskal-Wallis H test. c Chi-square test.d Fisher's Exact Test.A
TABLE 2
performance of the optimal Rss model and the optimal R FF model for different molecular receptor statuses discrimination.
P value: compared the performance between the optimal Rss model and the optimal R FF model in the training/validation cohort and test cohort of each discriminative task.Significant values (P < 0.05) are presented in bold.HR, hormone receptor; HEBC: human epidermal growth factor receptor 2 enriched BC; TNBC: triple-negative breast cancer.AUC, area under the receiver-operating characteristic curve; SEN, sensitivity; SPE, specificity; ACC, accuracy. | 2023-11-24T16:18:36.595Z | 2023-11-21T00:00:00.000 | {
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259035523 | pes2o/s2orc | v3-fos-license | Pilot ‐ scale Evaluation of an Anaerobic/Anoxic/Oxic Process for Nitrogen Removal from Sewage Using Metagenomic Sequencing
.
Introduction
With the rapid development of the chemical industry, pollution from chemical manufacturing processes will cause significant harm to humans and the environment [1]. During the production of hydrogen peroxide, the treatment of effluents that contain a large amount of inorganic and organic pollutants, such as ammonium salts, 2-ethylanthraquinone, 1,3,5-trimethylbenzene, and trioctyl phosphate, is challenging [2]. Traditional biological treatment does not meet the new local standards; chemical oxygen demand by dichromate (CODcr <60 mg/L, NH4-N <8.0 mg/L) for direct discharge [3]. Consequently, several biological processes, such as anaerobic-anoxic-aerobic (AAO), anaerobic-anoxic-aerobicaerobic (AAOO), and anaerobic-aerobic-anoxic-aerobic (AOAO), have emerged [4].
The AAO process, a system combining the traditional activated sludge, nitrification, denitrification, and biological phosphorus removal processes, has been widely used for chemical wastewater treatment plants (CWWTPs) in China owing to its effectiveness and low cost compared to other biological treatment strategies [5,6,7]. The removal of nitrogen in the AAO process is commonly related to low chemical oxygen demand/total nitrogen (COD/TN) ratio, biomass, temperature, influent load fluctuation, and sludge bulking [8]. First, a lower COD/TN ratio in CWWTPs results in unsatisfactory performance because of it lacks sufficient carbon for the denitrification process [9,10]. Second, the process conditions to satisfy the requirement for biodegradation has been reported to be related to the mass of sludge in the reactor, as well the sludge age [11]. In addition, a combination of high impact resistance with a membrane bioreactor process with high sludge age is more effective concerning reduction of sludge production than with a conventional activated sludge process at lower sludge ages [12].
On this basis, some improvements were made to the AAO process to improve the efficiency of wastewater treatment. Using an internal reflux system in the deoxidation zone, a step-feed AAO process for treating low-carbon and highnitrogen wastewater by distributing carbon sources from the anaerobic zone has been developed to improve the treatment response [13]. To reduce the carbon source requirement, the practicability of mixing free acid sludge treatment and dissolved oxygen (DO) management to achieve partial nitrification-denitrification in a continuous flow system (an aerobic-anoxic-oxic process) using real wastewater was assessed [14]. These studies only targeted process performance and improvement, whereas the microorganism community was majorly overlooked. However, modifications in control and operating conditions have also been reported, such as extended hydraulic retention time (HRT) and solid retention time (SRT) [15]. Nonetheless, the longer HRT would diminish the sewage sludge treatment efficiency per unit of time, and the longer SRT would reduce the nitrogen removal efficiency. Therefore, the AAO process requires a modification to allow for low temperatures and carbon/nitrogen (C/N) inflow.
Significant nitrogen loss may occur in the anoxic zone owing to the concentrations of organic carbon and favorably low DO level [16,17]. Biofilms effectively enrich low-growth biomass, thereby enhancing retention; this is attributed to the specific bacterial niche [18]. In addition, the enrichment of bacteria might improve the overall nitrogen removal performance of CWWTPs, especially for wastewater with an insufficient influent carbon source. [19]. Thus, it is more significant to explore the nitrogen removal effect of suspended carriers with mature biofilms in anoxic zone in pilot scale installations.
This study integrated two sequencing batch reactors (SBRs) with anoxic-carrier biofilms into the AAO process. They were observed for over 280 days with a low COD/TN ratio of actual chemical industry wastewater. We aimed to (1) investigate the performance of the AAO system and modified pilot-scale plant for treating low C/N chemical wastewater; (2) use metagenomic sequencing and quantitative reversetranscription PCR (qPCR) to analyze the abundance and composition of the microbial community structure in flocculent sludge in the anoxic and oxic zones and anoxiccarrier biofilms, and (3) to elucidate the basic mechanism of nitrogen removal. Compared with assembly-based species annotation, reads-based metagenomic species annotation methods are more comprehensive and accurate. We believe our study would provide novel insights into potential strategies for optimizing chemical sewage treatment plants.
Pilot-scale Experimental Reactor Operation
This study investigated the CWWTP located in Changzhou City, southeast China. The typical AAO process was used as the primary treatment for removing organic matter and nutrients. The pilot-scale experimental reactor contained two tandem SBRs (40L) simulating anoxic and oxic zones, named SBR-anoxic and SBR-oxic, respectively. Anoxic carriers were added into the anoxic zone to form mature biofilms and ensure that the suspended carriers and sewage water were fully in contact. The experiment lasted 280 days and was divided into two phases. In Phase I (days 1-52), the activated sludge was inoculated into the SBR according to the frequency of inoculated activated sludge entering the AAO process (activated sludge was collected from a water treatment plant in Changzhou). The real sewage contained NO3--N, NO2--N, NH4+-N and complex organic salt. The HRT was approximately 12 h, including 2 hours, 3 hours, and 7 hours under anaerobic, anoxic, and oxic conditions, respectively. The SRT was set to approximately 16 days. The temperature ranged from 10.7 °C to 25.2 °C. In Phase II (days 53-280), the suspended carriers from the anoxic zone were added into the SBR-anoxic reactor with a filling ratio of 40%. Influent and effluent samples of pilot-scale experimental reactor were collected daily.
Analytical Methods
All samples were filtered through 0.45-µm filters. The COD and concentrations of ammonium and total nitrogen were measured according to standard methods [20]. The MLSS and MLVSS from different zones were measured following standard methods [20].
High-throughput Sequencing and Microbial Community Analysis
Flocculent sludge and anoxic carriers were taken from the anoxic and oxic zones in August 2022 and freeze-dried (Free Zone 2.0; Labconco Co., Kansas City, MO, USA). The extraction was carried out using the cetyltrimethylammonium bromide method. The purity and integrity of the extracted DNA samples were analyzed using agarose gel electrophoresis for metagenomic sequencing. DNA purity was assessed by Nanodrop (OD 260/280 ratio). DNA concentration was accurately quantified with Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNA samples were fragmented by sonication and then end-polished, A-tailed, and ligated with a full-length adaptor for Illumina sequencing with further PCR amplification. After the library preparation was completed, an Agilent 2100 bioanalyzer system (Santa Clara, CA, USA) was used to detect the size of the inserts. Then, sequencing was performed using an Illumina PE150 platform (San Diego, CA, USA) [21]. KneadData software was used to conduct quality control (based on Trimmomatic) and dehosting (based on Bowtie2) of the original data [22]. Before and after using KneadData, Fast QC was used to test the rationality and effect of quality control. Kraken2 and the self-built microbial nucleic acid database, which contains the screened sequences belonging to bacteria, fungi, archaea, and viruses in the NCBI Nucleotide and RefSeq genome-wide database, were used to calculate the number of sequences of species in the sample [23]. Bracken was then used to estimate the actual abundance of species in the sample [24].
Quantitative Reverse-Transcription PCR
Anoxic carriers and flocculent sludge were collected in August 2022 from the anoxic zone and freeze-dried (Alpha 2-4 LSCbasic, Christ, Osterode am Harz, Germany). The biomass was collected thrice. Extracted triplicate DNA samples were pooled together to create one single DNA sample. The concentrations of the pure DNA samples were measured by a NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA). To determine the variations in the abundance of denitrification and anammox bacteria, the gene copy numbers of narG, nirG, narH, norB, hzsB, and hdh were measured by qPCR using an MA-6000 Real-Time PCR system (Agilent, Stratagene, Santa Clara, CA, USA) stained with fluorescent SYBR-Green. PCR amplification was performed in 10.8 μL reaction mixtures, consisting of 10 μL of 2× SYBR real-time PCR premixture (Vazyme Biotech, Nanjing, China) and 0.4 μL of both the forward and reverse primers (10 μmol/L).
Statistical Analyses
All data were expressed as mean ± SD analyzed by GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA, USA). A T test was used to evaluate the significance of all pairs. The following terminology denotes the statistical significance: * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.
Chemical Oxygen Demand Concentration and Nitrogen Removal Performance
The SBRs were fed with real chemical sewage and made consistent with AAO without changing the Phase I (Days 1-53) operational parameters; this included the HRT, SRT, and nitrifying liquid reflux ratios. Maintaining stability between the water output index of the two tandem SBR devices from day 33 resulted in an average ammonium removal efficiency of 96.1%, with an average effluent ammonium concentration of 2.6 mg/L (Fig. 1B). These results indicated optimal nitrification performance during this process. However, the average effluent TN was 37.1 mg/L, with an average TN removal efficiency (NRE) of only 51.4% owing to the low C/N ratio, resulting in incomplete denitrification (Fig. 1C). Meanwhile, the average effluent COD concentration was 50.5 mg/L with a removal efficiency on average was 81.2% (Fig. 1A). In Phase II (days 54-280), The carriers were inoculated in SBR-Anoxic without changing the operational parameters on day 54. During days 54-108, the anoxic biofilm activity gradually recovered; however, no noticeable effects were observed. During days 54-280, in the following stable operating period, the average effluent ammonium concentration was stable at 5.4 mg/L, and TN decreased from 37.2 to 13.4 mg/L. The NRE was stable at 82.6%, and the effluent COD concentration was 48.3 mg/L. Adding anoxic carriers could increase the NRE from 51.4% to 82.6%.
Analysis of Phyla and Class of Bacteria in Anoxic and Oxic Zones
The microbial community structure and diversity composition in the anoxic zone, anoxic-carrier biofilms, and oxic zone biofilms of the AAO system were investigated using the Illumina PE150 sequencing platform. A total of 6000 effective sequences were obtained by quality filtering steps and producing approximately 31000 sequences in all samples within the finish. These results confirmed the reliability and repeatability of sequencing.
Microbacterium was the most abundant in oxic zones, suggesting its critical role in hydrolyzing different types of organic matter. Notably, Pseudomonas (average 4.32%) and Acinetobacter (2.14%) can hydrolyze some organic matter, particular proteins, and high-saline wastewater. They were present in the activated sludge of Chemical wastewater treatment plants based on the AAO process [36,37].
Analysis of Bacterial Community in Anoxic-Carrier Biofilms and flocculent Sludge
The top eight most relative abundance of anoxic-carrier biofilms and flocculent sludge were compared (Fig. 3). Proteobacteria was the dominant bacterial phyla in the anoxic-carrier biofilms and flocculent sludge, accounting for 67.54% and 93.27%, respectively; this is consistent with the findings of a previous study showing that it was the most abundant phylum in most CWWTPs [38]. The microbial community of anoxic-carrier biofilms contained a significantly higher abundance of Actinobacteria (p<0.0001), Bacteroidetes (p<0.001), and Firmicutes (p<0.0001). Relevant studies have found that Actinobacteria play a vital role in the carbon and nitrogen cycle, especially in sewage treatment and aeration, where the abundance increases significantly [39]. Bacteroidetes and Firmicutes play a crucial role in nitrogen removal; the genera in these phyla have the functions of nitrification, denitrification, and even phosphorus removal [40]. The increased abundance of related phyla strengthens the ability of microorganisms to degrade nitrogen and organic matter in sewage. During the biological nitrogen conversion in the CWWTPs, typical microorganisms include sulfate-reducing bacteria (e.g., Desulfococcus) and microalgae aggregating bacteria (e.g., Microbacterium); some denitrifying bacteria (e.g., Thauera) (Fig. 4) were observed in this study [41]. Although these bacteria were found to be lower in abundance, they were found to be significantly higher in the anoxic-carrier biofilms than in the flocculent sludge. Furthermore, the metagenomic sequencing revealed the key microorganisms and related carbohydrateactive enzymes. Anammox bacteria (e.g., Planctomycetes (p<0.0001)) were observed in anoxic-carrier biofilms (Fig. 3); these favorably metabolize without DO or an organic carbon supply [42].
Potential Mechanism of Nitrogen Conversion Via Anoxic-carrier Biofilms
A LEfSe difference comparison analysis at the KEGG functional module level revealed 164 pathways that were significantly different in anoxic flocculent sludge and anoxic biofilms. Among these pathways, 97 were enriched in anoxic flocculent sludge and 67 in anoxic biofilms. Notably, genes related to nitrogen metabolism-related pathways (map00910) were significantly enriched in anoxic biofilms, suggesting enhanced nitrogen metabolism function (Fig. 5).
To further study the genes associated with nitrogen treatment in wastewater, we generated datasets related to nitrogen metabolism, including all genes in the nitrification, denitrification, and anammox pathways, and compared them with our metagenomic gene abundance results. "Deep" genetic mining revealed that the abundance of narG, nirS, narH, norB, hzsB, and hdh in anoxic-carrier biofilms was much higher than in flocculent sludge, the enrichment of denitrifying bacteria and anammox bacteria was proved successfully. qPCR results also confirmed that the key denitrification and anammox gene abundance in anoxiccarrier biofilms was significantly higher than that in flocculent sludge (narG: p<0.0001; nirS: p<0.01; narH: p<0.01; norB: p<0.0001; hzsB: p<0.01; hdh: p<0.01) (Fig. 6). This indicated the potential rates of denitrification and anammox significantly increased in anoxic-carrier biofilms than in flocculent sludge, which was probably due to the enrichment of typical denitrifying bacteria and anammox bacteria on the anoxic carrier biofilm. Moreover, the functions of several anammox bacteria to facilitate a nitrite loop might be essential for enhancing overall nitrogen removal performance in the bioreactor [43]. Thus, the results of this study corroborate previous findings that nitrate-to-nitrite conversion and anammox process could play a vital role in nitrogen metabolism [44].
Potential Mechanism of Nitrogen Conversion Via Anoxic-carrier Biofilms
For the treatment of chemical wastewater via moving bed biofilm reactor (MBBR), most previous studies have confirmed it is an efficient technology for removing organic matter and nitrogen from industrial and urban wastewater. However, most have focused on the oxic zone owing to its high efficiency for ammonium oxidation and organic matter removal [45,46]. Fewer studies have applied suspended carriers to the anoxic zone, compared with the oxic zone, to improve the nitrogen removal efficiency of CWWTPs [47,48], especially at low C/N ratio [49]. Consequently, the microbial community composition and functional transformation of anoxic-carrier biofilms remain poorly understood. Thus, we evaluated the removal of COD and nitrogen in modified SBRs during long-term operation and comprehensively analyzed the microbial community, metabolism-related pathways, and functional genes. The results suggest that abundant denitrifying and anammox bacteria were immobilized on the anoxic-carrier biofilms, which positively contributed to nitrogen removal.
Furthermore, the anaerobic zone is worth investigating due to the similar environmental conditions (e.g., pH, salinity, temperature, and DO). In the future, the effect of carrier biofilms in the anaerobic zone (e.g., microbial community composition, metabolic pathways, and the shift of microbial community composition) deserves further detailed research [50]. The modified system integrating AAO with SBRs exhibited considerable nitrogen removal. Although this case study shows the potential contribution of anoxic-carrier biofilms, further research on other possible microbial pathways, including nitrite ammonification, nitrous oxide denitrification, and their contribution to nitrogen removal [51] should be considered. The relationship among the physicochemical and biological processes, physicochemical characteristics, and environmental parameters in the biosystem and the effect on nitrogen removal must be further assessed using dynamic simulation [52].
Conclusion
The enhanced nitrogen removal in a CWWTP with anoxiccarrier biofilms, an AAO system, and SBRs was investigated for 280 days. Some characteristic bacteria were detected in the anoxic-carrier biofilms, and the significant inner associations were revealed. Our microbial community structure analysis indicates that some functional bacteria in the anoxic-carrier biofilms partially contribute to nitrogen removal. The abundance pattern of narG, nirS, narH, norB, hzsB, and hdh indicates a higher potential for denitrification and anammox pathways in anoxic-carrier biofilms than in the flocculent sludge. Overall, this study demonstrated a successful combination of SBRs with the AAO system. However, further efforts are required to clarify their complex interactions and potential metabolic pathways. | 2023-06-03T15:14:41.723Z | 2023-05-20T00:00:00.000 | {
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245041465 | pes2o/s2orc | v3-fos-license | Cognitive impairment in two subtypes of a single subcortical infarction
Abstract Background: Single subcortical infarction (SSI) is caused by two main etiological subtypes, which are branch atheromatous disease (BAD) and cerebral small vessel disease (CSVD)-related SSI. We applied the Beijing version of the Montreal Cognitive Assessment (MoCA-BJ), the Shape Trail Test (STT), and the Stroop Color and Word Test (SCWT) to investigate the differences in cognitive performance between these two subtypes of SSI. Methods: Patients with acute SSIs were prospectively enrolled. The differences of MoCA-BJ, STT, and SCWT between the BAD group and CSVD-related SSI group were analyzed. A generalized linear model was used to analyze the associations between SSI patients with different etiological mechanisms and cognitive function. We investigated the correlations between MoCA-BJ, STT, and SCWT using Spearman's correlation analysis and established cut-off scores for Shape Trail Test A (STT-A) and STT-B to identify cognitive impairment in patients with SSI. Results: This study enrolled a total of 106 patients, including 49 and 57 patients with BAD and CSVD-related SSI, respectively. The BAD group performances were worse than those of the CSVD-related SSI group for STT-A (83 [60.5–120.0] vs. 68 [49.0–86.5], P = 0.01), STT-B (204 [151.5–294.5] vs. 153 [126.5–212.5], P = 0.015), and the number of correct answers on Stroop-C (46 [41–49] vs. 49 [45–50], P = 0.035). After adjusting for age, years of education, National Institutes of Health Stroke Scale and lesion location, the performance of SSI patients with different etiological mechanisms still differed significantly for STT-A and STT-B. Conclusions: BAD patients were more likely to perform worse than CSVD-related SSI patients in the domains of language, attention, executive function, and memory. The mechanism of cognitive impairment after BAD remains unclear.
Introduction
Cognitive impairment is common after acute stroke. [1] While conceptually this is more likely to occur after large or strategically located areas of cerebral infarction, studies suggest that half of the survivors of first-ever lacunar infarction have cognitive deficits that are severe enough to impair daily activities. [2,3] Underlying cerebral small vessel disease (CSVD) is another pathophysiological explanation, in which domains of executive function, attention, memory, processing speed, and verbal fluency are prominent, [4] yet memory loss is the most commonly impaired cognitive domain after lacunar infarction. [5] While processing speed is one of the earliest and most prominent progressive cognitive impairments associated with CSVD, lesions of the frontal interhemispheric and thalamic projection fiber tracts that involve the frontal-subcortical neuronal circuits are also predictors of processing speed performance in age-related CSVD. [6] Thus, CSVD-related cognitive impairment is likely to depend on lesion location, particularly in the internal capsule, thalamus, caudate nuclei, anterior thalamic radiation, and forceps minor. [7] Through in vivo visualization of proximal culprit plaques in the penetrating arteries of the middle cerebral artery, we propose that branch atheromatous disease (BAD) is a distinct nosological entity of single subcortical infarction (SSI) that may guide management and prognosis. [8] The differences in cognitive performance between the two subtypes of SSIs can be used to distinguish their different etiological mechanisms. The present descriptive investigation compared cognitive performance between patients with BAD (atheromatous plaque of the parent artery at the orifice of the perforating artery) and CSVD-related SSI (lacunar infarction from intrinsic CSVD pathologically characterized by lipohyalinosis and fibrinoid degeneration). Based on the comparison of the differences in the cognitive function of SSI patients with different etiological mechanisms, the correlations between different cognitive function assessment scales in these patients were further analyzed. We also provided reference data for SSI patients using the Shape Trail Test A (STT-A) and STT-B to assess impairment in cognitive function.
Ethical approval
The study was approved by the Ethics Committee of West China Hospital (No. 2020 [324]), and the informed consent was obtained from all participants.
Patients
We prospectively recruited consecutive patients (age, 18-80 years) admitted to West China Hospital between July 2017 and November 2020 with first ever acute ischemic stroke due to a SSI (basal ganglia, corona radiata, internal capsule, and thalamus) identified by diffusion-weighted imaging (DWI) performed within 14 days of symptom onset. Patients were excluded if they had a history of other neurological or psychiatric diseases or pre-existing cognitive dysfunction; hearing or communication disorder, color blindness, or severe paralysis that would impair performance on tests; evidence of prior stroke on brain imaging; coexistent ≥50% stenosis in any of the ipsilateral internal carotid, middle or anterior cerebral, vertebral, basilar, or posterior cerebral arteries on computed tomography angiography (CTA); multiple lesions on magnetic resonance imaging (MRI) DWI; nonatherosclerotic vasculopathy (eg, dissection, vasculitis, and moyamoya disease); and evidence of any potential source of cardioembolism (eg, atrial fibrillation, recent myocardial infarction, dilated cardiomyopathy, valvular heart disease, or infective endocarditis).
Baseline characteristics including age, sex, years of education, dominant hemispheric infarction, lesion location (based on the strategic subcortical infarcts potentially affecting cognitive function in previous studies), cardiovascular risk factors (hypertension, diabetes mellitus, hyperlipidemia, coronary artery disease, current alcohol consumption, and smoking status), and time from symptom onset to admission were systematically recorded. The severity of neurological impairment was measured using the National Institutes of Health Stroke Scale (NIHSS) score. All patients underwent 24 h of electrocardiographic monitoring and/or Holter monitoring and transthoracic echocardiography to exclude those with cardioembolism. The Beijing version of the Montreal Cognitive Assessment (MoCA-BJ), Trail Making Test (TMT), and Stroop Color and Word Test (SCWT) were administered during hospitalization within 14 days after symptom onset. The patients were divided into two groups according to lesion size on DWI: BAD was defined as a SSI lesion (diameter ≥15 mm) in ≥3 consecutive axial slices; CSVD-related SSI was defined as a SSI lesion (diameter <15 mm) in less than three axial slices. [9] CSVD MRI markers Lacunes were defined as round or ovoid lesions (>3 mm and <20 mm diameter) occurring in the basal ganglia, internal capsule, centrum semiovale, or brainstem, with cerebrospinal fluid signal intensity on T2 and fluidattenuated inversion recovery (FLAIR), generally with a hyperintense rim on FLAIR and no increased signal on DWI, [10] and defined as single or multiple. [11] Enlarged perivascular spaces (EPVSs) were defined as small (<3 mm) punctate (if perpendicular) and linear (if longitudinal to the plane of scan) hyperintensities on T2 images in the basal ganglia or centrum semiovale. According to a validated semiquantitative scale of 0 to 4, [12] EPVSs in the basal ganglia were categorized as moderate to severe (grades 2-4). [11] Deep and periventricular white matter hyperintensities (WMH) were coded from 0 to 3 on the Fazekas scale [13] and categorized as either (early) confluent deep (score 2 or 3) or irregular periventricular extending into the deep white matter (score 3). [11] Two experienced neurologists blinded to patient data manually assessed the number of lacunes, EPVS, and WMH severity, with 10 patients randomly selected for assessment of the reproducibility of measurements. Any discrepancies between the two observers were resolved by consensus.
Cognitive assessments
The MoCA-BJ was used to assess cognition, as it is a widely accepted, popular, and brief standardized measure of cognition for use after stroke, [14] with a cut-off score of 26 showing excellent sensitivity (90.4%) and fair specificity (31.3%) for mild cognitive impairment (MCI). [15] The TMT is another sensitive and popular test used to identify MCI and dementia, with the variant STT for Chinese consisting of two parts [16] : Part A, in which the participant is asked to connect 25 pre-instructed digits, and Part B, in which the participant is required to alternately connect 25 pre-instructed digits, each appearing twice in both a circle and a square. In practice, derived scores usually remove the speed (in seconds) component from performance to provide a more refined measure of executive control. [17] However, Zhao et al [16] developed an index measure, "STT-B-1 min," defined as the number of correct responses within the first minute, to improve efficiency and performance. Receiver operating characteristic curve (ROC) analysis indicated area under the curve (AUC) values ranging from 0.816 to 0.913 for the STT-A and STT-B, with acceptable sensitivity and specificity.
The SCWT is widely used to evaluate basic human executive functions, particularly attention and informational processes. [18] It consists of neutral or incongruent colored words presented to participants who are asked to connect the correct name of a given color (card A, black wording) with the color (card B). Card C features the names of colors but with competing color names (eg, the word "green" written in red). Scores are derived from the difference in completion times (Stroop interference effects [SIE] time consuming) and correct numbers (SIE right numbers) between cards C and B, in which the larger the SIE, the lower the interference suppression efficiency. [19] Statistical analysis One-sample Shapiro-Wilk tests were used to assess data normality. Continuous variables with normal distribution were expressed as means ± standard deviation, while those with skewed distributions were expressed as medians (interquartile range). Significance testing was performed using an independent t test and Mann-Whitney U test as appropriate. Categorical variables were shown as numbers and percentages (%) and compared using chi-squared and Fisher's exact tests. A generalized linear model was used to examine the association between SSI patients with different etiological mechanisms and their cognitive function after adjusting for age, years of education, NIHSS, and lesion location. The correlations between MoCA-BJ, STT, and SCWT were analyzed using Spearman's correlation analysis. ROC analysis was used to assess sensitivity, specificity, and cut-off scores, with the AUCs used as an overall index of performance. In ROC analysis, we used a MoCA-BJ score of <26 as the "gold standard" to classify patients with SSI as cognitively impaired or cognitively normal. All analyses were two sided, and statistical significance was set at P < 0.05. All analyses were performed using IBM SPSS Statistics for Windows, version 26.0 (IBM, Armonk, NY, USA).
Results
This study enrolled a total of 106 patients, including 49 and 57 patients with BAD and CSVD-related SSI, respectively. CSVD-related SSI patients were more likely to have hypertension and were current smokers, while BAD patients were more likely to have hyperlipidemia and higher baseline NIHSS scores [ Table 1]. Table 2 After adjusting for age, years of education, NIHSS score, and lesion location, the performance of BAD patients on STT-A and STT-B remained worse than that of CSVD- Table 3].
Discussion
The results of our study showed that cognitive performance after BAD was significantly worse than that after CSVD-related SSI. We found significant differences in STT-A and STT-B between the groups but not the MoCA-BJ between the groups, which likely reflects its insensitivity to higher levels of cognitive function. These data provide insights into the mechanisms of cognitive impairment after SSI.
The STT is based on the TMT, which was developed for people who speak Chinese as their first language. The test assesses both "rapid visual search" and "visuospatial sequencing" factors, as well as the ability of "set shifting." A previous study demonstrated that the STT-A reflected language and attention, while the STT-B more reflected executive function and memory. [16] Hence, BAD patients are more likely to perform worse than CSVD-related SSI patients in terms of language, attention, executive function, and memory.
Screening tests for dementia are insensitive to the detection of mild cognitive dysfunction. The SCWT assesses the ability to inhibit cognitive interference, which occurs when the processing of a stimulus feature affects the simultaneous processing of another attribute of the same stimulus. [20] Kramer reported slower information processing in patients with subcortical ischemic vascular disease. [21] The SCWT can be used to evaluate the behavior control functions using the conflict between perception and speech. [22] Poor performances on difficult tasks such as the Stroop-B and Stroop-C are more likely to reflect genuine impairment. [23] When exploring the different cognitive status between patients with BAD and CSVD-related SSI at baseline, only Stroop-C (correct) demonstrated a statistically significant difference, indicating that the correct numbers are more sensitive than the time in this test. Nevertheless, the behavior in the incongruous condition (eg, Stroop-C) may be affected by difficulties that are not directly related to an impaired ability to suppress the interference process, which may lead to misinterpretation of the patient's performance. [24] Consequently, when assessing inhibition capability, the performance in the incongruous condition should be related to word reading and color naming abilities. [24] Most clinicians have difficulty in distinguishing between BAD and CSVD-related SSI. We previously found that the number of axial lesion slices (≥3), although with marginal significance, provided a better appreciation of the discrepancy of infarct compared to axial lesion diameter for predicting the mechanism of recent subcortical infarction. High-resolution MRI showed that patients with plaques presented larger infarction lesions and more proximal lesions compared to those patients without plaque, which was consistent with the imaging features of BAD. [25] The present study defined BAD as having a larger infarct diameter and more infarct layers compared to CSVD-related SSI as the infarct volume of BAD is theoretically larger. Therefore, BAD may involve more strategic regions than CSVD-related SSI, resulting in more serious cognitive impairment. While the NIHSS score is an established predictor of functional outcomes after stroke, [26] it lacks a cognitive component, [27] and its relationship with cognitive outcomes is controversial. [28,29] Yamamoto reported a higher initial NIHSS score in patients with BAD than that in patients with lipohyalinotic degeneration. [30] Fure et al [2] found that a neurologic deficit according to NIHSS was related to common cognitive variables in a bivariate analysis but not in the multivariate model, partly due to the relatively low NIHSS scores in patients with lacunar stroke. In our study, the NIHSS score at admission was also higher in the BAD group than that in the CSVD-related SSI group, which may partly explain the worse cognitive status of BAD patients. However, we observed no significant differences in CSVD MRI markers between the two groups. In other words, the burden of CSVD in patients with BAD remained substantial. We performed correlation analyses to study the relationships between the MoCA-BJ, STT, and SCWT. The MoCA-BJ was significantly correlated with the STT and SCWT, except for STT B/A. The high correlations between STT and global cognition were consistent with those reported by a Chinese study. [31] Our data showed that STT-B was most related to global cognition in patients with SSI [ Table 4]. The results of our analysis also revealed that STT-A and STT-B correlated well (r = 0.863). However, the Stroop-C (correct) correlated only moderately with the STT-A (r = À0.524) and STT-B (r = À0.586), suggesting that they measure somewhat different functions.
In our study, a higher correlation was found between STT (especially STT-B) and MoCA-BJ in SSI patients. Therefore, we established reference data for STT-A and STT-B in patients with SSI. The AUCs of the ROC curves in the BAD group were 0.821 and 0.824 for STT-A and STT-B, respectively, while the AUCs of the ROC curves in the CSVD-related SSI group were 0.701 and 0.729 for STT-A and STT-B, respectively. In addition, the sensitivity and specificity were acceptable.
Both BAD and CSVD-related SSI patients had low NIHSS scores (5 [2][3][4][5][6][7] vs. 2 [1][2][3][4], P = 0.001). Although the difference in NIHSS scores between the groups was statistically significant, the clinical manifestations of SSI patients were mainly pure motor or pure sensory deficits, and cognitive function was not generally affected by the disease itself. Patients with mild stroke present a new challenge for rehabilitation specialists because their primary deficits are more subtle than the typical stroke symptoms that are more overt. [32] Despite rehabilitation training, the greatest concern is the degree of physical dysfunction and not cognitive dysfunction. However, cognitive impairment after a mild stroke can severely impact an individual's ability to function in everyday life and perform meaningful occupations. [33][34][35] Early identification of post-stroke cognitive impairment may contribute to a favorable outcome; thus, clinical interventions in the acute phase may be beneficial for the quality of life of patients with mild ischemic stroke.
This study has several limitations. First, the sample size was small, limiting our statistical power; thus, large-scale studies are needed to verify our results. Second, we excluded patients with hearing disorders, communication disorders, color blindness, color weakness, and severe paralysis, which might have affected the accuracy of the executive tests. Third, we did not evaluate cerebral microbleed because some patients failed to complete the relevant MRI sequences; however, none of the patients had a history of cognitive dysfunction. Fourth, we lacked a control group for comparing the MoCA-BJ, STT, and SCWT between healthy people and patients with SSI. Fifth, we cannot fully explain why the cognitive impairment in BAD patients was more severe than that in CSVD-related SSI patients. Interpreting the cognitive impairment mechanisms underlying the two types of SSIs requires further research. Finally, follow-up of cognitive and functional outcomes is warranted to investigate the role of STT and SCWT in the prediction of long-term cognitive and functional outcomes after SSIs.
In conclusion, the results of our study indicated that BAD patients were more likely to perform worse than CSVDrelated SSI patients in the domains of language, attention, executive function, and memory. In addition, the STT-B was most related to global cognition in patients with SSI, suggesting the sensitivity of this test in detecting executive dysfunction and global cognition impairment. Future research is needed to fully elucidate the cognitive impairment features after BAD, which may contribute to the prevention rather than the treatment of PSCI. | 2021-12-12T16:36:20.344Z | 2021-12-08T00:00:00.000 | {
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252393450 | pes2o/s2orc | v3-fos-license | A Comprehensive Assessment of Self-Reported Post COVID-19 Symptoms Among Beneficiaries of Hospital Employee Scheme at a Tertiary Healthcare Institution in Northern India
Purpose With millions of people being affected by COVID-19, people living with post COVID-19 clinical symptoms (PCS) are expected to rise further. The primary aim of the study was to comprehensively assess self-reported PCS and its associated risk factors among beneficiaries of Hospital Employee Scheme of a tertiary healthcare institution in Delhi. Patients and Methods An online cross-sectional study was conducted using a semi-structured questionnaire developed by employing nominal group technique among individuals aged 18 years and above who were novel SARS-CoV-2 positive from January to April 2021. Participants were telephoned first, before sending the online survey link. Socio-demographic data, information on PCS along with potential risk factors, pre-existing morbidities, vaccination status, severity of acute illness and management were collected between June and July 2021. PCS was presented as relative frequency; Chi-Square test and odds ratio; adjusted values were used to rule out any association between PCS and predictors. Results In total, 773 of 1801 eligible participants responded to the survey (completion rate 42.9%), with a median age of 34 years (IQR 27–44). Males accounted for 56.4% and PCS was present in 33.2%. The most prevalent symptoms were fatigue (79.3%), arthralgia (33.4%), myalgia (29.9%), hair loss (28.0%), headache (27.2%), breathlessness (25.3%), and sleep disturbance (25.3%). The prevalence of PCS was reduced to 12.8% at 12 weeks. Female gender, older age, oxygen supplementation, severity of acute illness, and pre-existing co-morbidities were positively associated with PCS. Vaccination (second dose) reduced the odds of developing PCS by 39% compared to unvaccinated participants (aOR 0.61; 95% CI 0.40–0.96). Conclusion PCS affects almost all organ systems of the body, regardless of the severity of acute COVID-19 illness. Two doses of vaccine hel reduce the development of PCS.
Introduction
The World Health Organization declared COVID-19 a pandemic on March 22, 2020, approximately 3 months after the first case of the disease was identified. 1,2 Since then, the disease continues to spread in an unprecedented manner across the world causing the loss of millions of lives. As of July 4, 2022, more than 546 million people were affected and nearly 6.3 million people lost their lives due to the disease. 3 In India, approximately 43.5 million people have been infected with the virus and more than 525,000 of them have died. 4 including self-reported and systemic reviews, have illustrated that around 50-87% of the hospitalized patients experience at least one or more post COVID-19 symptoms (PCS) for several weeks. [5][6][7][8][9] For example, a self-reported online survey in Korea reported that 52.7% of the respondents have shown at least one persistent COVID-19 symptom. 6 Another study showed that 20% of the patients experienced PCS lasting more than 3 months. 10 A self-reported Swedish web questionnaires study reported 1 out of 10 with long-term symptoms for at least 4 weeks. 8 Such persisting and debilitating symptoms following COVID-19 mean that the adverse consequences of the pandemic do not end with recovery, and continue much beyond the acute phase of illness. 5 As the pandemic continues to spread globally, the number of people living with PCS will increase over time. Although the natural history of COVID-19 is not completely known, it is now well recognized that COVID-19 is a multiorgan systemic disease with broad spectrum of manifestations. 5 With millions of individuals recovering, the longterm consequence of COVID-19 is likely to become an additional burden on the healthcare delivery system, especially in low-and-middle-income countries. Understanding PCS and associated risk factors is crucial to reorient the model of care to make it more responsive to the emerging needs. Hence, information on PCS is essential to guide development of appropriate infrastructure and manpower, thereby designing management strategies and patient care plans in hospitals and rehabilitation facilities. The present study aimed to comprehensively assess self-reported PCS and associated risk factors among beneficiaries of Employee Hospital Scheme of a tertiary healthcare institution in Northern India.
Study Population
This cross-sectional study was conducted among adult population (those aged 18 years and above) who were beneficiaries of Hospital Employee Scheme (EHS) of a tertiary healthcare institute in Northern India. The institute is one of the biggest tertiary canters in the country with more than 15,000 employees and more than 3100 beds expanding across various medical subspecialties. The EHS was established in the institute to provide free healthcare services to hospital employees and their dependents. During the COVID-19 pandemic, the hospital staff and their dependents have received free COVID-19 care services for both outpatients and in-patients, including COVID-19 vaccination. The inclusion criteria for the study were positive report of SARS CoV-2 test from January 1, 2021 to April 30, 2021, and having recovered from the illness. This list of COVID-19 recovered individuals (2037) was obtained from the Department of Hospital Administration of the institution during the month of June 2022. From this list, we excluded participants aged below 18 years, individuals who could not contact telephonically before sending the link and participants not using or not having knowledge of WhatsApp. The variables recorded were age, sex, date of diagnostic testing for COVID-19, and relevant contact details (like telephone numbers). The required minimum sample size was estimated based on the seroprevalence among adults of 47.2% in megacities in December 2020, 11 with a relative precision of 10%, power of 80%, 95% confidence interval and a nonparticipation rate of 20%. The final estimated sample size calculated was 773 individuals.
Study Definitions
Post COVID-19 clinical symptoms: Symptom(s) that persisted beyond 4 weeks from the date of SARS-CoV-2-positive test conducted using either Reverse Transcriptase Polymerase Chain Reaction (RTPCR) or Cartridge Based Nucleic Acid Amplification Test (CBNAAT). Further, according to the timeframe, post COVID-19 symptoms were classified as shortterm post COVID-19 symptoms (ST-PCS): symptoms present beyond 4 weeks after the SARS-CoV-2-positive test and lasting less than or up to 12 weeks; and long-term post COVID-19 symptoms (LT-PCS): symptoms present beyond 12 weeks after the SARS-CoV-2-positive test.
Data Collection
A semi-structured questionnaire was developed for the study. The questionnaire was digitized using Google Forms. 12 Google Forms is an online data collection tool that is commonly used for surveys. 13,14 A nominal group technique was used to develop the questionnaire. One researcher developed the first draft of the study tool after extensive review of the relevant published papers to ensure all possible PCS. One expert each was requested to review the tool from the specialties of endocrinology, internal medicine, psychiatry, ophthalmology, and pulmonary medicine, respectively. The questionnaire was revised based on the feedback received from the experts during the group discussions (in person and https://doi.org/10.2147/IJGM.S381070
DovePress
International Journal of General Medicine 2022:15 online). The questionnaire included demographic information, and various possible risk factors for post COVID-19 manifestations. The final section of the questionnaire consisted of a list of possible post COVID-19 symptoms that were categorized based on organ systems. Before standardization, pre-testing of the questionnaire was done among non-study participants who had recovered from COVID-19. The English language was translated into Hindi by a translator, and back translated into English to ensure the accuracy of the translation.
The data were collected between June 16, 2021 and July 30, 2021. A minimum gap of 4 weeks was ensured between data collection and COVID-19 testing date of participants. All participants meeting the inclusion criteria were contacted telephonically and were requested to participate in the survey. The respondents who were using WhatsApp TM platform (Facebook Inc, Cambridge, MA, USA) were sent the survey link using the same number. If any participant did not use WhatsApp TM on their phone, they were requested to provide an alternative mobile number with WhatsApp TM installed. This was done to enhance the participation rate. If any respondent could not provide a WhatsApp number, or could not read both the English and Hindi languages, we requested the participants for a suitable time to call for telephone-based data collection if they are willing to participate.
WhatsAppTM is a popular medium for communication and has been used in many studies as an electronic platform for communication. [15][16][17] Once the survey link was shared, the participants were requested to save the sender number first and then to activate the survey link. Prior to inclusion in the survey, participants were required to offer informed e-consent. A total of three reminders at a gap of 1-2 days were sent to participants who did not respond to the survey link sent to them earlier.
Ethics Statement
The present study was reviewed and has been approved by the Institute Ethics Committee of All
Data Management and Analysis
The responses were automatically collected in a Google spreadsheet linked to the data collection form. The data were analysed using STATA version 15 (StataCorp 2015, Stata Statistical Software: Release 15, College Station, TX: StataCorp LP). The participants were divided into two groups, namely participants with post COVID-19 symptoms and participants without symptoms for further analysis.
Descriptive analysis was performed to summarize the findings. Continuous variables were presented as the mean and standard deviation for normal distribution, whereas median and interquartile range (IQR) were used for variables that were not distributed normally. Post COVID-19 manifestations were presented as relative frequency. Furthermore, post COVID-19 symptoms were categorized into 15 different groups according to different organ systems. The duration of symptoms was estimated from the date of confirmed test and date of response of the participants. Categorical data were presented as absolute counts, percentages and were further compared using the Chi Square and Fisher's exact test. Continuous variables were compared using t-test. Univariable and multivariable logistic regression models were used to explore explanatory variables associated with post COVID-19 symptoms. The results were presented as odds ratios and adjusted odds ratios (ORs and aORs), along with 95% confidence intervals. Variables that were significant at p<0.10 in the univariable regression analysis were included in the multivariate model. Since it was a web-based survey, the checklist for Reporting Results of Internet E-Surveys (CHERRIES) was followed while reporting the data. 18 graduate or above in education. A total of 134 (17.3%) respondents smoked or chewed tobacco products and 229 (29.6%) consumed alcohol (Table 1).
One-quarter (24.7%) had received the second dose of the vaccine at the time of the survey. Approximately three-fourths of participants (75.2%) reported that they had either asymptomatic or mild acute COVID-19, and 2.71% reported that they experienced severe acute COVID-19 (Table 1). Approximately 169 (21.9%) participants reported that they were managed in the hospital. Of these, 23 (2.9%) required oxygen supplementation during management. Furthermore, 8.3% of the participants considered themselves to have poor or very poor overall health status following COVID-19 illness.
Respiratory Symptoms
Considering the respiratory manifestations, approximately 25.3% of the participants reported breathlessness, followed by cough (24.9%). One person reported with chest tightness and heaviness.
Cardiovascular Symptoms
Among the cardiovascular symptoms, 16.0% of the respondents reported that they had palpitations followed by feeling cold and chills in the body and lower limbs (5.06%), 4.3% reported sweating at night and 4.7% for swelling of the lower limbs and another 3.1% reported hypertension. Three participants (1.2%) reported tachycardia, and one each reported chest pain and stroke as post-COVID-19 symptoms.
Musculo Skeletal Systems
There were few musculoskeletal manifestations experienced by the participants. The most common were arthralgia (33.46%) and myalgia (30.35%), followed by low back pain (0.78%) and leg pain (0.78%).
Gastrointestinal, Endocrine and Hepatobiliary and Kidney Symptoms,
A wide range of gastrointestinal symptoms was reported after recovery from COVID-19. Among them, loss of appetite (15.9%), diarrhea (13.2%), abdominal pain (10.1%), nausea and vomiting (8.94%) were the most prevalent manifestations. Other symptoms were acidity (2.72%) and an increase in appetite (0.78%) and constipation (0.78%). While 3.5% of the individuals reported liver function disorders, one participant reported abnormalities in kidney function tests.
Neurological Symptoms
Several post-COVID-19 neurological manifestations have been reported as shown in Table 2. Among them, the most frequently reported symptoms included headache (27.2%), sleep disturbance (25.3%), and inability to concentrate (18.7%), followed by loss of memory (14.0%). While 8.17% of the participants experienced dizziness, one had a tingling sensation in the body.
Dermatological Symptoms
A variety of post-COVID-19 dermatological manifestations were being noted among participants. Of them, hair loss (28.79%) and skin rashes (7.39%) were the most common symptoms, followed by discoloration of fingers or toes (3.5%). The least frequent dermatological manifestations were acne, itching of the whole body, dryness of skin, and nail discoloration ( Table 2). One person reported with Herpes zoster.
Dental Symptoms
Several oral manifestations have been reported among patients who have recovered from COVID-19. Dry mouth (8.95%) and pain in the gum or teeth (5.45%) were the most frequent post-COVID-19 oral health problems. Other less frequent oral manifestations were oral ulcer (1.17%), sensitivity to cold water (0.78%) and bleeding in the gum (0.78%).
Behavioral Problems
There were reports on changes in behaviors after recovery from COVID-19 (Table 2). Among them, the most prevalent were the increased use of electronic gadgets (24.9%) and loss of motivation (6.61%). Other less common post COVID-19related behavioural changes were loss of interest in interacting with friends or peer groups (4.28%), new onset alcohol consumption and smoking or use of tobacco products (0.39%).
Pre-Existing Comorbidities Among Participants
Among the participants, 33.8% reported that they had at least one or more associated comorbidities before the COVID-19 disease (Figure 3). Hypertension (10.1%) was the most common comorbidity, followed by diabetes mellitus (6.33%).
Factors Associated with Post-COVID-19 Manifestations
The univariable logistic regression model showed that several factors were associated with post-COVID-19 manifestations (Table 3). Younger age groups (absence of null value in 95% CI of OR; 2.94, 2. The multivariate analysis (Table 3) showed that PCS was 1.57 times more likely to develop in pre-existing comorbid participants than in healthy participants, 4.07 times more likely in patients with oxygen supplementation during treatment from non-oxygenation patients. Further, about 2.23 times for mild and 2.66 times for moderate COVID-19 illness are more likely to develop PCS than in asymptomatic patients. Smokers compared to nonsmokers were found to have a protective effect in univariable analysis, but in the multivariable analysis, after adjusting for other explanatory variables, smoking was not associated with PCS. One of the important findings in the multivariable regression model was that the odds of developing PCS were lower among individuals who received a second dose of COVID-19 vaccine in comparison to unvaccinated individuals (aOR 0.61; 95% CI 0.40-0.96, Table 3). The multivariable logistic regression model revealed that older age group, female gender, healthcare staff, oxygen supplementation during COVID-19 management, cognitive or memory impairment, severity of acute COVID-19 illness, and unvaccinated status were independent risk factors for PCS (Table 3).
Discussion
The current study was conducted among SARS-CoV-2 positive individuals who tested positive and recovered 4 weeks or more before the date of inception of data collection. They were asked about the status of COVID-19 illness during the telephonic interview to ensure that the information collected was for PCS. As of now, it is practically challenging to compare the findings and the prevalence of PCS across various studies due to differences in assessment time since recovery, and variability in the duration while defining post COVID-19 symptoms. Therefore, the prevalence of PCS, to date, has ranged from 27.8% to 95.0% depending on the time of data collection after recovery. 6,7,19,20 The present study showed that the prevalence of PCS was 33.2%, irrespective of the severity of COVID-19. This indicates that one in three individuals have persistent PCS after 4 weeks or more following the positive test. Furthermore, it is evident that many patients continue to experience persistent symptoms after COVID-19, irrespective of disease severity during acute illness and the requirement of hospitalization. Studies have reported multiple PCS in nonhospitalized patients after several months of recovery from COVID-19. 21,22 Although individuals reporting the severity of PCS were fewer in number, PCS ranged from mild to severe.
In the current study, the prevalence of PCS was slightly higher in males (56.4%) as compared to females (43.6%); however, females were more likely to develop PCS than males. The difference in physiological and socio-cultural factors may be possible explanation for this, but further study is warranted to explore the underlying reasons. A female preponderance of PCS has also been reported in other studies. 21,23 The present study also indicated that older individuals were more likely to develop PCS than younger age groups (Tables 1 and 3). Several studies also noted that increasing age is a risk factor for PCS. 19,20,24 Education, alcohol consumption, body mass index, and blood groups did not have any significant effect on the development of PCS.
The study noted that individuals working in the healthcare sector had a higher risk of developing PCS than individuals who were working in other sectors. In the multivariable regression analysis, non-healthcare staff are at lesser risk of having PCS than employees working in the hospital (OR: 0.65, Table 3).
Participants with moderate-to-severe COVID-19 illness are at a higher risk for PCS after recovery as shown in univariate analysis. A longitudinal follow-up study will be helpful to assess the duration of persistence for PCS among participants with severe acute illness. In the present study, fatigue was the most common symptom among all the PCSs, reaching up to 80.3%. In the studies conducted elsewhere, including systemic reviews and meta-analyses, fatigue was the most frequently reported symptom with a prevalence ranging from 30% to 82.9%. 6,21,25,26 The remaining symptoms, such as arthralgia and myalgia, hair loss, headache, shortness of breath, sleep disturbance, cough, loss of smell and taste, were noted in between 20% and 34% of the participants. The pathophysiology behind such a wide range of manifestations is not yet clear, however, it indicates multiorgan involvement as in the acute phase of the illness. The possible immunological mechanism involving the multi-organ system following SARS-CoV-2 infection has been illustrated to explain the appearance of these longlasting symptoms in other literature. 6,27 The present study also showed that the odds of having PCS were reduced by 39% in individuals who had received two doses of COVID-19 vaccine compared to persons who did not receive any dose (Table 3). This is an additional beneficial finding for the COVID-19 vaccine that has already been found to reduce the risk of SARS CoV-2 infection and the severity of acute illness. Therefore, vaccination against COVID-19 should be encouraged among the eligible population as early as possible.
A small percentage of recovered patients also experienced both near and distance visual impairment, and dry eyes. It is not clear whether symptoms could be side effects of medication that were commonly used during the acute management or related to weakness of the ocular muscles. The study also indicated a strong association between pre-existing comorbidities and the presence of PCS. Similar pre-existing co-morbidities such as hypertension, chronic respiratory diseases, and diabetes mellitus have shown to be determinants of the prolonged COVID-19 symptoms as illustrated in other studies. 24,28,29 Furthermore, our study showed that participants who were managed during the acute phase of COVID-19 illness with oxygen supplementation were more likely to develop PCS than participants who were managed without oxygen. A similar finding was shown in another study. 24 Among psychosocial behavioral changes, a sizeable number of recovered subjects also reported anxiety, and mood disorders, including panic and depression in the current study. Various changes in behavioral aspects in the present study were noted, such as a substantial increase in the use of electronic gadgets being the most common, among the survivors. A link with mental and behavioural problems among recovered patients has also been reported in other studies. 6,25,28 Overall, we observed that the PCS involved almost all organ systems of the body. Such multiorgan involvement in PCS has been described in other literature. 6,14,26 Similar results were reported in a mobile phone app-based study in the United Kingdom. 27 This implies that managing such widely variable symptoms requires a holistic multidisciplinary approach involving multiple specialties, including hospital and community-based rehabilitation programs to support healthcare and psychological needs for an extended time rather than organ-specific management.
To date, it is not yet fully understood how long the PCS will persist among COVID-19 survivors. In addition to our findings, various previous studies reported that SARS-CoV-2 can still infect individuals despite vaccination, although the severity of the disease is reduced. PCS can also affect individuals regardless of the severity of the acute phase of COVID-19 illness. Therefore, there is a need to develop a PCS care model that is suitable for resource limited countries. One such model is being run to manage PCS in the United Kingdom. 28,29 Individuals with PCS who have pre-existing comorbidities need a proper follow-up strategy because such persons are potential candidates for developing significant disabilities. In addition, a few patients who had recovered from COVID-19 also had newly detected diabetes, abnormal renal, liver, and thyroid function tests and stroke as in other studies. 24 These patients need to be investigated thoroughly per se, including those who report poor or very poor health following COVID-19 and need to be managed accordingly. Followup is necessary to know whether such conditions could be reversible in due course of time.
The limitations of our study include first, we relied on self-reported data so there may be potential for socially desirable responses or recall bias in participants. Since it was a cross-sectional study, we cannot rule out the casual relation of some of manifestations whether it was due to COVID-19 or persisting health problems. Second, we did not access the investigation records of the respondents. Therefore, the rating of severity of acute illness and management information were based on participants' responses. Furthermore, further study is warranted to assess any co-relation with metabolic disorders and cardio-respiratory symptoms. Third, our study may not be truly a representative of all communities since we procured the list from one institute, and we also could not collect data from the participants who were not using either smartphones or WhatsApp TM . | 2022-09-21T15:24:14.981Z | 2022-09-01T00:00:00.000 | {
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233251521 | pes2o/s2orc | v3-fos-license | IMPACT OF VAGAL NERVE STIMULATION ON QUALITY OF LIFE IN DRUG-RESISTANT EPILEPSY
Background and objective. Vagal nerve stimulation (VNS) represents an alternative therapy for intractable epilepsy. The aim of this study is to analyse seizure reduction and the life quality of these patients. Material and methods. We prospectively examined 28 adult patients treated with VNS that were followed-up at least 6 months after the surgery and we recorded the number of seizures and any other changes. 16 epilepsy patients completed the Quality of Life in Epilepsy-31 questionnaire (QOLIE-31). Results. Our data revealed that 64% of patients were responders with more than 50% seizure reduction. According to McHugh classifi cation of seizure freedom, 36% patients are in class I (80-100% seizure reduction), 29% class II (50-79% reduction), 21% class III (less than 50% reduction), 7% class IV (magnet benefi t only) and 7% class V (no improvement). Only 8 patients presented mild adverse effects, such as hoarseness, fatigue and cough. Life quality has improved for 68% patients. There is a strong correlation between life quality and health and a mild positive relation with the seizure reduction. Conclusions. VNS improves life quality for more than half of patients and is a therapy to consider in refractory epilepsy.
INTRODUCTION
The failure in keeping seizures under control can lead to a signifi cant decrease in life quality of epileptic patients. Up to one third of patients who suffer from the disease have intractable epilepsy. The International League Against Epilepsy (ILAE) defi nes epilepsy as refractory in case of inability to stop the seizures with at least two complete medical schemes (1).
Vagal nerve stimulation (VNS) is an alternative for medical or surgical refractory epilepsy and it is used in the case of failure or imposibility in performing an antiepileptic surgery, in the case of not obtaining clinical results after at least two medication plans, or in the case of medication intollerance of any kind. It has been used both for partial or generalized seizures, children or adults, for more than 30 years (2).
The device is similar to a cardiac pacemaker and it is attached subcutaneously in the left subclavicu-lar area, while the electrods are placed in the left vagus nerve. Although arrhythmias ar extremely rare, it is connected only the the left vagal nerve to even diminish that risk. The generator delivers intermittent electrical signals of low frequency to the nerve pathways (3). After the surgery, some parameters are noninvasively adjusted, including the intensity (output current), frequency, on time and off time. Patients also receive a magnet which can suspend the electrical stimulation while it is held in the generator area, thus interupting the seizure at its beginning (5).
The mechanism of action is not completely elucidated because of the diffi culty in animal studies, but it is based on the change in neurotransimitters such as serotonin, noradrenalin and GABA. The tenth cranial nerve has wide projections over some structures of the brain that are important in epileptogenesis, such as thalamus, amygdala, raphe nucleus, locus coeruleus. The electrical impulses mi-ght transform synchronous cortical activity to desynchronous and reduce the cortical excitability (4).
Due to the complex pathophysiology, VNS is indicated as an alternative to several diseases in case of medical failure. It is a possible therapy for medical refractory epilepsy, antiepileptic drug severe adverse effects, the impossibility in performing a curative antiepileptic surgery, uncontrolled status epilepticus, chronic headache, neurodegenerative disease, schizophrenia, autism, medical refractory depression (6)(7)(8).
Early complications such as bradycardia, asystole, hematoma, infections, vocal cord paralysis are related to the surgery technique, while the most frequent late complications include hoarseness, dyspnoea and coughing (9).
In an evidence-based guideline which evaluated 1274 studies, it was concluded that 55% of patients had at least a 50% seizure reduction with improvement over time, with the best response for generalized seizure type. The anti-depressive effect is an additional benefi ce. Changes in stimulation parameters are not completely accepted by all authors to increase the effect. The effi cacy of the magnet varies according to different studies from 25 to 75% (10). Another meta-analysis fi nds that only 30% had a 50% seizure reduction (11), but all literature data agrees that VNS has a signifi cant lower hospitalisation rate and it has a benefi ce on epileptic patients.
MATHERIAL AND METODS
Our study included 28 adult patients from Cluj-Napoca Neurology Clinic with the medium age of 32 years old, which were regularly followed-up after the VNS implantation. We recorded the clinical parameters of the patients, the number of seizures and adverse effects for a medium period of 20 months.
The inclusion criteria were the presence of vagal nerve stimulation therapy for epilepsy in adult patients. We excluded all children and the patients who withdrew from the study or deceased from other causes.
For 16 patients we evaluated the Quality of Life in Epilepsy-31 questionnaire (QOLIE-31) consisting of personal ratings of overall life quality, emo-tional well-being, seizure worry, energy, cognitive function, medication effects and social life. For each category we calculated the fi nal score which refl ects the life quality on that specifi c fi eld, and also the total score. Based on a group of 304 epilepsy patients, it was developed a table with pairing between any specifi c or fi nal score and a T-score. T-scores are linear transformations of the scores with the mean of 50 and a standard deviation of 10, representing that a T-score of 50 is correlated with the mean of the standard cohort (12). Values are expressed in mean± standard deviati on (range) or n (%).
Seizure frequency
For the patients in our study, the seizure frequency is extremely varied from one to 150 episodes per month, as it is presented in Fig. 2. At least 6 months after VNS implantation, we can observe in Fig. 3 a different distribution of frequencies with a general lower severity.
Seizure frequency reduction
For each patient we calculated the reduction rate according to the declared frequency anterior and after VNS implantation and we analysed the data using the McHugh and modifi ed Engel classifi cations.
After a medium period of 20 months, the majority of patients presented important improvements in epileptic episodes. According to McHugh classifi cation from Fig. 4, 10 patients were in class I with a reduction rate of more than 80%, 8 patients were included in class II with a reduction of 50-79%, 6 were in class III with less than 50% reduction, 2 presented no improvement and 2 had some benefi c effects of the magnet.
In modifi ed Engel classifi cation from Fig. 5, complete seizure freedom was declared by 6 patients, one presented with more than 90% reduction, 11 with 50-90% reduction and 1-patients had less than 49% reduction in seizure frequency.
Regarding the gender, our evaluation revealed no differentiation between females and males in the effi cacy of the VNS therapy. The type of epilepsy might be a predictor of the outcome, idiopathic etiology remaining with the best response.
3.The overall response to VNS
We classifi ed all 28 patients in responders(with a reduction in seizure frequency of more thena 50%) and non responders (<50% reduction). 64% of patients responded to this form of treatment and 36% did not declared a signifi cant effi cacy (Fig. 6).
Adverse effects
A majority of 20 patients had only temporary adverse effects, but 6 patients had a persistent dysphonia, one presented cough and one patients had fatique.
Overall life quality and health
The 16 patients from the study who completed the questionnaire accorded a grade from 1 to 10 for the overall life quality they consider to be characteristic most of the time.
To analyse the correlation between the overall life quality and seizure frequency reduction, we used Pearson correlation (Fig. 7). With an r of 0,39 there is a mild positive correlation between the two variables.
The way patients consider their overall health is very strong related to their life quality. In a Pearson correlation between overall life quality grade and the health grade (Fig. 8), we fi nd a strong positive linear relation with a r=0,76.
QOLIE-31 scores
We calculated the T-scores for all categories, a score equal to 50 refl ecting the mean of the responses from the standard epilepsy cohort. The results were very closed to the 50 value, in general lower. The mean from all T-scores from worries about a new seizure was 43,1, overall life quality had a T-scores mean of 46,1, emotions 46, energy 48,9, cognitive function 49,8, medication adverse effects 51,3, social implications were the most affected with a mean of 40.
We evaluated a possible correlation between the fi nal T-score and seizure reduction rate using Pearson correlation (Fig. 9). With a result of 0,13, there is not any signifi cant correlation between seizure reduction and the score which represents the life quality considered by the patient.
Life quality changes
We asked the 16 patients in the questionnaire to evaluate how VNS therapy has changed their lives, Overall life quality and seizure reducti on rate correlati on by choosing one of the predefi ned answers. 10 patients declared a moderate improvement, one considered a total life changing treatment, one has been a little infl uenced and 4 noticed no difference. From these patients, 11 consider a favourable treatment.
DISCUSSION
Vagal nerve stimulation is an accepted alternative treatment for refractory epilepsy. It has a documented role in reducing seizure frequency and intensity in time and interrupting the seizure at its beginning.
A large number of literature studies are focused on the reduction rate and its effi cacy, but only a few evaluate the impact of VNS therapy on life quality. The contribution of our study consists in analysing a therapeutic method not as well known and which, although in use for many years in other countries, it is still just at its beginning in our region. Also, studying QOLIE-31 on patients with vagal nerve stimulation represents a less common approach.
The limitations of the study are based on the small number of subjects, due to the extremely rare
CONCLUSIONS
Our data show that 64% of total 28 patients had more than 50% response in seizure reduction and the adverse effects were minor.
The quality of life scores in QOLIE-31 applied to the 16 patients were similar to the mean value of epileptic patients, with a lower tendency. The data show no correlation between the QOLIE-31 fi nal score and the seizure reduction rate, which suggests that vagal nerve stimulation is a possible therapy for reducing the number of seizures, but it is not necessarily related to the patient's perception about their life.
Finally, vagal nerve stimulation is an effective option for medical and surgical resistant epilepsy. | 2020-05-07T09:06:13.236Z | 2018-09-30T00:00:00.000 | {
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49342969 | pes2o/s2orc | v3-fos-license | Health worker led breast examination in rural India using electro-mechanical hand-held breast palpation device
Breast cancer is the most common female cancer worldwide representing nearly a quarter (23%) of all cancers in women.1,2 The global burden of breast cancer is expected to cross 2million by the year 2030, with growing proportions from developing countries.3 Although age-standardised incidence rates in India are lower than in the United Kingdom (UK) (25.8 versus 95per 100,000), mortality rates are nearly as high (12.7 versus 17.1 per 100,000, respectively) as those of the UK.1 Breast cancer incidence rates within India display a 3–4-fold variation across the country, with the highest rates observed in the Northeast and in major metropolitan cities such as Mumbai and New Delhi.4
Introduction
Breast cancer is the most common female cancer worldwide representing nearly a quarter (23%) of all cancers in women. 1,2 The global burden of breast cancer is expected to cross 2million by the year 2030, with growing proportions from developing countries. 3 Although age-standardised incidence rates in India are lower than in the United Kingdom (UK) (25.8 versus 95per 100,000), mortality rates are nearly as high (12.7 versus 17.1 per 100,000, respectively) as those of the UK. 1 Breast cancer incidence rates within India display a 3-4-fold variation across the country, with the highest rates observed in the Northeast and in major metropolitan cities such as Mumbai and New Delhi. 4 Diagnosis at advanced stages of disease contributes to a high mortality rate among women due to breast cancer, which can be attributed to low levels of awareness, ineffective screening methods, cumbersome referral pathways to diagnosis, limited access to effective treatment at regional cancer centres and incomplete treatment regimens. 3,5-10 With the rising breast cancer incidence in India 4 and disproportionately higher mortality. 11,12 it is essential to increase awareness and develop effective screening modalities which can work in large scale settings.
Mammography led screening is virtually impossible in India, with limited radiology manpower and competing healthcare priorities. While CBE (CBE) is affordable (Int $793 per life year saved), its effectiveness is limited (3). Also, training health workers to perform consistently high quality breast examination is challenging (4).
In LMICs, community health workers (CHWs) often represent an affordable resource for the education and provision of preventive, primary and promotive healthcare. Low-cost, user-friendly technology can help equip minimally trained CHWs to administer standardized breast exams without any special infrastructure. The low-cost technology must perform with better detection sensitivity than CBE (higher than 50%) and equally high specificity as CBE (94%) to accurately and effectively identify breast lumps in need of further diagnostic follow-up (diagnostic ultrasound, breast biopsy) without clogging the under-resourced infrastructure with false positives due to typical benign breast features (tissue variability, lumpiness and nodularity).
With this background, a study was designed to assess the feasibility of iBreastExam (iBE) in a community setting with the help of existing healthcare resources such as community health workers (CHWs) and social workers workers from Aastha Breast Cancer Support Group and Lions Club (Pune, India) were trained to operate iBE device to perform bilateral breast examinations.
Informed consent was taken in the local language before enrolling the patients for this study. Subjects were not offered any incentive for participating in the study. Subjects were asked to lay on a medical bed, after privately disrobing from the waist up. The operator performed the iBE in a clock-wise manner.
iBE comprises of an array of piezoelectric sensors that electronically palpate the breast and can differentiate between varying tissue elasticity. The "electronic palpation" was entirely automated and controlled internally. Following the iBE exam, patients underwent a routine Clinical Breast Examination (CBE) by an experienced doctor. The doctor was unaware of the iBE findings while performing the CBE. Patients who were either positive on iBE, CBE or both, underwent a breast ultrasonography exam (USG). If USG test was positive, patients were subjected to a breast FNAC or biopsy. IRB & Ethical committee clearance for this study were taken at Sasur Hospital in Pune.
Results
1300 subjects were enrolled in the study over 6months. The average age was 42.37years. iBE and Expert CBE was done for all patients.
Out of the 1300 subjects, iBE and Expert CBE were both negative in 1,139(87.6%) women. In 161(12.4%) women, either iBE or Expert CBE or both were positive. iBE was positive in 130(10%) women and Expert CBE was positive in 101(7.7%) women. 15 women from this group, refused to obtain a follow-up breast USG. USG was done for women either positive on iBE, CBE or both.
Of the 130 women that were positive on iBE, 103cases were confirmed by breast USG and 27cases did not show any signs of breast abnormalities as per breast USG. Of the 138 women that were positive on CBE, 101cases were confirmed by breast USG and 37cases did not show any signs of breast abnormalities as per breast USG.
Of the total 146 women positive (excluding the ones that refused follow-up), breast USG was positive in 101(69.2%) women for clinically relevant breast lesions. Of these, 18 women were recommended breast FNAC/biopsy and 3(0.23%) cases were diagnosed positive for breast cancer (Table 1-4).
Discussion
Electro-mechanical hand-held breast palpation device (iBreast Exam) uses piezoelectric tactile sensor invented at Drexel University. The patented ceramic sensor can assess the differences in tissue elasticity from the surface of the breast, non-invasively by creating micro-palpations that are driven from within the sensor. Using direct/ reverse piezoelectric effects, the piezoelectric sensor array enables "electronic palpation", from within the sensor and independent of the community health worker. And hence, in real-time, the sensor can assess the changes in the elastic modulus between normal breast tissue and abnormal masses that are stiffer.
The device (Figures 1A-D) has two major components; a scanner probe with multiple piezoelectric tactile pressure sensors and a computer tablet with proprietary software. The iBE device is battery powered and wireless; the software in the tablet displays compression data recordings. At the end of the test, a report is generated instantly showing normal areas in green colour and abnormal areas in red colour.
This study was conducted in rural environment where it was not feasible to provide standard of care imaging tests to every participant. For this reason, women that were negative on both iBE and CBE were not followed-up by any other imaging modality.
With these limitations, this study was primarily designed to validate the feasibility of iBE in rural settings and it's ability to identify clinically relevant breast lumps and possibly breast cancer, in asymptomatic women in rural areas.
Expert CBE is known to have high specificity (92-94%), meaning low false-positive rate. In the study, there were 2 cases that were positive on iBE and negative on CBE, while there were 1,139 cases that were negative on both tests. In this regard, iBE demonstrated a low false positive rate, comparable to that of Expert CBE.
Screening mammograms in USA have shown sensitivity as low as 54% to detect invasive breast cancer, especially if the women are young and have dense breasts. [4][5][6] Clinical study by Broach et al reported iBreastExam sensitivity of 86% and specificity of 89% to detect breast masses as compared to standard of care imaging tests such as mammography and breast ultrasound. 13 to standard imaging tests. 15 iBreastExam has reported comparable performance characteristics to standard of care imaging viz. screening mammography.
iBE was well accepted and used with comfort by the women in the study. It was found that it is easier to convince women to take iBE test against mammography on account of pain and radiation associated with mammography. Clinical breast exam is a subjective manual test in comparison with iBE, which is a standardized device based test which eliminates human subjectivity while performing breast examinations.
Conclusion
As a hand-held, radiation-free, painless and easy to use electromechanical tool, iBreastExam can potentially be a powerful screening tool for use in the developing world with limited resources where mammography and CBE by a trained physician is not readily available. From care pathway standpoint, Positive findings by the iBE can allow for triaged patients to be examined by targeted breast ultrasound at primary care level and before referring them to a larger medical centres where FNAC/biopsy as well as surgical excision could be performed. Training lay people on the use of the device could potentially provide screening to rural and underserved areas of underdeveloped nations where women historically have had no access to breast cancer early detection.
The iBE device is currently being piloted in a rural area in India where due to limited economic resources, women have essentially no access to mammography and present with late-stage breast cancers. As described in the literature, late-stage breast cancers have poorer prognosis than early stage breast cancers. 16 Due to the significantly lower cost of the iBE compared to conventional mammography, we see this device as a very useful adjunct to breast cancer detection worldwide in low-and-middle income countries (LMICs) with limited medical economic resources and a need for improved breast cancer early diagnosis. | 2019-03-18T14:03:48.455Z | 2018-06-20T00:00:00.000 | {
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265208476 | pes2o/s2orc | v3-fos-license | Comparative Efficacy and Safety of Statin Monotherapy and Statin plus Ezetimibe Combination in a Real-World Setting
Background: The objective of this study was to conduct a comparative evaluation of the effectiveness of ezetimibe in combination with statins or statin monotherapy in patients with hypercholesterolemia in a real-world setting. Methods: It was a retrospective multicenter observational study conducted in Russia. We included patients who received statins or a combination of statins with ezetimibe for ≥3 months. The primary endpoint of this study was the frequency of achieving low-density lipoprotein cholesterol (LDL-C) goal levels at the time of enrollment in the study (%). Results: The full analysis set consisted of 1000 patients: 250 subjects in the statin monotherapy group and 750 subjects in the combination group. The groups did not differ in clinical, demographic, or laboratory variables, except for a higher prevalence of hypertension and higher baseline lipid values in the statin monotherapy group. During treatment, the LDL-C concentration decreased by 1.10 ± 1.04 mmol/L (change of −27.5 ± 28.5% from baseline) in the statin monotherapy group and by 1.55 ± 1.17 mmol/L (change of −38.2 ± 25.6% from baseline) in the combination therapy group, p < 0.001. The target LDL-C level was achieved in 22.4% of the patients in the monotherapy group compared with 28.8% of the patients in the combination therapy group, p = 0.049. Conclusions: In real-world clinical practice, statin/ezetimibe combination therapy demonstrated a more frequent achievement of target LDL-C levels compared with statin monotherapy. The addition of ezetimibe to statin therapy increased the probability of achieving LDL-C level goals by 29%.
Introduction
Cardiovascular disease (CVD) remains one of the leading causes of death in many countries.Hypercholesterolemia is the main causal atherosclerosis risk factor that is poorly controlled worldwide [1].The first and main option for low-density lipoprotein cholesterol (LDL-C) control is a statin at a maximally tolerated dose.However, numerous studies demonstrated the low frequency of LDL-C goal attainment with statin monotherapy for high and very high cardiovascular risk persons.The use of ezetimibe in combination with a statin is a more effective choice for a significant decrease in LDL-C concentration [2].One large randomized clinical trial demonstrated a significant cardiovascular outcomes reduction with statin plus ezetimibe compared to statin-alone treatment in patients after acute coronary syndrome [3].These data allowed for providing the IA class and level of evidence for ezetimibe treatment for LDL-C goal achievement [2].Despite these guidelines, recent large cohort studies showed the low frequency of combination therapy in real practice in European countries [4,5].The latest paradigm is to start treatment of very high risk subjects with a combination of statin with ezetimibe [6].
Russia is on the list of very high cardiovascular risk countries due to a high population level of cardiovascular mortality [7].One of the reasons for the poor statistics is the high prevalence of lipid disorders among adults [8].Moreover, most people are unaware about their health status and risk factors profile, including the presence of elevated total cholesterol [9].Poor adherence to statins is a worldwide problem and another reason for the non-achievement of LDL-C goals.Ezetimibe combined with any statin should be considered as an essential step for the better treatment of patients with a high probability of cardiovascular events development [10].A large simulation study of six countries including Russia provided confirmation that both the free-and fixed-dose statin plus ezetimibe combination will substantially allow for the prevention of major adverse cardiovascular events [11].
The results obtained in randomized clinical trials may differ from real-world clinical practice.In this regard, post-marketing clinical studies are necessary to obtain the efficacy and safety of lipid-lowering therapy in the real world.Any new data obtained from different countries should enlarge the body of evidence for the best clinical practice.
The objective of this study was a comparative evaluation of the effectiveness and safety of ezetimibe in combination with statins or a statin monotherapy in real clinical settings in Russia.
Study Setting
This study was conducted at clinical sites involved in the routine management of patients with dyslipidemia.After a preliminary assessment of 109 clinical sites, 48 centers in various regions of the Russian Federation agreed to participate in this study.
Participants
During the period from 29 June 2021 to 25 November 2021, all the study sites included 1000 patients.The inclusion criteria were as follows: 1. Age > 18 years.2. Lipid-lowering therapy with a statin or statin in combination with ezetimibe in a stable dosing regimen for three or more months and no more than two years before the study enrolment.4. A willingness and ability to sign an informed consent form to participate in the study.5.The availability of the primary medical documentation, which allows for an assessment of all the parameters necessary for this study from the moment of initiating lipid-lowering treatment.
This study did not include patients with established or suspected familial hypercholesterolemia, intake of fibrates and omega-3 polyunsaturated fatty acids, and treatment with methods of extracorporeal filtration and/or plasmapheresis.Clinically significant hepatic and/or renal impairment, hypothyroidism, ezetimibe monotherapy, and statin intolerance at any dose were also exclusion criteria.All drugs were prescribed in accordance with the recommendations given by the attending physician as a part of routine clinical practice and in accordance with the routine practice of the study site.All therapy was administered prior to data collection and prior to enrollment in this study.There were no study dropouts due to the retrospective design.All the patients signed an informed consent form.All the patients completed this study.
Study Design
The UNISON study is a retrospective observational study of the efficacy and safety of statin monotherapy or a statin in combination with ezetimibe in patients with hypercholesterolemia. Considering a retrospective observational study design, which meant only the collection of data contained in medical documentation, no specific procedures for the patients were conducted, except for signing the informed consent form.Before signing the informed consent form, the study physician evaluated the criteria eligibility for this study.The information from the outpatient medical record was evaluated from the initiation of lipid-lowering therapy (3-24 months prior to the enrollment) up to the date of enrollment (Visit 1).The data collected from the medical records were recorded in the electronic case report form (eCRF) by the investigators.
Ethical Approval
This study was organized in accordance with the principles of good clinical practice, the Declaration of Helsinki, and applicable to local regulations.This study was initiated and conducted by the Russian National Atherosclerosis Society (RNAS) and the Contract Research Organization Ligand Research under the sponsorship of JSC AKRIKHIN.Every patient was informed about all aspects of this study and had an opportunity to have any study-related question answered before signing the informed consent form.This study was approved by the Independent Interdisciplinary Committee for ethical expertise of clinical trials (Moscow, Russia), protocol #7 dated 23 April 2021.This study was registered at ClinicalTrials.gov(NCT04895098).
Study Procedures
The assessments for the patients provided routine demographic data, including age, sex, height, body weight, waist circumference, family history of CVD, personal CVD and its duration, prior revascularization procedures, concomitant diseases, and current therapy, including lipid-lowering drugs.The following parameters of the lipid profile were analyzed at baseline and over time: total cholesterol, TG, HDL-C, and LDL-C.Also, at baseline and over time, the safety of lipid-lowering therapy was determined based on the levels of transaminases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)), as well as the level of creatine kinase (CK).
Information about adverse drug reactions (ADRs) was collected as a part of the routine practice of the doctor.For the safety analysis, the data specified in the outpatient records or other patient records were used.ADRs were coded according to the Medical Dictionary for Regulatory Activities (MedDRA).
Endpoints
The primary endpoint of this study was the frequency of achieving the target levels of LDL-C (in accordance with the recent European dyslipidemia guidelines) at the time of enrollment in the study (%) [2].The secondary endpoints included the mean change in LDL-C from the start of statin therapy to the study enrollment (absolute difference and percent change from baseline); the mean change in the total cholesterol level from the start of statin therapy to enrollment in the study (percent change and absolute difference); the mean change in the HDL-C level from the start of statin therapy to enrollment in the study (percent change and absolute difference); and the mean change in the TG level from the start of statin therapy to enrollment in the study (percent change and absolute difference).
Statistical Methods
The data of all the patients included in this study were entered into an electronic database via an electronic statistical analysis, which was carried out using R version 4.1.2(The R Foundation for Statistical Computing, Vienna, Austria).Descriptive statistics were used, and the following values were calculated: the arithmetic mean, 95% confidence interval (CI) for the mean (unless otherwise indicated), standard deviation, median, interquartile range, and minimum and maximum for continuous data.The nominal/discrete data were processed by calculating the proportion of the absolute number of observations.The Shapiro-Wilk test was used to assess the normality of the distribution of the quantitative data.The qualitative data were presented as the absolute frequencies (number of observations), proportions (percentages), and 95% confidence interval (CI) (unless otherwise indicated).The discrete data were compared across the treatment groups using the chi-squared test/Fisher exact test, and the continuous data were compared using the unpaired Student t-test for normally distributed data or the non-parametric Mann-Whitney test for non-normally distributed data.Comparisons with set levels were performed using the Wilcoxon test.All the tests were two-sided and performed at a 5% level of significance.In general, the substitution of missing data was not performed, i.e., missing data were not replaced by the calculation or creation of new data but were handled as 'missing' in the statistical evaluation.
Study Population
The full analysis set consisted of 1000 patients (100%): 250 participants in the statin monotherapy group and 750 in the ezetimibe plus statin therapy group.Due to the retrospective design, there were no patient dropouts and protocol deviations.The groups did not differ in clinical, demographic, or laboratory variables, except for a higher prevalence of hypertension and higher baseline lipid values in the statin monotherapy group.(Table 1).At the enrollment, most patients were receiving atorvastatin (54.2%) and rosuvastatin (41.1%) (Figure 1).
HDL Cholesterol
During therapy, there was a slight increase in HDL-C: by 0.020 ± 0.376 mm statin monotherapy group (+5.0 ± 33.1% from baseline) and by 0.036 ± 0.397 m ± 38.5% from baseline) in the combination therapy group, p < 0.001.
LDL-C
During treatment, the LDL-C concentration decreased by 1.10 ± 1.04 mmol/L (change of −27.5 ± 28.5% from baseline) in the statin monotherapy group and by 1.55 ± 1.17 mmol/L (change of −38.2 ± 25.6% from baseline) in the combination therapy group, p < 0.001.
Total Cholesterol
The total cholesterol reduction was 1.25 ± 1.12 mmol/L (change of −20.4 ± 19.0% from baseline) in the statin monotherapy group and 1.76 ± 1.27 mmol/L (change of −27.7 ± 18.0% from baseline) in the combination therapy group, p < 0.001.
Target Achievement in CV Risk Differentiated Subgroups
An analysis of the subgroups formed based on the level of cardiovascular risk and the intensity of statin therapy revealed that the LDL-C target achievement proportion in patients at high cardiovascular risk, who received high-intensity statin therapy, was higher in the subgroup of patients treated with ezetimibe and statins (32% vs. 25% in the statin monotherapy group, p = 0.492), while these proportions in the subgroup of patients at very high risk were 24% and 16%, respectively (p = 0.059).Also, the target levels were more frequently achieved in the subgroup of patients at very high risk, who received moderate-intensity statin therapy: 33% in the ezetimibe plus statin group vs. 15% in the statin monotherapy group (p = 0.035).This finding was not confirmed in the other subgroups due to the small sample sizes (Table 2).
Target Achievement in CV Risk Differentiated Subgroups
An analysis of the subgroups formed based on the level of cardiovascular risk and the intensity of statin therapy revealed that the LDL-C target achievement proportion in patients at high cardiovascular risk, who received high-intensity statin therapy, was higher in the subgroup of patients treated with ezetimibe and statins (32% vs. 25% in the statin monotherapy group, p = 0.492), while these proportions in the subgroup of patients at very high risk were 24% and 16%, respectively (p = 0.059).Also, the target levels were more frequently achieved in the subgroup of patients at very high risk, who received moderate-intensity statin therapy: 33% in the ezetimibe plus statin group vs. 15% in the statin monotherapy group (p = 0.035).This finding was not confirmed in the other subgroups due to the small sample sizes (Table 2).
Target Achievement in CV Risk Differentiated Subgroups
An analysis of the subgroups formed based on the level of cardiovascular risk and the intensity of statin therapy revealed that the LDL-C target achievement proportion in patients at high cardiovascular risk, who received high-intensity statin therapy, was higher in the subgroup of patients treated with ezetimibe and statins (32% vs. 25% in the statin monotherapy group, p = 0.492), while these proportions in the subgroup of patients at very high risk were 24% and 16%, respectively (p = 0.059).Also, the target levels were more frequently achieved in the subgroup of patients at very high risk, who received moderate-intensity statin therapy: 33% in the ezetimibe plus statin group vs. 15% in the statin monotherapy group (p = 0.035).This finding was not confirmed in the other subgroups due to the small sample sizes (Table 2).
Safety
During the study, 17 adverse events (AEs) were reported in 16/1000 (1.6%) patients.Of these, two (33%) events in the monotherapy group and six (55%) events in the combination therapy group were mild.Three (50%) AEs in the former group and four (36%) AEs in the latter group were moderate.One severe AE was observed in each of the groups: one (17%) and one (9%), respectively.
Discussion
This is the first large-scale study of the use of ezetimibe in real-world clinical practice in Russia aimed to analyze the achievement of the new LDL-C targets specified in the latest clinical guidelines.This multicenter, retrospective, comparative, observational study of the efficacy and safety of statin monotherapy and the combination of statins with ezetimibe demonstrated that the LDL-C target achievement rate in the statin monotherapy group was only 22.4%.Combination therapy with statins and ezetimibe is associated with a more frequent achievement of LDL-C targets with good tolerability and no increase in the incidence of adverse events.Our data are very similar to the European DA VINCI study that showed that high-intensity statin monotherapy in very high risk primary and secondary prevention patients allowed for achieving LDL-C targets in 17% and 22%, respectively [4].
Several real-world studies in different countries demonstrated the comparative advantage of ezetimibe added to statins to reach LDL-C target levels [12][13][14][15].In a Chinese retrospective cohort study of 1727 ASCVD patients, the addition of ezetimibe to a statin produced the achievement rates of LDL-C below 1.8 and 1.4 mmol/L over the first year, as high as 50.6 and 25.6%, respectively.However, in the second and third follow-up years, these rates decreased to 31.3, 30.3% and 15.5, 16.5%, respectively.The multivariable analysis showed that male sex, the combined use of atorvastatin or rosuvastatin with ezetimibe, better adherence, and smoking cessation were associated with a higher achievement rate [12].A Spanish retrospective, observational study included 1570 ASCVD patients.They were treated with ezetimibe combined with atorvastatin (47.8%) or rosuvastatin (52.2%) in a high-intensity regimen.Despite these combinations, LDL-C below 1.4 mmol/L was not reached in about 70% of the participants [13].The Korean retrospective study analyzed electronic medical records from 4252 patients treated between 2015 and 2017 in two tertiary care medical centers.Only those who switched to the statin/ezetimibe combination after statin monotherapy were enrolled.Enhancing the lipid-lowering therapy provided additional significant LDL-C level reduction by 31-41% depending on statin intensity and the achievement of the LDL-C levels < 1.8 mmol/L in about 90% of the subjects.A subgroup analysis was performed of the better efficacy of rosuvastatin/ezetimibe than the atorvastatin/ezetimibe combination within the same statin intensity [14].A retrospective analysis of the electronic medical records of 311,242 very high risk outpatients showed that the addition of ezetimibe in patients already prescribed a statin reduced LDL-C by an additional 24%, with a greater reduction by 28% with a fixed dose compared to a free combination (19.4%; p < 0.0001) with the achievement of an LDL-C level of <1.8 mmol/L in 31% and 21% of the cases, respectively [15].Even the latest data from an observational, prospective SANTORINI study of around 9000 patients at high or very high risk between 2020 and 2021 in 14 European countries documented that the landscape of lipid-lowering therapy remained unchanged [5].Considering that 24% were on combination therapy, 54% were receiving statins monotherapy, and 22% were not treated at all, the current European LDL-C goals were achieved only in each fifth subject [5].
The efficacy in terms of lipid metabolism, tolerability, and the safety of ezetimibe, both in monotherapy and in combination with statins, including the fixed-dose combination of simvastatin or rosuvastatin or atorvastatin at different doses, was extensively evaluated in randomized clinical studies.Combination therapy with ezetimibe and atorvastatin at a dose of 10 mg was associated with a 53% reduction in LDL-C (comparable with the effect of atorvastatin monotherapy at a dose of 80 mg, 54%) [16].The addition of ezetimibe to rosuvastatin 40 mg resulted in a 70% reduction in LDL-C (compared to a 57% reduction in LDL-C in the rosuvastatin 40 mg group) [10].The addition of ezetimibe to ongoing statin therapy for 6 weeks resulted in an additional 25% decrease in LDL-C, while the decrease in patients who received a placebo and continued statin therapy was only 3.7% [17].
The data from an 8-week, double-blind, multicenter, randomized, controlled phase III study (I-ROSETTE) showed that the average change in the level of LDL-C in all groups of combination therapy with rosuvastatin plus ezetimibe was more than 50%.The number of patients who had achieved LDL-C targets at week 8 was significantly greater in the ezetimibe plus rosuvastatin group (180 (92.3%) of 195 patients) than in the rosuvastatin monotherapy group (155 (79.9%) of 194 patients) (p < 0.001) [18].Thus, combination therapy with statins and ezetimibe provides an additional 18-25% reduction in LDL-C and significantly increases the number of patients achieving the LDL-C target.
In contrast to the fixed-dose combinations of statins and ezetimibe, ezetimibe has an advantage of its possible addition to any statin at any dose.In our study, the patients treated with combination therapy were significantly more likely to achieve the target lipid levels, 28.8% vs. 22.4% in the monotherapy group, and the LDL-C concentration decreased more significantly in the combination therapy group.
The results of the UNISON study add new information to the available evidence from randomized studies and real-world settings and confirmed a rather low LDL-C target achievement rate in patients with high and very high cardiovascular risk.The study demonstrated superior efficacy of combination therapy with statins and ezetimibe compared with statin monotherapy in terms of more patients achieving target levels of LDL-C.These conclusions are supported by an additional analysis, which demonstrated that the addition of ezetimibe to both high-intensity statin therapy and moderate-intensity statin therapy led to statistically significant results in achieving lipid targets, changes over time, and a percent reduction in LDL-C.Combination therapy was more effective when used in patients with very high cardiovascular risk.It should also be mentioned that the addition of ezetimibe to the therapy did not increase the risk of adverse reactions (the proportion of adverse reactions was the same in both groups).Meanwhile, our study provides important and additional information received from almost 50 clinical centers in Russia and from thousands of patients that statins with ezetimibe represent the optimal paradigm for improving the treatment of LDL-C.
Study Limitations
Our study has several limitations.First, it has a retrospective design with the inclusion of patients receiving statin monotherapy and a combination of various statins with ezetimibe in a ratio of 1:3, with a total number of 1000 treated patients.We have data from a randomized controlled trial, according to which the addition of ezetimibe to a statin improves the clinical outcomes of patients with coronary heart disease due to better control of LDL-C levels [3].However, there is not enough data from the real world.Most studies evaluating the benefits of ezetimibe in addition to statins have also been retrospective [12][13][14][15].Insufficient use of ezetimibe in many countries depends more on the inertia of physicians and the resistance of patients to current methods of treatment of dyslipidemia.Second, because the retrospective data from patient records were analyzed, there was no control over the patients' adherence to therapy.It was shown that in the case of the free combination, the chance of missing tablets of either the statin or ezetimibe is increased [15].If some of our patients on combination therapy had low adherence to it, the real number of those on LDL-C targets could be higher.Third, several patients had non-optimal starting doses of statins that were not adjusted in a timely manner.But again, this fact demonstrates that in routine practice many physicians have concerns regarding maximal doses of statins.The DA VINCI study also provides evidence that high-intensity statins are used only 22% and 42% in primary secondary prevention groups, respectively [4].Considering the similarity in the results of the DA VINCI and UNISON studies, we recognize that there is significant room for improvement in high and very high cardiovascular patients' management both in Europe and in Russia.At the same time, because of the much higher cardiovascular morbidity and mortality in Russia, greater efforts should be provided by our healthcare system to increase both general practitioners' and their patients' awareness and education on the current opportunities of hypercholesterolemia management.If the free combination of a statin with ezetimibe will be used widely and in accordance with the current dyslipidemia guidelines, approximately 342,000 major adverse cardiovascular events may be prevented in Russia over 5 years compared with the continuation of clinical practice as of the beginning of 2020 [11].
Despite all the limitations, our study provides new and valuable information on the effectiveness and safety of statins in combination with ezetimibe therapy in a real-world setting.
Conclusions
In real-world clinical practice, statin/ezetimibe combination therapy demonstrates more frequent achievement of target LDL-C levels compared with statin monotherapy.The addition of ezetimibe to statin therapy increases the probability of achieving LDL-C goals by 29%.
Figure 1 .
Figure 1.Treatment regimen in the study.
3. 2 . 1 .
Achievement of the LDL-C Target The primary efficacy endpoint was the frequency of achieving the target LDL-C (according to the 2019 ESC/EAS Guidelines for the management of dyslipidemia a as local guidelines) at the time of study enrollment.The LDL-C target was achie 22.4% (56/250) of the patients [95% CI 17.4-28.1]in the monotherapy group versus of the patients (216/750) [95% CI 25.6-32.2] in the combination therapy group (Fig The differences between the groups in the frequency of achieving the LDL-C targe statistically significant: OR 0.714 (0.499-1.009), p = 0.049.
Figure 1 .
Figure 1.Treatment regimen in the study.
3. 2 .
Efficacy of Treatment, FAS (Full Analysis Set) Population 3.2.1.Achievement of the LDL-C Target The primary efficacy endpoint was the frequency of achieving the target LDL-C levels (according to the 2019 ESC/EAS Guidelines for the management of dyslipidemia as well as local guidelines) at the time of study enrollment.The LDL-C target was achieved in 22.4% (56/250) of the patients [95% CI 17.4-28.1]in the monotherapy group versus 28.8% of the patients (216/750) [95% CI 25.6-32.2] in the combination therapy group (Figure2).The differences between the groups in the frequency of achieving the LDL-C target were statistically significant: OR 0.714 (0.499-1.009), p = 0.049.
Figure 3 .
Figure 3. Lipid profile changes in the treatment groups, % change from baseline.
Figure 4 .
Figure 4. Lipid profile changes from baseline in the treatment groups, mmol/L.
Figure 3 .
Figure 3. Lipid profile changes in the treatment groups, % change from baseline.
Figure 3 .
Figure 3. Lipid profile changes in the treatment groups, % change from baseline.
Figure 4 .
Figure 4. Lipid profile changes from baseline in the treatment groups, mmol/L.
Figure 4 .
Figure 4. Lipid profile changes from baseline in the treatment groups, mmol/L.
Table 2 .
LDL-cholesterol level goals achievement depending on risk category and treatment regimen. | 2023-11-15T16:12:02.854Z | 2023-11-13T00:00:00.000 | {
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228612271 | pes2o/s2orc | v3-fos-license | Identification of Driver Genes Regulating the T-Cell–Infiltrating Levels in Hepatocellular Carcinoma
The driver genes regulating T-cell infiltration are important for understanding immune-escape mechanisms and developing more effective immunotherapy. However, researches in this field have rarely been reported in hepatocellular carcinoma (HCC). In the present study, we identified cancer driver genes triggered by copy number alterations such as CDKN2B, MYC, TSC1, TP53, and GSK3B. The T-cell infiltration levels were significantly decreased in both HCC and recurrent HCC tissues compared with the adjacent normal liver tissues. Remarkably, we identified that copy number losses of MAX and TP53 were candidate driver genes that significantly suppress T-cell infiltration in HCC. Accordingly, their downstream oncogenic pathway, cell cycle, was significantly activated in the low T-cell infiltration HCC. Moreover, the chemokine-related target genes by TP53, which played key roles in T-cell recruitment, were also downregulated in HCC with TP53/MAX deletions, suggesting that copy number losses in MAX and TP53 might result in T-cell depletion in HCC via downregulating chemokines. Clinically, the T-cell infiltration levels and chemokines activity could accurately predict the response of sorafenib, and the prognostic outcomes in HCC. In conclusion, the systematic analysis not only facilitates identification of driver genes and signaling pathways involved in T-cell infiltration and immune escape, but also gains more insights into the functional roles of T cells in HCC.
INTRODUCTION
Hepatocellular carcinoma (HCC) is a highly aggressive cancer with an increasing incidence, accounting for the majority of all primary liver cancer cases (Kung-Chun Chiu et al., 2020). Prior hepatitis B and/or hepatitis C infection is often considered a major risk factor of HCC, along with alcohol consumption, tobacco use, and obesity (Grandhi et al., 2016). Clinically, limited early symptoms in HCC patients could lead to delayed diagnoses, which greatly undermines the survival outcomes of HCC patients (Sun et al., 2020). For patients with advanced stage HCC, whose conditions are usually not suitable for surgical resection, immunotherapies are considered as an effective and promising strategy (Caraballo Galva et al., 2020).
The association between a dysregulated immune system and the development of HCC has been demonstrated in many studies, and changes in the abundance or function of tumor-related immune cells, such as self-reactive cytotoxic T cells, CD4 + T cells, regulatory T cells, and natural killer (NK) cells, are observed in HCC (Ding et al., 2018). T lymphocytes are among the most critical players in the inhibition of tumor cells (Thompson et al., 2010). It has been reported that cytotoxic T lymphocytes and CD4 + T cells could affect antigen recognition and DNA-based immunization (Grimm et al., 2000). To our knowledge, tumorinfiltrating lymphocytes are an essential component of tumor microenvironment (TME), and a previous study has assessed the clinical significance of tumor-infiltrating NK cells in HCC, successfully relating NK cell abundances to several immune checkpoint proteins (Wu et al., 2020).
As higher T lymphocyte-infiltrating rates are considered to be associated with favorable prognoses in many cancers (Chaoul et al., 2020), it is critical to explore what potentially drives T-lymphocyte infiltration in HCC patients. Notably, genomic aberrations, such as copy number alterations (CNAs) and neoantigen load, are also observed to be associated with immune infiltration in other cancers (Safonov et al., 2017). As new computational methods have enabled the identification and interpretation of cancer driver genes at multi-omics, it is now possible to explore mechanism behind CNA-related cancer driver genes and the T-lymphocyte infiltration and to examine the association between different T lymphocytes infiltrating levels and response to antitumoral drugs and their impacts on HCC prognosis. Therefore, in order to identify the CNA-triggered driver genes and unveil their underlying molecular mechanisms involved in T-cell infiltration, we conducted a systematic data analysis and anticipated to identify the driver genes associated with T-cell infiltration, and link them to drug treatment response and prognostic outcomes.
Data Acquisition
The discovery datasets of gene expression and CNAs in The Cancer Genome Atlas (TCGA) cohort were collected from UCSC Xena database (Goldman et al., 2019), which curated preprocessed TCGA data including genomics, transcriptome, and proteome. Moreover, we also downloaded normalized gene expression data of GSE109211 (Pinyol et al., 2019), GSE56545 (Xue et al., 2015), and GSE14520 (Roessler et al., 2010) from Gene Expression Omnibus (GEO) database to evaluate the associations of T-cell infiltration levels with recurrence, response of sorafenib treatments, and prognostic outcomes. The normalized gene expression data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) HCC cohorts were collected from previous study (Gao et al., 2019). We also collected 242 well-known driver genes including 127 oncogenes and 115 tumor suppressors from previous study (Mayakonda et al., 2018).
The Correlation Analysis of CNAs and Gene Expression Levels
The segmented CNAs were used as the input of GISTIC v2.0 (Mermel et al., 2011) (Genomic Identification of Significant Targets in Cancer). The recurrent CNAs identified by GISTIC were used for the correlation analysis. For each gene, Pearson correlation analysis was conducted on the log2 copy number ratio and log2-FPKM (fragment per kilobase of exon model per million reads mapped)-based gene expression. Pearson correlation coefficient of 0.4 was used as cutoff threshold to determine whether the gene expression was triggered by the CNAs. The resulting driver genes should also be curated in the 242 well-known driver genes.
The Infiltration Levels of T Cells and Cell Cycle or Chemokine Activity
The T-cell-specific marker genes, marker genes of the seven-step cancer-immunity cycle, and gene expression data of HCC cell lines were collected from previous studies (Zheng et al., 2017;Xu et al., 2018;Qiu et al., 2019). Subsequently, the T-cell-specific marker genes were refined by excluding those with expression levels higher than 1 FPKM in HCC cell lines. We conducted single-sample gene set enrichment analysis (ssGSEA) to estimate the T-cell infiltration, cell cycle, and chemokine activity by their signature genes. The ssGSEA is a rank-based method to measure the relative abundance for a gene list of interest and has been widely used to estimate immune-cell infiltration and validated in a series of in vitro and in silico tests by previous studies (Senbabaoglu et al., 2016;Xue et al., 2019;Yu et al., 2019;Zuo et al., 2020). The ssGSEA was implemented in R GSVA v1.36.2 package (Hanzelmann et al., 2013) with gsva function.
The Anticancer Immune Response of Cancer-Immunity Cycle First, we collected signature genes from previous study , which defined the cancer-immunity cycle as seven steps of anticancer response. Second, we selected T cell-related gene signatures (Supplementary Table S2) and estimated the anticancer activity of the seven steps by ssGSEA.
Gene Set Enrichment Analysis
The GSEA was conducted against Reactome pathways (Jassal et al., 2020) on the preranked gene set based on the t statistics, which were calculated by comparing the samples of high T-cell infiltration with those of low T-cell infiltration. The GSEA was implemented in R fgsea v1.14.0 package with fgsea function.
Principal Component Analysis and Support Vector Machine Model
The principal component analysis (PCA) and support vector machine (SVM) were conducted on the infiltrating levels of the five T cells. The PCA was implemented and visualized in R FactoMineR and factoextra packages. The R e1071 and ROCR package were employed to build the SVM model (Sing et al., 2005), visualize the receiver operating characteristic (ROC) curves, and calculate the area under the curve (AUC) values.
Survival Analysis
The infiltrating levels of the five T cells and chemokine activity were used as the predictors of the overall survival (OS). The chemokine activity was estimated by the ssGSEA. The Cox proportional hazards regression model was employed to perform univariate and multivariate analyses for OS. A multivariable Cox model was built based on the infiltrating levels of T cells and chemokine activity, which were selected by stepwise method. The Cox model was trained in TCGA cohort and used to predict the risk scores for the samples from GSE14520 and CPTAC HCC cohorts using the corresponding T-cellinfiltrating levels and chemokine activities. The median value of the risk scores was used as cutoff thresholds to plot the KM curves, and the statistical significance was evaluated by the log-rank test.
Statistical Analyses
All the statistical analyses were performed in R (version 4.0.0). The Wilcoxon rank-sum test or t-test was employed to compare the means of the two samples. Multiple sample comparison was tested by analysis of variance (ANOVA). The symbols of * , * * , * * * , * * * * represent the statistical significance at 0.05, 0.01, 0.001, and 0.0001, respectively.
Discovery of Driver Genes Associated With T-Cell Infiltration
To discover the driver genes associated with T-cell infiltration, we first estimated the infiltrating levels of five representative T cells including cytotoxic CD4 cells, effector memory CD8 + T cells, naive CD4 + T cells, mucosal-associated invariant T (MAIT) cells, and naive CD8 + T cells for the TCGA and GSE56545 samples by ssGSEA. Specifically, the normal tissues had significantly higher T-cell-infiltrating levels than the both primary and recurrent tumor tissues (Figure 2A, Wilcoxon rank-sum test, P < 0.05). Moreover, we also found that the T-cell-infiltrating levels were significantly lower in recurrent tissues compared with the primary tissues ( Figure 2B, P < 0.05). In addition, we observed that the infiltrating levels of cytotoxic CD4 + and effector memory CD8 + cells were higher in stage I and were decreased with the degree of the disease stage ( Figure 2C). Exceptionally, naive CD4/CD8 and MAIT cells were observed to be highly infiltrated into stage IV tumors, but these cells did not have anticancer effects (Supplementary Figure 1A). These results indicated that the T-cell-infiltrating levels were significantly lower in primary HCC and reduced in recurrent and advanced-stage HCC as compared with the primary tumors.
With the infiltrating levels, we classified the TCGA HCC samples into three subgroups, with high, intermediate, and low T-cell-infiltrating levels by hierarchical clustering analysis (Figure 2D), respectively. The comparative analysis of the 19 driver genes in the samples with distinct levels of T-cell infiltration or copy numbers of driver genes revealed that only two genes, TP53 and MAX, were significantly downregulated in samples with either low T-cell infiltration or deletion of the corresponding genes ( Figure 2E, ANOVA and textitt-test, P < 0.0001). Consistently, the two genes were also observed to be downregulated in CPTAC HCC samples with CNV loss of TP53 or MAX (Figure 2F, P < 0.0001). These results indicated that the TP53 and MAX might be the candidate driver genes that regulate the T-cell infiltration.
Signaling Pathways Regulating the Infiltrating Levels of T Cells in HCC
To further investigate the signaling pathways that potentially regulate the T cells infiltrating into the HCC tissues, we compared the gene expression profiles of low T-cell infiltration subgroup with those of the high T-cell infiltration subgroup. The GSEA revealed that cell cycle progression-related pathways including mitotic prophase, mitotic prometaphase, S phase, DNA replication, G2/M DNA damage checkpoint, G2/M Checkpoints, and cell cycle checkpoints were significantly upregulated in low T-cell infiltration subgroup, whereas the activities of TCR signaling, interleukin-1 signaling, and interleukin-1 family signaling were observed higher in high T-cell infiltration subgroup (Figure 3A, Benjamini and Hochberg adjusted P < 0.05). Moreover, as TP53 and MAX were involved in regulating the cell cycle checkpoint, the activity of cell cycle pathway was significantly enhanced in both TCGA and CPTAC HCC samples with TP53 or MAX deletions ( Figure 3B, P < 0.0001). Furthermore, we also conducted ssGSEA to estimate the T-cell-mediated anticancer activity of the seven steps based on the signature genes by previous study and found that high T-cell infiltration subgroup had higher activities of all the seven steps than the low T-cell infiltration subgroup (Figure 3C), indicating that the reduced anticancer activity mediated by T-cell infiltration in HCC might be caused by a systematic dysfunction of the entire process.
Furthermore, as the immune-cell recruitment might be regulated by the chemokines secreted by the tumor cells in TME (Chow and Luster, 2014), we then investigated whether CNV loss of TP53 and MAX could regulate the expression of the cytokines. We then collected 14 genes encoding chemokines including CCR7, CCL3, CCL4, CCL19, CCL21, CXCL10, CXCL11, CCL7, CXCL1, CXCR4, CCL1, CCL17, CCR4, and CCL28, which were also transcriptionally regulated by the transcription factors involved in cell cycle such as TP53 and MAX, and found that these genes were highly enriched in the downregulated genes in HCC with TP53/MAX deletions (Figures 4A,B; false discovery rate (FDR) < 0.05), indicating that the CNV loss in TP53/MAX might downregulate their target genes, thereby weakening the immune-cell recruitment.
T-Cell-Infiltrating Levels and Chemokines Are Associated With Treatment Response in HCC
As the immune cells infiltrating into tumor tissues could enhance the chemotherapy response (Rusakiewicz et al., 2013;Hugo et al., 2015;Cai et al., 2019), we then investigated whether the two driver genes, T-cell-infiltrating levels and chemokines, were also associated with treatment response in HCC. We collected another gene expression dataset with sorafenib treatment response (GEO accession: GSE109211). As expected, TP53 and MAX were expressed higher in responders than nonresponders (Supplementary Figure 1B, P < 0.0001). Similarly, we estimated the infiltrating levels of five T cells and chemokine activities for the HCC samples by ssGSEA and corresponding signature genes and built two SVM models using the T-cell-infiltrating levels and chemokine activity to predict the response of sorafenib treatment, respectively. The infiltrating levels of the five T cells were higher in the responders than nonresponders (Figure 5A). The ROC curves revealed that the T-cell infiltration could accurately predict the response of both sorafenib treatment with AUC values of 0.92 (Figure 5B), respectively. Moreover, we also observed chemokine activity was higher in HCC responders than nonresponders (Figure 5C, t-test, P < 0.0001). The ROC analysis revealed that chemokine activity could also predict the response of sorafenib treatment in HCC Frontiers in Genetics | www.frontiersin.org at a high performance (Figure 5D, AUC = 0.86), indicating that chemokine activity might be associated with sorafenib sensitivity in HCC. These results indicated that the T-cell infiltration and chemokine activity in HCC could predict the response of sorafenib treatment in HCC.
The Prognostic Significance of T-Cell-Infiltrating Levels and Cell Cycle Activity in HCC
To systematically evaluate the prognostic value of the T-cellinfiltrating levels, chemokine activity, and the two driver genes, TP53 and MAX, in HCC, we tested their association with OS in TCGA, GSE14520, and CPTAC cohorts (Gao et al., 2019). First, the cytotoxic CD4 cells, effector memory CD8 + T cells, MAIT cells, and chemokine activity were selected as the best combination for risk prediction by the stepwise method, and we built the multivariable Cox model based on the T-cell-infiltrating levels and chemokine activity in TCGA cohort ( Table 1). The high-risk group had significantly shorter OS than the low-risk group ( Figure 6A, P < 0.0001). Moreover, the risk scores of samples from the two testing cohorts, GSE14520 and CPTAC, were predicted based on the trained model and corresponding T-cell-infiltrating levels and cell cycle activity. Consistently, the OS in high-risk group was still worse than the low-risk group in both of the cohorts (Figures 6B,C; P < 0.05). With this sample stratification, the differences of recurrence-free survival (RFS) between the high-and low-risk groups were also tested. The RFS was also observed shorter in high-risk group than the low-risk group of the two testing cohorts (Figures 6D,E; P < 0.05). These results indicated that the T-cell-infiltrating levels and cell cycle activity could serve as potential prognostic biomarkers in HCC.
DISCUSSION
T-cell infiltration into the TME is an important feature for the therapeutic activity and prognostic prediction (Spranger and Gajewski, 2018). However, the driver genes and signaling pathways regulating the T-cell infiltration have not been completely discovered in HCC. In the present study, we identified a series of cancer driver genes triggered by CNAs such as CDKN2B, MYC, TSC1, TP53, and GSK3B, which were enriched in the frequently amplified and deleted regions, respectively, and well-characterized in several cancers (Zack et al., 2013;Tokheim et al., 2016;Bailey et al., 2018). With the T-cell infiltration levels in liver normal tissues, HCC, and recurrent HCC tissues, we found T-cell infiltration levels were decreased progressively, showing consistency with previous studies that reduced T-cell infiltration might be associated with poor prognosis (Ye et al., 2019;Pinato et al., 2020). Particularly, we observed that deletions of MAX and TP53 were significantly associated with reduced T-cell infiltration in HCC. Loss of p53 function has been confirmed to decrease T-cell infiltration in breast cancer (Pinato et al., 2020), Accordingly, the downstream oncogenic pathway, cell cycle, was significantly activated in the low T-cell infiltration HCC, suggesting that the deletions in MAX or TP53 regulated T-cell infiltration by enhancing uncontrolled cell cycle. As chemokines secreted by the tumor cells played vital roles in immune-cell recruitment in TME (Chow and Luster, 2014) and were transcriptionally regulated by transcription factors such as TP53 (Blagih et al., 2020), consistently, we found that these genes were highly enriched in the downregulated genes in HCC with TP53/MAX deletions, which gave us a hint that the CNV loss in TP53/MAX might downregulate their chemokine-relayed target genes, thereby weakening the immune-cell recruitment. As multiple biological processes were involved in T-cell-mediated anticancer activity, interestingly, we found that all the seven steps were reduced in low T-cell infiltration subgroup, indicating that cancer immunotherapy required drug combinations targeting these biological processes (Ott et al., 2017).
Furthermore, the T-cell infiltration levels and chemokines might be used as clinical biomarkers for the treatment response and prognostic prediction. Using the T-cell infiltration levels of five T cells and chemokine activity, we found that the T-cell infiltration levels and chemokine activity could accurately predict the response of sorafenib treatment. Similarly, immune cell abundance has been reported as an indicator for sorafenib response (Cabrera et al., 2013). In addition, the T-cell infiltration levels and chemokines were identified as favorable prognostic biomarkers in HCC, further suggesting that the T-cell infiltration levels were promising biomarkers to be applied in clinical practice.
In conclusion, we performed a systematic analysis to identify driver genes and signaling pathways involved in T-cell infiltration, which not only revealed the underlying mechanism that regulating T-cell infiltration, but also improved the understanding of the functional roles of T cells in HCC. The difference of overall survival probabilities between two risk groups in GSE14520 and CPTAC cohorts. The differences of the recurrence-free survival between the two risk groups in GSE14520 (D) and CPTAC (E) cohorts.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.
AUTHOR CONTRIBUTIONS
YC, ZY, and JP contributed to the conception and design of the research. YC, YT, and JW contributed to the acquisition of data and analysis and interpretation of data. YC, YT, and JW contributed to statistical analysis. YC, YT, JW, WW, and QT contributed to drafting the manuscript. YC, YT, JW, LL, ZL, WL, YL, JP, and ZY contributed to revision of the manuscript. All authors contributed to the article and approved the submitted version. | 2020-12-14T14:08:51.549Z | 2020-12-14T00:00:00.000 | {
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8121556 | pes2o/s2orc | v3-fos-license | Dinuclear clathrochelate complexes with pendent cyano groups as metalloligands †
a Dinuclear clathrochelate complexes with two, three, four, or fi ve cyano groups in the ligand periphery were prepared following two distinct synthetic strategies: (a) zinc( II )- or cobalt( II )-templated poly-condensation reactions of CN-functionalized arylboronic acids and phenoldioximes, or (b) postsynthetic cross-coupling reactions of polybrominated zinc( II ) clathrochelates with 4-cyanophenylboronic acid. The new clathrochelate complexes were used as metalloligands for the construction of heterometallic Zn 2+ / Ag + and Co 2+ /Ag + coordination polymers (CPs), which were characterized by single crystal X-ray di ff rac-tion and FT-IR. A one-dimensional CP was observed for ditopic clathrochelates, whereas two-and three-dimensional CPs were generated from tetra-and pentatopic metalloligands. The three-dimensional network is unique as it displays an unprecedented network topology with the point symbol (8·10 2 )(8 2 ·10 4 ) (8 2 ·10)(8 3 ·10 3 ). Furthermore, it is a self-catenated net with an extremely high topological density.
Introduction
Silver(I) ions tend to form labile complexes with a flexible coordination number and geometry. 1 These features are attractive for the preparation of coordination polymers (CPs) 2 because the reversible and malleable coordination chemistry facilitates crystallization, and thus characterization. A sizable fraction of the silver(I) CPs reported to date is based on polycyano ligands. Various ligands have been used in this context, including compounds with two, 3 three, 4 four, 5 and more 6 cyano groups. In addition to purely organic ligands, the utilization of metalloligands with cyano groups in the ligand periphery has been explored. Some selected examples are shown in Fig. 1.
The groups of Englert, Hosseini, Burrows and Mahon used homo-and heteroleptic metalloligands containing 3-cyanoacetylacetonato ligands for the construction of silver(I) CPs. 7 Tritopic metalloligands were obtained with iron(III), 7h aluminum(III), 7c and chromium(III), 7g whereas ditopic metalloligands were formed with copper(II) 7h and palladium(II). 7b Carlucci and coworkers have used β-diketonate ligands with two benzonitrile groups to make hexadentate metalloligands. The complexes display an overall charge of zero or minus one, depending on the central metal ion. Very similar three-dimensional CPs were obtained with these metalloligands, despite the difference in overall charge. Notably, it was possible to perform anion exchange in single crystal to single crystal processes. Schulz and coworkers have prepared silver(I) CPs with a diamond-like topology using the tetrahedral [Al(OC 6 H 4 CN) 4 ] −1 anion. 8 Due to network interpenetration, large channels or pores were not observed.
We have recently started to explore the potential of dinuclear clathrochelate complexes as metalloligands. 9,10 These complexes can be synthesized in metal-templated one-pot reactions from easily accessible starting materials. Their characteristics make them interesting building blocks for structural supramolecular chemistry: they are rigid, relatively large, luminescent (for M = Zn), and anionic. Furthermore, the metalloligands display an unusual trigonal bipyramidal geometry. So far, efforts have focused on clathrochelates with pendent pyridyl 9 or carboxylic acid groups. 10 Below, we show that clathrochelates can also be decorated with cyano groups. Following two distinct synthetic strategies, we were able to prepare clathrochelates containing two, three, four, or five cyano groups which are oriented in a divergent fashion. The utility of these new metalloligands is demonstrated by the synthesis of one-, two-, and three-dimensional coordination polymers, in which the clathrochelate ligands are linked by Ag + ions.
Results and discussion
Direct synthesis of CN-functionalized clathrochelates Dinuclear clathrochelate complexes with boronate ester capping groups are generally prepared by polycondensation of a boronic acid with a phenoldioxime in the presence of a divalent metal ion (Mn 2+ , Zn 2+ , or Co 2+ ). 9-12 In order to introduce cyano groups in apical position, we have performed a similar condensation reaction using commercially available 4-cyanophenylboronic acid in combination with 2,6-diformyl-4-tert-butylphenol dioxime (L1). As metal salts, we have employed either [Co(H 2 O) 6 ](NO 3 ) 2 or Zn(OTf ) 2 . The reactions proceeded in a clean fashion, and the desired clathrochelates 1 and 2 could be isolated in high yield following addition of tetraethylammonium hydroxide (Scheme 1). Both complexes were analyzed by Fourier-transform infrared spectroscopy (FT-IR) and high-resolution mass spectrometry. The diamagnetic zinc complex 1 was also analyzed by NMR spectroscopy (DMSO-d 6 ). Only one set of signals for the protons of the phenolatodioximato ligand were observed, corroborating the expected pseudo-C 3 symmetry of the complex. The FT-IR spectra showed a characteristic band at 2220 cm −1 , which can be assigned to the absorption frequency of the cyano groups. In line with earlier observations, 9,10 complex 1 was found to be luminescent with an emission maximum at 445 nm (DMF, λ ex = 335 nm, Fig. S15 †).
Previously, we had investigated the magnetism of dinuclear Co clathrochelates with pendent carboxylic acid groups using the Evans method. The data suggested that the Co(II) centers have a high spin configuration. For complex 2, we have now performed a more detailed analysis using a superconducting quantum interference device (SQUID) magnetometer. A plot of the magnetic susceptibility vs. temperature reveals an increase of susceptibility as temperature decreases and a broad maximum around 87 K below which a significant decrease of the susceptibility occurs (Fig. 2).
Given that the pair of cobalt ions are well separated from other magnetic ions, the inter-cluster interaction can be neglected and the Hamiltonian of the system can be written simply as H = −2J·S 1 ·S 2 , where S 1 and S 2 are spins of two cobalt ions that form a pair and J is the intra-cluster exchange interaction. The room temperature value of the effective moment per Co ion reaches 5.0µ B , close to the theoretically maximum value for a high-spin cobalt(II) state with L = 3 orbital momentum included. The data has been analyzed using MagSaki software 13 which takes into account the magnetic exchange coupling J, spin-orbit coupling λ, an orbital reduction factor κ and the axial-splitting parameter Δ. Very good agreement with measured data has been achieved with J = −20(3) cm −1 , λ = −110(10) cm −1 , κ = 1(0.1) and Δ = 286(30) (see red line in Fig. 2, left). These parameters correspond to significant g-factor anisotropy, g x = 4.9(0.3) and g z = 3.0(0.3), which is in agreement with preliminary ESR measurements. These values are similar to those reported for other di-nuclear Co-based compounds with much larger intra-cluster magnetic interaction. 14 In order to prepare clathrochelates with cyano groups in lateral position, we have prepared the phenoldioxime ligand L2. The synthesis of this ligand was accomplished by Pd-catalyzed cross-coupling of 4-cyanophenylboronic acid and 2,6-diformyl-4-bromophenol in presence of SPhos and Pd 2 (dba) 3 , 15 followed by treatment of the crude dialdehyde with hydroxylamine hydrochloride (Scheme 2).
The tri-and pentatopic clathrochelates 3-5 were then obtained by using L2 in combination of either 4-bromophenylboronic acid or 4-cyanophenylboronic following the standard protocol (Scheme 3). To the best of our knowledge, complexes 4 and 5 represent first examples of polycyano ligands with trigonal bipyramidal geometry. Zn-based clathrochelates 3 and 4 are luminescent, with emission maxima at 450 nm (DMF, λ ex = 335 nm, Fig. S15 †). Their emissions are thus slightly redshifted compared to what was observed for 1. The analytical data for 3-5 are in line with the expected structures. The magnetic properties of 5 were expected to be similar to what was found for 2 and additional SQUID measurements were not performed.
Synthesis of CN-functionalized clathrochelates by postsynthetic cross-coupling reactions
Recently, we have shown that brominated clathrochelate complexes of Zn 2+ and Co 2+ are sufficiently stable to be used as substrates in Pd-catalyzed cross-coupling reactions, allowing the preparation of elongated polypyridyl ligands. 9a We anticipated that a similar strategy could be used to prepare clathrochelate complexes with pendent benzonitrile groups. Indeed, the two-fold coupling of the previously described clathrochelate 6 featuring 3-bromophenylboronate ester caps 9a with 4-cyanophenylboronic acid gave the desired complex 7 in 91% yield (Scheme 4). With regard to potential applications, it is worth noting that ditopic cyanoliogands with a bent shape have been used extensively for the preparation of silver(I) CPs. 3a-g Compared to the ligands used previously, clathrochelate 7 stands out because of its size and its negative charge. Attempts to prepare a linear dicyano ligand by cross-coupling of a clathrochelate with terminal 4-bromophenylboronate ester groups were unfortunately not successful. Even though we were able to detect the desired product by mass spectrometry, we were not able to achieve a complete reaction, and separation of the side products was found to be difficult. This result indicates that coupling reactions in para position to the boronate ester function are more problematic.
To further test the scope of the cross-coupling procedure, we prepared the tetrabrominated clathrochelate complex 8 using 3,5-dibromophenylboronic acid and dioxime L1. The subsequent four-fold coupling with 4-cyanophenylboronic acid in the presence of Pd 2 (dba) 3 and SPhos was remarkably clean, providing the tetratopic complex 9 in 89% yield (Scheme 5).
The elongated clathrochelates 7 and 9 are soluble in polar organic solvents such as DMSO, nitromethane and DMF, and the analytical data match the proposed structures. Both complexes are luminescent, with an emission maximum at 445 nm (DMF, λ ex = 335 nm, Fig. S16 †).
Coordination polymers with silver(I)
After having established efficient protocols for the synthesis of CN-functionalized clathrochelate complexes, we started to explore their utilization as metalloligands in CPs. As outlined in the introduction, polycyano ligands are particularly suited for the preparation of silver(I) CPs, and we thus focused on reactions with Ag + salts. For some clathrochelate complexes, we were able to obtain crystalline CPs upon reaction with silver salts, and the results are detailed below.
Single crystals of the CPs 10 and 11 were obtained by layering a solution of AgNO 3 in MeOH on top of a solution of 1 or 2 in nitromethane (Scheme 6). Crystallographic analyses revealed that both CPs are isostructural compounds having stoichiometry [Ag(1)(CH 3 OH)](CH 3 OH) 1.5 (CH 3 NO 2 ) for 10 and [Ag(2)(CH 3 OH)](CH 3 OH) 1.25 (CH 3 NO 2 ) 1.5 for 11. The terminal cyano groups coordinate to the Ag + ions in a slightly bent fashion (N-Ag-N = 154°and 155°) forming an infinite chain. The Ag + ions display a trigonal pyramidal geometry, with one of the coordination sites being occupied by a methanol ligand. The fourth coordination site is occupied by the oxygen atom of an adjacent boronate ester group. It should be noted that coordination of a metal ion to the oxygen atom of clathrochelate complexes has not been observed before. As a result of this cross-linking, we observe an unusual double chain architecture (Fig. 3). 3d A summary of the bond distances of the connecting silver complexes is given in Table 1.
The structures of 10 and 11 do not contain nitrate anions from the silver salt because the charge compensation is achieved by the metalloligand itself. It should be noted that for previously reported silver(I) CPs, the anion derived from the silver salt was often found to influence the final structure. 3d,4a,d,5b,g, 16 We were also successful in preparing a CP using the tetratopic metalloligand 9. Layering a solution of AgOTs in MeOH on top of a solution of 9 in nitromethane led to the formation of transparent, plate-like crystals of coordination polymer 12 (Scheme 7).
Single crystal X-ray analysis of 12 revealed the formation of a two-dimensional network structure with the stoichiometry [Ag 2 (9) 2 ](CH 3 OH) 1.5 (CH 3 NO 2 ) 3 . A graphical representation of the structure is depicted in Fig. 4. One can observe two distinct clathrochelate metalloligands in the structure. The first one is coordinated via all of its cyano groups to silver ions Ag(1) and Ag(2), thereby forming an infinite double chain. The second clathrochelate is coordinated via only one of the four cyano groups to silver ions Ag(2). The latter display a trigonal pyramidal geometry (Ag(2)-N av. = 2.281 Å), with the fourth coordination site being occupied by the oxygen atom of a boronate ester group from an adjacent clathrochelate (Ag(2)-O = 2.587(6) Å). The Ag(1) ions display approximate trigonal planar geometry, with coordination of Ag(1) to two cyano groups (Ag(1)-N av. = 2.173 Å) and one oxygen atom of a boronate ester (Ag(1)-O = 2.390(5) Å). The Ag-O-B linkages connect the double chains, resulting in an overall two-dimensional 3,6-coordinated network structure, where silver atoms act as three-connected nodes and clathrochelates as six-connected nodes (through four cyano and two boronate groups). The underlying net of this CP has the kgd topology (kagome dual, given in terms of the RCSR notation; 17 see ESI, Fig. S19 †) which is the most widespread topology for 3,6-coordinated two-dimensional CPs. 18 As it was observed for CP 10 and 11, the charge compensation is achieved by the metalloligands, and tosylate anions from the silver salt are not found in the structure.
Silver(I) CPs based on pentacyano ligands with a trigonal bipyramidal geometry areto the best of our knowledgenot known. We were thus particularly interested to obtain CPs with the pentatopic metalloligands 4 and 5. After several attempts, we were able to crystallize CP 13 using metalloligand 5 in combination with AgClO 4 in the presence of MeOH, DMF and 1,2-dichlorobenzene as solvents (Scheme 8). Single crystal X-ray analysis of 13 showed that a three-dimensional network with the stoichiometry [Ag 3 (5) 2 (OMe)](C 6 H 4 Cl 2 )(MeOH)(sol.) n had formed (Fig. 5). The asymmetric unit of the crystal contains three silver ions and two clathrochelate complexes 5. Although the ratio of Ag to 5 is equal to 3 : 2, the overall network is neutral due to presence of disordered methoxy groups which are coordinated to Ag(2) ions. Besides, the Ag(2) Scheme 7 Synthesis of the heterometallic coordination polymer 12. ions act as linkers between two cobalt-containing metalloligands (Ag(2)-N av. = 2.119 Å). The Ag(1) and Ag (3) ions, on the other hand, are coordinated in a trigonal planar fashion to three cyano groups, with Ag(1)-N distances varying from 2.11(2) to 2.30(2) Å. Both metalloligands act as four-connected nodes through both of the apical and two of the three lateral cyano groups (one cyano group is 'free'). The first crystallographically independent metalloligand coordinates to one Ag(2) and three Ag(1) ions, and the second one to one Ag(2) and three Ag (3) ions. As a result, the Ag(2) ions links two interpenetrating CPs of the stoichiometry [Ag (5)] and the utp topology (widespread among 3-coordinated CP nets) 19 to form a three-dimensional net. A topological analysis of the resulting architecture by means of the ToposPro package 20 revealed that the net has a point symbol (8·10 2 )(8 2 ·10 4 )(8 2 ·10)(8 3 ·10 3 ). To the best of our knowledge, such a network topology has not been observed in crystals before. The structure was deposited to the Topos TTD collection under the abbreviation epf1.
An intriguing structural feature of 13 is that all eight-and ten-membered circuits of the nodes are crossed by other rings, resulting in self-catenation ( Fig. S20 and S21 †). Self-catenated structures are single networks with regions in which chains from the same net pass through the smallest topological circuits in a fashion similar to that of interpenetrated systems. The observation of self-catenation in 13 is unusual because most of reported self-catenated nets are constructed from flexible ligands. 21 The catenation in 13 is very dense from a topological point of view: the eight-membered rings are crossed by 42 other rings and the ten-membered rings are crossed by at least 57 other rings. The topological density of the net can be given as TD 10 = 3127, which is the number of nodes within the first 10 coordination spheres of a given node. According to the RCSR database, 17 this value represents the highest topological density among all known 3,3,4,4-coordinated nets, and is almost as high as TD 10 = 3245 for a recently published 3,4-coordinated mhq-z net. 22 Despite the high topological density, CP 13 displays a very large solventaccessible volume of 53% according to calculations with Mercury. 23 These voids are occupied by highly disordered solvent molecules, some of which could not be located from difference Fourier maps. The attempted desolvation of CP 13 resulted in rapid degradation of the material.
Conclusions
We have shown that dinuclear clathrochelate complexes with a trigonal bipyramidal shape can be decorated with cyano groups in apical and/or lateral position by using cyanofunctionalized starting materials for the clathrochelate synthesis. Alternatively, we have been able to prepare elongated clathrochelates with pendent benzonitrile groups by post-synthetic modification of brominated clathrochelates in Pdcatalyzed cross-coupling reactions. The functionalized clathrochelates can be used as metalloligands for the preparation of heterometallic Co 2+ /Ag + and Zn 2+ /Ag + coordination polymers, as evidenced by the synthesis of one-, two-, and three-dimensional CPs in reactions with silver salts. Due to the negative charge of the metalloligands, the CPs are devoid of anions derived from the silver salt. A structure-directing role of the anion, as observed for many silver(I) CPs, is thereby avoided. The three-dimensional CP 13 is of special interest because it displays an unprecedented network topology and a very high topological density.
Potential applications of the CN-functionalized clathrochelate complexes described above are not restricted to the formation of polymeric networks. Polycyanoligands have also been employed for the constructions of molecularly defined nanostructures using coordination-based self-assembly, 24 and our new metalloligands should be useful building blocks in this context as well.
Materials and general procedures
Clathrochelate 6 and 2,6-diformyl-4-tert-butylphenol dioxime ligand (L1) were synthetized according to literature. 10,11 1 H and 13 C NMR spectra were obtained on a Bruker Avance III ( 1 H: 400 MHz, 13 C: 100 MHz). 1 H and 13 C chemical shifts are reported in parts per million δ ( ppm) referenced to the internal solvent. All spectra were recorded at RT. Electrosprayionisation MS data were acquired on a Q-Tof Ultima mass spectrometer (Waters) operated in the negative ionization mode and data were processed using the MassLynx 4.1 software. APPI-FT-ICR experiments were performed on a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT-ICR MS, Thermo Scientific, Bremen, Germany) equipped with a 10 T superconducting magnet (Oxford Instruments Nanoscience, Abingdon, UK). Data analysis was carried out using XCalibur software (Thermo Scientific, Bremen, Germany). IR spectra were recorded on a Perkin Elmer Spectrum One Golden Gate FTIR spectrometer. Emission spectra were recorded with a Varian Cary Eclipse spectrometer. Magnetization and magnetic susceptibility measurements were performed with a Quantum Design MPMS-XL 5 T superconducting quantum interference device (SQUID) magnetometer. The powder samples were contained in plastic capsules which were incorporated into two plastic straws as sample holder. The measurements were collected as a function of the applied field of 1 T and temperatures ranging from 5 to 350 K with Zero Field Cooled method (ZFC). | 2017-11-11T06:55:50.330Z | 2016-10-04T00:00:00.000 | {
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230586532 | pes2o/s2orc | v3-fos-license | Efficacy of site of pallor to detect anemia and its correlation with etiology in under five children
Background: The diagnosis and management of anemia largely depends on clinical assessment for pallor. Objective was to evaluate the usefulness of clinical pallor to detect anemia, to correlate pallor with grades and etiology of anemia. Methods: This case control study included 300 children in the age group of 6months to 5years. Pallor was assessed in four sites conjunctiva, tongue, nailbed and palm. Children with pallor at any one site were taken as study group (n=150) and without pallor at all 4 sites as controls (n=150). Hemoglobin estimation and other relevant investigations were done. Anemia was diagnosed according to WHO criterion (Hb<11 g/dl in 6 months-5 years) and graded as mild, moderate and severe. Results: Both groups were comparable in characteristics of age and gender (p value>0.05). In pallor group, 119 had anemia, whereas non pallor control group had 45 anemics. Sensitivity and specificity of pallor for anemia detection were 72.6% and 77.2% respectively. Maximum sensitivity, specificity and predictive values were found for palmar pallor. Tongue turned out to be least sensitive for identifying pallor. All the four sites were found to have statistically significant correlation in identifying mild, moderate and severe grades of anemia. Among causes of anemia; iron deficiency anemia was the etiology in 81.1% of cases. Pallor at each site showed no statistically significant correlation with etiology. Conclusions: Pallor is useful in detecting anemia. Multiple site examination is suggested as its increases the sensitivity. No positive correlation observed between pallor and its etiology.
The diagnosis and management of anemia largely depends on clinical assessment for pallor. Assessment of pallor for anemia is an important part of general physical examination of every patient. Sites used are conjunctiva, tongue, face, lip, nailbed, and palm. Usefulness of pallor in severe anemia is well established but not in mild anemia. The integrated management of childhood illness (IMCI) strategy developed by the World health organization recommends the use of palmar pallor as the initial screening tool. 3 Even while describing pallor, vague terms like mild, probable etc. are used. There are hardly any studies assessing the accuracy of pallor for detection of anemia in Indian pediatric population. This study was undertaken with the objectives of evaluating the usefulness of pallor in four anatomical sites to detect anemia, tocorrelate pallor with grades of anemia and with the etiology of anemia.
This case control study was done in Father Muller
Medical college, Mangalore from October 2012-March 2014. Purposive sampling technique was used. Parental consent was obtained from all the study participants. 300 children in the age group of 6 months to 5 years were included in the study. Children were excluded if they did not meet age criteria, those with shock and if they were already diagnosed with anemia. Detailed history and examination were done. History was collected from the mothers as well as child.
Pallor was assessed in four sites namely conjunctiva, tongue, nail bed and palm under day light. Conjunctiva was examined by everting the lower palpebral conjunctiva. Pale conjunctiva is those with very less or no evidence of red color on the anterior rim, which matched the fleshy color of the posterior aspect of palpebral conjunctiva. Tongue was examined on the dorsal surface. Nailbeds without pressing was looked for nailbed pallor. Palmar surface and creases were compared with examiner's palm to detect pallor. Children with pallor at any one site were taken as study group (n=150) and without pallor at all four sites as controls (n=150).
After history and physical examination, blood sample was taken for haemoglobin estimation and other relevant investigations. All samples were collected within 3 hours of physical examination. Anemia was diagnosed according to WHO criterion (Hb<11 g/dl in 6 months-5 years). 4 Anemia was divided into mild (Hb: 10-10.99 g/dl), moderate (Hb: 7-9.99 g/dl) and severe (Hb: <7g/dl). Anemic patients were further investigated to find out the etiology. Study was approved by Institutional Ethics Committee. Data were expressed as frequency and percentages. Chi square test was used to calculate association between variables, and sensitivity and specificity. P value<0.05 was considered significant. Statistical analysis was performed using SPSS v21.
RESULTS
Three hundred patients were included in the study of which 150 were assigned as cases (pallor-study group) and 150 as controls (no pallor group). Both the groups were matched in terms of age and gender. There were 91 male and 59 females in pallor group whereas 83 and 67 in nonpallor group. Age distribution as<1 year, 1-3 years and 3-5 years in 32%, 30% and 38% respectively in pallor group. In control group these were 26.7%, 42.7% and 30.7%.
Out of 150 patients with pallor, 119 had anemia, whereas non-pallor control group had only 45 anemics. Sensitivity and specificity of pallor for anemia detection were 72.6% and 77.2% respectively. Positive predictive value and negative predictive values stand at 79.33% and 70.00% respectively. The mean hemoglobin in pallor group was 9.34±2.2 g/dl and in non-pallor group it was 11.43±1.04 g/dl.
Identification of pallor in various sites
In the study group (with pallor), pallor was identified in 104 (69.4%) in conjunctiva, 86 (57.30%) in tongue, 108 (72%) in nailbed and 120 (80%) in palm. Sixty-one cases had pallor in all 4 sites. The sensitivity, specificity and predictive values of each site are given in Figure 1. Maximum sensitivity, specificity and predictive values were found for palmar pallor. Tongue turned out to be least sensitive for identifying pallor.
Pallor was correlated with grades of anemia in all four sites (Table 1). All the four sites were found to have statistically significant correlation with anemia (p value<0.001). Sensitivity of pallor in all the four sites was found to have positive correlation with severity of anemia. To detect severe anemia sensitivity of conjunctival pallor was 100% ( Table 2).
Etiology of anemia and relation with pallor
Iron deficiency anemia was the etiology in 81.1% of cases. Hemolytic anemia and leukemia in 1.8% each, chronic diseases and malaria in 3%, others were the causes in 9.1%. Other causes included megaloblastic anemia, hypothyroidism, autoimmune hepatitis, CMV infection and acute bleeding. Pallor at each site was tried to correlate with etiology (Table 3). However, no statistically significant correlation was found.
DISCUSSION
Anemia is common in this age group especially iron deficiency because of increased demands of iron and reduced oral intake. Bad feeding habits, especially during the weaning period, results in replacement of breast milk by foods that are poor in iron and other nutrients, including vitamin B12 and folic acid which exacerbate the problem. 5 In 2012 a study on anemia was conducted in rural Maharashtra by Kumar et al and observed that maximum anemia cases were in age group 1-5 years. 6 According to NFHS-III survey, almost 7 in 10 children aged 6-59 months are anemic, including 40 percent who are moderately anemic and 3 percent who are severely anemic. 7 The NFHS-IV survey showed some improvement with 58% anemics compared to earlier 70%. 2,8 In our study among 300 children, 65.7% were less than 3 years. No statistically significant correlation was found with age and pallor although increased pallor occurrence was observed in <3years. Pallor and anemia were found to be more in males. Difference may be because of different growth patterns resulting in increased demand. Many studies found no association between anemia and gender whereas other authors reported that anemia is more common in boys. 5,9- Out of 150 children with pallor, 119 had anemia. Like most of the studies pallor was strongly associated with anemia. 6,12 Sensitivity of pallor for anemia is found to be 72.6% and specificity 77.2%. Pallor was found to be more specific than sensitive. Most of the studies indicate that pallor at each site is associated with significantly lower hemoglobin concentration. The relative performance of different anatomical studies was not consistent among studies, sensitivity varied from 81% to 29% in different population. Among 150 children with pallor, palmar pallor was identified in maximum cases (80%), tongue pallor in minimum cases (57.3%). All four sites had statistical correlation with anemia.
Palmar pallor was found to be the most sensitive and specific site for pallor followed by nailbed. Sensitivity was the least for tongue pallor and specificity wasthe least for conjunctiva and nailbed. Over all pallor was found to be more specific test rather than sensitive. Specificity ranged from 89-92%, sensitivity 45-60%. Out of 164 anemic patients 45 (27.4%) were not clinically detected. As pallor is considered as a screening test where sensitivity should be good, multiple site examination is advisable. Because overall sensitivity of pallor for anemia is much more (72%) compared to each site (45-66%).
A meta-analysis of 11 studies was done by Chalco et al concluded that none of the clinical signs were highly accurate for the diagnosis of anemia, but pallor was found to be more specific than sensitive. 13 Pooled estimates of sensitivity ranged from 29.2 to 80.9% and estimates of pooled specificity varied from 67.7 to 90.8%. They concluded that that pallor correlates well with the Hb estimation as only 7.5% of the anemic children were not detected clinically. One study reported sensitivity, specificity, and positive predictive value (PPV) of palmar pallor as an indicator for anemia at 50%, 93%, and 92%, respectively which is close to the values observed in present study. 14 Conjunctival pallor was missed in many patients because of the congestion associated with febrile illness. Also because of the congestion associated with crying while palpating. Tongue also will be congested in many infections, which probably can be attributed to low positive predictive value. Pigmentation largely affects sensitivity of pallor sites especially palmar pallor.
Because of racially homogenous sample, that variation was not studied by us. There is variation in looking for palmar pallor. It is recommended to look at palmar creases for pallor. In this study palmar surface and creases was compared with examiner's palm to look for pallor. In one Bangladesh study, the site of the palmar pallor was assessed over the thenar eminence without extending the fingers and found that palmar pallor did not work as well as conjunctival pallor for the detection for severe or some anemia. 15 In clinical approach many times pallor is graded as two or three. Such grading of pallor into 2 or 3 was done in some studies. 12,15,16 Pallor at each site was correlated with various etiologies like iron deficiency, malaria, leukemia, thalassemia. However, no significant correlation was found with any etiology. Few studies have found correlation with malaria and thalassemia. Study by Kalter and associates reported that anemia was more easily diagnosed in children with malaria. 15 Yalcin and colleagues reported that pallor of the conjunctiva is the most accurate in the cases of beta thalassemia with good sensitivity and specificity regardless of age and gender. 3 Another study concluded that palmar pallor is easy to recognize and might be helpful for health workers as an indicator not only for anemia but also for malarial parasitaemia whereas this clinical sign cannot replace thorough laboratory diagnostics. 17 A study was done in Kenya to correlate palmar pallor with parasitic infestations and to establish palmar pallor as an indicator of anthelminthic treatment. They concluded that palmar pallor is associated with anemia but not with intestinal helminth infection. 14
Limitations
The sample population consisted of hospitalized patients. Whether it has any influence because of high prevalence of anemia in hospitalized patients is not known. Another limitation is subjective variation of clinical pallor, which is common is not included here.
CONCLUSION
Pallor was found to be very useful in detecting anemia especially in moderate and severe cases. The best predictor of anemia in this study was palmar pallor. Multiple site examination is suggested as its increases the sensitivity. However, pallor was found to have no correlation with etiology of anemia. | 2020-12-10T09:06:04.022Z | 2020-12-14T00:00:00.000 | {
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258331866 | pes2o/s2orc | v3-fos-license | AI-assisted coding: Experiments with GPT-4
Artificial intelligence (AI) tools based on large language models have acheived human-level performance on some computer programming tasks. We report several experiments using GPT-4 to generate computer code. These experiments demonstrate that AI code generation using the current generation of tools, while powerful, requires substantial human validation to ensure accurate performance. We also demonstrate that GPT-4 refactoring of existing code can significantly improve that code along several established metrics for code quality, and we show that GPT-4 can generate tests with substantial coverage, but that many of the tests fail when applied to the associated code. These findings suggest that while AI coding tools are very powerful, they still require humans in the loop to ensure validity and accuracy of the results.
Introduction
Recent developments in artificial intelligence, particularly through large language models, have enabled the automated generation of computer code (Chen et al. 2021; Bubeck et al. 2023). In particular, GPT-4 has enabled human-level performance on a set of coding challenges that are outside of the training set of the model (Bubeck et al. 2023). In addition, automated coding assistants (particularly Github Copilot) have become integrated into commmon devlopment environments and are widely used, with some evidence that they can signficantly improve coding productivity. The performance of these models is also raising important questions regarding coding education, given that the current models can easily complete most coding problem sets using in introductory programming courses (Finnie-Ansley et al. 2022).
In the present paper we explore some of the implications of AI-assisted coding using GPT-4, in a more qualitative way than previous benchmarking assessments. First we examine the experience of interactive coding and debugging using the ChatGPT interface to GPT-4 on a set of data science coding problems. This experiment is meant to approximate the experience of a researcher with minimal expertise in prompt engineering, assessing the success and amount of effort required to perform these coding tasks. Second, we assess the ability of GPT-4 (using the OpenAI API) to refactor and improve the quality of existing code. This experiment is meant to assess the degree to which AI coding assistants might improve coding quality when used by researcers. Third, we assess the ability of GPT-4 to write tests for its own code, using a set of test prompts from several scientific domains. We conclude with an overall assessment of the utility of AI coding assistants for scientific researchers.
A fully reproducible workflow for this manuscript is available at https://github.com/poldrack/ai-coding-experiments.
Coding with GPT-4
Our first set of experiments examined the ability of GPT-4 (via the ChatGPT interface) to generate usable code for a set of data science problems. The prompts were generated manually (by author RP) and are listed in Appendix 1. Each prompt was submitted in a separate chat session; the human examined the resulting code, and issued additional prompts to try to fix problems. If the human was not able to obtain working code within about 5 minutes of effort or less, the problem was deemed unsolved. The results of this experiment are primarily qualitiative and subjective, but are meant to index the degree to which GPT-4 is a useful tool for a researcher with minimal prompt engineering skills. Figure 1 shows the proportion of successful outcomes as a function of the number of prompts required. 72% (23/32)) of attempts were successful in relatively quickly solving the problem; 37.5% (12/32) were successful on the first prompt. In cases where additional prompting was required, a common problem was the use of outdated functions or datasets from Python libraries.
The causes of unsuccessful outcomes were varied. In some cases it was due to the outdated nature of the GPT-4 training data. For example, in one case (prompt #12) ChatGPT could not successfully implement a solution that was compatible with the latest version of PyTorch. In another case (prompt #27), the labels used to query the NOAA API were incorrect, and the correct labels were not easily identifable upon further examination. In other cases, it was not immediately clear what was wrong with the code, and the researcher did not dig deeply enough to identify the root cause.
One of the examples highlights the contining challenges that ChatGPT has with mathematical processing (as outlined by Bubeck et al. (2023)). Prompt #4 asked ChatGPT to generate code to simulate a drift diffusion model (a common model for response times in cognitive psychology) and then fit the model parameters using the EZ-diffusion model (Wagenmakers, Van Der Maas, and Grasman 2007), which is a closed-form solution to estimating these parameters using response time and accuracy statistics. The initial code generated by ChatGPT attempted to fit a diffusion model through numerical optimization. After being prompted to generate a closed-form solution based on the original paper, ChatGPT did so, but the formula bore little resemblance to the actual formula from the paper. This is an example of a "hallucination" which is commonly seen with LLMs (Ji et al. 2023), and highlights the ongoing need for automatically generated code to be validated by a human.
Another example also highlights the need for sophisticated domain knowledge in assessing the outputs of ChatGPT. Prompt #18 asked ChatGPT to implement a hurdle model, which is a statistical model for zero-inflated count data that combines a binary model with count model using a truncated distribution. In general, this model is fit by performing maximum likelihood estimation on the combined likelihoods of the binary and count models. ChatGPT generated a solution that separately estimated a binary model and a count model, and then combined the predictions from the two models; this incorrect approach can be found in a number of examples from Github. This model fit the test data nearly as well as a reference implementation of the hurdle model (impmented in R), but is incorrect in comparison to the reference implementation. This highlights the need for close attention to detail in the implementation of any numerical methods, as incorrect implementations present on Github can result in incorrect outcomes.
Refactoring code using GPT4
The quality of research code is important for scientific reproducibility and transparency, as well as for code reusability and maintainability. In our initial explorations of code generated by GPT-4, we noted that the automatically generated code appeared to be substantially more readable than research code that we have encountered (or written) in the past. This led us to ask whether GPT-4 could improve the quality of existing code through refactoring (Fowler 2019), by which we mean modifying code to make it more readable or maintainable without changing its behavior.
To assess this, we downloaded more than 2000 examples of Python code from Github using the Githb Code Search API. Only one code file was allowed from any single repository. We further filtered these files, based in part on the criteria used by Chen et al. (2021) to select code for training of the OpenAI Codex model. Exclusion criteria included: • Presence of non-Python code (as guessed by guesslang.Guess()) • Presence of non-English language in the code (according to pycld2.detect()) • Presence of potential markers of automatic generation (e.g. strings such as "autogenerated", "generated by Django", etc) • Presence of potential obfuscation (the "if x -y:" pattern) • Lack of at least one function definition • Number of GPT tokens greater than 2500 or less than 200 • Maximum line length > 1000 • Mean line length > 100 • Maximum file size > 1MB The 274 code files passing these criteria were submitted to further analysis. Analysis of syntax errors and coding style was performed using the flake8 static code analysis package. Files were further excluded on the basis of any syntax errors identified by flake8.
The number of warning and error messages emitted by the flake8 linter was substantially reduced for the refactored code compared to the original (median 0.23 messages/line for original vs 0.09 messages/line for refactored, Cohen's d = 0.50; see Figure 2). While tools exist to perform automatic reformatting to ensure standard-compliance, this shows that GPT-4 generates code that is substantially more standards-compliant than the average programmer; given that the files sampled from Github were heavily filtered, this result is probably an underestimate compared to the broader population of Python code on Github. Figure 3 provides an overview of which errors were most common in the original code and how their prevalence changed after refactoring.
We further examined a set of code quality metrics, which were computed using the radon Python package. Metrics extracted from these outputs for further analysis included: • Logical lines of code • Number of comments • Mean cyclomatic complexity (a measure of the number of execution paths) • Maintainability index (a holistic metric for code maintainability, based on a composite of several metrics including Halstead volume Halstead (1977), cyclomatic complexity, lines of code, and % of comments) • Halstead "difficulty" (a metric of how difficult the code is to read, based on the number of distinct operators and the ratio of total number of operands to number of distinct operands) • Halstead "bugs" (a metric meant to estimate the number of bugs in delivered code) Comparisons between metrics for the original and refactored code are shown in Figure 4, and means, effect sizes, and p-values for the comparison (using a paired t-test) are shown in Table 1. Each of these metrics differed between the origin and refactored code (p < .05 after false discovery rate correction across all hypotheses). However, the effect sizes were all in the small to medium range, with Cohen's d values ranging from 0.13 to 0.33.
Automatically generated code and tests
Given the importance of validating AI-generated code using software tests, we next assessed the ability of GPT-4 to generate tests for its own code. We first used GPT-4 to generate 20 prompts for each of 5 different areas of research, using the following prompt: "Please generate 20 prompts to ask a chatbot to create Python code to solve a variety of {statistical and data science, physics, theoretical computer science, ecology, economics} problems." For each of the generated problems, we created a prompt to generate the code along with tests for the resulting code, such as the following: Write a Python program to simulate predator-prey interactions using the Lotka-Volterra equations, given the initial populations, growth rates, and interaction coefficients. Please embed the code within an explicit code block, surrounded by triple-backtick markers. Generate realistic values for any examples, and do not use input() commands. Create code that is modular and well-commented. Then, generate a set of pytest tests that exercise each of the functions, embedded in a separate code block.
We first examined whether each generated script would execute without failure; of the 100 generated scripts, 97 executed successfully. We then examined test coverage using the Coverage.py tool. As shown in Figure 5, the majority of files had test coverage of 100%, with 94% showing a coverage of at least 50% ad a minimum coverage of 40%. There was a weak but statistically significant negative relationship between the number of statements and the level of coverage (Spearman r = -0.23, p = .02). A median of three tests were generated for each file.
Running the tests required minor automated modification to the code, since the tests required importing the relevant functions but the names of the output files were not known to the LLM. After fixing this issue, 45 of the 100 tests completed successfully. The most common source of test failure was failure of a value assertion (47/100); in these cases, it was not immediately possible to determine whether the test or code was incorrect, without additional debugging. In ten cases, the test failed because the code did not raise the error expected by the test. In six cases, other errors were raised (Type, Index, Value, and ZeroDivision errors).
In summary, our analyses of automatic test generation demonstrate that GPT-4 can successfully generate testing code with good coverage, but that these tests fail often, requiring additional debugging to determine the root cause of the test failure.
Conclusions
Our analyses demonstrate that GPT-4 has strong Python code generation abilities, confirming the results of Bubeck et al. (2023). At the same time, the prevalence of errors in the generated code suggests that humans must remain in the loop in order to ensure the accuracy of any resulting code. Our interactive prompting experiment showed that a relatively novice prompt engineer can successfully solve coding problems within a small number of prompts the majority of the time; however, a sizeable minority of problems would have required significant human debugging in order to solve. An open question is whether re-prompting in a new context may have led to more successful outcomes in these cases.
Comparisons of Python code refactored using GPT-4 to the original code demonstrated that GPT-4 improved the quality of the code, at least as measured by common metrics of software quality and standards compliance. It should be emphasized that these results do not assess the accuracy of the code; rather, they suggest that GPT-4 can help programmers achieve code that is cleaner and potentially more maintainable than the original. Given that GPT-4 refactoring did not eliminate all standards compliance issues, the combination of GPT-4 with other code formatting tools (such as black) would likely result in even further improvements.
The examination of test generation by GPT-4 demonstrated that it was able to generate tests with a high degree of test coverage, but those tests failed a majority of the time. Such test failures require additional human effort to diagnose, Test coverage (percent) Figure 5: Percentage of test coverage as a function of the number of statements in the automatically generated code.
since it is not immediately clear whether the failure is due to inaccurate code, and inaccurate test, or both. These results suggest that while GPT-4 is a very useful tool for generating testing frameworks for new code, the specific test examples should be designed and implemented by a human with domain expertise to ensure that the tests are accurately assessing the intended behavior for the code.
There has been substantial speculation regarding the continued role of human programmers in the face of AI coding tools. The present results suggests that even with the latest generation of AI systems (i.e. GPT-4), human involvement is essential to ensure validity and accuracy of the resulting code. This seems to be especially the case when programming of mathematical concepts is involved. The lack of confidence calibration of tools like GPT-4 means that they will present answers in the same way regardless of the degree of support for the answer.
The prompts used in the present research are almost certainly suboptimal, and thus may be underestimating the potential performance of the model. For example, recent work has shown that chain-of-thought prompting can substantially improve the perfomance of LLMs on complex problems requiring reasoning (Prystawski, Thibodeau, and Goodman 2022; Wei et al. 2023), and this seems to extend to coding as well 1 . Further work is needed to examine the degree to which such improved prompting techniques might improve the performance of LLMs on complex coding problems, and at this point our results should be taken as a lower bound on the performance of these models.
Acknowledgments
Thanks to Mark Chen, David Coats, and Noah Goodman for helpful comments and discussion during the development of this work.
3 Appendix 1: Prompts used for interactive coding with GPT-4 Note: The number in brackets denotes the number of prompts necessary to be judged successful; NS denotes that the problem was abandoned before success. 1.
[4] Create python code that generates a new class called LinearRegressionStats which extends sklearn.linear_model.LinearRegression() to compute the t statistic and p-value for each regressor in the model. 2.
[1] Create python code to create an abstract base class called AbstractPublication to represent publications, with a method called from_pubmed(). Then create a class called Article that inherits AbstractPublication. The from_pubmed() method should take in a pubmed record downloaded using the biopython.Entrez tools, and should convert the author list to a variable called authors and the title to a variable called title. In the main function, perform a pubmed search for the query "cognitive control" and convert each record into an instance of the Publication class, saving them to a dictionary that uses the pubmed ID as the key. 3.
[1] Create python code to download 1000 abstracts from pubmed matching the query "cognitive control", clean up the text by removing standard English stopwords and lexicalizing the words, and apply topic modeling using latent Dirichlet allocation to find 10 topics. Print the highest scoring words for each topic.
[NS]
Generate python code to simulate a drift diffusion model of response times. Simulate 1000 trials from this model, and estimate the drift rate, boundary separation, and starting point parameters using the EZ-diffusion model.
5.
[1] Create a python application takes in a set of csv files, each of which includes two columns: one called "rt" that contains a response time in miiliseconds, and another "correct" that is binary (1/0) denoting whether the response was correct or not. there should also be another variable, called "compatible", that contains binary values (0/1) with half of the rows set to zero and half to 1. combine these files into a single data frame, using the individual file names as an index variable. then perform a linear mixed model that computes the effect of the compatible factor, with a random intercept and slope for the different file names. also create a test that automatically generates a set of data files with random values, and then tests the function to assess whether it properly returns the true values. 6.
[7] Create python code to find a set of articles matching the query "cognitive control" using pubmed. For each article extract the first and last author and their affilations. Then find their institutional web site and scrape their email address from the web site. 7.
[1] Create python code that uses the github api to download the 100 most recently committed python files. 8.
[8] create python code to load each python file from a directory called "python_files", and for each file compute cyclomatic complexity, maintainability index, and halsted complexity metrics for each file using the radon package.
9. [2] Generate a python script that performs crossvalidation with feature selection incorrectly performed outside of the crossvalidation loop. 10.
[1] Please create code to generate some test data for this code (#9) 11.
[NS] Write a python function that takes as input a textfile, then create an animation that: [1] Displays X lines of the textfile at a time, with Y characters per line. If the text file has more than Y characters in a line, then move to the n ext line. 32.
[1] Create a simulation to demonstrate the use of randomization to obtain a null distribution for hypothesis testing. First, generate a bivariate dataset with a sample size of 100 and a correlation of 0.1 between the two variables. Compute the correlation coefficient and obtain a p-value against the null hypothesis of r=0. Then, randomly shuffle one of the variables 5000 times and compute the correlation each time. Compute an empirical p-value for the actual R value based on the null distribution. | 2023-04-27T01:15:49.135Z | 2023-04-25T00:00:00.000 | {
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"pdf_src": "Arxiv",
"pdf_hash": "ce5e257229923ce41ab9f87b7ea4f16b65da6e5c",
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256868717 | pes2o/s2orc | v3-fos-license | Prioritized offline Goal-swapping Experience Replay
In goal-conditioned offline reinforcement learning, an agent learns from previously collected data to go to an arbitrary goal. Since the offline data only contains a finite number of trajectories, a main challenge is how to generate more data. Goal-swapping generates additional data by switching trajectory goals but while doing so produces a large number of invalid trajectories. To address this issue, we propose prioritized goal-swapping experience replay (PGSER). PGSER uses a pre-trained Q function to assign higher priority weights to goal swapped transitions that allow reaching the goal. In experiments, PGSER significantly improves over baselines in a wide range of benchmark tasks, including challenging previously unsuccessful dexterous in-hand manipulation tasks.
INTRODUCTION
Reinforcement learning (RL) has been used to great success in a variety of tasks, from gaming Mnih et al. (2013); Frazier & Riedl (2019); Mao et al. (2022) to robotics Brunke et al. (2022); Nguyen & La (2019). Typically RL is used in a setting where the agent learns to optimize behavior for one specific task. To allow for a more general RL solution, goal-conditioned RL (GCRL) Liu et al. (2022b); Chane-Sane et al. (2021); Andrychowicz et al. (2017) learns a policy that can reach arbitrary goals without needing to be retrained. Training GCRL agents can be difficult due to the sparsity of rewards in GCRL tasks, forcing the agent to explore the environment, which can be unfeasible or even dangerous in some real-world tasks. To utilize RL without environment interactions offline RL allows learning a policy from a dataset without putting real environments at risk ; Prudencio et al. (2022). Offline goal-conditioned RL (offline GCRL) combines the generalizability of GCRL and the data-efficiency of offline RL, making it a promising approach for real-world applications Ma et al. (2022).
Although offline goal-conditioned reinforcement learning (GCRL) is an appealing concept, it faces some challenges. The first is that the offline dataset only covers a limited state-goal-action space, which can cause incorrect value function estimations for out-of-distribution observations. This can lead to compounding errors in policy deviation ; Prudencio et al. (2022). Additionally, each state can have multiple goals, making it hard to learn from a limited state-goal observation space Chebotar et al. (2021). To deal with this issue, prior works have applied hindsight labeling to generate goal-conditioned observations in sub-sequences Ghosh et al. (2019); ; Ma et al. (2022); Andrychowicz et al. (2017). However, this often leads to overfitted goal-conditioned policies Chebotar et al. (2021). Furthermore, the goal-chaining technique proposed by Chebotar et al. (2021) is not able to handle noisy data properly, while inefficiently swapping goals Ma et al. (2022). In order to achieve successful skill learning across multiple trajectories, a solution is needed that can make agents effectively learn from the limited data. This paper presents Prioritized Goal-Swapping Experience Replay (PGSER), an approach for offline Goal-Conditioned Reinforcement Learning (GCRL) tasks. PGSER provides two main benefits: (1) allowing the agent to learn goal-conditioned skills across different trajectories, and (2) maximizing offline data utilization. The process of PGSER is illustrated in Figure 1. During the offline training stage, random goal-swapping augmentation is used to generate new goal-conditioned transitions ζ aug ; a pre-trained Q function is then used to estimate the priority of each ζ aug , and these transitions are stored in an additional prioritized experience replay buffer β aug . During training, data is sampled from both the original dataset buffer β and the added buffer β aug , which helps to improve the accuracy and effectiveness of the training process and enables the agent to learn goal-conditioned skills across different trajectories. We evaluated PGSER on a wide set of offline GCRL benchmark tasks. The experimental results show that PGSER outperforms baselines. Figure 1: An illustration of prioritized goal-swapping experience replay. During the offline training stage, we conduct random goal-swapping augmentation to create new goal-conditioned transitions ζ aug . A pre-trained Q function estimates the priority w of corresponding ζ aug in order to increase the priority of augmented transitions which are more likely to reach the goal. We store both ζ aug and w into an additional prioritized experience replay buffer β aug . During training the final policy data will be sampled from both the original dataset buffer β and the prioritized goal-swapping buffer β aug for goal-conditioned skill learning.
PRELIMINARIES
Goal-conditioned Markov decision process. The classical Markov decision process (MDP) M is defined as a tuple < S, A, T , r, γ, ρ 0 >, where S and A denote the state space and the action space, ρ 0 represents the initial states' distribution, r is the reward, γ is the discount factor, and T denotes the state transition function Sutton & Barto (2018). For goal-conditioned tasks, an additional vector g specifying the desired goal is included. This augmentation of MDP is referred as the goal-conditioned MDP (GC-MDP), < S, G, A, T , r, γ, ρ 0 , p g > Liu et al. (2022b). This GC-MDP includes a goal space G and a goal distribution p g , as well as a tractable mapping function φ : S → G to map the state to the corresponding goal. The state-goal pair (s, g) forms a new observation, which is used as the input for the agent π(a|s, g). The objective of GC-MDP can be formulated as: Two value functions are defined to represent the expected cumulative return in a goal-conditioned Markov Decision Process (GC-MDP): a state-action value Q and a state value V . The V π (s, g) function is the goal-conditioned expected total discounted return from the observation pair (s, g) using policy π, while the Q π (s, a, g) function estimates the expected return of an observation (s, g) for the action at for the policy π. Additionally, the advantage function A π (s, g, a) is another version of the Qvalue with lower variance, defined as A π (s, g, a) = Q π (s, g, a) − V π (s, g). When the optimal policy π * is obtained, the two value functions converge to the same point, i.e. Q * (s, g, a) = V * (s, g) Sutton & Barto (2018). In this work, we define a sparse reward where ||φ(s), g|| is a distance metric measurement, and is a distance threshold. We set the discount factor γ = 1. In such case, V (s, g) represents the expected horizon from state s to goal g, and Q(s, g, a) represents the expected horizon from state s to the goal g if an action a is taken. This setting yields an intuitive objective: finding the policy that takes the minimum number of steps to achieve the task's goal. This setting produces an intuitive objective: Find the policy that takes the minimum number of steps to achieve the task's goal.
Offline goal-conditioned reinforcement learning. In offline reinforcement learning (offline RL), an agent must work with a set of static, pre-existing data rather than interacting with an environment to collect data. This data is often collected by unknown policies Prudencio et al. (2022). In the offline goal-conditioned setting, the objective is the same as in online goal-conditioned RL, as defined by Equation 1. The offline data consists of goal-conditioned trajectories ζ stored in a dataset D : η is the state's corresponding goal representation calculated using η t = φ(s t ). The task goal g i is randomly sampled from p g and the initial state s 0 ∼ ρ 0 . Note that some trajectories can be unsuccessful (η i T = g i ). Prioritized experience replay. Prioritized experience replay (PER) Schaul et al. (2015) allows reinforcement learning from a diverse set of experiences. PER uses the temporal difference (TD)-error of each experience to determine the priority of that experience in the replay buffer. By prioritizing experiences with higher TD-errors, the agent can focus on those experiences that are most likely to reinforce its learning. This technique can be used to improve the convergence of reinforcement learning algorithms, allowing them to learn more effectively. Figure 2: The generalizability problem of offline GCRL: the agent needs to learn how to reach a goal from a state without a trajectory for this state-goal pair. Each color represents an individual goal-conditioned trajectory.
GENERALIZATION PROBLEM OF GOAL-CONDITIONED RL
Goal-Conditioned Reinforcement Learning (GCRL) aims to learn a general policy that can reach arbitrarily goals Liu et al. (2022b). However, the offline dataset state-goal pairs only cover a limited space of the goal-conditioned MDP. In other words, if we train a policy with the offline dataset, the policy learns to reach goals within a single trajectory in the dataset. This solution is not general but undesirably specific. Fig. 2 shows a simple visual example. In Fig. 2, each color represents a goal-conditioned trajectory. If we use the dataset in Fig. 2 for offline RL, we eventually get an overfitted policy that can only achieve a single goal starting from a given state. Ideally, we want an agent that can achieve as many goals as possible (the blue trajectory).
Several techniques have been proposed to learn a generalized and effective goal-conditioned policy. Actionable Models (AM) Chebotar et al. (2021) use a goal-chaining technique that augments the dataset by assigning conservative values to the augmented out-of-distribution data for Q-learning. However, the performance of AM is limited when the dataset contains noisy data labels Ma et al. (2022). Hindsight Experience Replay (HER) Andrychowicz et al. (2017); relabels trajectory goals to states that were actually achieved instead of the task-commanded goals, efficiently utilizing the data within a single trajectory. However, HER is not able to connect different trajectories as goal-chaining does. Contrastive Reinforcement Learning (CRL) Eysenbach et al. (2022) conducts contrastive learning on the goal-conditioned task by swapping the future observations between different goal-conditioned trajectories to generalize the skills. However, CRL focuses more on representation learning rather than addressing the generalizability of offline goal-conditioned tasks.
METHOD
To solve the challenges discussed in the previous section, we propose reusing the pre-trained Q function to generate better goal-swapping augmentation samples. A schematic illustration of the method inside the RL framework is in Fig. 1. We first conduct goal-swapping data augmentation to generate new state-goal pairs for Q value estimation. In the second stage, we use the pre-trained Q function to filter out the low-quality augmented samples and store the reachable samples for agent learning. In this work, we assume that in the same environment, all goals are reachable from all states. Our approach can be used with off-policy offline RL methods (e.g., TD3BC Fujimoto & Gu (2021), etc.), which aim to approximate the optimal Q value from the offline dataset. As discussed in Section 3, the offline goal-conditioned dataset contains only a limited number of state-goal observations. Therefore, training with offline data often results in overfitted policies. An illustrative example is in Figure 3(a). In this example environment, the goals g a and g b are (forward) reachable from three states s a 0 , s b 0 , and s c 0 . However, the original conditioned trajectories τ a , τ b , and τ c limit the agent's exploration to the known state-goal pairs s a 0 → g a , s b 0 → g b , and s c 0 → g c . To maximize the efficiency of our agent, we propose a goal-swapping augmented experience replay technique (detailed in Algorithm 1). This technique randomly samples two trajectories and swaps their goals, creating a virtually infinite amount of new goal-conditioned trajectories. As reinforcement learning is a dynamic programming method, this allows us to connect the state-goal pairs across different trajectories. By doing so, we are able to expand the range of achievable goals significantly while also increasing the speed and accuracy of our agent's learning process.
Let's continue the example in Figure 3(a), where the goals g ∈ [g a , g b , g c ] are reachable from the states s ∈ [s a 0 , s b 0 , s c 0 ] although they are not explicitly present in the offline dataset. However, if the goals are swapped between the original three trajectories [τ a , τ b , τ c ], the augmented goal-conditioned tuples shown in Figure 3(b) become available. The Q-learning-based approaches can leverage dynamic programming to chain the new trajectories and backpropagate their state-goal (state-goalaction) values to the previous pairs. An illustration of this can be seen in Fig.3(b), where the state s t is the "hub state" shared by the three original trajectories and from which all goals g i ∼ [g a , g b , g c ] are reachable. The values of these goal-conditioned states, Q(s t , a i t , g i ), can be estimated and recursively backpropagated to Q(s 0 , a i 0 , g i ), i ∈ [a, b, c]. The purpose of goal-swapping augmentation is to create as many state-goal pairs as possible so that the Q-learning (temporal difference learning style) can backpropagate values over all trajectory combinations. Alg. 1 provides an illustration of how this is done. By swapping goals between trajectories, the offline RL methods can extend the dataset and make use of diverse information. This ensures that the agent can explore and discover more accurate and robust policies.
REACHABLE TRANSITIONS IDENTIFICATION
Although temporal-difference learning can connect goals across trajectories, applying the goalswapping augmentation technique can be tricky. The reason is that the goal-swapping process is random, creating many non-optimal state-action pairs. Those augmented state-action pairs may not even have a solution (the augmented goals are not reachable) within the offline dataset. This is demonstrated in Fig. 3(c). To alleviate the inefficiency problem of random data augmentation, we Algorithm 1 Random goal-swapping experience replay Require: Denote the dataset as D, goal-conditioned tuples as ζ, state-goal mapping function g i = φ(s i ), and reward function r = R(φ(s), g). 1: Sample goal-conditioned transitions ζ from D: ζ i = {g, s, a, r, s } ∼ D. 2: Sample random goals: g rand ∼ D 3: Generate τ rand by replacing g with g rand in ζ: ζ aug = {g rand , s, a, r aug , s } where r aug = R(φ(s ), g rand ) 4: Return ζ and ζ aug aim to design a mechanism that makes the agent remember the reachable goal-conditioned transitions (positive samples) and ignore the unreachable ones (negative samples). In this work, we use a pre-trained Q-function as a "past-life" agent to improve the efficiency of random goal-swapping. Figure 4: Consider a deterministic MDP, the reward and discount factor is defined in Sec 2, and the maximum horizon H max = 4. The agent is trained with Q-learning with above transitions.
Based on Eq.1, the Q value represents the expected horizon to reach the goal when γ = 1. Denote the maximum horizon of the task as H, the reachable goal as g, and the unreachable goal as g . The Q value estimation should result in Q(s 0 , g, a) ≥ −H, indicating that g is reachable, and Q(s 0 , g , a) < −H for an unreachable goal. As is shown in Fig. 4, given a set of observed deterministic MDP transitions, the optimal state-goal-action value Q * (s, g, a) = −2 and the unreachable state-goal-action value Q(s, g , a) = −4. Such a simple example implies that the trained Q function can be used as an identifier to tell if the goal is reachable for a given state-goal-action pair. Now, we consider the random goal-swapping data augmentation process, denoting transitions in successful trajectories (goals that are reached) as positive transitions ζ P = {s P , g P , a P , r P , s P }, and random goal-swapping augmentation as negative training samples ζ N = {s N , g N , a N , r N , s N } (generated using Alg. 1). We also have a pretrained value function Q. For this data, Q will assign higher values to ζ P and, likely, lower values to ζ N when goals are not reachable. Consequently, this pre-trained Q can be used to identify how likely a goal-conditioned tuple {s, g, a, r, s } belongs to a reachable trajectory. In other words, the smaller the {s, g, a, r, s } q-value is, the less likely its goal is reachable. We provide estimated Q-value distributions in Sec. 5.2 to further illustrate this concept.
PRIORITIZED GOAL-SWAPPING AUGMENTATION
The random goal-swapping augmentation creates goal-swapped transitions at the same frequency, regardless of if the augmentation is positive or negative. To improve the efficiency of the augmentation, we introduce an additional experience replay buffer. This buffer is used to remember positive augmentations during offline training. As is discussed above, the Q function can be naturally used to identify if a state-goal-action is a goal-reachable observation. To assign priority values to the augmented transitions, we pre-train a value function, Q with Q-learning-based offline RL methods. After the pretraining, this function Q can estimate how likely the augmentations are positive. The higher the estimated Q value, the more likely the augmented transition is a positive augmentation, and it shall be sampled more frequently. We illustrate the process of creating an experience replay buffer in Fig. 1.
The additional experience replay buffer β aug is implemented in a PER style. β aug stores the goalswapping augmented transitions ζ aug = {s, g rand , a, r, s } and its prioity value Q(s, g rand , a). During the training, the agent samples the goal-swapped augmented transitions according to the their priority values. We illustrate our framework in Fig. 1 and provide a pseudo-code in Alg. 2. In this way, the positive goal-swapped augmentations will be sampled more frequently than the negative ones. In this way, we have a mechanism that can guide the agent efficiently use the goal-swapping augmentation to learn general goal-conditioned skills from the offline dataset.
Algorithm 2 Prioritized goal-swapping experience replay Require: The original offline buffer as β, the augmented buffer as β aug , goal-conditioned tuples as ζ, state-goal mapping function g i = φ(s i ), reward function r = R(φ(s), g), a pre-trained Q-function Q 1: Pre-train a off-policy value function Q(s, g, a) using random augmentation (Alg. 1). 2: Fill-in β aug using Q: a) Generate ζ aug = {g rand , s, a, r aug , s } using Alg. 1. b) Estimate priority weight w = Q(s, g aug , a). c) Store ζ aug into β aug and set its sampling priority using w. 3: Re-training the RL agent: a) Sample ζ ∼ β, Sample ζ aug ∼ β aug . b) Combine ζ and ζ aug for offline RL training.
EXPERIMENTS
The experiments in this section were designed to verify our main claims: 1) The pre-trained value function is able to identify if the goal is reachable; 2) The filtered goal-swapping experience replay can improve offline GCRL performances.
Tasks. Six goal-conditioned tasks from Plappert et al. (2018) were selected for the experiments. The tasks include four fetching manipulation tasks (FetchReach, FetchPickAndPlace, FetchPush, FetchSlide), and two dexterous in-hand manipulation tasks (HandBlock-Z, HandEgg). In the fetching tasks, the virtual robot should move an object to a specific position in the virtual space. In the dexterous in-hand manipulation tasks, the agent is asked to rotate the object to a specific pose. The offline dataset for each task is a mixture of 500 expert trajectories and 2,000 random trajectories. The fetching tasks and datasets are taken from . The expert trajectories for the in-hand manipulation tasks are generated similarly to Liu et al. (2022a).
Baselines.
For the experiments, we use TD3 Fujimoto et al. (2018) and TD3BC Fujimoto & Gu (2021) as they are built on the classical Q-learning approaches and solid baselines for many offline RL tasks . The HER ratio of 0.5 was used with all methods in all experiments. We trained each method for 10 seeds, and each training run uses 500k updates. The mini-batch size is 512. We used the cumulative test rewards from the environments as the performance metric in all experiments. Figure 5: Six goal-conditioned tasks.
PRE-TRAINING VALUE FUNCTION
To construct the prioritized goal-swapping experience replay, we first need to train a Q-function that can assign reasonable values to state-goal-action pairs. In this stage, several algorithms can be chosen to pre-train the value function, such as conservative Q-learning , actionable models Chebotar et al. (2021), TD3BC Fujimoto & Gu (2021), etc. In this work, we choose to use TD3BC Fujimoto & Gu (2021) as it has a minimal modification on the original Q-learning (its value function is not heavily modified as other offline methods). To ensure the Q function covers as much state-goal-action space as possible, we combined the random goal-swapping augmentation (Alg. 1) with TD3BC during the pre-training. We trained each value function for each task with 1000k updates, and each update's batch size is 512.
PRE-TRAINED Q VALUE DISTRIBUTION
We aim to validate our first research proposal: whether a pre-trained Q function can identify if transitions are goal-reachable. To do this, we created two classes of data: ζ N and ζ P . ζ N contains randomly augmented transitions with goal-swapping, while ζ P contains expert transitions. We used a pre-trained value function Q to evaluate all transitions in these two sets, and the results are displayed in Fig. 6. We believe that the differences in the Q-value distributions provide evidence to support our hypothesis that a pre-trained Q function can identify if transitions are goal-reachable. Overall, the Q-value distribution of ζ P notably differs from that of ζ N . Generally, ζ P has higher Q-values than ζ N , which validates our proposal in Section 4.2, that goal-reachable transitions will possess higher Q-values. This statement holds true for the tasks FetchPush, FetchPick, HandEgg and HandBlockZ. It is worth noting that for the simple task FetchReach, the Q-value of ζ N has a similar distribution as ζ P , suggesting that the random goal-swapping augmentation likely yields positive augmentations. Moreover, in task FetchSlide, the expert also provides unsuccessful trajectories, resulting in ζ P exhibiting two peaks in its Q-value (one on the minimal Q-value side and one on the maximum Q-value side). This is reflected in the Q-value distribution of ζ N , which mirrors the distribution of ζ P .
We also implement a set of simple linear classifiers that utilize the estimated Q-value to classify whether a given state-goal-action pair ζ = {s, g, a, r, s } is a reachable transition. The classification results are presented in Table 1. As shown in the table, the pre-trained value function Q is able to accurately estimate the goal-reachable transitions in the FetchPush, FetchPick, HandEgg, and HandBlockZ tasks. The results demonstrate that the pre-trained value function Q is able to identify the goal-reachable transitions.
PERFORMANCE
In this experiment, we aim to study the impact of prioritized goal-swapping experience replay on offline goal-conditioned reinforcement learning (RL). We used two deep Q-learning approaches, TD3 and TD3BC, to compare the performance of agents trained with prioritized goal-swapping experience replay (Alg. 2) versus the original algorithm and agents trained with random goal-swapping experience replay (Alg. 1). The comparison results are presented in Tab. 2.
Overall, the prioritized goal-swapping experience replay provided more robust performance improvements than the random goal-swapping augmentation for offline GCRL tasks. In particular, the Table 2: Evaluation of the goal-swapping augmentation in offline RL. The tested methods were trained without (*method*) and with (*method*-aug) the random goal-swapping augmentation (Alg. 1). The methods (*method*-mem) were trained with prioritized goal-swapping augmentation. The performance metric is the average cumulative reward over 50 random episodes. indicates that the augmented variant outperforms the original baseline according to the t-test with p-value < 0.05. text indicates that the (*method*-mem) variant outperforms the (*method*-aug) variant according to the t-test with p-value < 0.05. | 2023-02-16T02:15:49.618Z | 2023-02-15T00:00:00.000 | {
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216170263 | pes2o/s2orc | v3-fos-license | The effect of different levels of Fenugreek (Trigonella foenum-graecum L.) powder and extract on performance, egg quality, blood parameters and immune responses of laying hens in second production cycle
The present study was carried out to determine the effects of fenugreek powder (FP) and extract (FE) on performance, egg quality, blood parameters and immune responses of laying hens. One-hundred and fifty Leghorn laying hens were used in a completely randomized design with five treatments and five replicates for eight weeks. Treatments were various levels of FP and FE including zero (control; T1), 1.00% FP (T2), 2.00% FP (T3), 0.10% FE (T4) and 0.20% FE (T5). The results of this experiment showed that feed intake was increased linearly by the inclusion of FP compared to the control group. Supplementation of laying hens diet with 2.00% FP adversely affected feed conversion ratio (FCR). The FCR was decreased by 0.10% inclusion of FE compared to 0.20%. Egg yolk color was the highest when 1.00% FP added to laying hens diets compared to the other treatments. Serum metabolites and immune responses of laying hens were not affected significantly by fenugreek supplementation. From the results of the present study, it can be concluded that using 1.00% FP can improve feed intake by supporting FCR. Inclusion of 1.00% FP in laying hens diet enhanced egg yolk color of laying hens in the second production cycle.
Introduction
During the last decade and after banning the use of antibiotic growth promoters (AGP) in poultry nutrition, plant secondary metabolites and their derivatives have attracted a lot of attention for their potential role as alternatives for AGP. Recent studies showed that herbs and their derivatives can enhance growth performance and production properties and modulate the immune system of farm animals under normal or stress conditions. 1 Fenugreek (Trigonella foenum-graecum L.) a multifunctional herb belonging to the family of Fabacecae is known for its antifungal, antiviral, anticarcinogenic, antidiabetic and antimicrobial properties. 2 This plant is one of the oldest medicinal plants with excellent medicinal and nutritional properties. 3 Fenugreek contains numerous bioactive constituents such as alkaloids, flavonoids, steroid and saponins. 4 Alkaloids of this plant include trigocoumarin, nicotinic acid, trimeth coumarin, and trigonelline. 5 Fenugreek is an excellent off-season fodder and animal food supplement, due to having a high proportion of protein, fatty acids, and total carbohydrates. 4 Some researchers have studied the use of fenugreek in poultry feeding and their results have suggested that feed conversion ratios (FCRs) are affected positively by inclusion fenugreek into laying hens diets. 6 In this regard, researchers have reported that fenugreek extract in drinking water can improve product performance and immune system of laying hens, 4 however, others have noticed, unlike the previous observations that the use of fenugreek in layer diets in amounts of 1.00% and 2.00% has a negative influence on the egg production. 7 The objective of the present study was to confirm previous work on dietary supplementation of fenugreek powder (FP) or extract (FE) on performance, egg quality, blood parameters and immune response of laying hens.
Materials and Methods
Fenugreek was purchased from a local producer and the parts of the plant suitable for consumption were dried in shade and powdered. The voucher specimens were deposited at Khuzestan Agricultural Sciences and Natural Resources University Herbarium (KHAU), Ahvaz, Iran (voucher no. 262). For the preparation of extract, an adequate amount of fenugreek powdered material was macerated with ethanol (80.00%) in a proportion of 1:5 (w/v) and left for 72 hr at room temperature. The extract was then shacked, filtered and evaporated in a rotating evaporator until the solvent was disappeared and gave semi-solid mass yielded about 10.00% w/w. 8 Prior to feeding trial, the chemical composition of FP was determined by the Association of Official Analytical Chemists methods. Crude protein, total lipids, crude fiber, ash, and nitrogen-free extract content of fenugreek samples were 19.95, 7.50, 28.84, 13.83 and 19.22 of a percentage of dry matter, respectively. All procedures used in the current experiment were approved by the Committee on Poultry Research of the Agricultural Sciences and Natural Resources University of Khuzestan, Ahvaz, Iran (215-11.20.2016). One-hundred and fifty Leghorn (Hy-Line, W-36) laying hens in a second production cycle were used in a completely randomized design with five treatments and five replicates (n = 6) for eight weeks. Treatments were various levels of FP and FE including zero (control; T1), 1.00% FP (T2), 2.00% FP (T3), 0.10% FE (T4) and 0.20% FE (T5). The laying hens were fed a corn-soybean meal-based diet supplemented with FP by exposing of wheat bran or supplemented with FE on top of the basal diet (Table 1). Feed and water were offered ad libitum throughout the experimental periods. Egg weight (EW; g), egg mass (EM; g per hen daily), henday egg production (EP; %) and feed intake (FI; g) were measured daily and calculated for a whole experimental period. The FCR was also calculated as the ratio of FI per EM during the experiment. At the end of each week, 10 eggs were randomly collected from each treatment (two eggs per replicate) and individually weighted and external and internal egg quality traits were determined. The egg shape index was calculated by dividing egg width by egg length. Eggs Shell, yolk and albumen percentages were calculated by their weights. Egg shell thickness was measured at three different points (top, middle, and bottom) using a micrometer and the average shell thickness (mm) was obtained from the average values of these three parts. Eggshell strength was determined by mechanical resistive device (Karl Kolb GmbH & Co., Dreieich, Germany). Albumin height, Haugh unit, and yolk color were described by an automatic egg analysis machine (EMT-5200; Robotmation Co. Ltd., Tokyo, Japan). Blood samples were randomly collected from 10 birds per treatment from the wing vein into sterilized tubes.
The tubes were centrifuged (Hk 36; Hermle, Wehingen, Germany) at 3,000 rpm for 15 min and separated serum was stored in a freezer at -20 ˚C until the time of biochemical analysis. The sera were used for the colorimetric determination of the blood sugar, triglyceride and cholesterol using commercial kits (Pars Azmoon, Tehran, Iran) according to the manufacturer's protocols. For determination of immune response, at 5 th and 7 th weeks of the experiment, 0.50 mL 20.00% suspension of sheep red blood cells (SRBCs) was injected into breast muscle of two hens per replicate and blood samples were taken from wing vein one week after each injection. Serum was separated and evaluated for antibody titer against SRBCs by the hemagglutination method.
Statistical analysis. The present experiment was based on a completely randomized design. All results were statistically analyzed by General Linear Models, one-way analysis of variance, using SAS software (version 8.0; SAS Institute, Cary, USA). Significance between means was tested using Duncan multiple range tests. A probability value of p ≤ 0.05 indicated that the difference was statistically significant.
Results
The results of productive traits are given in Table 2.
The data indicate that EW, EM, and EP were not affected significantly by supplementation of FP or FE to the diets.
Hens fed a diet supplemented with 0.10% of FE, numerically produced about 5.70% more egg rather than a control group. Feed intake was affected significantly by using FP in laying hens diet and it was the highest at 2.00%. In terms of FCR, it was increased in T3 birds as a result of increasing FI in this group. Table 3 shows that the diet had no significant effect on egg quality traits such as egg shape index, Haugh unit, shell strength (kg per cm 2 ), shell thickness (mm) and shell, albumen and yolk weights (%). Only, egg yolk color was increased significantly by supplementation of hen diets with 1.00% FP.
Inclusion of FP and FE in experimental diets did not significantly affect the serum parameters such as glucose, triglycerides, and cholesterol throughout the experimental period (Table 4), (p > 0.05). However, serum triglycerides and cholesterol were decreased numerically in T4 rather than the control group (respectively 19.30 and 25.30%).
The effects of supplemental FP and FE on antibody titer against SRBCs of laying hens have been shown in Table 4. Results from this table represented that the inclusion of FP and FE had no significant effect on primary and secondary antibodies titer against SRBCs (p > 0.05).
Discussion
The FI was increased significantly in T2 and T3, but FCR was improved only in T2. As mentioned above, FCR was calculated by dividing FI to EM. In this study, FCR was increased by increasing FI as a result of supplementation of FP to laying hens diets. In 1.00% inclusion of FP, FI was increased, but FCR was the same as the control group. It was indicated that the FP at 1.00% can increase FI without changing FCR, thus in the equation, increasing FI without changing FCR resulted in EM improvement. In this study, FI increased linearly by supplementation of FP to laying hens diet. In the first step, increasing animal production depends on increasing FI. In this regard, it was shown that fenugreek contains bioactive components including biotin, trimethylamine, and neurin which can stimulate FI by their action on the nervous system. 1,4,9 Increasing hens FI by inclusion FP may result from the action of FP on digestive enzyme secretion. 1 In line with present results, other researchers have reported that inclusion plant secondary metabolites (essential oils and oleoresins) in diet can increase the pancreatic digestive enzymes. 1 Platel and Srinivasan have shown that fenugreek can increase pancreatic lipase activity in rats. 10 El-Mallah et al. have noted that increasing fenugreek seeds to turkey chicks' diet significantly increases nitrogen-free extract (NFE) digestibility. 11 El-Kaiaty et al. have reported that inclusion 0.50% fenugreek in laying hens diet has no significant effect on feed consumption compared to the control group. 12 In terms of EP and EM, the results of the current study were in agreement with El-Kaiaty et al. findings and in contrast with Abdalla et al. results. 12,13 Researchers have indicated that EP, EW, and EM of laying hens fed diets supplemented with fenugreek, cinnamon, fennel, and anise or their mixture are increased significantly. 13 These researchers have indicated that increases in EP, EW, and EM of laying hens may be due to the presence of unknown factors in herbs mixtures which have been considered essential for egg production. 13 Bayatizadeh et al. have reported that fenugreek as a good source of dietary protein and lipids can stimulate production parameters and improve FCR of laying hens. 4 Others have reported that FI and water consumption are not affected by addition of fenugreek seed aqueous extract to broiler water, but the weight of breast, thigh, and leg of broiler are significantly increased. 14 These researchers have concluded that the improvement in muscle weight in broiler may be due to antioxidant property of this plant which increases digestive enzymes and decreases bacterial activity. 14 Supplementation layer diet with FP and FE had no significant effect on egg quality except egg yolk color. Egg yolk color is one of the important egg quality characteristics and can affect egg marketing. A significant increase in egg yolk color by FP (T2) may be associated with the presence of carotenoids such as beta carotene in FP which can be deposited in egg yolk. 15 The enhancement of egg yolk color by medical plants has been observed in previous studies. 16,17 Although FI of laying hens was the highest in T3, but egg yolk color was not affected in this treatment. It has been demonstrated that dietary fibers (particularly soluble fibers) may increase digesta viscosity, disrupt lipid absorption and also impair bile acid reabsorption. 18 In the present study, the fiber content of T3 was the highest compared to T1 and T2 (crud fibers were 3.04, 3.22 and 3.40 respectively in T1, T2, and T3) and this may affect lipid (and its constituents such as carotenoids) digestion and absorption. Although, in the present study hens serum lipid constituents were not affected by different levels of FP. This inconsistency needs to be more investigated. El-Kloub has detected a numerical increase in egg shape index, yolk index and shell thickness when white laying hens were fed diets supplemented with 0.15% fenugreek from 40 to 59 weeks of age, but the differences were not significant, however, a significant decrease in Haugh units was detected. 19 Results of the present study were not in agreement with the findings of El-Kaiaty et al. reporting that fenugreek has a significant effect on yolk and albumen weights. 12 In the present study, it was shown that hen's serum constituents were not affected by fenugreek supplementation. Blood biochemical parameters represent the nutritional and physiological status and variation of these parameters usually reflects the health of an animal. 20 The results of this study are in contrast with others reporting a decrease in laying hens serum total cholesterol concentration and an increase in highdensity lipoprotein cholesterol concentration duo to fenugreek seed extract. 17 Also, El-Kaiaty et al. indicated that fenugreek seeds extract contains steroid saponins and reduce serum cholesterol. 12 The results of this study are in agreement with Weerasingha and Atapattu reporting that serum cholesterol levels are not affected by the dietary fenugreek seed powder in broiler chicken. 21 Previous findings have indicated that fenugreek contains bioactive components such as minerals, vitamins, lecithin and choline that help to dissolve cholesterol and fatty substances. 22 The previous study has indicated that fenugreek seed and its extract can reduce blood glucose levels. 23 The results from this study represented that the inclusion of FP and FE had no effect on antibody titer against SRBCs. In contrast with this results of Bayatizadeh et al. indicating that supplementation of FE to laying hens drinking water improves immune response (total antibody, immunoglobulin M and immunoglobulin G) of laying hens. 4 According to the results of the present study, dietary inclusion of FP and FE had no significant effect on performance parameters of laying hens, although, egg yolk color increased effectively by adding 1.00% of FP to the diet via transferring carotenoids from diet to egg yolk. | 2020-04-27T20:39:11.266Z | 2020-03-15T00:00:00.000 | {
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9131447 | pes2o/s2orc | v3-fos-license | Loss of Deacetylation Activity of Hdac6 Affects Emotional Behavior in Mice
Acetylation is mediated by acetyltransferases and deacetylases, and occurs not only on histones but also on diverse proteins. Although histone acetylation in chromatin structure and transcription has been well studied, the biological roles of non-histone acetylation remain elusive. Histone deacetylase 6 (Hdac6), a member of the histone deacetylase (HDAC) family, is a unique deacetylase that localizes to cytoplasm and functions in many cellular events by deacetylating non-histone proteins including α-tubulin, Hsp90, and cortactin. Since robust expression of Hdac6 is observed in brain, it would be expected that Hdac6-mediated reversible acetylation plays essential roles in CNS. Here we demonstrate the crucial roles of Hdac6 deacetylase activity in the expression of emotional behavior in mice. We found that Hdac6-deficient mice exhibit hyperactivity, less anxiety, and antidepressant-like behavior in behavioral tests. Moreover, administration of Hdac6-specific inhibitor replicated antidepressant-like behavior in mice. In good agreement with behavioral phenotypes of Hdac6-deficient mice, Hdac6 dominantly localizes to the dorsal and median raphe nuclei, which are involved in emotional behaviors. These findings suggest that HDAC6-mediated reversible acetylation might contribute to maintain proper neuronal activity in serotonergic neurons, and also provide a new therapeutic target for depression.
Introduction
Acetylation of the e-amino group of lysine is a reversible posttranslational modification mediated by acetyltransferases and deacetylases. This type of acetylation is not restricted to histones but also occurs on diverse proteins and affects functions such as DNA binding, protein-protein interaction, enzymatic activity, and stability. Therefore, lysine acetylation emerges as an important post-translational modification that regulates a wide range of cellular processes (reviewed in [1]).
Histone deacetylases (HDACs) are a family of enzymes with 18 isoforms in mammals, and are grouped into four classes by sequence homology [2]. HDAC6 belongs to class II, and has a unique structure with two catalytic domains and a C-terminal BUZ domain that binds ubiquitin. HDAC6 gene is located on X chromosome both in mice and human genome [3,4]. In mice, Hdac6 protein is broadly expressed in multiple tissues, particularly abundant in the brain and testis (Fig. S1) [5]. HDAC6 is known to be a multi-functional cytoplasmic deacetylase that controls cell motility [6][7][8][9], endocytosis [10], vesicle transport [11], glucocorticoid receptor maturation [12], autophagic protein degradation [13][14][15], and aggresome formation [16] by deacetylating atubulin, cortactin, and Hsp90. These cellular events are closely related to the acquisition and maintenance of proper function in neurons. For example, at synapses, vesicle transport and endocytosis are underlying mechanisms for neurotransmitter release and recycling. Glucocorticoid receptor maturation is necessary for the negative feedback regulation of stress at the hippocampus [17]. Autophagic protein degradation and aggresome formation are a part of the quality control of proteins, and their disturbance leads to neurodegenerative disorders [18]. Despite the fact that Hdac6 protein is abundantly expressed in mice brain, the physiological implications of Hdac6 as well as nonhistone acetylation in neural function are poorly understood. In this study, we found that Hdac6 is highly expressed in serotonergic neuron, and that loss of Hdac6 deacetylase activity leads to hyperactivity, less-anxiety and antidepressant-like behavior in mice. Our findings suggest that Hdac6-mediated non-histone deacetylation plays crucial roles in the expression of emotional behaviors.
Hdac6 is localized to serotonergic neurons in raphe nuclei
To study the physiological implications of Hdac6 in CNS, we first examined the distribution of Hdac6 in mouse brain by using specific antibody for mouse Hdac6. We found robust expression of Hdac6 in the medial and dorsal raphe nuclei (Fig. 1A), while (A-C) Hdac6 distribution in mouse brain. Hdac6 protein is visualized by an antibody to Hdac6 (green: each right panel) in the coronal sections of adult mouse brain spanning raphe nuclei (A), hippocampus (B), and motor cortex (C). Nucleus is visualized by DAPI staining (each left panel). Arrowheads indicate Hdac6-positive cells magnified in insets. (D) Coexpression of Hdac6 with Tph2 in dorsal raphe neuron. The coronal brain sections of the dorsal raphe from WT (upper panels) and Hdac6 KO mice (lower panels) were double immunostained for Hdac6 (red) and Tph2 (green), a marker of mature serotonin neuron. No signal was detected with anti-Hdac6 in Hdac6 KO mice. Merged images were shown in right panels (blue: DAPI staining). (E) HDAC6 expression in human brainstem. HDAC6 is visualized by antibody to HDAC6 in the horizontal sections of the postmortem human brainstem spanning raphe nuclei, locus ceruleus, and substantia nigra. DR, dorsal raphe nucleus; MnR, median raphe nucleus; Aq, aqueduct. Scale bars, 100 mm. doi:10.1371/journal.pone.0030924.g001 Hdac6 was expressed only weakly and sparsely in hippocampus (Fig. 1B), cerebral cortex (Fig. 1C), and other brain regions (data not shown). In the medial and dorsal raphe nuclei of the WT brain, Hdac6 signal was almost exclusively co-localized with tryptophan hydroxylase 2 (Tph2) that was used as a marker for serotonin neuron (Fig. 1D) [19]. No signal was detected with Hdac6 antibody in where Tph2 signal was positive in raphe nuclei of Hdac6 KO mice (Fig. 1D), confirming the specificity of our antibody. These results clearly indicated that mature serotonergic neurons highly express Hdac6 in mice. Similar distribution of HDAC6 was observed in human postmortem brain with additional expression of HDAC6 in substantia nigra and locus ceruleus (Fig. 1E).
The ascending serotonergic projections are derived largely from raphe nuclei and the serotonergic system contributes to regulate emotional behaviors. Indeed, the central serotonergic system is considered to be closely associated with the pathogenesis of psychiatric diseases such as depression, schizophrenia, or some anxiety disorders [20]. Therefore, these results raise the possibility that HDAC6 is involved in the expression of emotional behaviors.
Hdac6-deficient mice show emotional abnormalities
Hdac6-deficient (KO) mice are viable and fertile, and develop normally, regardless of the hyperacetylation of a-tubulin in most tissues [5]. Our histological examination revealed no obvious difference between wild-type (WT) and Hdac6 KO mouse brains (data not shown). To determine whether Hdac6 KO mice suffer from emotional abnormality, we performed a series of behavioral tests. In the open field test, total distance traveled, an index of activity, was significantly elevated in Hdac6 KO mice compared with that in WT mice (145% on average; p,0.001), while there were no significant differences between WT and KO mice in the number of entries into the center zone, which is an index of anxiety ( Fig. 2A). These data suggest that Hdac6 KO mice suffer from hyperactivity under novel environment. Next, we conducted an elevated plus-maze test, a commonly used test for assessing anxiety. Mice normally avoid the open arms (OAs) of the elevated plus-maze owing to anxiety. In this test, Hdac6 KO mice showed increased number of entries into the OAs (192% on average; p,0.05) and spent more time in OAs compared with WT mice (205%; p,0.05), but total distance traveled, an index of activity, was not significantly different between genotypes (p = 0.094) (Fig. 2B). These results suggest that Hdac6 KO mice have less anxiety.
To further examine the emotional aspects of Hdac6 KO mice, we employed the tail suspension test. This is a widely used test for assessing antidepressant activity by measuring the duration of immobility during a 6 min session; administration of antidepres- and spent more time in OAs (middle) compared with WT mice, but total distance traveled, an index of activity, was not significantly different between genotypes (right; n = 24 and 23 for WT and KO, respectively). (C) Antidepressant-like behavior of Hdac6 KO mice in the tail suspension test. Hdac6 KO mice showed decreased immobility compared with WT mice (n = 12 for WT and KO mice). (D) Normal home cage activity of Hdac6 KO mice. Home cage activity of KO mice in both light and dark periods was not distinguishable from that of WT mice (n = 5 and 6 for WT and KO, respectively). (E) Normal stress response of Hdac6 KO mice. No genotype differences were found in serum corticosterone levels in both basal and stressful conditions (n = 9, 10, 10, and 10 for control WT, control KO, +stress WT, and +stress KO, respectively). (F) Effect of fluoxetine on the immobility of WT and Hdac6 KO mice in the tail suspension test (n = 18, 18, 12, and 13 for saline WT, saline KO, fluoxetine WT, and fluoxetine KO, respectively). doi:10.1371/journal.pone.0030924.g002 sant decreases the immobility in rodents [21]. In this test, immobility time of Hdac6 KO mice significantly decreased in 75% of WT mice (p,0.05) (Fig. 2C), indicating that Hdac6 KO mice show antidepressant-like activity.
Behavioral abnormalities observed in Hdac6 KO mice here seem to be a consequence of emotional arousal. However, there is still some room for argument about the possibility that behavioral abnormalities in Hdac6 KO mice are merely due to increased locomotor activity, which is a key component of many behavioral tests. In order to assess whether basal locomotor activity was affected in Hdac6 KO mice, we recorded the home cage activity of WT and Hdac6 KO mice using an ANIMEX activity meter (ANIMEX, AB Farad) under a 12-h light/dark cycle for 24 h. Home cage activity of Hdac6 KO mice in both light and dark periods, however, was not distinguishable from that of WT mice (Fig. 2D) as well as circadian rhythms (data not shown). In addition, no significant difference between WT and Hdac6 KO mice was observed in neurophysiological screening (Table S1). These results indicate that the basal locomotor activity and the neurophysiological functions of Hdac6 KO mice are normal. It should be noted that the hyperactivity is only induced when Hdac6 KO mice is exposed to novel environment. This result implies that Hdac6 KO mice exhibit mental abnormality during unfamiliar environment.
Since stress is considered to be one of the important contributors in the etiology of depression, we investigated whether stress state and/or stress response is affected in Hdac6 KO mice. The hypothalamic-pituitary-adrenal (HPA) axis is a principal effector of the stress response, and is often activated during depression owing to impaired negative feedback regulation, which leads to an increase in serum glucocorticoid [22]. Therefore, we determined the concentration of serum glucocorticoid (corticosterone in mice) in both basal and stressful conditions induced by the tail suspension test. However, we found no difference between WT and Hdac6 KO mice (Fig. 2E). This suggests that the basal activity of HPA axis as well as stress response is normal in Hdac6 KO mice. Altogether, we concluded that behavioral abnormalities observed in Hdac6 KO mice arise from emotional arousal.
To obtain insight into the mechanism of emotional abnormalities of Hdac6 KO mice, we focused on the alteration in the content of serotonin, an important factor of etiology of depression [20]. Since Hdac6 is concentrated in serotonergic neurons in the dorsal and median raphe nuclei, Hdac6 depletion in these neurons would affect serotonin biosynthesis, resulting in change in the content of serotonin. To examine this, we measured the amount of serotonin and Tph2, a rate-limiting enzyme of serotonin synthesis in both WT and Hdac6 KO mice. For measuring serotonin concentration by immunoassay, we used the whole brain extract, because serotonergic neurons project to almost all regions of the brain. For evaluation of the expression level of Tph2, we used raphe nuclei extract. However, we found no obvious difference in both serotonin concentration and Tph2 level between WT and Hdac6 KO mice ( Fig. S2A and S2B). Moreover, the Tph2 distribution in the raphe nuclei of Hdac6 KO mice was also not distinguished from that of WT mice (Fig. 1D). These results suggest that serotonin synthesis and content in Hdac6 KO mice is normal.
Since acute administration of antidepressants reduces the immobility in the tail suspension test in mice typically by blocking serotonin reuptake and enhancing serotonin neurotransmission [20], it would be expected that Hdac6 KO mice have already been affected in this process. In an attempt to clarify this issue, we examined the sensitivity to antidepressant in Hdac6 KO mice. We injected fluoxetine, a selective serotonin reuptake inhibitor (SSRI), into Hdac6 KO mice, and evaluated its potency by the tail suspension test. As a result, fluoxetine significantly decreased the immobility of Hdac6 KO mice (48% vs. saline control; F (1,57) = 46.2; p,0.0001), and its potency for Hdac6 KO mice was similar levels to that for WT mice (56% vs. saline control) (Fig. 2F). Desipramine, a blocker of noradrenalin reuptake, and imipramine, a serotonin and noradrenalin reuptake inhibitor (SNRI), showed similar effects in Hdac6 KO mice (Fig. S3). These results suggest that loss of Hdac6 might not affect the monoamine reuptake targeted by SSRI/SNRI.
Administration of HDAC6 specific inhibitor leads to antidepressant-like behavior in mice
To explore whether antidepressant-like behavior in Hdac6 KO mice is simply due to loss of Hdac6 deacetylase activity, we adopted an approach to inhibit Hdac6 deacetylase activity in WT mice. For this purpose, we first tested the specificity of NCT-14b, one of a series of thiolate analogues that inhibit HDAC6 [23]. Since HDAC6 is localized exclusively in the cytoplasm and deacetylates a-tubulin [6,7,24], inhibition of Hdac6 activity would increase the amount of acetylated a-tubulin, but not that of acetylated histones. As expected, NCT-14b treatment of the HeLa cells increased the acetylation of a-tubulin but not that of histone H3, demonstrating the specificity of NCT-14b for HDAC6 (Fig. 3A). In contrast, sodium butyrate, a well-known class I HDAC inhibitor that does not inhibit HDAC6, only increased the acetylation of histone H3, while a non-specific potent HDAC inhibitor, trichostatin A (TSA), increased the acetylation of both atubulin and histone H3 (Fig. 3A).
In the tail suspension test, intraperitoneal administration of NCT-14b significantly reduced the immobility of WT mice (79% vs. saline-injected control; F (1,63) = 4.22; p = 0.044) (Fig. 3B). In contrast, the same amount of NCT-14b had little effect on the immobility of Hdac6 KO mice, indicating that NCT-14b targets Hdac6 to reduce immobility. It should be noted that the immobility-reducing effect of NCT-14b reached at almost equal to that of Hdac6 gene knockout (75% vs. WT mice; p,0.05; see Fig. 2C). This indicates that the antidepressant-like behavior of Hdac6 KO mice is not only due to the long-term effect of gene depletion, but also caused by the transient loss of Hdac6 deacetylase activity in the adult brain. To evaluate the inhibition potency of NCT-14b in vivo, brains of two mice from each group in Figure 3B were isolated, and then the acetylation level of a-tubulin was examined. As expected, NCT-14b treatment modestly increased the amount of acetylated a-tubulin in WT mice brain (Fig. 3C). To examine the local effects in the raphe nuclei of NCT-14b administration on a-tubulin acetylation , we injected same concentration of NCT-14b into WT mice, and evaluated the levels of a-tubulin acetylation in isolated raphe nuclei specimen. As shown in Figure S4, modest increase of a-tubulin acetylation was observed in raphe nuclei of NCT-14b-injected mice. These results confirm the validity of NCT-14b in in vivo application, and demonstrate that inhibition of the deacetylase activity of Hdac6 causes antidepressant-like behavior in mice.
Discussion
In the present study, we provided evidence that non-histone protein acetylation plays an important role in CNS by focusing on Hdac6, a multifunctional cytoplasmic deacetylase. We demonstrated that disturbance of Hdac6-mediated protein deacetylation both by gene knockout and by chemical inhibition of deacetylase activity results in behavioral consequences related to emotion in mice. Emotional features such as fear and depression are known to be closely associated with the serotonergic system. In good agreement with this, Hdac6 is most abundant in serotonergic neurons in the dorsal and median raphe nuclei, the origin of the ascending serotonergic fibers innervating the forebrain and amygdala [25,26]. Thus, it is plausible that Hdac6-mediated reversible acetylation in serotonergic neurons regulates its neuronal functions.
Recently, an antidepressant-like effect of HDAC inbitors, N-(2aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl) aminomethyl]benzamide (MS-275; classI HDACs inhibitor) and suberoylanilide hydroxamic acid (SAHA; class I and II HDACs inhibitor), in social defeat paradigms has been reported in mice [27]. Moreover, Hdac2 deficiency as well as chronic treatment with SAHA in mice leads to memory facilitation by increasing the synapse number in hippocampus CA1 region [28]. In these reports, transcriptional regulation controlled by the dynamic change of histone acetylation either in the nucleus accumbens [27] or hippocampus [28] is responsible for antidepressant-like effect or memory facilitation, respectively. In contrast, since the level of histones acetylation is not affected by both Hdac6 gene knockout (Fig. S2B) [24] and chemical inhibition of HDAC6 activity in Hela cells (Fig. 3A), it is clear that abnormality of emotional behavior caused by Hdac6 inhibition does not result from alteration of transcriptional regulation. To our knowledge, this is the first report to identify the association between non-histone protein acetylation and higher brain function.
The elevated plusmaze test and the tail suspension test, adopted in this study, are well-established behavioral experiments assessing the anxiety and depression, respectively. Although Hdac6 KO mice exhibited less anxiety and antidepressant-like activity in these tests, we should point out the possibility that such behavior is, in part, responsible for increased locomotor activity under a novel environment. In this regard, we consider that such noveltyinduced abnormal hyperactivity represents emotional problems.
Although, at present, the molecular mechanism of how inhibition of Hdac6-mediated protein deacetylation leads to hyperactivity, less-anxiety, and antidepressant-like activity still remains elusive, we have obtained some insight into this issue. First, the mechanism underlying antidepressant-like behavior observed in both Hdac6 KO mice and NCT-14b-injected WT mice might be different from that of current antidepressants, which target serotonin and noradrenalin transporters and enhance the neurotransmission [20]. Our results showing that the antidepressants are still effective in Hdac6 KO mice in the tail suspension test (Fig. 2F) and serotonin content is not affected in Hdac6 KO mice (Fig. S2A) support this view. Second, Hdac6 may contribute to regulate emotional behavior by deacetylating other Hdac6 substrate(s) besides a-tubulin. It should be noted that the amount of acetylated a-tubulin in NCT-14b-injected WT mice brain as well as dorsal raphe region does not reach the same level as that of Hdac6 KO mice (Figs. 3C, S4) even though NCT-14b fully affects the immobility in WT mice (Fig. 3B). Thus, there is a discrepancy between acetylation levels of a-tubulin and immobility in NCT-14b-injected mice, and it raises the possibility that Hdac6 has a specific substrate other than a-tubulin in serotonergic neurons. Further study is needed to find a specific target for Hdac6, which would be a key for serotonergic function associated with an antidepressant-like behavioral response.
Our findings also suggest that HDAC6-mediated reversible acetylation can become a new therapeutic target for depression. Since NCT-14b seems to act as a non-monoamine-based antidepressant, HDAC6 inhibitors may have potential as medicines to overcome the disadvantage of current antidepressants, namely, their requirement of at least several weeks for therapeutic action [20]. Identification of HDAC6 substrates and related cellular processes in serotonergic neurons associated with the expression of antidepressant-like behavior will bring new insights into the molecular basis of mood disorders.
Animals
Hdac6 KO mice [10] were backcrossed at least six times with 129SV background. Because Hdac6 gene is located on X chromosome [3], male mice carrying the mutant allele are null for Hdac6. Mice were maintained on a 12-h light/dark cycle (lights on at 7:00 AM) and were allowed access to food and water ad libitum. We used male mice at 3-12 months of age for all experiments. All experimental procedures in this study and housing condition were reviewed and approved by the animal experimentation committee of the Institute for Developmental Research in Aichi Human Service Center (Permit number: M-19). Antibodies Rabbit polyclonal antibody against mouse Hdac6 was as described previously [10]. Anti-acetylated lysine monoclonal antibody (clone AKL5C1) was generously provided by Dr. Minoru Yoshida (RIKEN, Saitama, Japan). Anti-histone H3 rat monoclonal antibody was generous gift from Dr. Hiroshi Kimura (Osaka University, Osaka, Japan) [29]. Commercially available antibodies for a-tubulin (Cedarlane), Lys40-acetylated a-tubulin (Sigma), human HDAC6 (C300, Santa Cruz Biotechnology), acetylated histone H3 (Millipore), tryptophan hydroxylase 2 (NB100-74555, Novus biologicals), and pan-actin (NeoMarkers) were also used in this study. For the double immunostaining procedure, rabbit polyclonal antibody against mouse Hdac6 was directly coupled with the Alexa Fluor 555 dye using the Alexa Fluor 555 Monoclonal Antibody Labeling Kit (Invitorgen) according to the manufacturer's instructions.
Chemicals
Fluoxetine hydrochloride, desipramine hydrochloride, and imipramine hydrochloride were purchased from Sigma. These drugs were dissolved in saline and injected intraperitoneally in a volume of 10 ml/kg body weight 30 min prior to testing. Drug doses were 25 mg/kg imipramine, 30 mg/kg fluoxetine, and 20 mg/kg desipramine. These effective doses were chosen on the basis of previous reports on mice [21]. Trichostatin A (TSA) and sodium butyrate were purchased from Sigma. An HDAC6selective inhibitor, the compound (S)-S-7-(adamant-1-ylamino)-6-(tert-butoxycarbonyl)-7-oxoheptyl-2-methylpropanethioate (NCT-14b) [23], dissolved in saline at 4.8 mg/kg, was injected intraperitoneally in a volume of 10 ml/kg body weight 24 h prior to testing. We compared the effects of NCT-14b treatment period either 30 min or 24 h by the tail suspension test and found that 24 h treatment more effectively reduced immobility of WT mice (our unpublished data).
Behavioral procedures
All behavioral procedures were undertaken during the light period under 100 lux light intensity. Before behavioral testing, mice were placed in an experimental room for 30 min prior to testing. For the open field test, a mouse was placed in the center of an open field apparatus (white wooden box: 40640620 cm, divided into 9 even-sized squares (13613 cm)), and recorded using a video camera positioned above the apparatus. The total distance traveled in the open field was recorded for 10 min to measure the horizontal activity. The number of entries into the square at the center of the field was counted to evaluate anxiety. For the elevated plus-maze test, a mouse was placed into the center of the plus-maze apparatus (Ohara-Ika Sangyo), which was raised 50 cm above the floor, consisting of two open arms (40 cm long by 10 cm wide) and two enclosed arms of the same size with walls 20 cm high, and recorded using a video camera positioned above the apparatus. The number of entries into the open arms was counted over a 10 min test session to evaluate anxiety. A video-tracking system with computer interface and video camera (Any-maze; Stoelting) was used to automatically collect behavioral data. In this system, the animal's entries into defined areas of the apparatus were counted when 80% of animal's entire area was onto respective zone. For the tail suspension test, a mouse was fastened by the distal end of the tail to a plastic clip (Yamashita-Giken) and was suspended in a white wooden box (40625625 cm). The presence of immobility, defined as the absence of any movement, was counted over a 6 min test session by a highly trained observer who remained blind to genotype and treatment. For the analysis of home cage activity, home cage with a mouse was placed on an ANIMEX activity meter (AB Farad) and mouse activity was measured for 24 h.
Immunohistochemistry
For immunohistochemistry, mice were anesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were removed and cryostat sections (10 mm thick, coronal sections) were prepared with standard protocols. For double immunostaining for Hdac6 and TPH2, the brain section was firstly reacted with anti-TPH2 antibody (1:200 dilution), followed by Cy2-conjugated secondary antibody, and then fixed by 3% paraformaldehyde in PBS. Next, the section was reacted with the Alexa Fluor 555 labeled antimouse Hdac6 antibody and 49,69-diamidino-2-phenylindole (DAPI) to visualize Hdac6 and the nuclei, respectively. The immunofluorescent signals were observed under confocal laser scanning microscopy (FLUOVIEW FV1000; Olympus). For the detection of human HDAC6 in postmortem human brain, paraffin sections (5 mm thick, coronal sections) were prepared with standard protocols. Sections spanning raphe and locus ceruleus were derived from adult human brain, and sections spanning substantia nigra were from a newborn infant brain. Paraffin sections were stained with anti-human HDAC6 antibody (1:200 dilution) followed by HRP-labeled secondary antibody, and were observed by light microscopy (BX61; Olympus). All experimental procedure using postmortem human brain have been reviewed and approved by the Human Ethics Committee of the Institute for Developmental Research in Aichi Human Service Center (approved number: 03-02).
Biochemical analysis
To confirm the specificity of NCT-14b, HeLa cells (purchased from ATCC) were treated with NCT-14b (1 mM), TSA (0.1 mM), or sodium butyrate (5 mM) for 1 h. Then, cells were lysed in Laemmli sample buffer and subjected to Western blotting with antibodies against acetylated a-tubulin (1:2000 dilution), acetylated histone H3 (1:1000 dilution), human HDAC6 (1:1000 dilution), and actin (1:2000 dilution). For the quantification of acetylated atubulin in the brain, each mouse was killed by cervical dislocation, and brain was quickly removed and homogenized in 5 ml of buffer (5 mM Tris-HCl pH7.6, 0.32 M sucrose, 1 mM EDTA, 1 mM TSA) with a glass-teflon homogenizer. The homogenates were centrifuged at 1000 g for 10 min to remove nuclei and debris. The supernatants (brain extract) were then subjected to Western blotting with an ECL detection system (GE Healthcare) as described previously [30]. The dorsal raphe-containing slice (2 mm thick) of mouse brain was cut out with razor blades directed by a chilled precision brain slicer (BS-2000C, Braintree Scientific, Inc.), and the dorsal raphe region were trimmed with a 1 mm biopsy punch (BP-10F, Kai Industries Co. Ltd) and processed for Western blotting. Serum corticosterone concentration was determined by immunoassay according to the manufacturer's instructions (AssayMax Corticosterone ELISA Kit, Assaypro). To evaluate the stress response, serum was prepared 30 min after the end of the tail suspension test, and was subjected to immunoassay. The serotonin concentration of both serum and brain extract was measured by immunoassay according to the manufacturer's instructions (Serotonin ELISA kit, GenWay Biotech).
Statistical analysis
Student's t test was used for statistical comparison (Figs. 2A-C, S1A). For multiple comparisons, groups were compared using twoway analysis of variance, followed by Bonferroni's post hoc test (Figs. 2E, 2F, 3B and S3). The data are presented as mean 6 s.e.m., n indicates the sample number, and p denotes the significance (*p,0.05, **p,0.01, ***p,0.001). Figure S1 Abundant expression of Hdac6 in the brain and testis in mice. Two micrograms of tissue extract was analyzed by Western blotting with anti-Hdac6 antibody. (TIF) Figure S2 Normal serotonin content in Hdac6 KO mice. (A) Serotonin concentration of both brain extracts and serum of Hdac6 KO mice was determined by immunoassay. Serotonin concentrations of Hdac6 KO mice were comparable to that of WT mice (brain extracts; WT, 18.861.2 ng/mg protein (n = 5), Hdac6 KO mice, 20.462.3 ng/mg protein (n = 6), serum; WT, 1154 ng643 ng/mg protein (n = 3), Hdac6 KO mice, 12746252 ng/mg protein (n = 4). (B) The expression levels of indicated proteins in dorsal raphe region of WT and Hdac6 KO mice brain were analyzed by Western blotting. Acetylation level of a-tubulin in Hdac6 KO mice was higher than that of WT mice. In contrast, the acetylation of histone H3 in Hdac6 KO mice was similar to WT mice. The Tph2, a rate limiting enzyme of serotonin synthesis in the brain, was abundant in dorsal raphe compared to cortex, and the amount of Tph2 in Hdac6 KO mice was comparable to that of WT mice. The amount of a-tubulin, histone H3 and actin were shown as loading controls. (TIF) Figure S3 Effects of imipramine and desipramine on Hdac6 KO mice in the tail suspension test. Effects of acute injection of imipramine (25 mg/kg) and desipramine (20 mg/kg) on the immobility of WT and Hdac6 KO mice in the tail suspension test was investigated. Imipramine significantly reduced both immobility times of WT (56% on average) and Hdac6 KO mice (59%) compared with each of saline-injected control mice (n = 18, 19, 18, and 16 for saline WT, saline KO, imipramine WT, and imipramine KO, respectively; F (1,67) = 39.47; p,0.0001). Desipramine showed similar results as reducing immobity of WT (62%) and Hdac6 KO mice (60%) (n = 18, 18, 11, and 12 for saline WT, saline KO, desipramine WT, and desipramine KO, respectively; F (1,55) = 76.52; p,0.0001). Data were presented as mean 6 s.e.m., and statistically analyzed by two-way analysis of variance followed by Bonferroni's post hoc test. (TIF) Figure S4 Effects of NCT-14b administration on tubulin acetylation in dorsal raphe. Amounts of acetylated a-tubulin (Ac-a-Tub) in cortex and dorsal raphe 24 h after NCT-14b administration were analyzed by Western blotting. Lower panel showed quantification of Ac-a-Tub normalized by a-tubulin (a-Tub). Although NCT-14b (14b) slightly increased the amount of Ac-a-Tub in dorsal raphe, it did not reach the same level as that of Hdac6 KO mice (KO). The effect of NCT-14b was more pronounced in motor cortex than in dorsal raphe. (TIF) | 2017-04-13T00:38:47.393Z | 2012-02-06T00:00:00.000 | {
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28429211 | pes2o/s2orc | v3-fos-license | Reversible Inhibitions of Gastric H’,K’-ATPase by Scopadulcic Acid B and Diacetyl Scopadol
Scopadulcic acid B (SA-B), a novel diterpenoid, is a main ingredient of the Paraguayan traditional medicinal herb “Typychi kuratii” (Scoparia dulcis L.). SAB and its debenzoyl derivative, diacetyl scopadol (DAS), specifically inhibit ATP hydrolysis of gastric H+,K’-ATPase. Both compounds inhibit the K+-dependent dephosphorylation step of the enzyme without any effect on the phosphorylation step. SA-B is a mixed-type inhibitor with respect to the activating cation, K+. SA-B lowers the affinity of H+,K+-ATPase to K+ and decreases the maximal velocity of ATP hydrolysis, whereas DAS is an uncompetitive inhibitor with respect to K+. Furthermore, the effects of SA-B and DAS on conformational states of the ATPase were studied by measuring the changes in the fluorescence intensity of the fluorescein isothiocyanate-labeled enzyme. The fluorescence study shows that SA-B primarily binds to the E2K form in the presence of Mg” and stabilizes the form and that DAS stabilizes the EzPK form. Therefore, the chemical modification of SA-B, debenzoylation, induced the changes in the pattern of inhibition of H’,K+-ATPase. Furthermore, the inhibition mechanisms of SA-B and DAS were different from those of omeprazole, which is an irreversible inhibitor, and SCH 28080, which is a reversible, competitive inhibitor with respect to K+. DAS also inhibited the K+-dependent p-nitrophenylphosphatase activity, and the inhibition was competitive with respect to K+, indicating that the K+-dependent p-nitrophenylphosphatase activity does not represent the partial reaction step of H’,K’-ATPase.
with respect to K+. DAS also inhibited the K+-dependent p-nitrophenylphosphatase activity, and the inhibition was competitive with respect to K+, indicating that the K+-dependent p-nitrophenylphosphatase activity does not represent the partial reaction step of H',K'-ATPase.
H',K'-ATPase is a proton pump for gastric acid secretion (l-5). Two types of compounds are well known to specifically inhibit H',K'-ATPase; one is substituted benzimidazoles such as omeprazole (6), picoprazole (7), and E3810 (8), and the other is substituted imidazo[l,Z-alpyridine such as SCH 28080 (9). Omeprazole is transformed to an active compound in an acidic compartment and modifies the essential cysteine residue of H',K'-ATPase (10)(11)(12)(13). Omeprazole inhibits not only the phosphorylation step of H+,K+-ATPase but also the K+-dependent p-nitrophenylphosphatase activity (11). SCH 28080 was reported to competitively bind to the K+ high * This study was supported in part by grant-in-aids for encouragement of young scientists (to S. A.), co-operative research (to N. T.), and scientific research (to N. T.) from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. affinity site of H+,K+-ATPase (14,15). SCH 28080 binds to the dephospho form of H',K+-ATPase, but its binding to the phospho form is controversial (16)(17)(18). SCH 28080 acts like K' and forms an enzyme-inhibitor complex, Ee. SCH 28080 or &(SCH 28080) (17). These H',K'-ATPase-specific inhibitors have given important insight into the reaction mechanism of the enzyme. However, how the enzyme actively transports the ions is still unknown.
To solve this fundamental problem, functional monoclonal antibodies and other specific inhibitors that have different inhibition mechanisms from those of omeprazole and SCH 28080 are also expected to be useful.
The inhibition of the H+,K'-ATPase activity caused by SA-B was antagonized by K' on the luminal side. SA-B and SCH 28080 commonly have a benzoyl group. Therefore, to determine the role of the benzoyl group in the inhibition mechanism, we synthesized a debenzoyl derivative of SA-B, diacetyl scopadol (DAS) (Fig. 1). DAS also specifically inhibits H',K'-ATPase. Interestingly, the inhibition was stimulated by K' on the luminal side. In this paper, we studied the inhibition mechanism of these compounds by measuring their effects on partial reactions of H',K'-ATPase (E, + EIATP + EIP + &P + &PK --, E,K + E,) and compared the inhibition points in the catalytic reaction of H+,K'-ATPase.
The inhibition mechanism of DAS was the simplest among known proton pump inhibitors such as omeprazole, SCH 28080, SA-B, and DAS.
EXPERIMENTAL PROCEDURES
Materials-SA-B was isolated from Scoparia dulcis L. as described elsewhere (19). It is a diterpenoid with a novel skeleton as shown in Fig. 1. DAS ( Fig. 1)
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This is an Open Access article under the CC BY license.
nm and an emission wavelength of 530 nm. Enzyme Phosphorylation and Dephosphorylation-Enzyme phosphorylation was measured as described by Maeda et al. (23). Fifty micrograms of lyophilized gastric vesicles were incubated in a solution containing inhibitor (SA·B or DAS) at the indicated concentration, 2 mM MgSO., and 40 mM Tris-HCI (pH 7.4) at 25°C for 20 min. Then, the mixture was incubated with [-y-32 P JAT P (1 X 10 5 cpm) and 5 p.M ATP at 25°C for 10 s. The reaction was stopped by the addition of 1 ml of ice-cold stop solution containing 10% trichloroacetic acid and 10 mM inorganic phosphate. The precipitated enzyme was collected on a Millipore filter (HAWP 0.45 urn) and washed thoroughly with the ice-cold stop solution, and the radioactivity of the filter was counted.
To study the effect of SA-B or DAS on the dephosphorylation step, H+,K+-ATPase in lyophilized gastric vesicles was phosphorylated in the presence or absence of SA-B or DAS as described above, and then the mixture was incubated with various concentrations of KCI at 25°C for 10 s. The K"-dependent decrease in the 32P·phosphoenzyme level was regarded as the parameter of dephosphorylation on the basis of the present result that neither SA-B nor DAS affected the phosphorylation step of the enzyme.
FITC Labeling of the Enzyme-Lyophilized gastric vesicles (750 I'g/ml) were incubated in 1 ml of solution containing 2 mM EDT A, 100 mM Tris-HCI (pH 9.2), and 5 I'M FITC for 30 min at 25°C (24). Then the solution was applied to a Sephadex G-50 column equilibrated with 40 mM Tris-HCI (pH 7.4) to remove unbound FITC.
Fluorescence Measurements-Fluorescence intensity of FITC was measured at 517 nm (exicted at 495 nm). The slit width was 2 nm for excitation and 10 nm for emission. The maximal volume of ligands added was 0.5% of the total volume in order to avoid dilution effects on the fluorescence intensity. Fig. 2 shows the effects of SA-B (A) and DAS (B) on activities of hog gastric H+,K+-ATPase and hog kidney Na+,K+-ATPase. Both compounds dose dependently inhibited the H+,K+-ATp· ase activity without any effect on the activity of Na+,K+-ATPase, which is a cation-transporting ATPase related to H+,K+-ATPase. The relative extent of inhibition of H+,K+-ATPase caused by high concentrations of SA-B was greater in tight vesicles than in leaky vesicles. The half-maximum inhibitory concentration (lC50) of SA-B was 28 p.M for tight vesicles and 54 ,uM for leaky vesicles. On the other hand, DAS was more potent in leaky vesicles than in tight vesicles. The half-maximum inhibitory concentration ofDAS in lyophilized vesicles was 17 ,uM, i.e. DAS is 3-fold more potent than SA-B. In tight vesicles, DAS even at 50 p.M inhibited the activity by 40% (Fig. 2B), and we could not measure the effect of DAS at 100 ,uM, because DAS at concentrations higher than 50 p.M was insoluble in the assay medium. The H+ concentration in tight vesicles is greater than in leaky vesicles. Since SA-B and DAS are stable compounds, neither protonation nor acid activation of these compounds is considered for the cause of difference in their potency between the two kinds of vesicles.
Specificity of SA-B and DAS to H+,W-ATPase-
The inhibition of the H+,K+-ATPase activity by SA-B or DAS was restored by dilution (data not shown), indicating that the inhibition by SA-B or DAS is reversible as in the case of SCH 28080 (14).
Effects of K+ on the Inhibition of H+,K+-ATPase by SA-B or DAS-K+ is transported actively from the luminal (intravesicular) side to the cytosolic (external) side of gastric vesicles in exchange for H+, i.e. K+ acts as a substrate of the ATPase. We studied the effect of the K+ concentration in the medium on the inhibition caused by SA-B or DAS of the K+-ATPase activity in lyophilized vesicles. The inhibition by SA-B was reduced as the K+ concentration increased. In contrast, the inhibition caused by DAS was enhanced as the K+ concentration in the medium increased. Preparations-Gastric vesicles enriched in H+,K+-ATPase were prepared from mucosa in the fundic region of hog stomachs by differential and density gradient centrifugation as described elsewhere (5). When leaky vesicles were needed, the vesicle suspension was diluted 10 times with distilled water, followed by immediate freezing in liquid N 2 • Then the vesicles were lyophilized and resuspended with the original volume of distilled water.
Microsomes of hog kidney abundant in Na+,K+-ATPase were prepared from the red outer medulla of hog kidney by differential centrifugation as described elsewhere (20).
K+-dependent p-nitrophenylphosphatase (K+-pNPPase) activity was measured in 1 ml of reaction mixture containing gastric vesicles (10 p.g of protein), 6 mM MgSO., 6 mM p-nitrophenyl phosphate, 40 mM Tris-HCI (pH 7.4) in the presence or absence of 15 mM KCI. The mixture was incubated at 37°C for 10 min, and the reaction was stopped by the addition of 0.5 N NaOH. Released p-nitrophenol was measured at 410 nm.
Proton Transport into Gastric Vesicles-Proton transport into gastric vesicles was monitored by measuring the quench of acridine orange fluorescence (22). The vesicles were incubated in either of the following two ways. The present inhibition mechanisms of SA-B (a mixed type) and DAS (an uncompetitive type with respect to K+) are different from a competitive type found for SCH 28080 (14,15).
Effect of SA -Bon Proton Uptake into Gastric Vesicles-We
studied the effect of SA-B on proton uptake into tight gastric vesicles. Fig. 4A shows the effect of SA-B on proton uptake into gastric vesicles in the presence of 150 mM KCl and 10 /lg of valinomycin. The quenching of acridine orange fluores- 37 'C. Then the ATPase activities were measured as described under "Experimental Procedures." The K+-ATPase activity of lyophilizedvesiclesin the absenceof inhibitors was 33.8 ± 0.6 /lmol/mg/h, and the valinomycin-stimulated K+-ATPse activity of nonlyophilizedvesicleswas 24.9 ± 1.9 /lmol/mg/h. The Na+,K+-ATPase activity in the absence of inhibitors was 11.5 ± 0.7 /lmol/mg/h. Data shown are averages ± S.E. for three observations. Fig.3A show that the inhibition of H+,K+-ATPase by SA-B is a mixed type and that SA-B lowers not only the affinity of H+,K+-ATPase to K+ but also the maximal velocity (V rnox ) of the H+,K+-ATPase reaction. Thus, the fact that the H+,K+-ATPase activity of tight vesicles was more sensitive to SA-B than that of lyophilized vesicles ( Fig. 2A) can be explained by this protective effect of K+. In lyophilized vesicles, the intravesicular K+ concentration can be assumed to be close to that of the reaction medium. In tight vesicles, the former is lower than the latter, since the KCl conductance across the vesicle membrane even in the presence of valinomycin is limited (25). The plots in Fig. 3B show that DAS is an uncompetitive inhibitor with respect to K+, indicating that DAS binds to the enzyme-substrate complex (26), that is DAS binds to the Kt-bound form of Ht.Kt-A'I'Pase (E 2K or E 2PK) and forms a stable inhibitory complex. Generally, in the case of uncompetitive inhibition, the degree of inhibition increases when the substrate concentration increases (26). This finding explains the above finding that H+,K+-ATPase was more sensitive to DAS in lyophilized vesicles than in tight vesicles (Fig.2B). vesicular K+ concentration. DAS dose dependently inhibited the K+-pNPPase activity. Its IC 50 values were 8.9, 30, and 56 /-1M in the presence of 2, 15, and 150 mM K+, respectively. In contrast with the results of the H+,K+-ATPase activity and its dephosphorylation step, the DAS-induced inhibition of the K+-pNPPase activity was antagonized by K+. Fig. 7 shows Lineweaver-Burk plots between the K+-pNPPase activity and the KCI concentration. The DAS-induced inhibition of the K+-pNPPase activity was competitive with respect to K+. Because we used gastric vesicles as the enzyme preparation of H+,K+-ATPase, we cannot rule out the possibility that this preparation includes K+-dependent phosphatase other than H+,K+-ATPase. We measured K+-pNPPase activity in the presence of 10 mM NiCb to remove a possible contribution from 5' -nucleotidase activity (35). Even in the presence of NiCb, the inhibition by DAS was also competitive with respect to K+ (data not shown).
Effects of SA-B and DAS on Change in FITC Fluorescence
cence reflects proton uptake into vesicles (3,22). SA-B dose dependently inhibited the rate of proton transport, its IC50 being 20 /-1M as determined from the initial slopes. SA-B at 100 /-1M inhibited the rate of proton uptake by 94%. Fig. 4B shows the effect of SA-B on proton uptake into gastric vesicles preincubated sufficiently in a solution containing 150 mM KCl in the absence of valinomycin for 12 h at 4 'C and then for 2 h at 25 'C. In this case, the proton uptake reached the maximal value rapidly compared with the case shown in Fig. 4A, and the effects of SA-B on the rate of proton uptake were less effective. SA-B at 100 /LM inhibited the rate of proton uptake by about 55%. This fact indicates that the K+ concentration in the vesicles preincubated for 1 min in the presence of 10 /-Ig of valinomycin plus 150 mM K+ (Fig. 4A) was much smaller than that in the vesicles incubated sufficiently in the 150 mM KCI solution (Fig. 4B).
Effects of SA-B and DAS on Phosphorylation of H+,K+-ATPase-Hereafter, we studied the effects of SA-B or DAS on partial reactions of H+,K+-ATPase to know the inhibition mechanism of these inhibitors. Here we studied the effects of SA-B or DAS on the formation of the phosphorylated intermediate of H+,K+-ATPase in lyophilized vesicles. When the enzyme was phosphorylated from 5 /-1M ATP in the absence of K+ for 10 s, the phosphoenzyme level was 1140 pmol of EP / mg of protein, indicating that 22% of the catalytic subunit was phosphorylated under the assumptions that H+,K+-ATPase constituted about 60% of the vesicle protein (27) and that this ATPase was composed of equivalent 114-kDa subunits (28,29). Neither SA-B even at 100 /-1M nor DAS even at 50 /-1M affected the steady state level of phosphoenzyme in the absence of K+ (data not shown). Therefore, these compounds did not affect the phosphorylation step of H+,K+-ATPase (E l -+ EIP plus E 2P). Effect of DAB on K+ -pNPPase Activity-K+-pNPPase activity has been regarded as the dephosphorylation step of H+,K+-ATPase (32). It is reported that K+-occluded conformation of Na+,K+-ATPase, E 2(K), has the phosphatase activity (33,34). Fig. 6 shows the effect ofDAS on the K+-pNPPase activity in gastric vesicles. In this experiment, we used lyophilized vesicles as an enzyme preparation to regulate the intra- Intensity of Ht Kr-A'I'Pase-s-Ber«, we studied the effects of SA-B and DAB on the conformation of the ATPase by using a fluorescence probe, FITC. FITC has been used for Na+,K+-ATPase and Ca 2+-ATPase, because its fluorescence level changes in response to ligand-induced conformational changes of these ATPases (36)(37)(38)(39)(40). FITC covalently binds to the lysine residue (Lys-518 in hog gastric H+,K+-ATPase) in or near the ATP binding site of the enzyme (24,29). The addition of K+ (1, 5, (Figs. 8 and 9). The decrease reflects the conformational change from the E 1 form to the E 2K form. (Fig. 8B) to the vesicle solution. SA-B dose dependently increased the fluorescence level of the enzyme (Fig. SA), suggesting that binding of SA-B to the E 2K conformation forms an inhibitory conformation different from E 2K. On the other hand, DAS did not reverse the quenching (Fig. 8B), indicating that DAS does not react with the E2K conformation. When the SA-B-treated enzyme was reacted with 5 mM Mg2+ and 15 mM K+ successively, the fluorescence change induced by K+ was dose dependently inhibited by SA-B (Fig. 9). The final fluorescence level at each SA-B concentration was almost the same as the corresponding fluorescence level observed in Fig. SA. That is, the final inhibitory products seem to be the same irrespective of whether SA-B is added before or after the formation of E 2 K. We name this inhibitory product E 2KI ("I" means SA-B). When the FITC-labeled enzyme preincubated in a solution containing 15 mM K+ was reacted with SA-B in the absence of Mg2+, SA-B did not affect the fluorescence level of E 2K (data not shown). Therefore, SA-B did not bind to the E 2K form in the absence of Mg2+. In contrast, successive additions of DAS, Mg2+, and K+ formed the E 2K conformation (data not shown), indicating that DAS did not bind to the E 2K form of the enzyme. DISCUSSION Several drugs have been reported to specifically inhibit gastric H+,K+-ATPase. For example, omeprazole (6), E3810 (8), and SCH 28080 (9) are very potent inhibitors of H+,K+-ATPase. In this study, we have shown that a natural compound, SA-B, and its debenzoyl derivative, DAS, have inhibitory effects on the H+,K+·ATPase. We will discuss their inhibitory mechanisms by comparing them with each other and with those of omeprazole and SCH 28080.
Effects of SA-B and DAS on the Dephosphorylation Step of
Omeprazole was reported to be transformed into an active compound in acidic compartment (10)(11)(12)(13). Complete inhibition of the enzyme activity was caused when about 2 mol of omeprazole/mol of phosphoenzyme bound to the H+,K+-ATPase (41)(42)(43). Recently Morii et al. (13) reported that omeprazole specifically modified Cys-322 of hog gastric H+,K+-ATPase. The inhibitory mechanism of omeprazole seems not to be specific to a particular reaction step, since omeprazole inhibits the H+,K+-ATPase activity, formation of phosphorylated intermediates, and the K+-dependent p-nitrophenylphosphatase activity (11).
The inhibition of H+,K+-ATPase by SCH 28080 is kinetically competitive with respect to the activating cation, K+ (14,15). Studies of fluorescence quenching of FITC-labeled H+,K+-ATPase have shown that SCH 28080 binds to the dephospho form of the enzyme and forms an inhibitory conformation of H+,K+-ATPase, E 2-SCH 28080 (17,18), which prevents rephosphorylation from ATP. SCH 28080 as a true K+ site inhibitor would be expected to block reaction steps in the catalytic cycle of H+,K+-ATPase, especially the K+-stimulated dephosphorylation of the E 2P form of the enzyme.
Wallmark et al. (17) reported that SCH 28080 inhibited the steady state level of phosphoenzyme intermediate in the absence of K+,but not the observed rate constant of phosphorylation. However, in their rapid reaction experiments 10 pM SCH 28080 did not affect the K+-stimulated dephosphorylation of the enzyme (17). On the other hand, Keeling et ol. (16) reported that SCH 28080 binds to both phospho and dephospho forms of H+,K+-ATPase. They proposed that this discrepancy was due to slow binding of SCH 28080 to H+,K+-ATPase (especially at low temperature) (18).
SA-B and DAS also specifically inhibit the H+,K+-ATPase activity. The kinetic pattern of inhibition of SA-B is a mixed type with respect to K+ (Fig. 3A); it inhibits the ATPase activity by lowering the affinity of the enzyme to K+ and also the Vmax value of the enzyme. The pattern of inhibition of DAS is an uncompetitive type with respect to K+ (Fig. 3B). The extent of inhibition increases as the K+ concentration increases, indicating that DAS reacts with the K+-bound enzyme (E 2K or E 2PK form).
In the experiments to clarify the inhibitory reaction step(s) of SA-B and DAS, we found that they specifically inhibit the K+-dependent dephosphorylation step of H+,K+-A'I'Pase without any effect on the steady state level of phosphoenzyme formation (E l -ElP plus E 2P) (Fig. 5, A and B). The inhibition by SA-B is dose dependently antagonized by K+, but that by DAS is enhanced by K+.
The fluorescence level ofFITC-labeled H+,K+-ATPase decreased when E 2K was formed from E l in the presence of K+ and Mg2+ (24). We studied the effects of SA-B and DAS on this fluorescence change of FITC. SA-B bound to the E 2K conformation of H+,K+-ATPase and formed an inhibitory complex E 2KI, the fluorescence level of which was different from that of E 2K (Fig. 8A). This inhibitory complex was not formed in the absence of Mg2+. The present results suggest that SA-B forms a stable inhibitory complex E2KI in the presence of Mg2+. On the other hand, DAS does not bind to the E2K form (Fig. 8B).
Present results show that SA-B can bind to both the phospho and dephospho form of gastric H+,K+-ATPase. The binding of SA-B to the phospho form of the enzyme inhibits H+,K+-ATPase by inhibiting its dephosphorylation step (Fig. 5A), lowering the affinity of the ATPase to K+. The binding of SA-B to the dephospho form inhibits H+,K+·ATPase by stabilizing the nonphosphorylated, K+ high affinity form (E 2KI).
In Na+,K+-ATPase, ouabain can bind to both the phospho and dephospho form of Na+,K+-ATPase (44). The binding of ouabain to the dephospho form of the enzyme results in inhibition of phosphorylation, whereas binding of ouabain to the phospho form blocks the K+-stimulated dephosphorylation by stabilizing the phosphorylated, K+ high affinity form (E 2PI) (36,(45)(46)(47). Ouabain does not affect the gastric H+,K+-ATPase. Therefore, SA-B is a counterpart of ouabain for H+,K+-ATPase.
Taking all above results on DAS into consideration (Figs. 3B, 5B, and8B), we suggest thatDAS inhibits H+,K+-ATPase by binding to the E 2 PK form of the enzyme and forming an inhibitory complex of E 2PKI. This is different from the case of ouabain, which binds to the E 2P form of Na+,K+-ATPase as stated above.
The K+-pNPPase activity has been regarded as a partial reaction step (dephosphorylation step) of Ht.K" -ATPase (32), because the K+-pNPPase and H+,K+-ATPase activities are copurified in the same fraction (48,49), and because both enzymes have the same sensitivity to cations and some inhibitors. In fact, SCH 28080, which binds to the K+ high affinity site of H+,K+-ATPase inhibits both enzyme activities. However, Ray and Nandi reported that Na" inhibits the K+-pNPPase activity without affecting the H+,K+-A'I'Pase activity (50), and reported that spermine inhibits the K+-pNPPase activity by competing with K+ without affecting the H+,K+-ATPase activity (51). From these and other related findings, they proposed the presence of three distinct K+ sites on H+,K+-ATPase; one for the H+ and Kt-trensporting ATPase reaction and two for the K+-pNPPase reaction (51), and that K+-pNPPase does not represent a partial reaction step of the H+,K+-ATPase activity (51). In this paper, we have shown that DAS inhibits the dephosphorylation step of H+,K+-ATPase (E 2PK -+ E 2K) and the K+-pNPPase activity. The inhibition of the H+,K+-ATPase activity is uncompetitive with respect to K+, whereas the inhibition of the K+-pNPPase activity is competitive. These results strongly indicate that the Kt-binding site of K+-pNPPase is different from that of the H+,K+-ATPase activity and that the K+-pNPPase activity does not represent the partial reaction step of K+-and H+transporting processes.
The inhibitory mechanisms of SA-B and DAS are summarized in the reaction scheme ofH+,K+-ATPase in Fig. 10. SA-B binds to both the phospho and dephospho form (EzKI). DAS specifically binds only to the phospho form (EzPKI).
DAS is a debenzoyl derivative of SA-B. The present study shows that the diterpenoid structure in both compounds is essential for inhibition of the H+,K+-ATPase activity, although the benzoyl group of SA-B partially engages in recognition of the K+ high affinity site. | 2018-04-03T01:10:10.890Z | 1990-12-25T00:00:00.000 | {
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267776242 | pes2o/s2orc | v3-fos-license | Medial or Lateral, That Is the Question: A Retrospective Study to Compare Two Injection Techniques in the Treatment of Knee Osteoarthritis Pain with Hyaluronic Acid
Background: Mild-to-moderate knee osteoarthritis (KOA) can be successfully treated using intra-articular hyaluronic acid (IA-HA). The medial infrapatellar (MIP) approach and lateral infrapatellar (LIP) approach are two of the most used techniques for performing IA-HA, but it is still not clear which one is preferable. Objectives: The study aims to find the best knee injection technique between MIP and LIP approaches. Methods: In total, 161 patients were enrolled, divided into two groups (MIP or LIP). Each technique was performed once a week for three weeks. Patients were evaluated using the Numeric Rating Scale (NRS), Knee Injury and Osteoarthritis Outcome Score (KOOS) and Roles and Maudsley Score (RMS) at T0 (before the first injection), T1 (one week after the third injection) and T2 (six months after). Results: NRS, KOOS and RMS showed a statistically significant improvement in both groups at all the detection times, without significant differences. No differences were detected between the groups in terms of systemic effect effusions, while the MIP group presented a mildly higher number of bruises in comparison with the LIP group (p = 0.034). Conclusions: Both the IA-HA techniques are equally effective in measured outcomes. The MIP approach seems to produce some local and transient side effects. So, the choice of the LIP or MIP approach depends on the operator’s skill and experience.
Introduction
Osteoarthritis (OA) is a chronic degenerative articular disease [1][2][3].Knee osteoarthritis (KOA) is the most frequent since the knee joint is particularly exposed to mechanical overloads [4], causing chronic pain and severe motor impairments, which lead to disability and loss of independence in carrying out the activities of daily living [5].
There are many therapies for KOA treatment, from drugs and new nutraceutical products to relieve pain [6,7] to prosthetic surgery during the most severe stages [8].
Hyaluronic acid (HA) is a glycosaminoglycan that occurs naturally in the knee synovial fluid.In KOA, due to decreased HA production, degradation and increased clearance, synovial fluid HA concentration is lower than in healthy knees.The aim of HA intraarticular injections (IA-HA) is the restoration of its viscoelastic properties, preventing cartilage degradation, promoting its regeneration and reducing chronic pain [9].IA-HA represents a valid and effective option for mild-to-moderate KOA management, as well as for severe non-surgical management [10].
There are many approaches to performing knee IA-HA injections.Maricar et al. identified eight different knee injection sites for the palpation-guided technique.Nevertheless, physician experience largely influences the accuracy of injections.A high parapatellar approach is preferred for fluid evacuation, while two of the most used are the lateral infrapatellar (LIP) approach and the medial infrapatellar (MIP) one [11].In both cases, the IA-HA injection is performed with the knee flexed at 90 • , accessing the knee joint by passing next to the patella and the related tendon with the medial or lateral patellofemoral approach.
KOA most often affects the medial tibiofemoral compartment.As a consequence, patients frequently report pain located in the medial compartment of the knee [12].Consequently, among patients, it is commonly thought that the MIP approach could be more effective due to the needle placement nearer the pain site.
Although previous studies have been conducted to evaluate the most effective needle placement into the knee intra-articular space, to our knowledge, none of the previous investigations compared these two approaches in terms of effectiveness and local side effects.
Since there is still no evidence available that the medial approach grants better outcomes or whether one of the two techniques is more valid than the other, a retrospective study was carried out to compare the effectiveness of these two techniques in terms of clinical outcomes and local side effects for treating KOA with HA injections.
Materials and Methods
This study is an observational retrospective one.It was carried out according to the Declaration of Helsinki Principles, and it received the approval of the human ethical committee of the General Hospital of Bari, Italy, protocol number 1402/CEL, 13 December 2023.
The written informed consent of each enrolled subject was originally collected as an express acceptance to undergo the injection treatments and to allow the use of the data for scientific research purposes.
Study Population
We retrospectively enrolled 161 patients (74 men and 87 women) affected by KOA who attended the Physical Medicine and Rehabilitation outpatient service of the Bari General Hospital from January 2019 to March 2023.
The inclusion criteria were as follows: • Diagnosis of KOA confirmed by a clinical medical evaluation and by an X-ray taken within the previous 12 months; The patients were retrospectively divided into two different groups according to the injection site.Group A consisted of 79 KOA patients treated once a week for three consecutive weeks with three HA MIP injections; group B consisted of 82 KOA patients treated with three HA LIP injections once a week for three consecutive weeks.All injections were performed by an expert physiatrist who had 5 years of experience in knee IA injections.
Intervention
The patient was positioned supine with the hip flexed at approximately 45 • and the knee flexed at approximately 45 • .Before each injection, a meticulous skin disinfection was performed using sterile gauzes soaked in povidone iodine solution.The same highmolecular-weight (>1500 kDalton) HA was used for each injection using a 2.0 in (5.1 cm) 21-gauge needle.Each vial contained 30 mg of HA in 2 mL.The performed injection techniques were the standard LIP and MIP techniques delivered in an ultrasound-assisted way (Figure 1).
The patients were retrospectively divided into two different groups according to the injection site.Group A consisted of 79 KOA patients treated once a week for three consecutive weeks with three HA MIP injections; group B consisted of 82 KOA patients treated with three HA LIP injections once a week for three consecutive weeks.All injections were performed by an expert physiatrist who had 5 years of experience in knee IA injections.
Intervention
The patient was positioned supine with the hip flexed at approximately 45° and the knee flexed at approximately 45°.Before each injection, a meticulous skin disinfection was performed using sterile gauzes soaked in povidone iodine solution.The same high-molecular-weight (> 1500 kDalton) HA was used for each injection using a 2.0 in (5.1 cm) 21-gauge needle.Each vial contained 30 mg of HA in 2 mL.The performed injection techniques were the standard LIP and MIP techniques delivered in an ultrasound-assisted way (Figure 1).The preliminary ultrasound assessment is useful for evaluating the anatomical structures and for establishing the correct needle direction [13].In the LIP technique, the needle is inserted about 1 cm below and 1 cm lateral to the inferior lateral margin of the patella, and then it is directed diagonally, going from the lateral side behind the patella (Figure 2).The preliminary ultrasound assessment is useful for evaluating the anatomical structures and for establishing the correct needle direction [13].In the LIP technique, the needle is inserted about 1 cm below and 1 cm lateral to the inferior lateral margin of the patella, and then it is directed diagonally, going from the lateral side behind the patella (Figure 2).
In the MIP technique, the needle is inserted about 1 cm below and 1 cm medially to the inferior medial aspect of the patella, and then it is directed obliquely, going from the medial side behind the patella (Figure 3).
Timing
All the involved patients were evaluated by three different physiatrists at the following detection times: • T0: at the enrolment, which overlapped with the date of the first injection; • T1: one week after completing the IA-HA cycle, three weeks after the first injection; • T2: six months after the first IA-HA injection.
The first injection was administered at T0, the second one a week after the first and the third one a week after the second.In the MIP technique, the needle is inserted about 1 cm below and 1 cm medially to the inferior medial aspect of the patella, and then it is directed obliquely, going from the medial side behind the patella (Figure 3).
Timing
All the involved patients were evaluated by three different physiatrists at the following detection times: • T0: at the enrolment, which overlapped with the date of the first injection; • T1: one week after completing the IA-HA cycle, three weeks after the first injection; • T2: six months after the first IA-HA injection.
The first injection was administered at T0, the second one a week after the first and the third one a week after the second.
Outcome Measures
The aim of this study was to assess the best clinical knee injection approach between MIP and LIP techniques in terms of knee pain and functional improvement and local side effects.At T0, T1 and T2, after a medical and ultrasound evaluation of the treated knee, In the MIP technique, the needle is inserted about 1 cm below and 1 cm medially to the inferior medial aspect of the patella, and then it is directed obliquely, going from the medial side behind the patella (Figure 3).
Timing
All the involved patients were evaluated by three different physiatrists at the following detection times: • T0: at the enrolment, which overlapped with the date of the first injection; • T1: one week after completing the IA-HA cycle, three weeks after the first injection; • T2: six months after the first IA-HA injection.
The first injection was administered at T0, the second one a week after the first and the third one a week after the second.
Outcome Measures
The aim of this study was to assess the best clinical knee injection approach between MIP and LIP techniques in terms of knee pain and functional improvement and local side effects.At T0, T1 and T2, after a medical and ultrasound evaluation of the treated knee,
Outcome Measures
The aim of this study was to assess the best clinical knee injection approach between MIP and LIP techniques in terms of knee pain and functional improvement and local side effects.At T0, T1 and T2, after a medical and ultrasound evaluation of the treated knee, each patient was evaluated with the Numeric Rating Scale (NRS) and Knee Injury and Osteoarthritis Outcome Score (KOOS).The NRS is a validated pain scale with a score ranging from 0 (no pain) to 10 (maximum pain) [14].The KOOS is a 42-item questionnaire useful for assessing self-reported progress in knee functions [15].At T1 and T2, the Roles and Maudsley Score (RMS) was also collected for each patient.The RMS is a subjective patient assessment of pain, and it was used as an instrument to evaluate the satisfaction with the treatment in terms of effectiveness, discomfort related to the execution and the procedure's side effects.At T1 and T2, for each patient, the examining physiatrist filled out a diary with all the local effects reported after the injections.The side effects were recorded after an inspection and palpatory examination to highlight bruises, hematomas and sites of pain; then, they were further investigated with an ultrasound evaluation.
Statistical Analysis
Data were analyzed using Stata MP18 software.We expressed continuous variables as mean ± standard deviation (SD) and range; categorical data were expressed as proportions.The Skewness and Kurtosis test was used to compare normal distribution of continuous data, and, whenever possible, we created a normal model for those data not normally distributed.For parametric data, a Student's t-test was used to compare continuous variables between the two groups.The continuous variables were compared between the two groups using the Student's t-test for independent data or using the Wilcoxon signed-rank test for non-parametric data.An ANOVA test was used for repetitive measures, comparing different timing.The Chi-squared test was used to compare categorical variables between groups.A multivariate linear regression was used for the analysis of the relationship between the NRS and KOOS outcomes at T0 and T2, and between sex, age, BMI and groups.Confidence interval was set at 95%, while a p-value was considered statistically significant if <0.05.
The ANOVA test for repeated measurements showed a statistically significant difference for all the aforementioned outcome measures in the comparison between times.The Roles and Maudsley Score, assessing procedure satisfaction as a self-reported outcome, showed a minimum decrement between T1 and T2 and a difference between groups (p = 0.042), both not statistically relevant.Every single outcome is also visually represented as line graphs in Figures 4 and 5, showing the evolution over time for each scale.Figure 6 displays the Roles and Maudsley Score at each evaluation time.The figure underlines the difference between the two groups, but, as outlined in Table 2, the p-value is >0.05; therefore, it is not statistically significant.
Roles and Maudsley Score
Group A -1.9 ± 0.5 1.8 ± 0.5 0.051 0.042 0.849 outcome, showed a minimum decrement between T1 and T2 and a difference between groups (p = 0.042), both not statistically relevant.Every single outcome is also visually represented as line graphs in Figures 4 and 5, showing the evolution over time for each scale.Figure 6 displays the Roles and Maudsley Score at each evaluation time.The figure underlines the difference between the two groups, but, as outlined in Table 2, the p-value is > 0.05; therefore, it is not statistically significant.scale.Figure 6 displays the Roles and Maudsley Score at each evaluation time.The figure underlines the difference between the two groups, but, as outlined in Table 2, the p-value is > 0.05; therefore, it is not statistically significant.Tables 3-8 describe the multivariate linear regression analyses by single outcome.Sex, age, BMI and knee side were identified as potential confounders and included in the multivariate linear regression analysis to investigate any influence on every single outcome measure.Tables 3-8 describe the multivariate linear regression analyses by single outcome.Sex, age, BMI and knee side were identified as potential confounders and included in the multivariate linear regression analysis to investigate any influence on every single outcome measure.In Table 9, the side effect prevalence is explained.No systemic adverse events and no allergic reactions (skin rash, hives) were reported.Only local adverse events were recorded, such as bruises and effusions.The presence of bruises was observed in 23 subjects (14.3%), and there was a statistically significant difference between groups (group A: 16, 20.3% vs. group B: 7, 8.5%; p-value = 0.034).For 23 (14.3%) patients, there was effusion, without statistically significant differences between the groups (group A: 15, 19.0% vs. group B: 8, 9.8%; p-value = 0.094).
Discussion
The efficacy of IA-HA injections for treating KOA is already well known [16].In fact, it represents a simple and safe procedure which grants short-and medium-term pain relief with a positive effect on joint functionality.Currently, there is weak evidence in the literature about the long-term effects of IA-HA for pain relief, but some studies demonstrated that IA-HA can delay knee arthroplasty surgery [17].
The best approach for knee injection is still uncertain; the procedure choice is often based only on the physician's experience.The goal is to deliver an adequate quote of medication in the IA space, to improve the technique accuracy and to reduce the risk that, during the injection, the needle may engage with the medial knee plica or the fat pad.
Our findings demonstrated the efficacy of IA-HA.In fact, both groups significantly improved between T0 and T2 both in terms of pain reduction according to NRS scores (p < 0.0001) and in terms of joint function, according to KOOS (p < 0.0001).
Particularly, the NRS decreased by approximately four points in both groups.These results are in line with the current scientific literature, which states the effectiveness of IA-HA injections in relieving knee pain during up to 6 months of follow up [18].Similarly, KOOS values improved in both groups and for each scale section.Also, these results are in line with the available evidence of IA-HA injections' effectiveness [19,20].
The multivariate linear regression analysis ruled out that these results were influenced by the determinants described in Table 1, except for two aspects.Particularly, sex seemed to slightly affect the NRS scores (p = 0.024) so that females seemed to have a better response to IA-HA.This gap may be due to a different experience of pain between men and women.NRS values slightly differ among people.Furthermore, women experience pain more frequently due to the role of sex and gender; this aspect could refer to many causes, ranging from factors related to biological sex to those related to psychosocial gender.Moreover, to our knowledge, the previous literature never reported or investigated gender differences in IA-HA effects, and it would be desirable for this aspect to be explored in future studies.The other apparently significant determinant is the BMI with respect to the trend of the KOOS Symptoms score (p = 0.025).In this case, an explanation could be the fact that people with a higher BMI usually have a lower KOOS Symptoms rate at baseline, and, therefore, they obtain a more marked increase in KOOS Symptoms rate between T0 and T2 due to the benefits derived from IA-HA.
No weight-related difference was found in pain scale between the two groups.Partially in contrast with our results, a study conducted by D'Alessandro et al. compared the accuracy of other injection techniques in overweight patients (BMI > 25) affected by KOA.They found no variation of injection-related pain in IA-HA between anterolateral and superolateral access.According to their research, an increase in BMI seems to be indicative of greater pain during anterolateral access.They explained this evidence as a consequence of a greater local production of adipocytokines, due to the augmented subcutaneous tissue in overweight patients, rather than to Hoffa's fat pad, whose volume seems to not be related to weight [21].
Most importantly for our research, there were no significant differences between the two groups according to NRS and KOOS.Based on our results, MIP and LIP approaches are equally effective as minimally invasive KOA treatments so neither approach is preferable to the other.
In the available scientific literature, data are lacking in the specific comparison between MIP and LIP approaches.A comparison study by Toda et al. [22] deepened the accuracy rates of three different knee IA approaches, namely, LIP and MIP approaches with the patient in a seated position and the modified Waddell approach, an anteromedial approach with manipulative ankle traction at 30 degrees of knee flexion.Although the number of patients was small, no significant differences were detected between the three techniques for KL 2 and 3 patients (p > 0.05), in line with our results.
The anterolateral approach, both medial and lateral, seems to be more accurate and effective than the traditional superolateral one [23].In fact, the MIP and LIP techniques are useful when the knee is dry, without joint effusions, with no anatomical variations, and when the knee cannot be fully extended [24].Moreover, these approaches are easy to perform also with a palpatory landmark guide in the absence of an ultrasound guide [25].
Regarding RMS, at each detection time, both groups had a high satisfaction with the received injection treatment.Even though there was no statistically significant difference between the groups, we observed an interesting trend in favor of the LIP approach (p = 0.051).Although this is only a trend, this can probably be justified by the fact that the LIP technique seems to be more accurate when the knee is dry; therefore, with the same effectiveness, it can be less painful for patients as it allows for a more accurate infiltration [26].In fact, a study by Jackson et al. compared different IA knee injections using real-time fluoroscopic imaging with contrast material and affirmed that a lateral midpatellar injection (an injection into the patellofemoral joint) was the most precise one since it was intra-articular in 93% of cases [27].This finding is also validated in a paper by Park et al. that investigated the injection accuracy rate in three different knee sites with an ultrasound-guided approach.They stated that, in KOA, ultrasound-guided IA-HA injections in the mediolateral or superolateral space were more accurate than those through the medial space [28].
This reasoning could be extended also to the analysis of the findings regarding the local side effects.In fact, there were no systemic adverse effects, while the results obtained for bruises and joint effusions were different in statistical terms.No differences were detected between the groups in terms of effusions, while the MIP group (group A) presented a significantly higher number of bruises in comparison with the LIP group (p = 0.034).The higher frequency of bruises could be due to the fact that, in the MIP approach, there is a higher risk of crossing subcutaneous small veins.Our results are in line with a previous study published by Lussier et al. [29] that confirms that both techniques are safe, but the LIP one seems to be more accurate, making even minimal injection-related discomforts less frequent.
In conclusion, MIP and LIP techniques appear to be totally equivalent in terms of effectiveness.This evidence is far from obvious or without usefulness.On the contrary, it allows us to choose the approach based on the skills of the operator performing the injections or based on the clinical contingency.Sometimes, KOA determines an alteration of the joint anatomy and a deformity such as to force access from one side rather than another [30,31].Particularly, the medial knee compartment is more frequently altered by KOA, and the bone reshaping could be an obstacle to a correct and easy injection using the MIP technique [32,33].In these cases, the LIP approach could be preferred, as well as in cases where there is an increased risk of bleeding and bruising caused by the infiltration itself (for example, in patients taking anticoagulants).Similarly, when there are no preferences due to the physician's expertise or due to specific anatomical contingencies, the LIP technique could be more advantageous due to a lower risk of even minimal side effects.
The current study presents some limitations.First of all, pain is a difficult parameter to assess in an objective way.In fact, pain outcomes were self-reported, but it was a mandatory condition to evaluate it.Then, in the literature, various knee entry sites are described for injecting HA, but, as we said above, we chose the two most used techniques.Therefore, further studies are needed to investigate the best injective way, including also other techniques and different operators.We established a relatively long-term follow up (6 months); however, it would be interesting to better understand long-term efficacy to investigate the differences in terms of injection frequency between the two techniques in a perspective study.
Conclusions
MIP and LIP techniques seem to be equally effective and safe as IA-HA injection procedures for patients suffering from chronic pain related to KOA.Therefore, the choice of the technique to be performed can be based on the operators' practical experience, thus reducing the risk of side effects.
Anatomical variations and specific risk factors, such as coagulopathies, may make the execution of the LIP technique more suitable, just as the degree of patient satisfaction may require switching to one approach rather than another during the same injection cycle.
Further studies are needed to deepen these aspects and to continuously refine knee infiltration techniques in order to increase the patients' satisfaction and compliance with therapies.
Figure 2 .
Figure 2. LIP technique performed in the left knee.
Figure 3 .
Figure 3. MIP technique performed in the left knee.
Figure 2 .
Figure 2. LIP technique performed in the left knee.
Figure 2 .
Figure 2. LIP technique performed in the left knee.
Figure 3 .
Figure 3. MIP technique performed in the left knee.
Figure 3 .
Figure 3. MIP technique performed in the left knee.
Figure 4 .
Figure 4. NRS by group and detection time.
Figure 5 .
Figure 5. KOOS scale-Symptoms, Pain, Activity of Daily Life, Sport, Quality of Life-by group.
Figure 4 .
Figure 4. NRS by group and detection time.
Figure 4 .
Figure 4. NRS by group and detection time.
Figure 5 .
Figure 5. KOOS scale-Symptoms, Pain, Activity of Daily Life, Sport, Quality of Life-by group.
Table 1 .
Characteristics of the sample divided per group.
BMI = body mass index; SD = standard deviation.
Table 2 .
Average ± SD and range of outcomes per time and group.
Table 3 .
A multivariate linear regression model to analyze the NRS variations between T2 and T0.
Table 3 .
A multivariate linear regression model to analyze the NRS variations between T2 and T0.
Table 4 .
A multivariate linear regression model to analyze the KOOS variations in Symptoms between T2 and T0.
Table 5 .
A multivariate linear regression model to analyze the KOOS variations in Pain between T2 and T0.
Table 6 .
A multivariate linear regression model to analyze the KOOS variations in Activity of Daily Life between T2 and T0.
Table 7 .
A multivariate linear regression model to analyze KOOS variations in Sport between T2 and T0.
Table 8 .
A multivariate linear regression model to analyze KOOS variations in Quality of Life between T2 and T0. | 2024-02-22T16:08:46.699Z | 2024-02-01T00:00:00.000 | {
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237895112 | pes2o/s2orc | v3-fos-license | Hijab Trends during Covid-19 in the Study of Contemporary Fiqh in Indonesia
Various ways are done by the community to prevent the spread of the virus This situation makes the Indonesian people make safety a trend. Many trend changes have been made, including the hijab trend to provide safety and stay fashionable in Indonesia. The choice of hijab trends is one of the halal business opportunities with health considerations. Indonesia with a Muslim majority is very appropriate if one of the trends taken is the hijab trend, because the majority of people use the hijab and according to the changing model. Thus, this study was carried out using a method of deepening contemporary fiqh studies which was used to analyze how people wear, community business opportunities, and the form of hijab according to sharia. Furthermore, the data was refined using qualitative methods to obtain data from hijab businesses, hijab users, medical teams, and business observers. This study found the hijab trend in contemporary fiqh studies to be one way to find permissible income, and is one way for a person to save himself from the covid-19 virus that has been safely used by the public. However, it becomes something that is not allowed in contemporary fiqh studies if the trend reaches ishrof behavior, is redundant, and raises arrogance. cara keselamatan sebagai tren. Perubahan tren banyak dilakukan, termasuk tren hijab untuk memberikan keselamatan dan tetap modis di Indonesia. Pilihan tren hijab menjadi salah satu peluang usaha yang halal dengan pertimbangan kesehatan. Indonesia dengan mayoritas muslim sangat tepat jika tren yang diambil salah satunya dengan tren hijab, karena mayoritas masyarakat menggunakan hijab dan sesuai perubahan model. Dengan demikian kajian ini dilakukan dengan metode pendalaman kajian fikih kontemporer yang digunakan untuk menganalisa cara pakai masyarakat, peluang usaha masyarakat, serta bentuk hijab sesuai syariah. Selanjutnya data disempurnakan dengan menggunakan metode kualitatif untuk mendapatkan data dari pelaku usaha hijab, pemakai hijab, team medis, serta pengamat usaha. Penelitian ini menemukan tren hijab dalam kajian fikih kontemporer menjadi salah satu cara dalam mencari penghasilan yang diperbolehkan, serta merupakan salah satu cara seseorang dalam menyelamatkan diri dari covid-19 yang telah aman digunakan oleh masyarakat. Namun, menjadi hal yang tidak diperkenankan dalam kajian fikih kontemporer jika tren sampai pada prilaku ishrof, mubadzir, dan memunculkan kesombongan. to aurat and beautiful garments for adornment. garment of piety is best. are of of hopefully
A. INTRODUCTION
Indonesia is a country with various characters that develop and are dominant in Islamic characters. Islamic character is formed by the large number of Muslim population. This is commonly known by the public, when talking about Indonesia it will be identical with religion, namely Islam. 3 This fact shows that Indonesia and religion are like two sides of the same coin, which cannot be distinguished or separated at all. 4 The thick nuances of religion among the Indonesian people cannot be separated from many factors, one of which is the role of Kiai and Islamic boarding schools in Indonesian society, so that society is so thick with religious nuances. 5 Indonesian people are fairly fanatical about religion, thus making their behavior follow religious regulations. One of them is the procedure in appearance, whether male or female. This is related to the religious element that gives rules in appearance, especially in the aurat element. Men who dress to cover their aurat and women must also cover their aurat. In relation to women/women, it is more detailed, such as having to wear clothes that are not tight and cover their genitals, such as having to wear a hijab.
Women are part of a society, because they are partners with men in prospering the earth and realizing empowerment. Islamic teachings give great attention, as well as a respectable position to women. Muhammad al-Ghazali, one of the great scholars of contemporary Islam stated that "if we return to the past a thousand years, then we will find women enjoying privileges in the material and social fields that are unknown to women on the five continents. Their situation at that time was better, compared to the situation of Western women today, as long as freedom in dress and association is not made a comparison.
Women have the same rights as men in developing businesses. However, women's rights must be balanced with the ability to maintain religion. If we review again, the involvement of women in work in the early days of Islam was very much. We can see this from the historical fact that women at the time of the Prophet Muhammad were active in various fields of work. At this time women have become jewelry that needs to be protected with Islamic concepts.
Indonesian women are very familiar with Muslim clothing, especially the hijab. The majority of Indonesian Muslim communities use the hijab. The fact is that almost all women in Indonesia use the hijab in their daily appearance, both young and even old people also use hijab. It is a habit of women in Indonesia. Aside from being a tool to cover the genitals of Islamic religious regulations, the hijab also seems to be a necessity for Indonesian women, even in its development it has become a trend in people's lives.
This happens when there are certain events or celebrations such as weddings, khaul events and even Islamic holidays. The hijab trend is one of the most noticed by Indonesian women, such as buying the latest hijab to wear at these events. Even during this pandemic, the hijab remains a trend among the public. In its regulations, the government requires the use of masks for the public, so that it becomes a challenge for entrepreneurs to adapt the hijab to these regulations. In Indonesia, this turned out to be an opportunity for entrepreneurs by giving the feel of a hijab with a mask. The hijab trend provides a solution for people to remain veiled by wearing trending masks, while still adhering to health protocols.
In the Covid-19 situation, this hijab trend has been in the spotlight from various circles, both in the entrepreneurial community and in the business community because it creates an opportunity to get halal sustenance. However, the question is whether this trend can be declared halal by contemporary fiqh views. This study looks at various angles, both in terms of how to wear it, the nature of when to dress according to trends, the impact on health and aspects of entrepreneurship.
B. METHODS
This study was conducted using a qualitative research methodology with interviews, documentation and observation methods. Researchers conducted this study to obtain valid data and in accordance with the values raised, because this study has differences with several previous studies that have been mentioned in this study. As for the differences referred to in this study, that this study discusses the hijab trend in Indonesia, the hijab trend in question is a hijab that also functions as a protector from the corona outbreak, and this study was conducted on issues when the corona virus outbreak hit Indonesia which was studied with the concept of contemporary fiction.
Business Opportunities during Covid-19 Contemporary Jurisprudence Study
Covid 19 is an outbreak that has recently made a scene throughout the world, covid 19 which was first discovered in the Republic of China is a type of virus that first appeared in one of China's provences, namely in Wuhan China on December 31, 2019. Covid 19 (corona) virus disease 2019) is a disease caused by a new type of corona virus, namely Sars-CoV 2. 6 Outbreaks of this disease can cause symptoms of acute respiratory disorders such as having a fever above 38 o C, coughing and shortness of breath. In addition, it is also accompanied by weakness, muscle aches, and diarrhea, even for severe patients, it can cause pneumonia, acute respiratory syndrome, kidney failure and even death. The virus outbreak, for which no cure has yet been found, has become 6 "Hindari Lansia Dari Covid-19," Oktober 2020, http://www.padk.kemkes.go.id/article/read/2020/04/23/21/hindari-lansia-dari-covid-19.html. one of the most frightening outbreaks for humans, especially since this virus is also thought to be capable of killing people infected with the corona virus.
Furthermore, it was also explained on the Indonesian Ministry of Health website, that Covid 19 can be transmitted from one human to another through close contact and droplets (sneezing and coughing liquid splashes), not through the air. The shape of COVID-19 when viewed through an electron microscope (airway fluid/throat swab) and depicted again the shape of COVID-19 is like a virus that has a crown. 7 In this case, this virus is very likely to spread quickly if humans come into contact with one another, especially if there are a lot of people in one place, which is very concerning because it will be one of the causes of the Covid 19 virus outbreak that spreads across many countries. This has an impact on all sectors, including not only the health sector, but also on education and the economy. Trade in Indonesia is no exception, with one factor influencing the process of purchasing and selling headscarves in Indonesia. However, this does not completely stop traders and buyers from buying and selling; rather, it provides an alternative for both, namely by producing or selling the headscarf and its masks, either as a unit with the veil or separately in the same color as the veil purchased by the customer.
After approximately 10 months since the announcement of this case for the first time, and the restrictions on the lockdown and PSBB are felt by all people, from traders, daily workers, and other fields of work. However, behind some negative things that are felt by some circles, there are still some people who actually make it an opportunity. Opportunity is always there at any time, even in circumstances like the current Covid. Taking advantage of opportunities by innovating is very worth it, as long as the materials used are dense and fibrous and of good quality, they will also be able to protect themselves. Basically the function of the hijab is to protect the genitals by covering what should be protected to the chest. As long as it is in accordance with sharia, the hijab can still be used. Covid has a negative impact on the Indonesian economy in general, but for a digital-based economy it has a positive impact.
Covid has had a huge impact on the economy. With the limitation of activities outside the home, many businesses have closed, many employees have been laid off, and especially for families, it is very felt that many of the husband's agenda as a source of search in various institutions has been canceled, so that additional income during the pandemic has decreased drastically. 8 Likewise, Nurul Handayani stated that the pandemic had a very large impact on the Indonesian economy. The first is to make household consumption or people's purchasing power, which is the pillar of 60% of the economy, fall quite deeply. Second, the pandemic causes prolonged uncertainty so that investment also weakens and has implications for the cessation of businesses, for example, there are many layoffs in several companies. Third, the entire world is experiencing a weakening economy, causing commodity prices to fall and Indonesia's exports to several countries also stalling. The existence of a hijab trend business opportunity can be said to be a promising business opportunity and has very bright future prospects. Especially in our country, Indonesia, where the majority of the population is Muslim, the hijab business is very prospective. Actually, the business opportunity is for anyone who is able to run it. 9 Covid has a very negative impact on the Indonesian economy. This is evidenced by the entry of Indonesia into a recession because in the 3rd and 4th quarters of 2020 the Indonesian economy was below 0%. This is because people's consumption or purchasing power is reduced, due to layoffs and business bankruptcy. Non-culinary businesses are indeed suitable to be done during a pandemic, one of which is the hijab trend. That's great for helping the economy. 10 Covid is very influential on the Indonesian economy, especially the lower middle economy. For those whose economy is middle to lower, it has a very negative impact because for their economic indicators, between profit and loss, they experience more losses or do not return on investment. As for those in the upper middle economy, their negative impact is a slight advantage compared to before the existence of covid. 11 This covid-19 pandemic hit Indonesia like a perfect storm that had a big impact on the Indonesian economy. Among them are causing household consumption or people's purchasing power to decline, which is the 60% support for the economy, falling quite deeply. The pandemic caused prolonged uncertainty, so that investment also weakened and had implications for the cessation of business. The whole world experienced a weakening economy, causing commodity prices to fall and Indonesia's exports to several countries also stalling. During Covid, there are many business opportunities that are run by the community. One of them is the hijab trend, and it is considered good as long as it covers aurat, because this business really supports economic needs that are currently difficult, and protects from virus transmission because it is in accordance with health protocols. 12 The position of this pandemic requires business opportunities to improve the economy, and this economy can be interpreted as a value. Value in the general and general concept that we have about the term value is actually an economic concept. The relationship of a commodity or service to the goods that people are willing to pay for it gives rise to the concept of value. However, the meaning of values and value systems. The term value in this broad sense is applied to objects, as well as to humans and their behavior. The study of economics was born from assumptions that arise from awareness and understanding of the scarcity of resources and tools to satisfy needs, dealing with human needs that are not limited in quantity, variety, or quality. 13 9 Nurul Handayani, Interview Dosen Asal Jakarta "Tren Hijab Saat Pandemic Covid-19," January 22, 2021. 10 From this assumption, economics emerges, which considers how society must construct a system of production and distribution of goods for living necessities that continues to grow, due to population growth, demands for a higher standard of living, and the complexity of the problems encountered in maintaining and sustaining life. Dealing with the notion of economic assumptions that is generally accepted as a paradigm. The moral generated by the Qur'an is just the opposite, namely creating an understanding of the absence of a scarcity of sources of life satisfaction, because Allah's sustenance is always abundant, not only sufficient for humans, but also for other living creatures. 14 Therefore, Islam teaches its people to try to get a better life in this world and at the same time get a good life in the hereafter. Getting a good life in this world and in the hereafter can guarantee the achievement of physical and spiritual well-being (falah). This means that the pursuit of life in the world can not be done except in a lawful way through a pious charity movement. 15
Hijab Trends in Contemporary Fiqh View
Some of the business opportunities that are trending during the pandemic. One of them is the hijab business which is becoming a trend in Indonesian society. Hijab is equipped with a mask that is usually used by Indonesian Muslim women. Hijab is the clothing that is needed during the COVID-19 pandemic.
In Islamic law, all aspects of human life are regulated completely, including the dress code. The clothes worn by Muslims must follow the provisions of Islamic law. Clothing worn by humans has three main purposes, namely; cover the limbs, protect the body, as jewelry and beauty. 16 As Allah says in the Qur'an: Meaning: O son of Adam, indeed We have sent down to you garments to cover your aurat and beautiful garments for adornment. And the garment of piety is the best. Such are some of the signs of God's power, hopefully they will always remember.
In the content of the verse, Shaykh Abdul Wahab Abdussalam explained that God gave grace to the children of Adam, namely in the form of clothing of all kinds. That we have made for you two types of clothing, clothing worn to cover the human aurat, which is in the form of basic clothing, such as underwear, women's clothing, hijab, and other clothing that is used to cover the aurat. Clothing that serves as jewelry and beauty as clothing that complements human beauty. 17 However, Allah also reminds that the most important clothes for mankind are the clothes of piety and faith. 14 Abdul Aziz,116. 15 With both, humans are saved from several traits that are not justified by religion such as ishraf, mubadzir, and arrogant.
Arrogance is a trait that is strongly condemned and discouraged in religion. Arrogance is feeling yourself more noble and perfect than other people who arise due to the deception of lust. Outwardly, arrogance can be seen from the behavior that appears, where it greatly affects the inherent nature of the owner. 18 Regarding the nature that is so prohibited in the religion, then with the hijab trend there are hijab users or hijab connoisseurs, both to sellers and users. With the existing trend, users and sellers are not allowed to have these characteristics. The use of the hijab is as a tool to cover the genitals as a means of obeying religious orders, not as a means to feel better than others. With the hijab trend during the COVID-19 pandemic, namely the emergence of a hijab trend that is one with the mask, either one or separately with a matching color selection. This is a way for women to obey the orders of the State and in accordance with the demands of Islamic teachings.
For the people of Indonesia, Islam is not just a choice in choosing a religion, it is not just a reference for behavior in social and social life. More than that, Islam is also one of the elements of Indonesia's ethnic identity markers. Because the Indonesian people have made Islam an identity in their lives, it is not surprising that all Indonesian people are closely related to Islamic elements. 19 Indonesian people in all their activities also continue to use Islamic religious rules, both in behavior, or in other matters, whether universal or personal. In its development, religion affects many things, even culture also has religious elements.
Changes in its development if it is in the fashion debate which is a source of business for various entrepreneurial drivers. Likewise, some business drivers can innovate from the values of necessity in religion such as the hijab trend which was born from the obligation of a Muslim woman to cover her aurat which has become a business or business. So this development must also be responded to by contemporary fiqh law to provide legal certainty for every group, both businessmen and consumers. How should Muslims respond to changing times that demand conditions to always be up to date, especially in the business sector, and its users.
Socio-cultural changes due to development in addition to creating a gap between old values and new values also create problems for fiqh. These changes can be illustrated by changing villages into cities, changing an agrarian economy society into an industrial and trading economy, changing the pattern of mutual cooperation to an individualist life, and preferably changing people's perspective and behavior towards assets and technical interactions. 20 Changes in society are also influenced by certain conditions that require the community to respond. Such as during the Corona virus outbreak, which greatly changed several community conditions, such as not recommending face-to-face activities, carrying out activities from home, as well as lifestyles that must comply with health protocols. These changes are an opportunity to develop businesses from various groups, such as the business of medical equipment, food, and even the fishon sector.
In the fishon sector, there are many opportunities to follow the changing trends of society, such as the hijab trend which is widely used by the community. Like Ria Safitri's statement, hijab as fashion follows circumstances, including a pandemic that requires people to wear masks, so the hijab trend with masks emerged. It is possible that there are also Muslim women who wear the hijab for reasons of maintaining health. More protection from the spread of the virus by wearing a hijab when leaving the house. Hijab is an obligation for Muslim women, fashion trends can make Muslim women more confident and proud as Muslim women, so that they can be used as suggestions for Islamic symbols. The use of hijab trends is not a problem, as long as it is in accordance with syar'i elements. 21 The trend used is not allowed for a lavish in following the trend, in the study of fiqh the lavish activity is known as Isrof.
Isrof is an act of luxury in life, such as luxury in food, drink, and others. This nature of luxury is a bad nature in life, as happens to human beings who always live in luxury. However, not all are considered luxuries such as people who give alms, go on pilgrimage, and others are not included isrof. The above is as it is said "spending property for the needs of Hajj including alms in the way of God, where one dirham is multiplied to seven hundred times." 22 In relation to the hijab, it is not justified to use the hijab which is in the isrof category, because it is not justified by religion. Because of this, the hijab trend must also pay attention to the luxurious nature (isrof).
As long as the hijab is a shield for the mouth and breathing, it is allowed, only that what happens in the sea is that it is stated that the type of scuba mask is declared unable to protect against the corona virus. 23 As a result, masks that have been modified with hijab, or hijab masks, are only for business opportunities, because masks are now a trend, so new hijab masks appear to be a trend and are in demand by the public, particularly young mothers who are always concerned with their appearance. 24 Masks or face coverings can serve as a barrier to stopping the transmission of infection, rather than not wearing them at all. In addition, it is recommended that the mask has a triple layer opportunity that protects against moisture from outside and protects 95% of dust, pollen, bacteria, viruses and other airborne particles. Hijab khimar is appropriate for those who initially forget to bring a mask and usually wearing a mask feels a bit cramped. The advantages are different from ordinary masks, if khimar goes anywhere because there is already a mask in khimar's hijab, in addition to masks as part of contemporary fashion, he also functions as a cover for genitalia. The hijab trend has inspired many Muslim women who did not initially wear a hijab, so 21 they used the hijab. Because seeing the good hijab fashion in Indonesia and the enormous benefits for its users. 25 When the hijab trend is in accordance with fiqh studies to cover the genitals, then this trend is very positive. Because basically in hijab, Islam has its own provisions.
The conditions that must exist in Muslim clothing can cover all parts of the body, other than those that have been excluded by religion, such as the face and palms. Clothing should not be used as a means to decorate the body. The dress should be thick and not flimsy. Do not use perfume or fragrance in the clothes to be worn, do not resemble men's clothes, and do not resemble clothing that is often used by non-Muslim women. In addition, these conditions in Islam are also not allowed to use the hijab to the category of mubadzir.
Mubadzir is an act that is prohibited in the teachings of Islam. Several notions of mubadzir have been put forward by several figures. Mubadzir (extravagant) is spending wealth in an illicit place. Ibn Abbas ra. say that the meaning of "mubadzir" is to spend wealth in a place that is not allowed (syara '). Meanwhile, Ibn Mas'ud ra said that the word of Allah تبذيرا( تبذر )وال is to spend wealth in the wrong way. 26 In relation to the hijab trend, there is a ban on redundancy. It is something that must be considered, namely avoiding the occurrence of redundant or wasteful in using the hijab. Both among sellers or entrepreneurs and among customers or buyers in the use of hijab by the community. Especially in the conditions of the Covid 19 pandemic, the use or collection of the hijab at the same time with the mask partner is an option in avoiding the redundant act itself. So that it will help the community in the behavior of the hijab trend, and comply with the health protocols recommended by the government.
The hijab trend is simpler and less accessible, so it's comfortable to wear. Because besides being simple, buyers also pay attention to protection in addition to style matters. The sharia view considers hijab to be good, as long as it is polite and covers the genitals, and is not excessive, because a Muslim woman must look elegant and neat in syar'iyah glasses. 27 If the hijab mask material is thick or layered, it can reduce the risk of being exposed to covid. However, this needs further research. Because, if you can protect yourself from the spread of the virus, why are there still so many people in the Middle East who are wearing the veil who are infected with Covid? The hijab trend at the time of covid did make some progress, although not significantly. This can be seen from the hijab models that these people wear which also function as masks. 28 Hijab protection + masks can protect against virus transmission or not, back to the standards used in making the hijab masks. When viewed from the sharia aspect, such a hijab trend is very well used as a substitute for the veil to make it look less conspicuous. Very supportive in terms of sharia, while in terms of protection from viruses, it is better to return to the use of cloth that is used according to health standards. 29 The hijab trend effectively has two benefits. First, it is an economically acceptable business, both by the community, especially Muslim women. Second, it has medical benefits, because in the era of a pandemic, one of the ways to prevent transmission of the virus is in addition to maintaining direct contact interactions, it is also necessary to have personal protective equipment such as the trending hijab with masks or face coverings. In sharia, basically hijab is fashion clothing which is part of a cultural product. However, this culture happens to be in line with some religious values, especially regarding a woman's genitalia. Although there are some parties who still often argue about it. In simple terms, hijab is fashion or culture that is in line with religious guidance. 30 The hijab trend is one solution for the community to develop a business or even start a business during Covid. Behind the negative side, of course there is a positive side, one of which is for those who own hijab companies as well as masks. Hijab as well as masks are not completely a solution to reduce the spread of the virus, because they must be adapted to medical recommendations. This means that not all hijabs comply with medical recommendations in preventing transmission. The hijab trend when viewed from the shariah perspective is a positive thing, but on the negative side, when this trend has diminished, they will return to their previous state or not wear the hijab again. 31 In the business space, this is commonplace. In the health room, this cannot be said to be an effective mask in reducing the transmission of COVID-19. In the sharia category, of course, this is highly recommended for women, because basically covering one's face is a sharia rule whose law must be obeyed according to the views of some schools of thought. Regarding the use of the hijab, it is okay to use it because it does not violate the provisions of sharia. It's just that it's not yet effective, because the hijab material that is produced cannot be guaranteed to be clean or not in accordance with national health standards. 32 From an economic point of view, this opportunity provides a positive value, because covid has had a very large impact, mainly due to large-scale restrictions, restrictions on business activities which then have an impact on the economy. Especially in businesses that are offline. Some goods went up, while people's purchasing prices weakened due to layoffs. The hijab trend can be said to be a positive side of covid, many people have switched to hijab, even though the main intention is to protect themselves from the virus, just to cover part of the body, not the syar'i intention to cover the genitals. This then opens up opportunities for the market to run a hijab business. Economically, this is very profitable, because of the large market demand, especially hijab with masks. 29 The existence of Covid as a whole has a very large negative impact on the economy. However, as a business actor and producer, you must have creative ideas to make opportunities in business. The existence of Covid is a great opportunity for hijab players to produce hijab with the trend of hijab + mask, uniform hijab with masks, and hijab combinations according to masks. This trend provides many advantages for hijab businesses, because in addition to models that are in accordance with conditions, Indonesia itself is a Muslim majority with model trends that are always updated, so that hijab users feel comfortable and confident when using hijab trends according to the Covid period. 33 This trend is an opportunity for many market sectors that require masks to protect against virus transmission, so this trend is one way to protect lives. In contemporary Jurisprudence, protecting the soul must be upheld in this Covid condition. Keeping the soul in question is the protection of the physical and psychological life of humans and their safety. Which means that all things that hurt the physical self and distress the human psyche are prohibited by law and the responsibility to protect the soul is borne by the individual, including the community whose aim is to maintain human existence and prevent bloodshed.
The application of sharia principles as a rule of agreement based on Islamic law in economic business activities is basically a specification of the rules of ahkam almuamalah in the framework of Islamic law, especially a set of rules of ahkam aliqtishadiyah wa al-maliyah. 34 A series of transactions known as business or sale and purchase transactions. Sale and purchase is an agreement to exchange things (goods) that have value, on the basis of voluntariness between the two parties in accordance with the agreement or provisions allowed by the syara.' 35 This form of agreement that provides revenue or income to the community.
In essence, the business opportunity at the time of covid, one of which was the hijab trend, which was in great demand by Muslim women, both those who wore hijab and those who did not, became one of the solutions in overcoming the transmission of the virus, as well as being a business that was in accordance with sharia. The hijab trend is one of the businesses to maintain personal health so that it can also maintain offspring. It is a business to take care of offspring, keeping these descendants means maintaining the sustainability of future generations. 36 In addition, it is also a business that calls for the good and prevents the bad. Ma'ruf is something that people know that it is pleasing to God, whether it is a matter of obligation or sunnah that contains benefits for individuals and congregations, while munkar is something that is denied by God, forbidden by God, because it contains danger to individuals and society. 37 In Islam, the use of hijab is one of the obligations that must be fulfilled by a Muslim woman to cover her aurat. While the use of hijab and masks is an obligation during a pandemic to overcome the transmission of a virus outbreak that is very dangerous for oneself, even though in reality the use of the veil in Islam is not an obligation.
Judging from the review of sharia, the issue of the use of veils has no mandatory rules, either the Qur'an or hadith. But, if viewed from the point of view of Maqashid Syariah, the use of the hijab model as well as the mask can be used in the framework of hifzh al-nafs (keeping life), in the stage of tahsiniyah, not hajjiyah, let alone dharuriyah. That is, it is only a complementary tool in hifzh al-nafs, not the only tool. Because there are still masks or faceshilds or others that also work the same as the hijab of the mask. In a health review, hijab masks are less effective in protecting the virus, because they tend to be thin and not double-stitched, and most hijabs are made of thin cloth. However, on the positive side, many cover their aurat and look Islamic, even though the intention is only for health. However, on the other hand, it can reduce the view of men to the opposite sex. This is a land of da'wah to justify untrue intentions. 38 This hijab trend is an opportunity for da'wah for the Islamic community to introduce the rules for maintaining aurat which has the aim of protecting oneself, both in terms of safety and health.
D. CONCLUSIONS
The corona virus outbreak has not made creative ideas from the business sector recede, even this outbreak has been used as a new opportunity by some business people who are sensitive to the needs of the community. Despite the fact that this epidemic poses a threat to the majority of the community, both in the health sector, harmony, and the business sector. Many people lose their jobs and opportunities to generate income which puts them in a very deprived position. With the existence of several protocols that must be obeyed by the community to achieve the elimination of the chain of the spread of the corona virus, it is causing difficulties for the community's economy today. There are several things that must be obeyed by the community, such as maintaining distance, washing hands, staying at home, and wearing masks.
The use of masks by the Indonesian people is carried out with various tools and models as desired. So that there are models of masks that are used by the Indonesian people, making the needs of the community during the pandemic increase, and businesses are trying to meet the increasing demand for masks. One form of mask model that is also a trend is the use of masks that have been integrated with the hijab, as well as masks that are uniform with the hijab. This trend has its own charm for women to use trendy hijabs and mask motifs that match the hijab. The use of the hijab trend has a positive impact on businesses in increasing income during a pandemic. Although the use of the hijab mask trend is not optimal in preventing the transmission of the corona virus if the cloth used as a mask material is too thin and does not meet health standards. However, if the material used is thick and meets health standards, then this hijab mask trend is very beneficial.
When viewed from contemporary fiqh studies, besides the hijab trend, it has benefits for business actors, as well as users. It also has the value of da'wah that is in accordance with sharia, because with the widespread use of the hijab trend, it provides a special attraction for people who initially did not cover their aurat, they became interested in wearing the hijab trend. As for business studies in contemporary fiqh, the hijab trend is one of the legalized businesses because the goods being traded are halal goods according to sharia, and are included in the category of businesses that maintain personal health, so that they can also maintain offspring. In addition, business calls for ma'ruf and forbids what is evil. | 2021-09-28T01:15:59.430Z | 2021-06-18T00:00:00.000 | {
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58929833 | pes2o/s2orc | v3-fos-license | Exploring the Role of Forestry Sector on Economic System of Gunungkidul District in 1993 - 2008
This study was conducted to explore the role of forestry sector in the economic system of Gunungkidul district. The Location Quotient (LQ) Analysis, Income Multiplier Effect Value, and Klassen Typology Analysis were employed to analyze the role of the forestry sector. The data were regional income of Gunungkidul district and Yogyakarta Province from 1993 to 2008, including the economic crisis period from 1997 to 1998. The result showed that forestry sector was an important sector in economic development of Gunungkidul district. LQ analysis indicated that forestry became a basic sector since pre-economic crisis period until post-economic crisis (1993 - 2008). Prior to the economic crisis, forestry sector generated the highest income multiplier effect value. However, the value dropped during and after the economic crisis. The economic crisis had an influence on the development pattern classification of forestry sector. Before economic crisis, forestry sector was classified as a developed sector (quadrant I) with the growth and shared to GDRP in Gunungkidul were higher than that in Yogyakarta Province. Meanwhile, since the economic crisis, forestry sector fell into the lower class as a stagnant sector.
I. INTRODUCTION
Forestry sector provides significant benefit for regional economic development in Gunungkidul district. The benefit could be classified into direct and indirect benefit. The direct among others benefit includes commercial and non-commercial use of wood, rattan, resin, and bamboo, while, environmental services are classified into indirect benefit (IIED, 2003).
In regional economic development, regional income is predicted by calculating the aggregation of the direct output value in each economic sector. This aggregated income value is called Gross Domestic Regional Product (GDRP). The income of forestry sector calculates the aggregated value of the direct forest output such resources. Later, the expansion of POF in Gunungkidul district is motivated by the price of wood product, especially teak wood, (Rohadi ., 2010).
Several studies on POF in various aspects have been conducted in Gunungkidul, such as the research conducted by Andayani (2005) and Milawati (2010). Andayani (2005) focused her research on the distribution of log and feasibility study of POF cultivation, while Milawati (2010) has clarified the people's income from agroforestry system in Mahogany-Teak-Gnetum pattern located in Patuk sub-district. Both studies were based on microeconomic sphere analysis, which were not presenting the role of POF in the regional economic development spheres of Gunungkidul district. Both research have proved the benefit of POF for people of Gunungkidul as individual (the POF grower), but they did not show the advantages of POF development for the whole Gunungkidul people. Therefore, the objective of this paper is to fill up the information gap by clarifying forestry sector's role in economic system in Gunungkidul district.
This study analyzed the role of forestry sector over middle term period from 1993 to 2008. For the sake of analysis, the periods were classified into three periods: pre economic crisis (1993 -1996), economic crisis (1997 -1999), and post economic crisis (2000 -2008). The objectives of this study were to clarify (1). the role of forestry sector by economic base theory; (2). the multiplier effect value of forestry sector; and (3). the development pattern of forestry sector by Klassen Typology.
A. Conceptual Framework
Forests ecosystem produces various benefits including three main benefits of economic, ecological, and social. The POFs also produce those three main benefits. In economic aspect, POFs generate direct benefit by selling wood and non wood products. POFs also produce many indirect benefits on ecological and social aspects such as micro climate regulation, carbon sequestration, water supply regulation, soil conservation, air pollution reduction, watershed protection, nutrient cycling, as well as education and research.
This paper focuses on the economic benefit of POF. Economic benefit of forest in regional economic sphere is depicted as forestry sector income on GDRP. The income was calculated of direct benefit of forestry sector which in supply timber and non timber forest product. The analysis was carried out to find out the role of forestry sector in the economic system of Gunungkidul.
To achieve the research objectives were presented above, the widely used and simple tools in regional economic analysis were employed i.e. : Location Quotient (LQ) Analysis, Income Multiplier Effect Value, and Klassen Typology. According to Tarigan (2009), the utilization of these tools is important to determine the superior and inferior sector in an economic development. In LQ Analysis, the superior sector called as the basic sector. Basic sector means the sector which generates more income than the same sector within the reference (larger) area. In this study, regional economic system of Gunungkidul district was analyzed and Yogyakarta Province was determined as the reference area. Then, Income Multiplier Effect Value was employed to scrutinize the multiplier impact of basis sector to the non-basic sector in the economic system of Gunungkidul. The Klassen Typology was examined to determine the position of forestry sector in the economic development stage of Gunungkidul district (Figure 1). In this method, the forestry sector explored whether it categorized as developed, stagnant, developing, or under-developed sector.
This study used various data including 1) output value for each sector of Gunungkidul district and Yogyakarta Province, 2) total output for whole sector of Gunungkidul district and Yogyakarta Province 3) output growth for each sector in Gunungkidul district and Yogyakarta Province. The data was collected from Statistical Centre Agency publication.
Location Quotient (LQ) Analysis
LQ Analysis was utilized to categorize forestry sector in economic system in Gunungkidul district into basic sector and non basic sector. While the basic sector indicate more than 1 (one) of LQ value, non basic sector indicate less than 1 of LQ value. The LQ formula employed in this study was referred to Bendavid (1974) with some modification. To calculate LQ value, Bendavid (1974) used employment level as a variable. Meanwhile, in this study, we were employed output value (sectoral income) as a variable. The similar modification also did by Kuncoro (2004) . Thus, the LQ value formula stated by: vxi = output value for x sector of Gunungkidul District vt = total output value for whole sector of Gunungkidul District Vxj = output value for x sector of larger area, Yogyakarta Province Vt = total output value for whole sector of larger area, Yogyakarta Province
Multiplier Effect Value
This study adopted coefficient of multiplier effect formula (Bendavid, 1974). The formula is expressed as below: M = coefficient of multiplier effect value Y = total output value of whole economic sector Yb = output value of basic sector
Klassen Typology
According to Sjafrizal (2008), the Klassen Typology was classifies the economic development phase into four groups, and the groups are divided into four quadrants. The sector classification was determined by two factors: the sector contribution to GDRP (Ys) and the growth C. Data Analysis rate in development sector (Rs). Table 1 shows the sector classification by Klassen typology. indicates the developed sector. This sector stage has two requisites: 1) the growth rate of forestry sector in local area/Gunungkidul district (Rsi) should be equal or more than the growth rate of the forestry sector in larger area (reference area/Yogyakarta Province) (Rsn) (Rsi ≥ Rsn), and 2) the contribution value of forestry sector to the GDRP in Gunungkidul district (Ysi) should be equal or more than the contribution value of forestry sector in Yogyakarta Province (Ysn) (Ysi ≥ Ysn).
points out the stagnant sector, which prerequisites: Rsi should be equal or more than Rsn (Rsi ≥ Rsn); Ysi should be less than Ysn (Ysi<Ysn).
indicates the developing sector, which prequisites: Rsi should be less than Rsn (Rsi < Rsn); Ysi should be equal or more than Ysn (Ysi ≥ Ysn). Lastly, points out the underdeveloped sector, which requisites: Rsi should be less than Rsn (Rsi < Rsn); Ysi should be less than Ysn (Ysi < Ysn).
Gunungkidul district is one of districts in Yogyakarta Province. The district is located in the south eastern part of the province area. Geographically, the district is located between 7 46' 8 09' South Latitude, and 110 21' 110 50' East Longitude. The north and east side of district area is neighboring to Central Java Province, whereas the south side is bordering with the Indian Ocean (Figure 1).
The district area is around 1,485.36 km (Statistics Office of Yogyakarta Province, 2008), and covers the largest area among other districts in Yogyakarta Province. The area is about half of Yogyakarta Province area. The district area is classified into agriculture and non agriculture lands. According to the data presented by Statistics Office of Yogyakarta Province (2008), agriculture land covers 112,935 ha (76 % of total district area) and the rest 35,601 ha (24 %) is non agriculture area. The agriculture area is divided into six land use types. Figure 2 shows the detail of the land use in Gunungkidul district.
The first quadrant
The second quadrant The third quadrant the fourth quadrant In terms of forest condition in Gunungkidul district, while state forest area is decreasing, the POF area is increasing (Utari, 2010
B. Basic Sector
The share of forestry sector to GDRP fluctuated from 1993 to 2008 (Figure 3). During pre-economic crisis period (1993 -1996), the contribution of forestry sector to GDRP was less than one percent. During economic crisis period (1997 -1999), the share raised to near 10 %. Meanwhile, the share of agriculture sector dropped to below 30 %. This change of the share indicated that people cut down their trees to fulfill their need during economic crisis period. On the other hand, the income shared of agriculture sector dropped due to the decline of demand on agricultural goods.
The analyzed data presented that LQ value of forestry sector from 1993 to 2008 was more than one ( Table 2). The fact indicated that forestry sector was very important in economic system of Gunungkidul district. The economic crisis has not changed the role of forestry sector as a basic sector in Gunungkidul district.
Goods provided by basic sector was traded in local market and also in regional or national market. As a basic sector, forestry has a high Source: Data processed Figure 3. The share of forestry and agricultural sector to GDRP of Gunungkidul District in 1993 2008 potential to generate income by selling output to the other districts or to the regional market in Gunungkidul district. It indicated that output from forest sector was very important to fulfill human need in local area as well as larger area in Gunungkidul district (Yogyakarta Province).
The income multiplier effect of forestry sector was relatively fluctuated. Before economic crisis period, the income multiplier effect value of forestry sector was the biggest in the economic system in Gunungkidul district. The value was more than one thousand, while the other ones were no more than sixty.
However, the value changed during and after ecocomic crisis that occurred in 1997. During these periods, the income multiplier effect value of forestry sector was dropped until ten. On the other hand, the value of others basic sectors were relatively constant. The income multiplier effect value of agriculture sector stayed constant at around three and mining quarrying sector also stayed constant at around fifty.
The income multiplier effect indicated that the income from basic sector could stimulate generating income from non basic sectors. As an C. Income Multiplier Effect Value example, the income multiplier effect value of forestry sector in 1993 was 1,382.93 (Table 3). These value means that when forestry sector generated income of US$ 1,000.00, it could stimulate to generate income from non basic sectors until US$ 1,382,930.00.
The process of generating income from non basic sector could be explained as follows. The basic sector produces output and trades it into regional market (larger market). The economic actors in Gunungkidul district gain more income from the trading. The economic actors will spend more income to fulfill human needs by consuming various product that also afforded from non basic sectors in the local area. The rising of consumption could stimulate non basic sectors to increase output.
The income multiplier effect value of mining and quarrying sector was higher than that of forestry sector as well as agricultural sector after economic crisis (Table 3). The implication of these values is that the developing mining and quarrying sector could generated more income from non basic sector. However, developing mining and quarrying sector should be noticed in terms of the negative environmental effect. Meanwhile, the developing of forestry sector The result of economic development pattern of forestry sector in Gunungkidul district is shown in Table 4. The forestry sector was classified as a developed sector during preeconomic crisis period. That pattern indicated that forestry sector rapidly grew and highly contributed to economic system in Gunungkidul district. The sector growth value and the sector contribution rate of forestry in Gunungkidul district were higher than that in Yogyakarta Province.
However, during and after the crisis period, forestry sector was down to stagnant level. This level indicated that the forestry sector growth rate in Gunungkidul district was less than that in Yogyakarta Province, yet the sector contribution in the District was higher than that in the province.
1. Forestry sector had a basic sector in economic system in Gunungkidul district since the preeconomic crisis period until post-economic crisis (1993 -2008). It indicated that forestry sector was important for generating income on economic development in Gunungkidul district. 2. Before the economic crisis, forestry sector generated the highest income multiplier effect value. However it had been dropping since the economic crisis up to 2008.
V. CONCLUSION
3. The economic crisis influenced the pattern of sector forestry development in Gunungkidul district. Before the economic crisis, forestry was classified as a developed sector with featured: forestry sector growth and contribution to GDRP in Gunungkidul district were higher than that in Yogyakarta Province. Meanwhile, during and after the crisis, the forestry sector fell to the lower class as a stagnant sector, with feature: lower forestry sector growth but higher contribution to GDRP in Gunungkidul district than the growth and contribution of that sector in Yogyakarta Province. | 2018-12-15T02:24:08.463Z | 2012-12-10T00:00:00.000 | {
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251726185 | pes2o/s2orc | v3-fos-license | The challenging management of borderline ovarian tumors (BOTs) in women of childbearing age
Borderline ovarian tumors (BOTs) account for approximately 15% of all epithelial ovarian cancers. In 80% of cases the diagnosis of BOTs is done at stage I and more than a third of BOTs occurs in women younger than 40 years of age wishing to preserve their childbearing potential; the issue of conservative surgical management (fertility-sparing treatment) is thus becoming of paramount importance. At early stages, the modalities of conservative treatment could range from mono-lateral cystectomy to bilateral salpingo-oophorectomy. Although cystectomy is the preferred method to promote fertility it can lead to an elevated risk of recurrence; therefore, an appropriate counseling about the risk of relapse is mandatory before opting for this treatment. Nevertheless, relapses are often benign and can be treated by repeated conservative surgery. Besides the stage of the disease, histological subtype is another essential factor when considering the proper procedure: as most mucinous BOTs (mBOTs) are more commonly unilateral, the risk of an invasive recurrence seems to be higher, compared to serous histotype, therefore unilateral salpingo-oophorectomy is recommended. In the appraisal of current literature, this review aims to gain better insight on the current recommendations to identify the right balance between an accurate staging and an optimal fertility outcome.
Introduction
Borderline ovarian tumors (BOTs) are a heterogeneous group of neoplasms with recognized potential malignancy, histologically defined by epithelial proliferation and nuclear atypia without recognizable destructive stromal invasion. Similar to carcinoma they can spread to the peritoneum and eventually to lymph nodes and in some patients can recur (1).
Peritoneal spread is present in 10% of BOTs and is divided into non-invasive (nearly 85% of implants) or invasive (2); the mortality rate for patients with non-invasive and invasive implants is 4.7% and 34% respectively (3).
BOTs are staged according to the FIGO staging system used for ovarian carcinoma (4). Most of the BOTs have a low potential for malignancy and are confined to the ovaries at presentation: unlike ovarian carcinoma in nearly 80% of cases the diagnosis is done at stage I with <1% of women diagnosed at stage IV (5).
Borderline ovarian tumors survival is 95% at 5 years, 90% at 10 years for women with FIGO stage I-III and nearly 77% at stage IV (6).
The vast majority of BOTs have serous or mucinous histotypes; about two-thirds are serous BOT ( Table 1). Other rare types (<5%) are clear cell, endometrioid and Brenner tumors. A description of the natural behavior of the different histotypes is essential for the selection of the most appropriate surgical strategy.
Serous borderline ovarian tumors (sBOTs) are bilateral in 15%-25% of cases and noninvasive peritoneal spread is present in 15%/40% of cases (7) (Figure 1). The risk of invasive peritoneal spread is very low in early-stage serous tumors; only in a small percentage of cases do the implants infiltrate the underlying subperitoneal tissue and should thus be considered, according to the 2014 WHO (World Health Organization), as low-grade serous carcinoma (8).
The micropapillary/cribriform pattern is a variant of the common sBOT. This lesion is defined by distinct morphological criteria and is more likely associated with a higher rate of bilateral ovarian involvement, recurrence, and invasive peritoneal implants compared with serous lesions without micropapillary patterns (9).
Mucinous borderline ovarian tumors (mBOTs) represent the second most common histologic subtype accounting for 30%-50% of all borderline ovarian tumors. Mucinous borderline tumors are nearly always unilateral and tend to be larger than sBOTs (average diameter of 20 cm) ( Table 1) (10). Patients with mBOTs relapse less frequently than patients with serous disease, but when an extraovarian relapse occurs, the risk of an invasive recurrence and possible death seems to be higher (11).
Methods
An electronic database search (Pubmed, Medline and Embase) was performed up to April 2022.
All pertinent articles evaluating the diagnostic and therapeutic approaches centered on fertility-sparing treatment of borderline ovarian tumors have been included in this review.
All original studies, meta-analyses, systematic reviews and case reports published in English were considered. The reference lists were systematically reviewed to identify other studies for potential inclusion in this narrative review.
Surgical approach
Fertility sparing surgery (FSS) has been defined as the preservation of the uterus and ovarian tissue in one or both (12). More than a third of BOTs affect women of reproductive age who wish to preserve their fertility potential.
In the management of early-stage BOTs, FSS is the mainstay of treatment, as an alternative to radical surgery (13).
In the advanced stages of the disease, the oncological safety of conservative treatment has still to be clarified (14). As a rule, patients with advanced-stage BOT should be considered not amenable to conservative surgical therapy in the presence of invasive implants or noninvasive implants not completely resectable (15)(16)(17).
Great attention has been recently focused on uterine preservation in the management of patients where preservation of healthy ovarian tissue is not feasible (18).
Indeed, ovarian tissue cryopreservation at the time of surgery, oocyte freezing, oocyte donation or a transfer of frozen embryos obtained before the surgical procedure are proposed to permit fertility (12,19,20).
FSS does not seem to affect the overall survival (21) however conservative treatment has been found to increase the relapse rate and therefore it is necessary to give full information to the patients about this risk.
Although the definitive diagnosis requires pathological evaluation after surgical excision, however preoperative knowledge of the specific ultrasonographic and macroscopic MRI features to differentiate BOTs subtypes can be extremely helpful to promote optimal patient management (22,23).
The Frozen section (FS) plays an additional important role in determining the appropriate surgical management however, the surgeon should be aware of the well-known limitations of FS. The diagnostic accuracy rate for FS remains high for benign and malignant ovarian tumors but is relatively low for BOTs.
Frozen samples tend to under diagnose BOT as benign tumors in 25%-30% of cases, and improperly identify BOTs as carcinoma in 20%-30% of cases (24,25). More caution in the use of FS in BOTs is needed, especially in cases of bulky tumors, where the intraoperative histology may lead to misdiagnosis of some features (e.g., microinvasion, papillary variant,intraepithelial carcinoma, stromal microinvasion) (26)(27)(28).
Although surgical staging does not have a significant impact on survival rate (29), nevertheless an initial complete staging appears to significantly reduce recurrence among BOTs patients (30). A complete exploration of the abdominal-pelvic peritoneal cavity, peritoneal washing, multiple peritoneal biopsies, infracolic omentectomy and complete resection of the implants for staging purposes are recommended (31).
A primary task for the surgeon is the complete removal of all peritoneal implants for both staging and therapeutic purposes with wide resection of surrounding tissue to allow the pathologist to discriminate non-invasive from invasive implants (32).
For the aforementioned reasons, surgical restaging should be considered in patients at higher risk of malignancy (mBOT, micropapillary variant, etc.) who underwent incomplete visual exploration of the abdominal pelvic peritoneum at the first surgery (33).
Lymph nodes involvement has a low prognostic value (34). In a retrospective analysis by Matsuo et al., no difference was found in survival rates in patients undergoing lymphadenectomy (35).
Lymphadenectomy is usually suggested only for cases with enlarged lymph nodes or invasive tumors detected on frozen examination (36,37).
Obtaining a biopsy for the histopathologic evaluation from a normal appearing contra-lateral ovary is not helpful to reduce the risk of recurrence; an accurate preoperative ultrasonographic examination and a careful intraoperative macroscopic inspection is considered adequate for this purpose (38).
The use of minimally invasive or traditional open surgery has been evaluated in the literature: whatever the approach used, rupture of an intact tumor during its dissection/removal could alter the FIGO staging and affect the risk of recurrence (36).
In the last years, the use of minimally invasive procedure has increased dramatically, because of their reduced postoperative complications, blood loss, shorter postoperative recovery and cosmetic results. However, the decision on the surgical approach for BOT patients should be based on preoperative diagnostic features, epidemiological aspect and the surgeon's skill.
Published data report an increased intraoperative tumor rupture during laparoscopy cystectomies and identifies tumor volume as the main predictor factor.
Indeed, in a retrospective study of 105 patients, the tumor rupture was significantly more frequent during laparoscopy compared to laparotomy (29.5% vs. 13.1%, p = 0.038) (39). The conversion laparotomy rates is reporting of approximately 30% for BOT patients (40).
In another retrospective analysis, adnexa larger than 10 cm in maximum diameter were associated with a 4-fold risk of surgical spillage with laparoscopic approach (54.5% vs. 12.1%) compared to open surgery (37).
The laparoscopy compared to laparotomy has not shown a negative impact in terms of the recurrence rate, the survival and the feasibility of surgical management of BOTs (41). If surgery without risk of tumor rupture is possible, then the laparoscopic approach could be considered feasible, safe and recommended over laparotomy (40).
Robotic surgery is a feasible alternative in managing ovarian cancer as long as there is careful consideration given to patient selection (42). Robotic surgery is considered an option for the treatment borderline ovarian tumors, however the haptic feedback allowing to measure tissue traction and avoid cyst rupture is present only in some robotic platforms (43).
Prospective randomized studies are needed to determine the relevance of robotic surgery in this context.
Another ultra minimally invasive approaches is the minilaparoscopy that represents a great challenge for adnexal disease. Gueli Alletti et al. (44) have described a successful case of conservative staging surgery through the use of 2.4 mm needleoscopic instruments concluding that this could prove a beneficial tool in borderline disease.
Adnexal surgery: What is best?
There is a lack of clear international guidelines on the optimal FSS procedure. FSS in stage I include unilateral/ bilateral cystectomy, unilateral salpingo-oophorectomy and unilateral salpingo-oophorectomy plus contra-lateral cystectomy.
The impact of the histological subtype and the presence of factors associated with poor prognosis (microinvasion, micropapillary pattern, peritoneal implants) on FSS approach is relevant (45). As most mBOTs are at high risk of invasive recurrence and co-existence with invasive cancer areas is possible, unilateral salpingo-oophorectomy is considered the preferred surgical treatment in these cases (46); cystectomy is admissible only in presence of bilateral mBOT or when controlateral cystectomy is the only method to preserve fertility in patients with previous salpingo-oophorectomy (47).
Concerning sBOTs, often bilateral and characterized by a relatively benign behavior compared to mBOTs, the theoretically reproductive advantage of cystectomy as opposed to unilateral salpingo-oophorectomy is still waiting for a definitive conclusion.
The reproductive outcome seems not different between unilateral oophorectomy and cystectomy (48) on the other hand, but the available literature has raised concerns about a higher recurrence rate after cystectomy (49).
In a French multicenter study, including 313 patients with stage I BOTs, the recurrence rates after cystectomy, unilateral salpingo-oophorectomy or bilateral salpingo-oophorectomy have been found as 30.3%, 11% and 1.7%, respectively (50).
These results have been confirmed in a recent systematic review reporting the rate of recurrence correlated with the type of conservative surgery with a higher rate after cystectomy (41).
As opposite, Palomba et al. report that the use of bilateral cystectomy compared with a unilateral salpingooophorectomy and a contralateral cystectomy (in patients with bilateral BOTs, mainly in serous subtype) increases the fertility rate without increasing the recurrence rate (51). Vasconcelos et al. confirmed these results in a meta-analysis showing that, in case of bilateral serous BOT, unilateral salpingo-oophorectomy + contra-lateral cystectomy did not obtain any advantage compared to bilateral cystectomy in terms of recurrence (26.1% vs. 25.6%) (52). Standing this unresolved dispute, it is wise to concludethat whenever cystectomy is the selected procedure then appropriate counseling is recommended about a possible higher risk of local and peritoneal recurrence compared with salpingooophorectomy.
Obviously, cystectomy or unilateral salpingectomy + contralateral cystectomy remains the only fertility-sparing option in case of bilateral sBOT and in rare cases of previous surgical salpingo-oophorectomy (53).
The overall risk of recurrence varies between 2% and 24% and the risk of invasive recurrence ranges from 0.5% to 3.8%. Recurrences are seen in the remnant ovary after cystectomy, or in contralateral ovary or as extraovarian peritoneal and omental implants (54).
Complete surgical eradication of ovarian tumors and peritoneal implants even if not visible macroscopically is the prerequisite in minimizing the risk of disease relapse; preand intra-operative ultrasounds are of invaluable help to accomplish this goal (55).
Twenty-five percent of recurrences are diagnosed after 5 years (56); however, the recurrence rate is time-dependent and relapses may occur 15 years after surgery.
During the first two post-operative years, recurrences seem to be more frequent; a close follow-up is needed for this period through a systematic clinical examination including transvaginal ultrasonography and serum markers (57); unfortunately, only 40% of women with stage I BOTs have elevated levels of Ca125 and can benefit of this diagnostic measure (58,59).
Most of sBOTs recurrences are in greater part borderline tumors, easily treated by repeated conservative surgery in patients desiring to preserve fertility (60).
Concerning mBOTs, if the risk of relapse is significantly lower compared with sBOTs, on the other side the risk of an invasive recurrence is higher (61).
Along with the histotype, some further clinicopathological factors, although not unanimously, are considered helpful to identify patients more prone to invasive recurrence (60,62).
Early-stage according to FIGO classification is a well-known independent risk factor for recurrence (63,64); indeed, the rate of extraovarian recurrence has been demonstrated higher in stage IC3 and grade 3 tumors and consequently such aspects should be recognized as limits of conservative management for oncology safety (65).
Other factors such as micropapillary pattern and stromal microinvasion are histological aspects featuring a high-risk group likely to develop an invasive recurrence (57,66).
Serous borderline ovarian tumors with micropapillary patterns seem to be more commonly associated with advanced stage, bilateral ovarian involvement, and invasive recurrence than the typical sBOTs (67,68).
Notably, serous BOT displaying a micropapillary pattern without implants (stage I) or with non-invasive implants (stage II and III) could have the same prognosis as serous BOT without a micropapillary pattern (69).
On this basis, in case of micropapillary serous BOT without invasive implants it could be reasonable to propose a conservative approach only if combined with a careful and long follow-up (70); radical surgery to avoid any recurrence should be considered in those patients who completed their reproductive plans after conservative surgery or in cases without follow-up opportunities (71).
Data from literature identifies, stromal microinvasion, defined as a lesion that invades the stroma to a depth of 5 mm or less as a predictor of relapses: In a case series evaluating follow up data of 171 borderline mucinous tumors,microinvasive pattern was associated with an higher reuccrence rate (p = 0.013). In particular, in the group without microinvasion the rate of recurrence was 1.7% (2 of 116 cases), whereas in the group with microinvasion 14.3% (4 of 28 cases). No significant association was reported between clinicopathologic variables of these tumor and recurrence (72).
Higher rate of recurrence were also reported in a a retrospective study conducted on 902 patients with BOTs. Patients with microinvasive BOT had a significantly higher rate of recurrence than patients without microinvasive BOT (17.4 vs. 7.8%, OR 3.55, 95% CI 1.091-11.59, p = 0.03) . In particular stromal microinvasion was found as a prognostic factor for significantly shorter disease free survival (26.7 vs. 11.9 months, p = 0.031) (73).
In addition, data from 209 patients confirm that microinvasive BOTs recurred earlier with respect to noninvasive BOTs, with the median time to recurrence of 10.5 months for the first one and 17 months for the latter .For these patients unilateral salpingo-oophorectomy instead of cystectomy seems to not prevent relapses in microinvasive BOTs, that were recorded in 27% of the patients (74). However, in the studies, the overall survival seems to not differ significantly from BOTs without microinvasion.
Regrettably, microinvasion is associated with high frequency with the micropapillary variant in the serous BOTs and this is a potential confounding factor to identify its exact role in the genesis of recurrence (75)(76)(77). Due to these uncertainties, also for these patients, fertility-sparing surgery may be a reasonable option in young patients with BOTs, only if an accurate and strict follow-up is possible (78). Frequency and types of exams to perform in the follow-up surveillance are not established (79).
Fertility outcomes after fertilitysparing surgery
Studies provide inconclusive findings about the impact of fertility-sparing treatments for BOTs on ovarian function (80) and an unanswered question remain whether pregnancy outcome is determined by the type of conservative approach (unilateral salpingo-oophorectomy/ovarian cystectomy). It is clear that ovarian surgery, especially after a second attempt, may reduce healthy ovarian parenchyma, increasing the risk of infertility. Moreover, the occurrence of postoperative adhesions might interfere with fallopian tube function (80)(81)(82)(83).
However, after fertility-sparing surgery, pregnancy outcomes are encouraging and most pregnancies are achieved spontaneously as early as 3 months after surgery (84).
To avoid pregnancies complicated by recurrent disease, many physicians recommend delaying pregnancy until a sufficient follow-up period after initial surgical treatment (85,86).
Little is known about the incidence and management of BOTs during pregnancy; however, expectant management could be a safe option in case of recurrences in pregnancy (87).
There is no specific data on the management of infertility following conservative treatment of BOTs and it is unclear if there is a potential impact of the use of fertility drugs on the recurrence rate of the disease (88).
Further data are needed on this topic considering that induction of ovulation and in vitro fertilization may be required in order to enhance the chance of conceiving.
Borderline ovarian tumors during pregnancy
Little is known about incidence and management of BOTs during pregnancy. In reported literature, Borderline tumors diagnosed in pregnancy contain features concerning for aggressive behavior if compared to those diagnosed in non pregnant patients. A higher incidence of advanced stage at presentation as well as an higher higher percentage of mucinous BOT with intraepithelial carcinoma and microinvasion, and serous BOT with micropapillary component have been reported (89).
Unfortunately, concerning the management of BOT during pregnancy,only limited data, based mostly on case reports, are present in the literature.
As such, the standardization of the management strategy during pregnancy is difficult and, at the moment, it is based on the gold standard treatment of non-pregnant women. An attitude of close surveillance should be adopted to exclude sign of malignant transformation (rapid enlargement of tumor, abnormal vascularization, presence of solid tissue) (90).
It is advisable that pregnancy and delivery are carried out in a tertiary center specialized in gynecology oncology. Technical difficulties of performing a complete staging for these patients at the initial surgery could necessitate a post partum completion staging or a debulking procedure and eventually an adjuvant chemotherapy (91).
Conclusion
Fertility-sparing surgery is a well-established strategy available for patients with BOTs who desire to preserve fertility. This procedure is characterized by an excellent reproductive outcome and long-term survival.
Invasive recurrences remain one of the most important parameters of the safety of FSS. Unfortunately, the paucity of available data does not permit a definite identification of the prognostic factors of recurrence and makes the extent of conservative surgery as well as the modalities of a careful and effective post-operative follow-up still matter of debate.
Additional well-designed prospective studies, with larger samples, are needed to clarify these unresolved issues.
Author contributions
AM, LDC, PS undertook the searches. PG, MB, CB contributed to data extraction and drafted the manuscript. AM, MP, FV, MCDA, MT participated in data analysis and interpretation, preparation of the manuscript and critically revising the paper, conceived the idea of the manuscript. All authors contributed to the article and approved the submitted version.
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. | 2022-08-23T13:51:06.654Z | 2022-08-23T00:00:00.000 | {
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5812375 | pes2o/s2orc | v3-fos-license | Role of sphingosine-1-phosphate phosphatase 1 in epidermal growth factor-induced chemotaxis.
Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF. To determine whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase, we used fumonisin B1, a specific inhibitor of ceramide synthase. Although fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF- or S1P-induced chemotaxis. EGF activated the epidermal growth factor receptor to the same extent in SPP-1-expressing cells, yet ERK1/2 activation was impaired. In agreement, PD98059, an inhibitor of the ERK-activating enzyme MEK, decreased EGF-stimulated migration. We next examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate Galpha(i) suppressed EGF-induced migration. Moreover, expression of regulator of G protein signaling 3, which inhibits S1P receptor signaling and completely prevented ERK1/2 activation mediated by S1P receptors, not only reduced migration toward S1P but also markedly reduced migration toward EGF. Collectively, these results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration.
The bioactive sphingolipid, sphingosine-1-phosphate (S1P), 1 is a ligand for a family of five specific G protein-coupled recep-tors (S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 ) (1) that are coupled to various G proteins. These receptors regulate diverse cellular processes and have been implicated in cytoskeletal rearrangement and regulation of cell movement (2,3). The founding member of this receptor family, S1P 1 , couples to a G i pathway (4), and its gene disruption in mice demonstrated an essential function for vascular maturation (5). More recently, several studies have established that S1P 1 is also essential for lymphocyte recirculation and that it regulates egress from peripheral lymphoid organs (6,7). In addition to its extracellular functions, S1P acts intracellularly to regulate cell survival (8), yet its direct targets have not yet been elucidated.
As with other potent lipid mediators, levels of S1P are low and tightly regulated in a spatial-temporal manner. Sphingosine kinase (SphK) catalyzes the synthesis of S1P (2), which can be irreversibly degraded by S1P lyase (9 -11) and reversibly converted back to sphingosine by S1P phosphatases (SPPs) (12)(13)(14)(15). Recently, two isoforms, designated SPP-1 (12,13,16) and SPP-2 (15), have been identified. Both belong to the family of type 2 lipid phosphate phosphohydrolases (LPPs) (17,18) which are magnesium-independent, membrane-associated, and N-ethylmaleimide-insensitive. Although specific biological roles for the LPPs have not yet been established, the Drosophila gene wunen, which encodes a homologue of LPP1 and LPP2, negatively regulates primordial germ cell migration (19); LPP3 mediates gastrulation and axis formation, probably by influencing the canonical Wnt signaling pathway (20). Except for the conserved residues within three domains present in the active sites of LPPs (17), the two S1P phosphatases have little overall homology to other known LPPs and, in contrast to the broad specificity of the other LPPs (18), are specific sphingoid base phosphate phosphatases.
Recently, it has been shown that SPP-1 and SPP-2 are localized to the endoplasmic reticulum (ER) where they degrade S1P to terminate its actions (13)(14)(15). These findings have far reaching implications, because the ER also contains the enzymes of ceramide biosynthesis, and suggest an important role for SPP in the regulation of de novo ceramide biosynthesis. Indeed, we have recently found that SPP-1 functions in an unprecedented manner to up-regulate biosynthesis of long chain C16-ceramide (14), which has been implicated in mitochondrial-dependent apoptosis (21).
Epidermal growth factor (EGF) plays an important role in proliferation and migration of diverse cell types and cancers. These processes are mediated through binding to the EGF receptor (EGFR), a transmembrane protein with tyrosine kinase activity, and the consequent regulation of various downstream targets (22,23). Of particular relevance to this study, many signaling events of G protein-coupled receptors (GPCRs) in diverse cell types transduced by potent lipids, such as LPA and S1P, are dependent on the function of the EGFR (24,25). EGFR transactivation is important for LPA-induced head and neck cancer cell proliferation and motility (26). Because a recent study suggested that S1P also activates ERK1/2 by transactivating EGFR (27), we examined the role of SPP-1 in EGFinduced cell locomotion. We found that overexpression of SPP-1 abolished the chemotactic effect of EGF, and, conversely, knockdown of SPP-1 enhanced migratory responses to EGF. Our results suggest that intracellular metabolism of S1P may play an important role in EGF-directed chemotaxis.
MATERIALS AND METHODS
Materials-[␥-32 P]ATP (3000 Ci/mmol) was purchased from Amersham Biosciences). S1P and fumonisin B1 (FB1) were from Biomol Research Laboratory (Plymouth Meeting, PA). Sphingosine was from Avanti Polar Lipids (Alabaster, PA). Serum and media were from Biofluids (Rockville, MD). G418 and EGF were obtained from Invitrogen. Collagen type I was purchased from BD Biosciences (Bedford, MA). Polycarbonate filters were from Neuro Probe (Gaithersburg, MD). PD98059 was purchased from Calbiochem (La Jolla, CA). Other chemicals were from Sigma.
To measure cellular S1P phosphohydrolase activity, HEK 293 cells (5 ϫ 10 5 ) were permeabilized with 50 M digitonin and incubated with [ 32 P]S1P or [ 32 P]phosphatidic acid (500,000 cpm, 1 M). Loss of membrane integrity was determined by the inability of cells to exclude trypan blue (Ͼ95% when permeabilized with digitonin compared with Ͻ5% in untreated cells). Lipids were extracted from 1 ml of medium with 2.7 ml of methanol/CHCl 3 /HCl (100/200/2, v/v). 1.2 ml of 2 M KCl and 1.2 ml of CHCl 3 were then added for phase separation. The organic layer was dried under nitrogen and resuspended in CHCl 3 /methanol (95/5, v/v). Lipids were separated by TLC and the remaining [ 32 P]S1P quantified as described above.
Chemotaxis Assay-Chemotaxis was measured in a modified Boyden chamber using polycarbonate filters (25 ϫ 80 mm, 12-M pore size) coated with collagen type I (50 g/ml in 5% acetic acid) as described previously (28). Briefly, chemoattractants were added to the lower chamber, and cells (5 ϫ 10 4 /well) were added to the upper chamber. After the indicated times, non-migratory cells on the upper membrane surface were mechanically removed, and the cells that traversed and spread on the lower surface of the filter were fixed and stained with Diff-Quik (VWR, Buffalo Grove, IL). Migratory cells were counted using a microscope with a ϫ10 objective. Each data point is the average number of cells in three random fields and is the mean Ϯ S.D. of three individual wells.
SPP-1 Regulates
Chemotaxis toward EGF-Similar to its effect on chemotaxis of many other cell types (29), low concentrations of S1P enhanced chemotaxis of HEK 293 cells (Fig. 1A). EGF also induced a dose-dependent increase in migration of HEK 293 cells, with an optimum effect at ϳ100 ng/ml (Fig. 1A). As expected, the effect of EGF was completely abolished by AG1478, a specific inhibitor of the intrinsic EGF receptor tyrosine kinase (Fig. 1B). Interestingly, pertussis toxin, which ADP ribosylates and inactivates G␣ i , decreased EGF-induced chemotaxis by almost 50% (Fig. 1B). Consistent with other studies (30 -32), migration toward S1P, but not fibronectin, was also inhibited by pertussis toxin (data not shown). These results suggest that EGF-directed migration of HEK 293 cells may involve, at least in part, activation of a G i -coupled receptor.
Recently, we reported that SPP-1 plays an important role in regulation of intracellular levels of S1P (14). Moreover, we found that although sphingosine kinase 1-generated S1P stimulates survival independently of S1PRs (8), it can act in an autocrine manner to stimulate the S1P 1 cell surface receptor and consequently regulate cytoskeleton rearrangement and focal adhesions (8,33,34) and chemotaxis (35). This "inside-out" signaling by S1P, which can regulate pathways downstream of S1P 1 receptors important for cell locomotion, prompted us to investigate the potential function of SPP-1 in chemotaxis. Surprisingly, we found that overexpression of SPP-1 drastically reduced migration of HEK 293 cells not only toward S1P but also toward EGF and serum ( Fig. 2A). In sharp contrast, IGF-1-stimulated migration was unaltered (Fig. 2B), suggesting that overexpression of SPP-1 does not disrupt all essential mechanisms of cellular locomotion.
To better understand the physiological functions of SPP-1, we also examined the effect of down-regulating its expression with siRNA on migration toward EGF. In agreement with a recent report (13), transfection with siRNA targeted to SPP-1, but not control siRNA, specifically reduced expression of SPP-1 as determined by semiquantitative RT-PCR (Fig. 3A). Importantly, knockdown of SPP-1 markedly enhanced EGF-directed motility but did not influence chemotaxis toward serum or fibronectin (Fig. 3B).
Role of Ceramide-Dephosphorylation of S1P to sphingosine by SPP-1 has been associated with a large increase of ceramide levels resulting mainly from acylation of increased sphingosine by ceramide synthase (14,16). Because ceramide inhibits signaling pathways downstream of the EGFR (36,37), it was of interest to determine whether the inhibitory action of SPP-1 on chemotaxis was mediated by changes in ceramide biosynthesis. To this end, we used FB1, a specific inhibitor of ceramide synthase. In agreement with our previous results (12), FB1 blocked ceramide production in SPP-1-overexpressing cells (Fig. 4A) and concomitantly increased sphingosine (Fig. 4B). It did not reverse the negative effect of SPP-1 expression on EGFand S1P-induced chemotaxis (Fig. 4, C and D), suggesting that, in fact, inhibition of chemotaxis by SPP-1 is not a result of ceramide or sphingosine accumulation but rather dephosphorylation of S1P. Moreover, treatment with FB1 did not influence migration of vector cells toward EGF yet somewhat reduced chemotaxis toward S1P (Fig. 4D). S1P Is Cleaved Intracellularly by SPP-1-Although SPP-1 is mainly localized in the ER (13,14), it is possible that similar to other LPPs (38) a small fraction could also be localized to the plasma membrane where it might act as an ecto-S1P phosphatase. To examine this possibility, we permeabilized the plasma membrane of cells expressing SPP-1 with digitonin, an approach developed by Brindley and co-workers (39) to determine ectophosphatase activity of the LPPs. If exogenous S1P were cleaved at the plasma membrane by ectophosphatase activity of SPP-1, then its rate of hydrolysis by cells overexpressing SPP-1 would be independent of permeabilization, as was shown to be the case for LPA hydrolysis by cells overexpressing LPP1 (39). In contrast to LPP1, we found that when HEK 293 cells expressing SPP-1 were incubated with [ 32 P]S1P there was no detectable hydrolysis over a 20-min period as long as cell integrity was maintained (Fig. 5A). However, when the plasma membrane was permeabilized with digitonin, S1P was rapidly dephosphorylated by SPP-1 transfectants compared with vector-transfected cells and almost completely disappeared within 20 min (Fig. 5B). In agreement with the substrate specificity of FIG. 6. SPP-1 overexpression abrogates EGF-induced ERK 1/2 activation but not EGFR tyrosine phosphorylation. HEK 293 cells stably expressing vector or SPP-1 were serum-starved for 16 h and treated without or with EGF (100 ng/ ml) for 5 min. A, cells were lysed, and equal amounts of proteins were immunoprecipitated with anti-EGFR and analyzed by Western blotting with anti-phosphotyrosine antibody (upper panel). Blots were then stripped and reprobed with anti-EGFR (bottom panel) as a loading control. B, equal amounts of total lysates were also analyzed by immunoblotting with anti-phosphotyrosine; activation of ERK (C) and p38 (D) was determined with phospho-specific anti-ERK1/2 and p38 antibodies, respectively. Blots were stripped and reprobed with ERK2 or p38 antibodies to demonstrate equal loading. Numbers indicate relative intensities determined by densitometry of scanned blots. (14), HEK 293 cells overexpressing SPP-1 and permeabilized with digitonin failed to cleave nonsphingoid base phospholipids, such as [ 32 P]phosphatidic acid (Fig. 5A). These results indicate that SPP-1 is unable to cleave S1P at the plasma membrane and is active only when S1P is produced inside the cells or transported there.
SPP-1 Expression Does Not Abrogate EGF-induced Tyrosine Phosphorylation of EGFR yet Inhibits ERK Activation-Be-
cause SPP-1 expression potently inhibited migration toward EGF, it was possible that this was caused by either reduction in EGFR expression, interference with activation of the EGFR, and/or inhibition of its downstream signaling. However, no significant changes in EGFR expression were detected in cells transfected with SPP-1 (Fig. 6A). Moreover, EGF stimulated tyrosine phosphorylation of EGFR to the same extent in vector and SPP-1 transfectants (Fig. 6A). We noticed, however, that expression of SPP-1 decreased levels of two tyrosine-phosphorylated proteins with molecular masses of 42 and 44 kDa, likely phospho-ERK1/2.
Previous studies suggest that S1P activates the EGFR through a protein kinase C-dependent pathway that links Ras signaling to the activation of ERK1/2 in Rat-2 cells (27). Although overexpression of SPP-1 did not significantly decrease EGF-induced tyrosine phosphorylation of EGFR (Fig. 6, A and B), it markedly reduced ERK1 activation and had a lesser effect on ERK2 phosphorylation (Fig. 6C) without affecting p38 phosphorylation (Fig. 6D).
Involvement of S1P Receptors in EGF-mediated Chemotaxis-Collectively, our data suggest that the intracellular level of S1P, regulated by SPP-1, is important for EGF-directed chemotaxis. Although it is well established that S1P acts as a ligand for S1P receptors to regulate cytoskeletal changes and migratory re-FIG. 7. Involvement of ERK1/2 in EGF-and S1P-induced chemotaxis. HEK 293 cells stably expressing vector (V) or SPP-1 were serum-starved for 16 h and treated without or with S1P (100 nM) for 10 min (A) or pretreated without or with FB1 (25 M) for 16 h and then treated with EGF (100 ng/ml) for 10 min (B). Cells were lysed and ERK activation was determined with phospho-specific anti-ERK1/2 antibodies. Blots were stripped and reprobed with ERK2 antibody (A) or anti-ERK1/2 antibodies (B) to demonstrate equal loading. Numbers indicate relative intensities determined by densitometry of scanned blots. C, naïve HEK 293 cells were treated without or with the indicated concentrations of PD98059 and then stimulated with EGF (100 ng/ml) for 10 min, and ERK1/2 activation was determined as above. D, HEK 293 cells were pretreated without or with PD98059 (5 M) and allowed to migrate for 4 h toward EGF (100 ng/ml) or S1P (10 nM). Data are means Ϯ S.D. of three individual determinations. Similar results were obtained in at least three independent experiments. *, p Ͻ0.05 by t test.
sponses (reviewed in Ref. 29), a few studies suggest that the inhibitory effect of S1P on chemotactic motility is likely mediated through intracellular actions rather than through cell surface receptors (42,43). However, the observation that PTX reduced chemotaxis toward EGF (Fig. 1B) strongly argued against this possibility. To further separate responses dependent on intracellular S1P from those that are receptor-mediated, we specifically blocked signaling of the heterotrimeric G proteins that S1PRs couple to and signal through. Regulators of G protein signaling, such as RGS3, function as GTPase-activating proteins for G␣ i and G␣ q subunits of heterotrimeric G proteins, resulting in their inactivation (44). It was previously shown that transfection of HEK 293 cells with RGS3 completely prevented signaling mediated by activation of S1PRs with exogenous S1P (45). In agreement, we found that S1P-induced ERK1/2 activation was blocked in HEK 293 cells overexpressing RGS3 (Fig. 8A). RGS3 also reduced ERK activation induced by EGF. Importantly, expression of RGS3 not only reduced migration toward S1P, it also markedly reduced migration toward EGF (Fig. 8B). DISCUSSION Our results suggest that SPP-1-regulated intracellular levels of S1P, acting through G protein signaling, plays an important role in EGF-directed cell motility. Several lines of evidence support this notion. First, overexpression of SPP-1, which decreases intracellular levels of S1P, suppressed motility toward EGF. Conversely, down-regulation of SPP-1 enhanced chemotaxis toward S1P and EGF. Third, PTX treatment, which inhibits G i signaling, markedly reduced motility toward EGF, although the EGFR is a tyrosine kinase and does not utilize G proteins for signaling. Finally, blocking signaling of all the heterotrimeric G proteins that S1PRs couple to and signal through also markedly reduced migration toward EGF. This receptor communication between EGFR and S1PRs is distinct from the well established transactivation of the EGFR by other GPCRs (24,25,46).
Although SPP-1 overexpression is also associated with an increase in ceramide that is inhibited by the ceramide synthase inhibitor FB1 (14), FB1 did not attenuate the inhibitory effect of SPP-1 on EGF-induced chemotaxis. These results suggest that SPP-1 acts mainly by reducing S1P accumulation in response to EGF, rather than by formation of sphingosine or by increasing its conversion into ceramide or further ceramide metabolites. SPP-1 can therefore modulate inside-out signaling mediated by S1P through S1PRs important for EGF-induced chemotaxis. In agreement, Obeid and co-workers (13) have recently shown that not only were intracellular levels of S1P increased by siRNA knockdown of endogenous SPP-1 in HEK 293 cells, it also induced an increase in secretion of S1P into the extracellular medium.
How ER-resident SPP-1 is able to reduce S1P levels at the plasma membrane and inhibit S1PR activation is an intriguing conundrum. It has been suggested that close contacts exist between the ER and plasma membrane (47). If so, it is possible that SPP-1 might dephosphorylate S1P at specific sites of contact between these two organelles. Alternatively, similar to calnexin and calreticulin, other ER-resident proteins that have been observed at the cell surface (48), it is also possible that SPP-1 can function at the plasma membrane as an ectophosphatase similar to other LPPs (39). There are several other possibilities that can explain how overexpression of SPP-1 inhibits exogenous S1P-induced chemotaxis and ERK activation. First, it is possible that overexpressed SPP-1 inhibits S1Pinduced chemotaxis not by dephosphorylating S1P at the cell surface but by decreasing intracellular S1P formed in response to activation of sphingosine kinase by S1P itself. Meyer zu Heringdorf et al. (49) showed that exogenous S1P induced calcium mobilization in HEK 293 cells through sphingosine kinase activation and that this was PTX-sensitive, indicating that intracellularly generated S1P was acting in an autocrine manner to activate cell surface G i -coupled S1P receptors. Secondly, it is also possible that there is a small fraction of the total SPP-1 that is expressed on the plasma membrane that is capable of reducing local concentrations of S1P near receptors, yet assays are not sensitive enough to detect such small changes. Consistent with this notion, we have shown previously that SPP-1-transfected HEK cells have a 3-fold increase in plasma membrane S1P phosphohydrolase activity compared with 5-fold in the ER and that the total activity is about 20% of that of the ER (14). In agreement, most of the increase in ceramide after S1P treatment in these cells occurred in the internal membrane fraction, although there was also a small but significant increase of ceramide in the plasma membrane (14).
How is it possible that S1P produced in an intracellular compartment such as the ER can play a role in EGF signaling? Although it was originally considered that activated FIG. 8. Overexpression of RGS3 in HEK 293 cells blocks migration toward S1P and EGF. HEK 293 cells stably expressing vector or RGS3 were serum-starved for 16 h. A, cells were treated without or with S1P (100 nM) or EGF (100 ng/ml) for 5 min and lysed. ERK activation was determined with phospho-specific anti-ERK1/2 antibodies. Blots were stripped and reprobed with ERK2 antibody to demonstrate equal loading. B, duplicate cultures were allowed to migrate toward S1P (10 nM) or EGF (100 ng/ml) for 4 h, and chemotaxis was measured. Data are means Ϯ S.D. of three individual determinations. Similar results were obtained in at least three independent experiments. *, p Ͻ0.05 by t test.
EGFR is rapidly internalized in early endosomes as a mechanism of receptor inactivation, accumulating evidence indicates that the EGFR remains active within early endosomes (50). Moreover, endosomal EGFR signaling was sufficient to activate the major signaling pathways, including Ras, ERK, and Akt, but not PLC␥, leading to cell proliferation and survival (51). A recent functional imaging study demonstrated that internalized EGFR travels to the ER, where the resident protein tyrosine phosphatase-1 catalyzes its dephosphorylation, leading to inactivation of EGF signaling prior to its degradation in lysosomes (52). ER-resident SPP-1 might act in a similar manner as an off switch to terminate EGF signaling by degrading S1P. Spatial and temporal partitioning of S1P formation and degradation within cells could regulate the duration of signals and provide an added layer of control to on-off signaling for receptors that utilize intracellularly generated S1P as a signaling molecule.
In many cell types, activation of ERK1/2 is required for motility induced by EGF (26,41) and S1P (53). Although overexpression of SPP-1 did not affect EGFR activation, it reduced EGF-induced ERK1/2 phosphorylation, suggesting that S1P production may be required for EGFR downstream signals. In agreement, we found that overexpression of RGS3, which inhibits signaling through all of the S1P receptors expressed in HEK 293 cells (45), reduced not only ERK1/2 phosphorylation induced by EGF but also its chemotactic effects. In this regard, EGF-activated ERK1/2 in ovarian theca cells was PTX-sensitive (54). We also found that EGF-induced cell motility is inhibited by pretreatment with PTX, suggesting that intracellular S1P, whose level is regulated by SPP-1, can activate a heterotrimeric G␣ i protein-coupled S1PR, likely by an autocrine pathway. This is consistent with the observation that decreased expression of SPP-1 increases secretion of S1P (13). Although S1P is secreted by several types of cells in response to various agonists (55), little is known about the mechanisms of its release. The ATP-binding cassette transporter ABCB1 (previously called MDR-1 and P-glycoprotein), which catalyzes movement of platelet-activating factor in addition to amphiphilic drugs from the cytosol to the extracellular environment (56), has recently been implicated in transport of S1P out of cells (57). Transport of S1P out of cells is an important issue to resolve because it impinges on S1P actions at the cell surface and possibly inside cells. | 2018-04-03T03:26:37.221Z | 2004-08-13T00:00:00.000 | {
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271302662 | pes2o/s2orc | v3-fos-license | A citizen science approach to develop a digital intervention to reduce HIV stigma and promote HIV self‐testing among adolescents and young adults: a mixed methods analysis from Kazakhstan
Abstract Introduction Kazakhstan has one of the fastest‐growing HIV epidemics in the world, with increasing rates among adolescents and young adults (AYA). Innovative strategies are needed to increase HIV testing uptake and decrease HIV stigma among AYA. Citizen science, defined as the active engagement of the general public in scientific research tasks, promotes and facilitates community engagement throughout the research process. This citizen science study used crowdsourcing to engage AYA in Kazakhstan to develop a digital intervention to reduce HIV stigma and promote HIV self‐testing. Our objectives in this paper are to describe the approach used, its feasibility and acceptability, and AYA motivations for and lessons learned collaborating on the study. Methods From October 2021 to July 2022, in collaboration with a Community Collaborative Research Board and a Youth Advisory Board, we developed an open call requesting multimedia submissions to reduce HIV testing stigma. Eligible submissions were separated by age group (13−19 or 20−29 years) and judged by a panel composed of AYA (n = 23), healthcare professionals (n = 12), and representatives from the local government and non‐governmental organizations (n = 17). Each entry was reviewed by at least four judges and ranked on a 5‐point scale. The top 20 open call contestants were asked to submit self‐recordings sharing their motivation for and experience participating in the contest and lessons learned. Descriptive statistics were calculated for quantitative data. Qualitative data were coded using open coding. Results We received 96 submissions from 77 youth across Kazakhstan. Roughly, three‐quarters (n = 75/96) of entries met judging eligibility criteria. Of the eligible entries, over half (n = 39/75) scored 3.5 or higher on a 5‐point scale (70.0%). The most frequent types of entries were video (n = 36/96, 37.5%), image (n = 28/96, 29.2%) and text (n = 24/96, 25.0%). AYA's primary motivations for collaborating on the study included a desire to improve society and help youth. The main challenges included creating content to address complex information using simple language, finding reliable information online and technological limitations. Conclusions Crowdsourcing was feasible and highly acceptable among AYA in Kazakhstan. Citizen science approaches hold great promise for addressing the increasingly complex health and social challenges facing communities today.
I N T R O D U C T I O N
Eastern Europe and central Asia (EECA) has the world's fastest-growing HIV epidemic with a 43.0%increase in incident cases of HIV acquisition from 2010 to 2020 [1] and for adolescents and young adults (AYA) rates are projected to increase 27.5% by 2030 [2].Within EECA, Kazakhstan has the largest increase in incident cases of HIV [1], with a 132.7% increase in HIV incidence among AYA from 2018 to 2020 [3] coupled with low HIV testing rates (in 2015, 22.0% female; 15.0% male AYA tested) [4,5].Low uptake of HIV testing is due to a number of factors including perceived low risk of http://onlinelibrary.wiley.com/doi/10.1002/jia2.26314/full| https://doi.org/10.1002/jia2.26314 HIV acquisition, inconvenient testing locations and fear of HIV stigma [6][7][8][9][10].Many in Kazakhstan are afraid to be tested due to concerns of severe discrimination if they test positive for HIV [7,[11][12][13].In Kazakhstan, HIV testing is traditionally administered at city AIDS Centres, where it is obvious individuals are receiving HIV services.HIV self-test kits recently became available in Kazakhstan, but are predominately targeted at men who have sex with men and the messaging may not resonate with AYA.Providing AYA with HIV self-test kits allows them to access testing in a private location, thereby reducing the fear of involuntary disclosure of perceived HIV serostatus or assumed related sexual behaviours or substance use.AYA-tailored messaging is needed to increase HIV testing in this group.
Innovative strategies are needed to generate tailored messaging and reduce HIV testing stigma among AYA in Kazakhstan.Citizen science is the active engagement of the general public in scientific research tasks [14].Citizen science operates under a horizontal approach where community members are considered competent in-the-field experts [15].It can engage vulnerable communities and promote health equity [16].Not relying solely on public health experts fosters innovation and greater inclusion of perspectives from diverse community members, increasing ownership, relevance and sustainability of interventions [17].
Citizen science utilizes participatory methods, such as crowdsourcing, which engages a group of people to develop and share solutions to a problem [18].Citizen science can be an effective way to develop community-based solutions for a wide range of societal and health challenges, including HIV stigma [14].Crowdsourcing often utilizes digital technologies, which have been shown to improve a variety of HIV-related outcomes, including promoting HIV testing [19] and antiretroviral therapy adherence [20].Digital technologies have also shown promise in reducing HIV stigma among healthcare providers [21] and internalized HIV stigma among people living with HIV (PLWH) [22].
The JasSpark Project (meaning "Young Spark" in the Kazakh language) is a citizen science study to engage AYA in Kazakhstan to develop a digital intervention to reduce HIV stigma and promote HIV self-testing.The objectives of this paper are to describe (1) the citizen science approach used, (2) the feasibility and acceptability of using this approach to develop a digital intervention to reduce HIV stigma, and (3) AYA's perspectives on their motivations and learnings collaborating in the study.
M E T H O D S
Our study used crowdsourcing to engage AYA in Kazakhstan to develop a digital HIV stigma reduction and HIV selftesting intervention package.To address the first objective, we describe the process of implementation, including modifications that occurred during the study.To assess feasibility and acceptability, we describe Community Collaborative Research Board (CCRB) and Youth Research Collaborative (YRC) participation and the number and quality of open call submissions received.To assess AYA perspectives, we describe the findings from video recordings solicited from contestants with crowd-sourcing entries ranked in the top 20.Descriptive statistics were calculated using SPSS (v28.0).
Description of approach used
We launched an online crowdsourcing open call among AYA across Kazakhstan to develop intervention materials in Russian and Kazakh languages.The study was informed by the Theory of Planned Behavior, which posits that the intention to test for HIV is influenced by attitudes about HIV testing (including stigmatizing attitudes), perceived need, and an evaluation of the risks and benefits of testing [23,24].We used a Citizen Science Framework [25] integrated with stigma manifestations from the HIV Stigma Framework [26] to guide the study (see Figure 1).
Establishment and meetings with the CCRB and YRC
AYA were involved in the JasSpark study through the team's YRC, which was comprised of two separate groups of youth.The first was recruited from non-governmental organizations (NGOs) focusing on HIV among youth to partner on the study as part of the CCRB.These were AYA who were living with HIV and/or were engaged in youth activism in Kazakhstan (n = 8).As part of their role, with the other CCRB members, these AYA were responsible for engaging in more high-level decision-making, including the co-development of study procedures, strategies for the open call, judging submissions and co-development of a dissemination plan.Due to high interest from AYA not on our CCRB, we expanded participation to additional youth volunteers (n = 25).This second group consisted of AYA collaborators who heard about the study via word-of-mouth from CCRB members and Global Health Research Center of Central Asia (GHRCCA) staff and through announcements about the study at local universities, youth NGOs and on social media.The majority of volunteers in this second group were not living with HIV.AYA volunteers collaborated on a number of day-to-day study development aspects, including managing study social media accounts, co-creating promotional materials for the study, providing feedback on the design of the submission portal and pilot testing it, and codesigning and testing crowdsourcing procedures.Some AYA volunteers also helped judge crowdsourcing entries.AYA who helped judge entries received compensation for their time spent judging (27,000 tenge, ∼$60 USD).AYA were not financially compensated for involvement in other study activities.
All AYA assisting with the study received a certificate of collaboration.
Our CCRB was comprised of the eight AYA mentioned above and representatives from youth local and international NGOs; Kazakhstan city, provincial and national AIDS Centres; youth health clinics; and media specialists working with youth.We had no strict selection criteria for the CCRB, but aimed to include a broad spectrum of professionals involved in working with youth.Given GHRCCA's long-standing research presence in the region, research staff had many existing connections with NGO, health agency and other organization staff that work with youth.Many CCRB members had previous experience serving on CCRBs or collaborating on research, though the majority of AYA CCRB members did not have prior research collaboration experience.CCRB members were offered 27,000 tenge (∼$60 USD) compensation for their time and effort.
We worked with our CCRB to co-create a solution that would allow for efficient collaboration and was mutually feasible and convenient for all.We had online meetings via Zoom and documentation was shared via email.We created a What-sApp group based on feedback from AYA CCRB members to provide another outlet for sharing ongoing feedback and collectively discuss research process issues.To facilitate cocreation with youth volunteers [15], we created a separate WhatsApp group and Telegram channel with the AYA volunteers and messaged them multiple times a week.
CCRB members and AYA volunteers received training on study procedures, defining stigma and judging processes.Attendance at meetings was tracked.Utilization of the diverse talents and strengths of our citizen collaborators greatly improved the development of study materials and the flow of study procedures.Citizen collaborators exhibited strong enthusiasm for the study, including significant in-kind contributions of time and skills, requests by organizations to share crowdsourced materials on their websites, and positive feedback from contestants and volunteers with requests to become involved in other studies.
Development of intervention materials: crowdsourcing open call
Our collaborative process (Figure 2) began with posting an open call on our study website [27] and various social media channels (e.g.Instagram, Tiktok, Facebook, WhatsApp, Telegram) and through youth events (in-person and online).We developed a national crowdsourcing open call, inviting AYA ages 13−29 years living in Kazakhstan to submit multimedia entries.YRC members created a video to promote the contest and managed study social media accounts.CCRB and YRC members provided feedback on the study website and participated in livestream events to promote and provide more information about the contest.The call focused on developing submissions to reduce HIV stigma to promote HIV self-testing among at-risk AYA in Kazakhstan.It contained a toolkit with basic information about stigma and HIV, as well as a list of free software and resources to aid in the development of materials.Due to the presence of stigmatizing content in early submissions, we added additional information about avoiding stigmatizing language based on guidelines from HIV-focused organizations (e.g.UNAIDS, UNICEF) to the open call instructions.
To be eligible to submit, AYA had to be 13−29 years old and live in Kazakhstan.They were allowed to submit multiple submissions, either individually or as a group.Eligible submission formats included video, audio, text, images/photos and other multimedia content (e.g.online games, webpages, crossword puzzles).Submissions could be in Russian or Kazakh.Prior to submission, all contestants had to complete a Multimedia Release Form (also signed by a parent for AYA under age 18) and Contestant Agreement providing their permission to use their content as part of a research study and in presentations and agreeing they would postpone publishing their materials until after completion of the scientific study.AYA in the YRC were eligible to submit entries to the crowdsourcing open call, but none submitted.
All submissions were screened for eligibility and the presence of stigmatizing content prior to judging.Each entry was first reviewed by two GHRCCA research staff (OB, DG), and then all entries were reviewed by the Kazakhstan-based PI (GM).Contestants who had entries with stigmatizing content were provided feedback by research staff via their preferred communication method (e.g.WhatsApp, email) and given a chance to revise and resubmit entries.Those who chose to resubmit had only their revised entry judged, while those who chose not to resubmit had their original entry judged.
Eligible submissions were divided by age group (13−19 years old or 20−29 years old) for evaluation by a judging panel consisting of Kazakhstani AYA (n = 23), healthcare professionals (n = 12), and representatives from the local government and NGOs (n = 17).Each entry was judged by two AYA in our YRC (volunteers and AYA CCRB members) and by two other CCRB community partners (i.e.AIDS Centre, youth clinic or NGO staff).To obtain diverse perspectives while easing judging burden, each judge rated a maximum of five entries, thus all entries were not rated by the same four judges.We distributed entries across judges to ensure each judge received a comparable mix of different content types (e.g.video, image).Entries were ranked on a 5point scale based on four judging criteria used in previous crowdsourcing studies [28,29]: (1) potential to reduce HIV stigma to increase HIV testing; (2) innovation; (3) relevancy to youth; and (4) overall impression.Entries were considered high-quality if they scored an average of 3.5 or higher on a 5-point scale between the four judging scores.All contestants received participation certificates.A virtual awards ceremony was conducted to honour awardees.First place was awarded for the top Russian and Kazakh language entries in each age category (13−19 years and 20−29 years) and received 220,000 tenge (∼$485 USD).Second-place entries in each age category received 132,000 tenge (∼$290 USD) and thirdplace entries received 67,000 tenge (∼$145 USD).Seven-teen contestants received honourable mentions and received 27,000 tenge (∼$60 USD).Multimedia content from winning submissions were combined to form the intervention package that would be tested in a subsequent randomized control trial.Final intervention materials were adjusted for clarity and to correct errors.
To explore the motivations and learnings of AYA collaborators, we messaged the top 20 open call contestants (determined via the 10 highest average judging scores in each age category) and asked them to submit self-recordings responding to the prompts: (1) Why did you decide to take part in this competition?(2) What new things did you learn while working on your content?(3) What was the hardest thing about creating content?Due to resource limitations, we were not able to gather feedback from all 77 contestants.The top 20 entries included entries in both Russian and Kazakh and across media types (e.g.video, image, text).Submission of self-recordings was optional.Interested contestants (n = 13) sent self-recorded videos via a messaging app to GHRCCA research staff.An initial coding structure was developed based on the prompts sent to the contestants, and then refined through an iterative review process by the research team.The coding of each recording was conducted by at least three members of the research team.The data collection process (from initial meetings with the CCRB to the sharing of self-recordings) was conducted between October 2021 and July 2022.All study procedures were reviewed and approved by Columbia University's Institutional Review Board and Al-Farabi Kazakh National University's Ethics Committee.
CCRB and YRC feasibility and acceptability
The CCRB (n = 25, including 8 AYA; 20.0% male, 80.0% female, age range 14−73) met twice before launching the open call to determine content and procedures.Attendance was high-96.0%(n = 24) during the first meeting and 80.0% (n = 20) during the second meeting.We held a third meeting with the CCRB to review judging procedures (attendance 80.0%, n = 20).Seven of the eight AYA CCRB members participated in the judging process.CCRB members were invited to attend the virtual awards ceremony following the judging process (52.0%attended, n = 13).CCRB members spent an average of 8−10 hours contributing to the study.Seven of the eight AYA CCRB members also served as AYA volunteers.AYA volunteers (n = 25, 60.0% male, 40.0%female, age range 14−31) were highly active in collaborating on the study; of the 25 volunteers, 23 (92.0%) helped conduct at least one component of the study (e.g.develop promotional materials, disseminate study information via social media, participate in judging).Among the AYA volunteers who were not CCRB members, six served as judges.
The time AYA volunteers spent collaborating on the study ranged widely.On the low end, some partners spent a few hours total on all activities, while on the high end, partners spent several hours each week over the duration of the study period.
Open call feasibility and acceptability
During the 4-month open call period, 3412 individuals visited the website.We received 96 submissions from 77 youth (28.6% male, 71.4% female) across Kazakhstan.Eleven youth submitted two entries and four submitted three entries.Nearly, two-thirds (64.6%, n = 62/96) of entries were from contestants between ages 13 and 19.
AYA collaborator motivations and learning
Thirteen out of 20 top contestants sent self-recordings.AYA described their motivations for participating, lessons learned from participation and challenges creating content.
Motivations for participation
Seven contestants expressed their desire to improve society or help others feel supported as a key motivation for participating in the contest.Additionally, six AYA contestants were artistic and expressed wanting to develop creative materials or use their skills.
Lessons learned from participation
All contestants reported learning something new about HIV, stigma and/or testing.A number of contestants reported learning that PLWH can live long and normal lives.Several contestants also mentioned learning about the ability for PLWH to give birth to children without HIV, indicating a persistent misperception in Kazakhstani society.Contestants also reported learning more about the challenges faced by PLWH, including children with HIV.Some contestants also reported using the knowledge and skills they gained from participating to design crowdsourcing projects to address other societal problems.
Challenges creating content
Many contestants discussed the difficulty in creating content that could convey complex information using simple, nonstigmatizing language.Contestants wanted their work to have a positive impact and struggled to develop compelling mes-saging.AYA also discussed the difficulty in sifting through stigmatizing information online to find reliable sources.Many AYA reported not being aware of HIV stigma themselves and needing to search for reliable information to become more informed.For some AYA, this included meeting with HIV specialists or other professionals.Contestants also reported some technological challenges in creating content.Although some AYA had extensive previous experience with video editing, audio and graphic software, other AYA had limited exposure to these types of tools and had to learn how to use them.
D I S C U S S I O N
The JasSpark Study used a citizen science approach to develop digital intervention materials to reduce HIV stigma and promote HIV self-testing among AYA.There is limited research on citizen science approaches to stigma reduction [28].Most research aimed at HIV stigma reduction has used education-based, skills-building and/or counselling approaches with public health experts [6,30,31].However, citizen science approaches have been used to address other issues facing AYA, such as school community wellbeing [32], barriers to physical activity [33], nutrition [34] and asthma [35].
The study serves as a useful model for designing inclusive methods to broaden public engagement in addressing stigma.Compared with other studies using crowdsourcing among diverse populations, we received a large number of submissions and a high percentage of high-quality submissions [18,36], indicating high acceptability.Our findings indicate that crowdsourcing is a feasible citizen science approach to use among AYA in central Asia.A challenge in citizen science projects is finding engaged volunteers.While some projects have hundreds of volunteers, in some studies, less than 10.0% actively make contributions [37].However, active participation among our AYA volunteers was high-greater than 90.0%, indicating citizen science approaches may be particularly wellsuited for engaging AYA, particularly on topics they consider important.Of note, our AYA volunteers received no monetary compensation, only certificates of collaboration.Given that many AYA are applying for colleges or jobs and such certificates are valuable for their resumes, this may have been a motivating factor for their collaboration.
Motivations for participating in citizen science projects can vary, but often include reasons related to values (e.g.humanitarian concerns for others), understanding (e.g.opportunity for learning new skills/knowledge), social (e.g.opportunity for interacting with others), career (e.g.obtain career-related benefits) and protective (e.g.reduce guilt over being more fortunate than others) [37].The majority of AYA citizen scientists in our study cited pro-social motivations.Many AYA had a strong desire to improve society and help youth or use their creative talents for good.Crowdsourcing provides AYA an opportunity to use their creative skills in a competitive forum, which may provide a way to engage them in an important topic they might not otherwise engage in.
Our study also highlighted the challenges associated with addressing stigma via citizen science approaches.Some crowdsourced materials developed by citizens could increase stigma, consistent with other literature [14].Nearly, a third of submissions contained stigmatizing content, indicating a need for vetting and refinement of community contributions.Approximately one-third of contestants revised submissions based on feedback, demonstrating a desire to learn.Open call submissions also served as a useful window to highlight where some sources of societal HIV stigma were stemming fromin this case, primarily around misperceptions and exaggerated fears about how HIV is transmitted.This is valuable for the design of future research studies and programmes to address HIV stigma in Kazakhstan and central Asia.
This study illustrates the promise of using a citizen science approach to develop HIV stigma reduction interventions.Strengths of this approach included strong participation from citizen scientists, including AYA; a large proportion of highquality submissions; and the development of highly creative and innovative intervention content.We also implemented strong quality control procedures; all submissions in our study were reviewed by at least three people to determine whether the material included stigmatizing content.
However, there are some limitations.First, because this study was implemented in a real-world environment, we were not able to fully control all study processes (e.g.number of submissions, quality of submission content).Second, we did not ask for feedback from contestants who did not submit high-quality entries due to limited resources.Youth who were not finalists may have had different experiences and motivations for participating in the crowdsourcing contest.Third, approximately two-thirds of the total entries were from the younger age group (13−19 years) and the majority were in Russian.This suggests that if one wants to engage diverse groups of AYA, multiple open calls may need to be developed that engage young adults and those who speak only Kazakh.Individuals who speak only Kazakh tend to be predominately located in rural areas of Kazakhstan compared to individuals who are bilingual or speak only Russian, so contestants may have been more urban as well.Finally, each entry was reviewed by four different judges.Inter-rater agreement was low, suggesting the need for novel approaches to reduce the judging burden and have reliable ratings from a diverse community of judges.Further analyses of study results are ongoing [38] and will be reported in future papers.
C O N C L U S I O N S
In summary, citizen science approaches hold great promise for addressing solutions for the increasingly complex health and social challenges facing communities today.Further work is needed to determine for which outcomes citizen science approaches are effective.In the often-challenging policy environments in EECA health systems, citizen science can be a tool for community change and make interventions more culturally relevant and innovative.Citizen science may also expand citizen knowledge of and trust in science and increase the inclusion of diverse communities.As investigators increasingly use citizen science approaches, it is important that details are shared across studies so that methods can be improved and best practices developed. | 2024-07-21T05:26:09.217Z | 2024-07-01T00:00:00.000 | {
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257440820 | pes2o/s2orc | v3-fos-license | Obligate role for Rock1 and Rock2 in adult stem cell viability and function
The ability of stem cells to rapidly proliferate and differentiate is integral to the steady-state maintenance of tissues with high turnover such as the blood and intestine. Mutations that alter these processes can cause primary immunodeficiencies, malignancies and defects in barrier function. The Rho-kinases, Rock1 and Rock2, regulate cell shape and cytoskeletal rearrangement, activities essential to mitosis. Here, we use inducible gene targeting to ablate Rock1 and Rock2 in adult mice, and identify an obligate requirement for these enzymes in the preservation of the hematopoietic and gastrointestinal systems. Hematopoietic cell progenitors devoid of Rho-kinases display cell cycle arrest, blocking the differentiation to mature blood lineages. Similarly, these mice exhibit impaired epithelial cell renewal in the small intestine, which is ultimately fatal. Our data reveal a novel role for these kinases in the proliferation and viability of stem cells and their progenitors, which is vital to maintaining the steady-state integrity of these organ systems.
Introduction
Harnessing the regenerative capacity of tissue stem cells represents a therapeutic avenue to restore damaged tissue seen in many human diseases. Successful implementation of this approach requires a deep understanding of the molecular mechanisms regulating stem cell viability, expansion and differentiation. Maintaining the proliferative capacity of stem cells and their progenitors is essential in tissues with high turnover, such as the intestinal epithelium and bone marrow (BM), where the continuous expansion and differentiation of these cells preserves organ function [1,2]. Identification of the factors controlling homeostatic stem cell renewal in these organ systems will greatly facilitate our approach to their therapeutic manipulation.
The cues mediating cellular proliferation stem from extracellular signals, such as growth factors, cytokines and lipids, that activate intracellular pathways controlling the morphological and mechanical properties of the cell. This is a dynamic and tightly regulated process requiring remodeling of the actin cytoskeleton. The Rho GTPases, members of the Ras superfamily, have an established role in mediating cytoskeletal rearrangement, and control a number of cellular processes including polarization, migration, viability, and cell division [3]. The most studied members of this family, RhoA, Cdc42, and Rac play key functions in modulating cell cycle by activating the assembly and organization of the actin-myosin cytoskeleton. In particular, RhoA plays an instrumental role during the final stages of mitosis, enabling the successful generation of two independent daughter cells [4]. Following chromosomal duplication, RhoA is activated, and signals for the formation of the actin-myosin contractile ring, which is necessary for membrane ingression and abscission during cytokinesis. Genetic studies reveal that cells devoid of RhoA display cell cycle arrest due to a failure in cytokinesis, causing apoptotic and necrotic cell death [5][6][7][8]. This is particularly detrimental to the hematopoietic system and gastrointestinal (GI) tract, where mice harboring null alleles of RhoA display severe pancytopenia and abnormal small intestine crypt architecture and function, resulting from a block in hematopoietic and intestinal stem cell proliferation respectively [6,8].
RhoA signals through a number of effectors, including Citron Kinase, mDiA and the Rho kinases, Rock1 and Rock2 [9]. We were particularly interested in investigating the contribution of the Rho kinases to cell proliferation given their established roles in the regulation of cytoskeletal dynamics [10]. Rock1 and Rock2 belong to the AGC Serine/Threonine kinase family, and are implicated in a multitude of physiological processes through their control of actin-myosin contractility [10]. The importance of this pathway in modulating the actin cytoskeleton is highlighted by the phenotype of individual germline knockout mice, which display severe defects during development, including aberrant closure of the ventral body wall, resulting in omphalocele [10]. Because ablation of either gene results in embryonic lethality, it has been difficult to study how each isoform contributes to the proliferative processes governing stem cell function and conservation of tissue structure [10]. While small molecule inhibitors of Rho-kinases suggest they are involved in cleavage furrow formation and cytokinesis, their broad activity against other AGC kinases and poor pharmacological properties have precluded their application in vivo [11][12][13]. Thus, to more thoroughly address the role for Rock1 and Rock2 in homeostatic stem cell function, we utilized inducible gene targeting to specifically delete these kinases in adult animals. Our system allowed us to circumvent the embryonic lethality of the germline knockouts, and assess the impact of combined Rock1 and Rock2 deficiency on stem cell viability and tissue integrity.
Ubiquitous expression of Rock1 and Rock2 in mouse tissue
We examined Rock1 and Rock2 gene expression using both single cell RNA-seq and qPCR. These analyses revealed overlapping and ubiquitous expression profiles of both genes in murine embryonic, neonatal, and adult tissues, and in sorted populations of hematopoietic cells (Fig. 1A-C). The comparable expression pattern and possible functional redundancy, suggested that simultaneous conditional gene targeting of Rock1 and Rock2 was needed to investigate their role in the adult tissues.
Global conditional deletion of Rock1 and Rock2 in adult mice is fatal due to cytopenia and intestinal dysfunction
To circumvent the developmental requirement for Rock1 and Rock2 and address their combined biological roles, we generated Table 1 Genotypes and treatments of each mouse strain. Abbreviations used in the text are listed next to the treatment conditions. floxed alleles for both genes, and intercrossed them to obtain Rock1 flox/flox ; Rock2 flox/flox mice ( Fig. 2A). These strains were bred to the Rosa26-Cre ERT2 (R26 Cre− ERT2 ) line to allow for conditional deletion with tamoxifen (TMX). The cohorts used, genotypes, and experimental design are listed in Table 1. For simplicity we refer to controls as WT, single knockouts as R1cKO, R2cKO, and the double knockout at R1;R2cKO. Analysis of splenocytes from each of the strains confirmed TMX dependent loss of Rock1 and Rock2 transcripts and protein ( Fig. 2B and C, Supplemental Figure 1). Interestingly, deletion of either gene alone did not lead to an upregulation of the other isoform, indicating sufficient expression of both genes to ensure compensation in each vital tissue. Given the lethality observed in mice with germline deletion of Rock1 or Rock2, we closely monitored the different strains following TMX injection for mortality. Contrary to their compulsory role in development, loss of Rock1 or Rock2 in adult mice did not affect survival, revealing a functional redundancy between the two kinases under homeostatic conditions (Fig. 3A). Conversely, ablation of both genes together was fatal with all mice dying within a week after the last of 5 TMX doses. Control groups were comparable to wildtype (WT) mice. As such, for the majority of studies, we used TMX treated Rock1 +/+ ;Rock2 +/+ ;R26 Cre− ERT2 as controls. We performed a blood chemistry assessment on the R1;R2cKO mice to understand what pathological features could be contributing to the mortality. Serum chemistry in R1;R2cKO mice revealed hypoalbuminemia and hypoglycemia, as well as decreased triglyceride (TG) and cholesterol levels indicative of inappetence and maldigestion, which is in agreement with the observed reduction in body weight (Figs. 3B and 4A). Likewise, elevated Blood Urea Nitrogen (BUN) levels, an indication of kidney failure, suggested the mice were suffering from dehydration due to intestinal loss of water (diarrhea). The lack of nutrient absorption, pointed to defects in the small intestines of these mice. Additionally, Complete Blood Count (CBC) analysis revealed the mice suffered from severe pancytopenia, with an absolute loss of all mature blood lineages, likely attributable to BM insufficiency (Fig. 4B). These data implicated the GI and hematopoietic systems were compromised by the absence of Rock1 and Rock2.
Rock1 and Rock2 deletion in adult mice leads to a block in hematopoiesis due to cell-cycle arrest and apoptosis
To investigate the cause of the pancytopenia, we examined the hematopoietic compartment of the R1;R2cKO mice. The process of hematopoiesis ensures the continuous replenishment of blood cells throughout the lifespan of an organism. Hematopoietic stem cells (HSCs) are a self-renewing population that gives rise to various progenitors that proliferate and differentiate into mature blood cells. : CD45 + B220 + , T cells: B220 -CD3e + CD90.2 + CD45 + , Neutrophils-: B220 -CD3e − CD90.2 -Siglec F − Ly6G + CD45 + , Eosinophils:B220 -CD3e − CD90.2 -Ly6G − Siglec F + CD45 + , Macrophages:B220 -CD3e − CD90.2 -Ly6G − Siglec F − CD11b − F4/80 + CD45 + ) from BM of WT or R1;R2cKO mice harvested on day 6 from the first TMX treatment. LSK cells and individual leukocyte populations were enumerated using FACS while total BM and splenocytes were counted on a cell counter. Data are represented as mean with S.D. n = 3-5 mice per group. *P < 0.05,**P < 0.005,***P < 0.005 t-test, relative to control. The rapid expansion of these progenitors is central to blood cell generation, particularly under conditions that impact mature cell viability, like infection or irradiation [14,15]. As a functional actin cytoskeleton is requisite for successful cell division, we hypothesized that the absence of the Rho-kinases could perturb this process, leading to a block in hematopoiesis. Indeed, Rock1 and Rock2 were detected broadly across hematopoietic cells, including multipotent precursors (Fig. 1C). In accord with the survival data, R1cKO, (caption on next page) A. Sambandam et al. and R2cKO single knockouts did not display severe perturbations in their hematopoietic compartment, with equivalent numbers of LSK progenitors and total BM cells and splenocytes to controls ( Fig. 5A and B). However, TMX treatment caused a time-dependent decrease in total cellularity within the BM and spleens of R1;R2cKO mice, with a significant effect on mature BM leukocytes ( Fig. 5C-E). The effect on BM cellularity indicated that Rock1 and Rock2 are necessary for maintenance of multiple blood cell lineages. In order to determine if pathway activity was required within hematopoietic precursors, we established radiation chimeras, where Rock1 flox/flox ; *P < 0.05,**P < 0.005 t-test, relative to control. and GFP in green (marking clones) at 7 days after heat shock-mediated clone induction (7d AHS, A-B ′ ) and at 28-30d AHS (C-D ′ ). (E) Clone size after heat shock mediated clone induction in the young and old control and Rok 2 LOF mutant intestinal stem cells in flies (at days 7 and 28-30) Boxed areas are magnified and displayed next to their respective images. Data are representative of 3-5 independent experiments. ***P < 0.005,****P < 0.005 t-test relative to control. Rock2 flox:flox ;R26 Cre− ERT2 BM was engrafted into irradiated hosts, followed by TMX treatment to delete Rock1 and Rock2 (Fig. 6A). The chimeras did not display enhanced mortality (Fig. 6B). When we examined host vs donor chimerism 10 days after the first TMX treatment of the engrafted animals, we observed a dramatic loss of the R1;R2cKO donor cells, that led to an increased frequency of residual host derived cells, which was most evident in the neutrophil population (Fig. 6C). This suggested that WT cells had a survival bias compared to R1;R2cKO derived BM cells. To dive into this further, we performed mixed BM chimeras where Rock1 flox/flox ; Rock2 flox:flox ;R26 Cre− ERT2 or Rock1 +/+ ;Rock2 +/+ ;R26 Cre− ERT2 BM cells were co-transferred with an equal number of BoyJ (CD45.1) WT cells (1:1) into irradiated recipients (Fig. 7A). This experiment allowed us to investigate whether R1;R2cKO BM cells could compete with WT BM cells in the engraftment of irradiated hosts. Upon TMX injection, we found that WT BM cells had a survival advantage over R1;R2cKO cells. Few to no R1;R2cKO donor derived cells were detected in the major lymphoid tissues of recipient mice (Fig. 7B). Moreover, the Lin-Sca-1 + c-Kit + or LSK cell population, that constitutes the most primitive cells of the hematopoietic system, was dramatically reduced (Fig. 7B) Given their indispensable role in blood cell generation, we focused on understanding the defect in LSK cells. We found that purified R1;R2cKO LSK cells were unable to engraft irradiated wildtype hosts (Fig. 7C). The total number of the Hematopoietic Stem Cells (HSCs) and Multipotent Progenitor (MPP) cells, which make up the LSK pool, were decreased in R1;R2cKO, and the bulk LSK population displayed heightened apoptosis ( Fig. 7D and E). The cytopenia was reminiscent of the phenotype reported in mice with conditional deletion of RhoA driven by Mx1-Cre [8]. These mice also present with acute hematopoietic failure due to a profound reduction in LSK cells. RhoA deficient LSKs exhibit cell cycle arrest due to defective cytokinesis, impacting viability. As Rho-kinases are RhoA effectors, we hypothesized that the observed reduction in LSK cells was also due to impaired proliferation. We assessed cell cycle distribution of LSK cells in the R1;R2cKO mice using short term in vivo BrdU incorporation, which labels cells synthesizing new DNA during S phase, and can be quantified by flow cytometry. R1;R2cKO LSK cells displayed a greater frequency of BrdU incorporation compared to controls, with R1;R2cKO LSK cells accumulating in S phase, signifying a block in cell cycle (Fig. 7F). Akin to the RhoA phenotype, the inability to progress through cell cycle leads to cell death. These results establish a cell-intrinsic role for Rho-kinases within hematopoietic cells, and because the defect affects the earliest precursors, the generation of all blood lineages is disrupted. Our results underscore an essential role for the RhoA-Rho kinase pathway in hematopoietic stem cell proliferation and viability.
Global deletion of Rock1 and Rock2 in adult mice affects intestinal stem cells survival and alters homeostasis
In addition to the maintenance of blood cells, preservation of the stem cell pool is critical for the steady-state regeneration of the small intestinal epithelium [1]. Epithelial cells within the small intestine undergo rapid turnover, which in mice takes approximately 3-5 days. Dead cells are shed from the villus tip into the GI lumen, and new specialized epithelial cells are formed from the differentiation of stem cells at the crypt base. Perturbation of this process leads to epithelial barrier dysfunction, loss of nutrient absorption and inflammation from leakage of gut microbiota into circulation. Given that R1;R2cKO mice presented with signs of small intestinal insufficiency, we investigated the role of Rock1 and Rock2 in intestinal epithelium homeostasis. Oral gavage of FITC-Dextran to measure homeostatic permeability across the GI tract revealed that R1;R2cKO mice had a breach in the intestinal epithelial cell barrier, with a significant amount of dye detected in the serum (Fig. 8A). In contrast, FITC-Dextran was not detected in the R1cKO, and R2cKO single knockouts, indicating that loss of either gene alone did not impact the intestinal barrier. We next examined histological changes to the intestine architecture over time in the R1;R2cKO strain using the same experimental design used for the hematopoiesis analyses (Fig. 5C). H&E staining revealed that R1;R2cKO mice begin to lose small intestine crypts soon after TMX injection, and that, by day 9, intestinal villi were attenuated and crypt architecture was profoundly disrupted (Fig. 8B). This was accompanied by widespread epithelial cell death, highlighted by abundant cleaved caspase-3 in this compartment (Fig. 8C). Furthermore, using ISH and qPCR, we observed a dramatic reduction of the intestinal stem cell compartment, marked by expression of the Lgr5, Axin2, and Olfm4 genes ( Fig. 8D and E). Similarly, BrdU incorporation revealed the absence of proliferating stem cells from the crypt compartment (Fig. 8F). The morphological changes and their kinetics were reminiscent of radiation induced enteropathy [16]. Interestingly, the kinetics of crypt demise, approximately 5 days, corresponds to the time required for turnover of the murine small intestine, and explains why the mice succumb so quickly following Rock1 and Rock2 deletion.
In light of the parallels in intestinal stem cell turnover between mammals and flies, and the ease of genetic manipulation in the latter species, we evaluated this pathway in the Drosophila system [17]. Using heat shock induced site-directed recombination and clonal analysis, we generated ISC clones that were homozygous for a strong loss-of-function Rok (the fly orthologue) mutation, Rok 2 [18]. This method also allows for lineage tracing of mutant ISCs by GFP expression. After inducing homozygous Rok 2 ISCs by heat shock, we quantified clone size over time. Control ISC clones grew to contain over 10 (7 days after heat shock, AHS) and 20 (30d AHS) cells over time whereas Rok deficient ISC clones remained small (1-5 cells) (Fig. 9A-E). These results indicate a significant ISC proliferation defect and highlight the importance and conservation of this gene in stem cell function.
Abnormal intestinal homeostasis observed in the absence of Rock1 and Rock2 is associated with radiation resistant cells
While many cell types contribute to crypt turnover, we chose to elucidate the immune contribution given the published role for leukocytes and their products in regulating intestinal crypt biology, including regeneration [19,20,21]. The acute sensitivity of BM-derived cells to Rho-kinase deletion suggested the intestinal phenotype in R1;R2cKO could in part be attributed to their depletion. To test this hypothesis, we performed BM reconstitution experiments, where irradiated Rock1 flox/flox ;Rock2 flox:flox ;R26 Cre− ERT2 mice were engrafted with WT BM, and treated with TMX to delete Rock1 and Rock2 in radiation resistant cells, referred to as WT (CD45.1)-> R1:R2cKO (Fig. 10A & Table 1). Intriguingly, the chimeras were afflicted with extensive intestinal damage and mortality, phenocopying the R1;R2cKO mice, underscoring the contribution of radiation resistant cells to crypt turnover (Fig. 10A-C). These data demonstrated WT BM was insufficient to preserve barrier integrity, however as we still detected host derived radiation resistant CD45.2 + cells in the blood and intestine of the WT (CD45.1)-> R1:R2cKO chimeras, we could not completely rule out the involvement of leukocytes in this process (Fig. 10D). These CD45.2 + cells were sensitive to Rho-kinase deletion, as seen by their reduction upon TMX treatment, raising the possibility that immune cell death could be a cause of tissue destruction (Fig. 10D) [22]. Thus, while BM derived cells were not able to rescue the intestinal phenotype, we could not exclude the involvement of tissue resident radiation resistant immune cells in sustaining crypt regeneration.
Epithelial deficiency of Rock1 and Rock2 impairs stress induced intestinal repair
The intestine relies on a local population of stem cells to generate the absorptive and secretory epithelial cells that make up the crypt. We hypothesized that akin to hematopoietic stem cells, intestinal stem cells (ISCs) would be sensitive to Rho-kinase deletion, (caption on next page) A. Sambandam et al. preventing crypt turnover. Moreover, radio-resistant ISCs have been described in epithelial barrier generation in the small intestine [23][24][25]. Crossing the Rock1 flox/flox ;Rock2 flox:flox strain to the Villin Cre-ERT2 allowed us to examine their function in all cells of epithelial origin, including intestinal stem cells [26]. TMX treated Rock1 +/+ ;Rock2 +/+ ;Villin Cre− ERT2 were denoted as WT Villin Cre− ERT2 , while Rock1 flox/flox ;Rock2 flox:flox ;Villin Cre− ERT2 as R1;R2 Villin Cre− ERT2 (Table 1). Surprisingly, we found that despite efficient deletion of Rock1 and Rock2 in the epithelial compartment, R1;R2Villin Cre− ERT2 mice exhibited only minor crypt loss and cell death that was not accompanied by animal mortality (Fig. 11A and B). As organoid cultures recapitulate many aspects of intestinal physiology, we used this system to examine epithelial budding and proliferation in R1:R2cKO derived ileal tissues where TMX was administered ex vivo [27]. Organoid budding and growth were not perturbed in the absence of Rock1 and Rock2 (Fig. 11C-E). These data reveal that basal epithelial proliferation and differentiation is not dependent on epithelial cell-intrinsic Rho-kinase activity.
Next we compared the phenotypes between the strains to elucidate why R1;R2Villin Cre− ERT2 mice were viable compared to R1: R2cKO and WT (CD45.1)-> R1:R2cKO radiation chimeras. We noted that a significant difference between the intestines from the R1; R2Villin Cre− ERT2 and the R1:R2cKO and WT (CD45.1)-> R1:R2cKO radiation chimeras, was that the latter two strains displayed extensive inflammation. This response was likely the consequence of cell death caused by Rho-kinase deletion, and/or a result of the disrupted epithelial barrier. We hypothesized that a similar cell-intrinsic role for Rock1 and Rock2 in the epithelium would become apparent in an injury setting. To determine whether damage to the GI tract would impair regeneration in the R1;R2Villin Cre− ERT2 , we used a short-term radiation model. Radiation damage to the small intestine causes cell death and subsequent tissue destruction, requiring activation of repair pathways, which in wildtype mice takes 3-4 days [24]. In contrast to controls which recover from irradiation, R1;R2Villin Cre− ERT2 displayed a profound defect in regeneration of the small intestine epithelium as seen by histology (Fig. 11F). We also examined the tissue repair response in these mice to Dextran Sodium Sulfate (DSS) treatment, which inflicts a similar insult to the GI tract, with perturbations to the epithelial barrier preceding an inflammatory response [28]. Compared to controls, R1;R2Villin Cre− ERT2 displayed a greater loss in body weight and colon length, two parameters indicative of disease severity in this model (Fig. 11G and H). Akin to the irradiation phenotype, the small intestines from the DSS R1;R2Villin Cre− ERT2 were more inflamed and had a disrupted crypt architecture (Fig. 11I). These data highlight an essential role for epithelial Rock1 and Rock2 in intestinal tissue repair in response to stress.
To elucidate the molecular pathways perturbed by Rho-kinase deletion, we performed RNA sequencing experiments on bulk ileal tissue from the different mouse strains on days 3 and 8 from the first TMX injection. We found significant differences between the WT and R1:R2cKO mice which were similar at both time points, and for simplicity show the data on day 8 (Fig. 12A). Comparison across the groups revealed common patterns of gene expression between R1:R2cKO and WT (CD45.1)-> R1:R2cKO chimeras, mirroring the similar intestinal phenotypes ( Fig. 12B and C). However, fewer analogous changes were observed between the R1:R2cKO and R1; R2Villin Cre− ERT2 . Pathway analysis revealed induction of genes involved in stem cell activity and inflammation in both the R1:R2cKO and WT (CD45.1)-> R1:R2cKO chimeras (Fig. 12D). Consistent with the nutritional deprivation resulting from the dysfunctional gut barrier, these mice also displayed reduced expression of metabolic pathways. Interestingly, we found higher expression of inflammatory genes, particularly those associated with granulocytes and tissue damage, such as Ly6g,Clma1, Cd11, Mmp3, Cd44 in the R1: R2cKO and WT (CD45.1)-> R1:R2cKO chimeras (Fig. 12E). We also found a reduction in genes involved in anti-microbial responses and repair, such as Reg3b. These data suggest that the failed repair is in part due to the excessive inflammation associated with Rock1 and Rock2 deletion.
Discussion
Manipulating cytoskeletal reorganization represents an attractive means to modulate cellular secretion, migration and proliferation in vivo. Multiple studies using isolated cell systems indicate that the RhoA pathway promotes F-actin assembly and myosin II activation, which are necessary steps for formation of the cytokinetic furrow and successful cell division [4]. Cells devoid of RhoA have impaired viability due a failure in cytokinesis leading to cell cycle arrest [5][6][7][8]. These studies demonstrated the absolute requirement for functional RhoA activity in cell proliferation, with particularly profound effects in stem cells. Using Cre deleter lines and BM chimeras, we identify an essential function of Rho-kinases in stem cells, with an obligate role in maintaining the integrity of the BM and small intestine in the adult. This is the first demonstration for Rho-kinases as central components of homeostatic tissue turnover. Our results are consistent with what has been reported with conditional deletion of the RhoA gene in the hematopoietic and epithelial cell compartments, and underscore the importance of this pathway in the preservation of the stem cell reservoir in the GI tract and BM, which is necessary for tissue homeostasis and survival [6,8,29]. In the hematopoietic system, we identify Rho kinases as a key component of LSK proliferation and subsequent differentiation to mature blood lineages. In contrast to the cell-intrinsic effect observed in the BM, perturbation of crypt viability was only observed in the intestinal epithelial specific conditional knockout following injury. The observed phenotype is similar to what has been reported for the epithelial cell knockout of RhoA, where the gene was ablated under control of the Villin Cre promoter [6]. Mice deficient in epithelial RhoA also display crypt damage and inflammation due to a loss in intestinal stem cells. Interestingly, and in agreement with our studies, these mice do not succumb to their intestinal injury, and recover over time.
Several mechanisms may explain the lack of an intrinsic role for this pathway in epithelial cells under homeostatic conditions. It is conceivable that other Rho proteins or effectors are able to compensate for the loss of this pathway within the ISC [6]. In particular, the RhoA target, Citron Kinase, has been shown to regulate cytokinesis in multiple cell types, although involvement in adult stem cells has not been described [30]. Furthermore, since the intestine is extremely resilient to insults, and can regenerate even after lethal radiation, a substantial amount of damage may be needed to cause a complete failure in repair. This could explain why profound intestinal destruction was seen in the R1:R2cKO and WT (CD45.1)-> R1:R2cKO chimeras, where multiple cell types are affected by Rock1 and Rock2 deletion, causing widespread cell death and inflammation. A similar phenotype was seen in epithelial deletion of FAK, which regulates proliferation, survival and motility through RhoA [31]. Epithelial FAK was found to be essential in wound repair only after DSS induced intestinal damage, emphasizing the role for numerous cell types in maintaining basal intestinal integrity [32].
The BM chimera data point to a Rho-kinase dependent radiation resistant population(s) in mediating tissue turnover in the intestine, however, the specific cell-type(s) and crypt function are unclear. A number of radio-resistant cells exist in the intestine, such as stromal cells, subsets of epithelial stem cells, neurons and immune cells, the latter in particular are significantly diminished following Rho-kinase ablation. It is possible that Rho-kinases are mediating activities in these cell types that have direct effects on crypt biology. Alternatively, the impaired regeneration could be an indirect consequence of uncontrolled inflammation caused by death of radioresistant cell types that are sensitive to Rho-kinase deletion.
As the systems we established do not allow us to readily resolve these hypotheses, additional studies using cell-specific inducible Cre deleter lines will be needed to tease apart the direct vs. indirect roles of radiation resistant populations. Furthermore, the contribution of other Rho family members and effectors will also need to be addressed using genetic studies to fully understand the relevance of this pathway in vivo. Moreover, due to the loss in body weight and rapid death of the animals, we were only able to focus our studies on the GI tract and hematopoietic system. It is likely Rho-kinases play a role in other cellular processes that could not be studied using this system because of the kinetics of animal demise.
Conclusions
Because of their role in mediating proliferative signals, substantial efforts are underway to identify inhibitors against Rock1 and Rock2, particularly to treat malignancies and fibrotic diseases [13]. Our data indicate that in designing Rho-kinase inhibitors, it will be essential to monitor for toxicities within tissues with high turnover, where homeostatic stem cell renewal is essential for organ function. This will be particularly important for application of Rho-kinase inhibitors to chronic diseases where continuous blockade of the pathway is needed to achieve therapeutic benefit.
Declaration of competing interest
Authors are/were employees of Genentech when the studies were conducted. [36]. C57BL/6 N C2 ES cells [37] were electroporated with 20 μg of either linearized targeting vector DNA, and cultured under drug selection essentially as described [38]. Positive clones were identified using long range PCR followed by sequence confirmation. Correctly targeted ES cells were subjected to karyotyping. Euploid gene-targeted ES cell clones were treated with Adeno-FLP to remove PGK neomycin and ES cell clones were tested to identify clones with no copies of the PGK neomycin cassette and the correct sequence of the targeted allele was verified. The presence of the Y chromosome was verified before microinjection into albino Bl/6 N embryos. Germline transmission was obtained after crossing resulting chimeras with C57BL/6 N females. Genomic DNA from pups born was screened by long range PCR to verify the desired gene targeted structure before mouse colony expansion.
Tamoxifen injection for in vivo studies
To delete Rock1 and Rock2 in 6-8 week old mice crossed to the Rosa26-Cre ERT2 and Villin Cre-ERT2 lines, intraperitoneally (i.p.) injections of Tamoxifen (TMX) (90 mg/kg body weight (BW), Sigma) were performed for 5 consecutive days.
Real Time PCR
RNA was isolated with an RNeasy Mini Kit (Qiagen). An ABI 7500 Real-Time PCR system (Applied Biosystems) and TaqMan One-Step RT-PCR Master Mix (Applied Biosystems) were used for real-time RT-PCR (custom-made primers and probes see Table, Applied Biosystems). Results were normalized to those of RPL19 and relative expression was calculated by change in threshold (DDCT method). For ROCK expression in multi tissue panel, cDNA from Takara bio and in house generated cDNA were used.
Host chimerism analysis
Rock1 flox/flox ;Rock2 flox:flox ;R26R Cre− ERT2 mice were lethally irradiated and given B6. SJL-Ptprca Pepcb/BoyJ BM cells. Irradiation and reconstitution are described above. Two months later, mice were divided into two groups, one receiving sunflower oil (control) and the other receiving TMX as described above. Ten days later, blood and the small intestine tissue were analyzed for host chimerism. Cells were stained for CD45.1 (donor Boy J marker) and CD45.2 (host marker) by FACS. Percentage of residual CD45.2 (host cells) were plotted as a measure of host chimerism.
Organoid Studies
Primary epithelial organoids were collected as follows: Small intestine tissue was isolated, flushed with PBS, filleted, and cut into ~1 cm by 1 cm pieces. After dissection, the tissue pieces were washed in PBS three times or until the PBS was clear to remove the mucus layer. Afterward, the tissue pieces were incubated for 5 min at 37 • C in PBS with 2.5 mM EDTA to dissociate epithelial cells. After incubation, the tissue pieces were mixed gently and then strained from the solution. The tissue pieces were then resuspended in PBS with 5 mM EDTA and incubated for 5 min at 37 • C. After incubation, the tissue pieces were shaken to dislodge epithelial crypts, the supernatant was filtered through a 100-μm filter, and the solution was spun down to pellet the cells. After centrifugation, the cell pellet was washed with 25 ml of DMEM without serum to remove excess EDTA and centrifuged once more to pellet the cells. After the final centrifugation, the cells were resuspended in a 50:50 solution of media and Matrigel, and 50 μl was plated into each well of a 24-well plate. The Matrigel solution was allowed to solidify at 37 • C for 30 min before adding 500 μl of stem cell media (STEMCELL Technologies, catalog no. 06005). Organoids were treated with (4-OHT) 50 ng/ml for the first three days, followed by regular stem cell media. Rock1 and Rock2 gene expression in the organoids were confirmed at day7 by qPCR. To measure organoid growth, total organoid area was analyzed at each time
Organoid imaging
Organoids were imaged with a 4 × Plan Fluor objective (NA: 0.13, Nikon) on a Nikon Ti-E inverted microscope equipped with a Neo scMOS camera (Andor, Oxford Instruments), a linear encoded automated stage (Applied Scientific Instrumentation), 37 • C/5% CO2 environmental chamber (Okolab), all run by NIS Elements software (Nikon). Brightfield images covering the whole matrigel plug (3 × 3 fields of view) were acquired and stitched into one large image (Nikon). To measure organoid growth, total organoid area was analyzed at each time point using a custom script (Matlab, Mathworks), then normalized to time zero.
Cell Cycle Analysis
Mice were injected with 200 μl of BrdU (10 mg/ml). After 4 h , a second dose of BrdU was injected, and 2 h after the second injection mice were euthanized. BM cells were harvested and stained for LSK cells and BrdU according to the BD Pharmingen protocol.
Histology and Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue sections 4 μm in thickness mounted on glass slides were used for immunohistochemistry and staining. Small intestines were harvested, and the duodenum was fixed and sectioned for staining with hematoxylin and eosin, Cleaved Caspase 3, (rabbit anti-Cleaved Caspase 3, D175, Cell Signaling Technology), BrdU (rat anti-BRDU, ab6326, Abcam). For analysis of DSS-induced colitis, colons were prepared for histology by the 'gut-roll' or 'Swiss-roll' technique (in which the rectal end of the intestine is placed in the center with the remaining tissue rolled up longitudinally around it). Tissues were then fixed and sectioned and then stained with hematoxylin and eosin.
In situ hybridization
ISH was performed using reagents and protocols from Advanced Cell Diagnostics. Briefly, the small intestine was longitudinally cut through the midline and fixed in 10% neutral buffered formalin for 24 h before being placed in 70% ethanol and processed for paraffin embedding. Sections at 6 μm thickness were cut through the midline and perpendicular to the lumen and then allowed to dry in a 60 • C oven for 1 h. Sections were rehydrated in two washes of xylene for 5 min each followed by two washes in 100% ethanol, one wash in 95% ethanol, and one wash in 90% ethanol, all for 1 min each. After rehydration, the samples were incubated in hydrogen peroxide, boiled in antigen retrieval buffer, and then digested with proteinase for 15 min at 40 • C. After digestion, the slides were washed twice for 1 min with ISH wash buffer and then hybridized with probes for 2 h at 40 • C. Following hybridization, amplification steps were completed according to Advanced Cell Diagnostics protocol. After the final amplification incubation, the slides were washed in ISH wash buffer and detected with horseradish peroxidase conjugated with DAB, counterstained with hematoxylin, and then baked in a 60 • C oven for 15 min before mounting with non-aqueous mounting media.
Western blot
Whole-cell lysates from spleen and small intestine tissues were prepared with a lysis buffer containing protease inhibitors and phosphatase inhibitors (Cell Signaling 9803). Lysates were run on Nu-PAGE 4-12% Bis-Tris gel and transferred according to standard protocols. After transfer, blots were incubated with either an anti-Rock1 (Cell Signaling 4035s), anti-Rock2 (ab125025), anti-Rabbit IgG-HRP (Cell Signaling 7074s) Anti-B actin (Cell Signaling 4970) antibody was used as the loading control. Table 1) were injected with TMX for 5 consecutive days. Small intestines were harvested on days 3 and day 8 from the start of TMX injections. Five replicate samples were collected for each time point and condition. RNA was extracted using RNeasy Kit (Qiagen) with on-column DNase digestion.
QC of total RNA was done to determine sample quantity and quality. The concentration of RNA was determined using NanoDrop 8000 (Thermo Scientific) and the integrity of RNA was determined by Fragment Analyzer (Advanced Analytical Technologies). 100 ng of total RNA was used as an input material for library preparation using TruSeq Stranded Total RNA Library Prep Kit (Illumina). Size of the libraries was confirmed using High Sensitivity D1K screen tape (Agilent Technologies) and their concentration was determined by qPCR based method using Library quantification kit (KAPA). The libraries were multiplexed and sequenced on Illumina HiSeq4000 (Illumina) to generate 30 M of single end 50 base pair reads.
RNA-seq data were processed using the HTSeqGenie R package (v. 4.8.0) [39]. Briefly, reads were aligned against the reference mouse genome (GRCm38) using GSNAP (v. 2013-11-01). Uniquely aligned reads that fell within exons of gene models specified by GENCODE (v. 27) were counted using the countFeatures method from the HTSeqGenie package. Genes with low expression were excluded from analysis using the filterByExpr method from the edgeR package [40]. We then used the voomWithQualityWeights method from the limma package to transform the data and determine sample and observation weights [12]. Differentially expressed genes were identified using limma comparing the WT samples against samples from R1;R2cKO, R1;R2 Villin Cre− ERT2 , or WT (CD45.1)-> R1:R2cKO chimera mice (see Table 1). Genes were considered significantly different if they varied by greater than 2-fold, and had a Benjamini-Hochberg adjusted p-value less than 0.05.
Single Cell RNA-sequencing
Data from the Mouse Cell Atlas (v. 1.1) were downloaded from (https://figshare.com/articles/dataset/HCL_DGE_Data/7235471) in AnnData format. Data were analyzed using the scanpy Python toolkit [41,42]. Cells with at least 200 detected features and features detected in at least 3 cells were included in the analysis. Raw count data were normalized and then subjected to log1p transformation. We then identified highly variable genes across the dataset. Data from these genes were scaled, and then PCA was performed on the scaled data. A k-nearest neighbor graph was then computed using k = 10 and the first 40 principal components. The neighborhood graph was then embedded using the Uniform Manifold Approximation and Projection (UMAP) method for dimensionality reduction to visualize gene expression data.
To evaluate the expression of Rock1 and Rock2 across blood cell populations (including HSCs), we created an integrated single-cell atlas consisting of the following datasets: 1) Isolated HSC cells from Ref. [43] annotated using a combination of author-provided cluster names and known sets of marker genes, 2) Bone marrow cells from the Mouse Cell Atlas, and 3) single-cell data from FACS-isolated HSC subpopulations (LT-HSC, ST-HSC, MPP2, MPP3, and MPP4) [44]. To make values comparable across datasets, each sample within each dataset was log-transformed and library sized corrected using the scater package. Low quality cells within each dataset were then removed by identifying cells that lied more than three standard deviations outside of the sample-wide distribution for fraction of mitochondrial reads, number of genes identified, and total number of reads per cell. Batch integration was performed using fastMNN.
Lineage Tracing using Mosaic Analysis with a Repressible Marker (MARCM)
The MARCM method allows for site-directed genetic recombination to generate and mark (by GFP) homozygous mutant cells for a gene of interest in an otherwise heterozygous genetic background 1 . Here, we used MARCM to induce ISC clones that were homozygous mutant for Rok 2 . The Rok 2 mutant allele is a strong loss-of-function, if not null, allele 2 . To generate Rok 2 clones, MARCM19 females were crossed to either neoFRT19A or Rok 2 neoFRT19A/FM7c males. Clones were induced in adult flies (5-10d old) by heat shock at 37 • C (waterbath) for 45 min in an empty fly vial. Flies were then maintained for 7d and 28-30d after heat shock (AHS) to measure clone growth over time. | 2023-03-12T15:32:13.517Z | 2023-03-01T00:00:00.000 | {
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88355563 | pes2o/s2orc | v3-fos-license | Prevalence of Road Traffic Accident (Non-fatal) in Children: Retrospective Study at Tertiary care Centre
1 Assistant Professor, Department of Forensic Medicine & Toxicology, Gajra Raja Medical College, Gwalior 2 Yoga Course .Department of Yogic Sciences, LNIPE, Gwalior 3 Professor & Head, Department of Forensic Medicine & Toxicology, TSM Medical College, Lucknow *Corresponding Author Dr Chandra Shekhar Wagmare Email: Drshekharr05@gmail.com, Mob No-9424678980 Abstract Fatal and Non-fatal road traffic accident in childhood contribute significant public health problem in world. As children grow and their path extends from home to world, they are exposed to hazards and risks. The fatalities from Road Traffic Accidents constitute a major cause of unnatural deaths among children in India. The present study observed that most of road injury fatalities in children involved males in the age group of 5-15 years, most commonly affected were travellers in two wheelers. The present study found that the maximum number of casualties occurred between 8 am –12 Noon which is comparable with findings of other parts of India. Most schools close after 4 pm therefore comparatively more children are present on the road at this time of the day thereby increasing their exposure to automobiles . One of the best ways to do it is to include road safety issues in school curriculum. Those children who ride bicycles should be made to wear helmets as it is expected to reduce the severity of injury to the head. Drivers should avoid talking on mobile phones while on roads. Keyword: Children, Road Traffic Accident, Tertiary Care centre.
Introduction
Fatal and Non-fatal road traffic accident in childhood contribute significant public health problem in world. As children grow and their path extends from home to world, they are exposed to hazards and risks (1) . Despite the fact that children use roads as pedestrians, cyclists, motorcyclists, vehicle passengers but the road environment is rarely developed with involvement of their needs and activities. At global level, road traffic injuries are one of the leading causes of death from unintentional injury, with the highest rates among 15 to 19 years age group of children and a major burden on the world's economy causing a yearly loss of about 518 billion dollar (2). The fatalities from Road Traffic Accidents constitute a major cause of unnatural deaths among children in India. The Epediomological data of Road Traffic Accidents concern with children are lacking in our country. It is also the foremost cause of unnatural deaths in children, contributing to an annual loss of more than 260,000 lives in the 0-19 year age group (3) . India contributes one-sixth of the world's population, 29.5% of which belongs to the 0-14 year age group. India has witnessed a 10-fold increase in the number of fatalities from 1970 to 2009 with one accident occurring every minute and one fatality every four minutes (4) . It has been reported that road traffic injuries are the second most frequent cause of death in the 5-14 year age group in India (4). . In 2015 alone, more than 400 children were killed in school bus related incidents; thousands were injured in car, motorbike and pedestrian accidents. According to data from the National Crime Records Bureau, 15,633 children were killed in road accidents across India in 2015, that is nearly 7 times more deaths caused due to road accidents than crimes against children like murder and foeticide (5). . Overpopulation, increased number of vehicles on roads, poor road conditions and disregard for traffic rules and regulations are major contributing causes of increased injuries and fatalities in India. Hence the present study was carried out to provide a basic baseline data to policy makers to plan for safer transportation routes and in setting up of health care facilities considering trauma in areas that report a higher number of accidents and injuries.
Material Method
The present study is a retrospective analysis of case details of 183 children who admitted in Causality department of GRMC Gwalior, India from period of Ist June 2015 to 31 st May 2016. This institute provides tertiary care to the people of more than half a dozen neighbouring district of central India. Data were collected from In Patient Registers and case sheets of all 183 cases from medical record section of centre. In this study children of age group 1 to 18 years are included who are admitted for RTA with various types of non-fatal injuries. The information regarding age and sex distribution, rural and urban distribution, time of the accident, non-fatal injury effecting to various body parts and organs and other socio demographic parameters were included under this study. Injuries were divided in to simple and complicated depending on requirement of surgical interventions, and simple injuries are defined as an abrasion that do not require intensive care or surgical interventions, and those which require intensive care of wound like suturing under anaesthesia, fractures, intracranial injuries, head injuries, lacerated facial injuries etc. were considered as complicated injuries under this study.
Observation and Results
Out of 183 children injured in road traffic accident, higher proportion were reported in boys (151(82.51%)) in compared to girls (32 (17.48%)) so proportion of Male to Female is 4.7:1 Among 183 children, 36 children have simple injuries and 147 have sustained complicated injuries. In 143(78.14%) cases, incidence took place in rural area and 32(22.28%) cases were noticed in urban area. The prevalence of road injury varied with socioeconomic condition and types of vehicle occupied by children for travel and for going to school. Children of different age group of rural population were more affected in compared to children reside in urban area. Children of 5-10 years age group were more involved followed by children of 10-15 years age group and then 15-18 years age group. In this study, the prevalence of road injury was highest among children who travel by two wheeler (80.87 %) in compared to travel by four wheeler & three wheeler (6.55% each) and 6.01% cases were reported in pedestrian. Under this study, injury sustained to the head and neck region of the body of children were more common (28.96%) followed by multiple region injuries (27.86%),) then chest injuries (14.20%) and abdominal injuries (11.47%).
Discussion
Road traffic injuries (RTI) have been increasing over the past twenty years and the situation in India is worsening in concern to children. In 2015, WHO research studies showed that in India vehicle ownership is 6 per 100 person but road traffic fatalities are 11 per 100 person, while compared to other countries where much higher vehicle ownership rates than India but lower road traffic injuries cases were reported (6). . Research in several countries shows that, 90 percent of all road traffic accident are caused by human error and only a small portions caused by vehicle defect, poor road design and inadequate maintenance In many situation driver impairment is the most crucial component of road traffic accident worldwide (7) . According to road safety data from transportation research and injury prevention programme (TRIPP) by Indian Institute of Technology Delhi in collaboration with WHO by Dinesh Mohan, Geetam Tiwari and Kavi Bhalla shows that vehicle population is increased to 140000 in 2015 from 20000 in 1970 (8) and prevalence of road traffic accidents is high among children of age group between 15 -18 years (31.8%) and high involvement with passengers of two wheelers (74.9%) in rural populations (73.3%). The contributing factors for this prevalence could be road conditions in Indian rural areas, less knowledge of driving skill and negligence in following traffic rules, carrying children in front part of vehicles is more common in rural India. Road traffic accident was the most common and most neglected non-infectious cause of morbidity and mortality in children of India. The present study observed that most of road injury fatalities in children involved males in the age group of 5-15 years, most commonly affected were travellers in two wheelers. The present study found that the maximum number of casualties occurred between 8 am -12 Noon which is comparable with findings of other parts of India. Daytime is the usual working time in India for offices, business institutions and schools so there is greater exposure of people to vehicles at this time (9). . Most schools close after 4 pm therefore comparatively more children are present on the road at this time of the day thereby increasing their exposure to automobiles (10). . The time period between 4pm -8 pm is generally the closing time of offices and more automobiles are present on the road during this time and moreover, this is also the time when children usually go out of home to play after completing their homework (11). . The findings of the present study are in accordance with the results of studies conducted nationally and internationally by WHO and government and non-government agencies.
Conclusion
The present study observed that most of the road injury fatalities in children involved males in the age group of 5-15 years.. Most commonly affected were two wheeler occupants of rural areas and most of the fatalities were seen during the daytime. To bring the case rate down, children, especially with rural background should be made aware about the importance of strict compliance to traffic rules and regulations. One of the best ways to do it is to include road safety issues in school curriculum. Those children who ride bicycles should be made to wear helmets as it is expected to reduce the severity of injury to the head. Drivers should avoid talking on mobile phones while on roads. The present study has highlighted the urgent need to frame road safety policies like separate lanes for different vehicles as the traffic in India in general and this region in particular consist of all kinds of automobiles including two wheelers, three wheelers and four wheelers thus increasing the chances of accidents. Installation of red lights and marking of zebra crossings on the roads particularly near schools and playgrounds would be a welcome decision. The government should also ensure that the vehicles must follow certain fixed speed limits at road where more children are exposed. | 2019-03-31T13:32:30.476Z | 2019-03-15T00:00:00.000 | {
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268902801 | pes2o/s2orc | v3-fos-license | The Application and Development of Image Recognition Technology in the Field of Computer Vision
. Image recognition is an important field of computer vision research. With the emergence of convolutional neural networks, image recognition has rapidly developed in many fields and has entered people's daily lives. This article reviews image recognition applications and future prospects in three representative fields: healthcare, intelligent driving, and security monitoring. In medical image analysis, image recognition technology can improve medical image interpretation speed and accuracy by automatically learning image features, providing doctors with more comprehensive information and decision-making support. Regarding intelligent driving, image recognition can help vehicles perceive and understand the traffic environment in real time, improving driving safety and automation level. In terms of security monitoring, image recognition plays a vital role in facial recognition, object detection, and behavior analysis, providing intelligent monitoring and security guarantees. However, these areas still have challenges, such as large data volumes, labeling difficulties, and privacy issues. In the future, people can look forward to combining image recognition technology with other technologies to improve models' interpretability and decision-making ability and further promote the development of these fields.
Introduction
Image recognition is an important research direction in computer vision, and its applications are gradually penetrating various fields.Image recognition technology can automatically analyze and understand information such as objects, scenes, and actions in images or videos, thereby achieving more intelligent and efficient applications [1].Therefore, both domestically and internationally, scientists have received widespread attention from image recognition technology.In recent years, significant progress has been made in image recognition technology due to the advancement of deep learning algorithms and the availability of large-scale image datasets.This paves the way for extensive practical applications in various fields and has broad prospects.
It is used for medical image analysis to achieve more accurate diagnoses and personalized treatment plans in healthcare.Doctors can use image recognition algorithms to analyze medical images and detect anomalies, achieving faster and more reliable diagnoses.In addition, image recognition can assist surgical procedures by accurately identifying target areas and guiding surgeons during the surgical process.
Image recognition plays a vital role in autonomous vehicles and intelligent transportation systems.It helps with object detection, lane detection, and traffic sign recognition, enhancing the perception ability of vehicles.This, in turn, enables autonomous vehicles to drive safely on the road, reducing the risk of accidents.In addition, image recognition can be used in traffic management systems to optimize traffic flow and improve overall efficiency.
Image recognition technology also contributes to the fields of security and monitoring.It can achieve facial recognition, object tracking, and behavior analysis, achieving more effective monitoring and threat prevention.Monitoring systems with image recognition algorithms can quickly identify suspicious activities, intrusions, or individuals, enhancing overall security measures.This article will focus on the application of image recognition in fields such as medical image analysis, intelligent driving, and security monitoring and explore the difficulties and challenges faced in these fields, as well as the potential and prospects of image recognition technology in solving these problems.The article also mentions the challenges and limitations in these fields and future development directions to provide references and ideas for researchers in related fields and promote further application and research of image recognition technology in these fields.
Medical Image Analysis
Medical image analysis refers to the analysis of medical images through image recognition technology.With the continuous development of image recognition technology, in addition to doctors manually and subjectively interpreting medical images, advanced image recognition technology can now play an essential role in medical image analysis.Medical image analysis has improved the interpretation speed and accuracy of medical images through artificial intelligence technology, providing doctors with more comprehensive information and decision-making support, further improving the level of medical diagnosis and treatment, and improving patients' healthcare outcomes.At present, image recognition plays a vital role in the diagnosis and treatment of lung cancer, breast cancer, and other cancers.According to statistics, lung cancer deaths are the highest among various cancers [2].Therefore, this article focuses on applying image recognition to lung cancer.
Lung CT is a commonly used imaging examination method that can be used to diagnose lung cancer.CT scanning can provide detailed lung images and detect abnormal structures and tissues.In clinical practice, the most common and popular method of lung cancer examination is CT scanning, which is also used to diagnose patients.Lung cancer is usually divided into four stages based on its severity and spread: stage I, stage II, stage III, and stage IV [3].However, there may be errors and misidentification of cancer stages in manually interpreting CT images.In order to ensure a correct diagnosis, image recognition is now needed.Classifying lung CT images into normal and different lung cancer stages is a standard classification problem.
In applying medical imaging technology in deep learning diagnosis, the automatic extraction of medical image features is one of the most crucial steps in the entire algorithm.Convolutional neural networks (CNN) can automatically learn features in images without manually designing complex feature extractors [4].CNN has become an effective tool for processing image data through its automatic feature learning, hierarchical feature extraction, and parameter-sharing advantages.With the increase of datasets and the improvement of computing resources, CNN has made significant breakthroughs and successes in fields such as image recognition, object detection, and image segmentation, which has sparked the rise of this field.CNN neural networks also exhibit excellent performance in image recognition of lung CT.N. Mohanapriya et al. obtained lung tumor classification accuracy rates of 85.33%, 84.51%, and 85.79% using three different DCNN classification structures [5].Shalini Wankhade et al. used CNN-RNN, a hybrid neural network, as a new method for cancer cell detection for early diagnosis of lung cancer and further improved the accuracy of diagnosis using advanced 3D convolutional neural networks (3D-CNN) [6].Recently, Sandeep Wadekar et al. used the CNN model VGG19 to modify the CNN model to a pre-trained visual geometry group 19 (VGG19) and used pre-trained and improved enhancement techniques to classify lung cancer biopsy images.The finely tuned VGG19 model showed high accuracy in the experiment, reaching 97.73%.The experimental results show that this method performs better than existing methods [7].
Intelligent Driving
Image recognition and autonomous driving are closely related.Image recognition technology is one of the cores of the auto drive system, which is used to perceive and understand the visual information in the environment in real time.Autonomous vehicles are usually equipped with multiple sensors, including cameras, radar, and LiDAR, among which the camera is responsible for collecting image data.Analyze and process these images using image recognition technology.
Image recognition in intelligent driving is divided into active and passive image recognition [8].Simply put, active image recognition is image recognition that obtains the required information through user guidance or interaction, while passive image recognition is the automated recognition and analysis of images.For intelligent driving, the main issue is how to make the computer understand the entire scene and guide the car to make the next decision.Overall, image recognition plays a crucial role in intelligent driving, helping the intelligent driving system make accurate decisions and plans through real-time perception and analysis of images and improving the driving safety and automation level of vehicles.Convolutional neural networks are widely used to achieve binocular 3D perception in autonomous vehicles.Among them, the CNN-based FlowNet framework proposed by Alexey Dosovitskiy et al. in 2015 is a commonly used method.FlowNet is an optical flow estimation framework based on convolutional neural networks, aiming to accurately estimate the optical flow in images by learning convolutional neural networks [9].The FlowNet framework can calculate the depth of different objects in the scene by analyzing and comparing the disparity information between binocular images, achieving perception and understanding of 3D scenes.FlowNet framework plays a vital role in intelligent driving.Through binocular vision depth perception and motion estimation, it has realized vital functions such as 3D scene perception, distance calculation and collision warning, visual odometer, obstacle detection and tracking, and attitude estimation, further improving the perception and decision-making ability of the auto drive system [8].In 2020, Debrina Dutta et al.
proposed a new CNN-based all-terrain autonomous driving model with an overall testing accuracy of 96.8%.The accuracy of this model is very impressive during the day but slightly decreases under nighttime, foggy, and rainy conditions [10].This is also where relevant researchers need to improve in the future.
Security Monitoring
Image recognition has played a significant role in security monitoring fields such as facial recognition, behavior analysis, object detection, and tracking.With the development of artificial intelligence, machine learning, and Internet of Things technology, the field of security monitoring has received widespread attention from scientists worldwide.
Face recognition and target detection are essential technologies in security monitoring that can accurately identify and locate suspects.The security system can identify individuals who have been blocked or have specific identities and can also effectively identify and accurately locate offending items through object detection, triggering alerts to notify security personnel.In 2021, Vidit Kumar et al. designed a face skeleton based on the YCBCR color space for feature point detection and extraction.The skeleton was used to detect Harris corner feature points and SURF feature points from each face, and a support vector machine classifier was trained to recognize faces [11].Experiments were conducted on public datasets, and the results showed that our method has reliability, accuracy, and robustness and can be successfully applied to multi-face recognition systems in the real world.
According to statistics, the crime rate at night is often higher than during the day.Night creates natural escape conditions for criminals, and infrared cameras are essential in the dark.Therefore, the research on surveillance cameras with infrared (IR) imaging function combined with image recognition has always been a current research focus [12].Fast R-CNN (Region Convolutional Neural Network) technology has become a vital algorithm in object detection in the real-time crime scene evidence analysis system.It has achieved remarkable results in many practical applications.In 2022, Meiyu Liang et al. proposed an improved Faster R-CNN infrared target detection algorithm with improved performance compared to the Faster R-CNN target detection algorithm, which has good effectiveness and feasibility [13].
For the identification of dangerous weapons, Ahmed Abdelmoamen Ahmed et al. used the Raspberry Pi 3 device in conjunction with the CNN model and Mask R-CNN deployed on Intel Neural Computer Rod 2 (NCS2) for classification, achieving an overall prediction accuracy of 94% for identifying dangerous weapons [14].With such high accuracy, it is already possible to efficiently screen hazardous materials from many items, significantly reducing the pressure of manual screening.
Overall, the importance of image recognition in security lies in providing more intelligent, efficient, and accurate monitoring and security guarantees.It can help people quickly identify and respond to potential security threats, enhance their sense of security, reduce criminal behavior, and provide adequate security management and resource allocation.
Comparison of Three Fields
The image recognition in the above three fields has different application objects, advantages and limitations, as shown in Table 1.
Medical Imaging Analysis
The existing deep learning models have achieved very high accuracy and credibility in judging a single disease.However, in the clinical medical decision-making of real doctors, it is often necessary to judge unknown diseases based on multiple information such as the patient's medical history, physical condition, laboratory tests, and imaging examinations.In the future, image recognition technology can combine natural language processing technology, integrate multiple aspects of image and language information, incorporate disease-related influencing factors into deep learning models, and improve the interpretability of the model is a focus of researchers.
Intelligent Driving
With the development of intelligent driving in recent years, many car manufacturers led by Tesla have equipped cars with functions such as automatic cruise control, lane keeping, and automatic lane changing.It can perceive the environment based on traffic signs and road markings, and judge and adapt to the road conditions ahead of the vehicle in real-time.However, at present, it is still necessary for drivers to remain vigilant and intervene at any time during autonomous driving to ensure safe driving and not fully autonomous driving.In the final analysis, the accuracy of intelligent recognition cannot reach the level that humans can believe.With the continuous development of image recognition technology and the continuous improvement of accuracy, it is believed that one day, fully autonomous driving will enter our lives and liberate drivers' hands.
Security Monitoring
With the further integration of technologies such as big data, cloud computing, and the Internet of Things, the application of image recognition in the field of security monitoring will become more extensive and in-depth.Nowadays, intelligent security monitoring has not yet been widely popularized.Reducing costs, improving technical accuracy, and strengthening privacy protection are important directions to promote the popularization of intelligent security monitoring.With the continuous progress of technology and the maturity of intelligent solutions, image recognition will face greater development opportunities in the field of intelligent security.This will bring more efficient, precise, and reliable security guarantees to urban security, improving the quality of life and sense of security for urban residents.
Conclusion
Image recognition technology has widespread applications and enormous potential, gradually penetrating medical imaging, intelligent driving, and security monitoring.In the future, image recognition technology will continue to develop, and accuracy and stability will continue to improve.Combining image recognition with other fields of technology, such as natural language processing, big data, and the Internet of Things, will further promote the intelligence and efficiency of image recognition.However, image recognition still faces challenges such as dataset quality, annotation difficulties, and environmental complexity.Addressing these issues requires in-depth research and exploration, strengthening data annotation and privacy protection.Overall, the application and prospects of image recognition are comprehensive.With the advancement of technology and the promotion of applications, image recognition will play a more critical role in various fields, bringing more convenience and security to people's lives and work. | 2024-04-05T18:05:58.049Z | 2024-03-13T00:00:00.000 | {
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38291151 | pes2o/s2orc | v3-fos-license | Efficacy of omeprazole and amoxicillin with either clarithromycin or metronidazole on eradication of Helicobacter pylori in Chinese peptic ulcer patients
AIM: One-week triple therapy with proton pump inhibitors, clarithromycin and amoxicillin has recently been proposed as the first-line treatment for Helicobacter pylori (H pylori) infection; however, data regarding the effects of this regimen in China are scarce. The aim of this prospective and randomized study was to compare the efficacy of clarithromycin and metronidazole when they were combined with omeprazole and amoxicillin on eradication of H pylori and ulcer healing in Chinese peptic ulcer patients. METHODS: A total of 103 subjects with H pylori-positive peptic ulcer were randomly divided into two groups, and accepted triple therapy with omeprazole 20 mg, amoxicillin 1 000 mg and either clarithromycin 500 mg (OAC group, n = 58) or metronidazole 400 mg (OAM group, n = 45). All drugs were given twice daily for 7 d. Patients with active peptic ulcer were treated with omeprazole 20 mg daily for 2-4 wk after anti-H pylori therapy. Six to eight weeks after omeprazole therapy, all patients underwent endoscopies and four biopsies (two from the antrum and two others from the corpus of stomach) were taken for rapid urease test and histological analysis (with modified Giemsa staining) to examine H pylori. Successful eradication was defined as negative results from both examination methods. RESULTS: One hundred patients completed the entire course of therapy and returned for follow-up. The eradication rate of H pylori for the per-protocol analysis was 89.3% (50/56) in OAC group and 84.1% (37/44) in OAM group. Based on the intention-to-treat analysis, the eradication rate of H pylori was 86.2% (50/58) in OAC group and 82.2% (37/45) in OAM group. There were no significant differences in eradication rates between the two groups on either analysis. The active ulcer-healing rate was 96.7% (29/30) in OAC group and 100% (21/21) in OAM group (per-protocol analysis, P>0.05). Six patients in OAC group (10.3%) and five in OAM group (11.1%) reported adverse events (P>0.05). CONCLUSION: One-week tr iple therapy wi th omeprazole and amoxicillin in combination with either clarithromycin or metronidazole is effective for the eradication of H pylori. The therapeutic regimen comprising metronidazole with low cost, good compliance and mild adverse events may offer a good choice for the treatment of peptic ulcers associated with H pylori infection in China. © 2005 The WJG Press and Elsevier Inc. All rights reserved.
INTRODUCTION
Helicobacter pylori (H pylori) infects the stomachs of more than 50% of people worldwide, and is responsible for most peptic ulcer diseases, gastritis and gastric malignancies [1][2][3][4] .According to the Maastricht 2-2000 consensus report [5] , eradication of H pylori infection is strongly recommended in duodenal and gastric ulcers, whether they are active or not.Cure of the infection not only promotes peptic ulcer healing but also reduces ulcer relapse.Recently, 1-wk triple therapy with a proton-pump inhibitor (PPI) and two antimicrobial agents (clarithromycin, amoxicillin, or metronidazole/tinidazole) has been shown to be one of the most effective regimens and is recommended as the firstline treatment of H pylori eradication due to its high cure rates and convenience [6][7][8] .However, as in many other infectious diseases, antibiotic resistance is the major cause of treatment failure.Metronidazole-resistant strains of H pylori have been reported to be increasing worldwide [9][10][11] .
Although clarithromycin is an excellent drug for treating H pylori infection overseas [12,13] , this drug has not been widely used in China due to its high cost.Therefore, we evaluated the efficacy of 1-wk triple therapy with omeprazole, amoxicillin and clarithromycin (OAC) for H pylori eradication and active peptic ulcer healing in Chinese population.We also compared the results of OAC regimen with a conventional traditional triple therapy with omeprazole, amoxicillin and metronidazole (OAM).
Eradication methods
Patients were randomly divided into two groups, and accepted triple therapy with omeprazole 20 mg, amoxicillin 1 000 mg and either clarithromycin 500 mg (OAC group, n = 58) or metronidazole 400 mg (OAM group, n = 45).All drugs were given twice daily for 7 d.Patients with active peptic ulcer were treated with omeprazole 20 mg daily for 2-4 wk after anti-H pylori therapy.Each patient was asked to return at the end of antibiotic treatment for a structured clinical interview to assess adverse events and compliance.
Evaluation of eradication therapy
Six to eight weeks after omeprazole therapy, all patients underwent endoscopies and four biopsies (two from the antrum and another two from the corpus of the stomach) were taken for rapid urease test and histological analysis (with modified Giemsa staining) to examine H pylori. Successful eradication was defined as negative results from both examination methods.The healing of active ulcer was also evaluated during endoscopic examination.
Statistical analysis
The results of treatment were evaluated with per-protocol (PP) analysis (which included only patients who completed the study) and intention-to-treat (ITT) analysis (which included also patients who did not complete the study).The demographic and clinical characteristics of the two groups were compared by 2 test.The results of treatment were compared by 2 test or Fisher's exact test.P<0.05 was considered statistically significant.
Demographic and clinical characteristics
The demographic and clinical characteristics of the 103 patients in the two groups are shown in Table 1.No significant differences in demographic and clinical characteristics were found between the two groups.2. There were no significant differences in eradication rates between the two groups.
Healing rates of active peptic ulcer
The active ulcer-healing rate on PP analysis was 96.7% (29/30) in OAC group and 100% (21/21) in OAM group.There were no significant differences between the two groups ( 2 = 0.71, P>0.05).
Adverse events and compliance
Completed questionnaires about the adverse events and compliance were obtained from all the 103 patients.Adverse events were noticed (Table 3) in six patients in OAC group, and five patients in OAM group, with no statistically significant differences between the two groups ( 2 = 0.02, P = 0.90).The symptoms of adverse events were mild and did not necessitate any additional treatment in both groups.
None of the other serious events such as hepatic or renal functional damages were found by means of biochemical examination in the two groups.All patients, except for two who had acute allergic skin rashes and one who had diarrhea, were able to take the study medication completely for the full study period.Thus, 100 patients (97.1%) had an excellent compliance.
DISCUSSION
Many authors have reported a correlation between H pylori infection and peptic ulcers [1,7,8] .Incidence of H pylori infection was higher in patients with gastroduodenal ulcers than in subjects without gastroduodenal disorders.The eradication of H pylori has been strongly recommended in all patients with peptic ulcer, including those with complications [5] .
Eradication of H pylori could assure rapid symptom relief and accelerate ulcer healing [14] , prevent ulcer relapse and reduce complications [7,8,[15][16][17][18] .Furthermore, eradication of H pylori could also improve the healing of intractable ulcers [19][20][21] .However, the survival capabilities of H pylori in the stomach made it difficult to be eradicated, and effective treatment required multi-drug regimens consisting of two antibiotics (usually selected from clarithromycin, metronidazole, amoxicillin, and tetracycline) combined with PPI or bismuth compounds [5,22,23] .Although the optimal treatment of H pylori infection is still a matter of debate, the effectiveness of PPI based 1-wk triple therapy has now been well established and remains one of the first-line therapies of choice [6,14,24,25] .Clarithromycin is a new generation of macrolide antibiotic that inhibits bacterial protein synthesis.Its antibacterial spectrum is similar to that of erythromycin, but it is more acid-stable, better absorbed, and is thought to be an effective drug for treating H pylori infection [7,12,13] .Among several eradication regimens, PPI with clarithromycin and amoxicillin is thought to be one of the most effective treatments of H pylori. Amoxicillin resistance was rarely reported [26] but clarithromycin resistance has increased year after year [27] , and eradication rates with clarithromycin-containing regimens decreased significantly [28] .The present study showed that the H pylori eradication rate in OAC group was 89.3% (50/56, PP analysis).The result is in accordance with previous reports from China and Spain [29,30] .However, in a study from Japan by Ogura et al [31] , eradication was achieved in 39/40 (98%) by PP analysis in clarithromycin-based triple therapy for non-resistant H pylori infection.These results indicate that the therapeutic effect of clarithromycin for H pylori eradication is not quite consistent.It may be related to different resistance to clarithromycin of infecting H pylori strains in various countries and regions.Widespread use of antimicrobial drugs has resulted in a worldwide increase in the prevalence of antibiotic resistance in H pylori, 5-11% of clinical H pylori strains isolated in China are resistant to clarithromycin [32,33] .Although clarithromycin was not available in China before 1996, the other members of macrolides such as spiramycin, erythromycin and roxithromycin have been widely used over the past years for the treatment of respiratory infection, sexually transmitted diseases and other infectious diseases.Thus, H pylori is able to develop resistance to clarithromycin rapidly after contact with it, as crossresistance exists between macrolides.Some studies have shown that clarithromycin resistance in H pylori substantially affected the success rate of eradication regimens containing clarithromycin [28] .In the present randomized study, there were no significant differences between OAC and OAM treatment groups in terms of H pylori eradication and ulcer healing, confirming that 1-wk triple therapy with omeprazole and amoxicillin in combination with either clarithromycin or metronidazole has the same effectiveness on eradicating the bacterium.Both eradication regimens were well tolerated and patient compliance was excellent.However, clarithromycin is too expensive to be widely used in China.
Antibacterial treatment of H pylori is difficult because of the very rapid development of resistance to antimicrobial agents, especially to nitroimidazoles, such as metronidazole and tinidazole, and clarithromycin [34] .The resistance of H pylori to metronidazole and clarithromycin strongly affected the success of regimens involving these drugs.The prevalence of resistance to these anti-microbial agents varied with gender, ethnic group and country of origin [34] .It was reported from Hong Kong (China) that almost 50% of pretreatment strains of H pylori were resistant to metronidazole and over 10% to clarithromycin [33] .Metronidazole resistance has been shown to reduce H pylori eradication rates in the regimens containing amoxicillin and metronidazole [35,36] .Several studies have shown a significantly higher rate of metronidazole resistant H pylori among women [37][38][39] , indicating that this drug can be widely used for pelvic inflammatory diseases in females [37] .In the current study, the number of men was absolutely more than that of women either in OAC or in OAM group.Whether the sex bias of patients was related to the better eradication in OAM group remains unknown.We did not test in vitro sensitivity to metronidazole and clarithromycin.Although Epsilometer (E) test has been recommended as the best and simplest method for routine testing of antibiotic sensitivity to H pylori, the technique is not yet widely available in China.On the other hand, the exact mechanism responsible for the development of H pylori resistance to metronidazole still remains obscure, antimicrobial effectiveness in vivo was poorly predicted by sensitivity in vitro [37] .This is largely because the current breakpoints, which are the in vitro concentrations defining the cut off between sensitive and resistant strains, do not correlate with levels required for eradication of infection from the gastric mucosa.
In the past, prevention of peptic ulcer recurrence was based on long term use of H 2 -receptor antagonists or PPIs.Since H pylori was recognized, it has been well understood that eradicating the bacterium could significantly reduce the recurrence of peptic ulcer diseases [8,[16][17][18] .In our study, the ulcer relapse rate during the 12-mo follow-up was 66.7% (4/6) in H pylori-positive patients and none of the 24 H pylorinegative patients relapsed (data not shown).In conclusion, 1-wk triple therapy with omeprazole and amoxicillin in combination with either clarithromycin or metronidazole is equally effective for eradication of H pylori and ulcer healing.Clarithromycin is the most expensive antimicrobial drug used to treat H pylori infection.Metronidazole with lower cost, good compliance and mild adverse events may offer a good choice for the treatment of peptic ulcers associated with H pylori infection in China.
Table 1
Baseline characteristics of patients in two groups Eradication rates of H pyloriOf the 103 patients enrolled in this study, 3 (2.9%)withdrew from the study because of drug-related adverse events.Of them, two patients (each from OAC group and OAM group) with skin rash and one from OAC group with diarrhea discontinued the treatment.As a result, 100 patients (97.1%, 56 patients in OAC group and 44 patients in OAM group) completed the entire course of therapy and returned for follow-up.The eradication rates based on PP or ITT analyses are shown in Table
Table 2
Eradication rates in two treatment groups
Table 3
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14957787 | pes2o/s2orc | v3-fos-license | The Gapeev-K\"uhn stochastic game driven by a spectrally positive L\'evy process
In Gapeev and K\"uhn (2005), the stochastic game corresponding to perpetual convertible bonds was considered when driven by a Brownian motion and a compound Poisson process with exponential jumps. We consider the same stochastic game but driven by a spectrally positive L\'evy process. We establish a complete solution to the game indicating four principle parameter regimes as well as characterizing the occurence of continuous and smooth fit. In Gapeev and K\"uhn (2005), the method of proof was mainly based on solving a free boundary value problem. In this paper, we instead use fluctuation theory and an auxiliary optimal stopping problem to find a solution to the game.
and the Laplace exponent is given by the Lévy-Khintchine formula (1.1) where µ ∈ IR, b 2 ≥ 0 and Π is a measure on (0, ∞) called the Lévy measure of X and satisfies (0,∞) The reader is referred to Bertoin [5] and Sato [15] for a complete introduction to the theory of Lévy processes. Denote by T 0,∞ the family of all [0, ∞]-valued stopping times with respect to F. We are interested in establishing a solution to a special class of stochastic games which are driven by spectrally positive Lévy processes. Specifically, for α ≥ 0 and β, q, K > 0, let L t := e −qt+Xt + t 0 e −qs (α + βe Xs )ds, and U t := e −qt (e Xt ∨ K) + t 0 e −qs (α + βe Xs )ds.
We are interested in the stochastic game consisting of two players and expected pay-off given by for x ≥ 0. The inf-player's objective is to choose some σ ∈ T 0,∞ which minimizes (1.2), whereas the sup-player chooses some τ ∈ T 0,∞ which maximizes this quantity. We are principally interested in showing the existence of a stochastic saddle point (also known as Nash equilibrium cf. Ekström and Peskir [8]). That is, we want to find τ * and σ * such that M x (τ, σ * ) ≤ M x (τ * , σ * ) ≤ M x (τ * , σ) for all τ, σ. for any τ, σ, i.e. τ * = 0 and σ * = 0 form a Nash equilibrium whenever In what follows, we assume (A): ψ(−1) < q.
In that case, the Laplace exponent ψ is well-defined on [−1, ∞) and moreover the Lévy-Khintchine formula can be extended to the interval [−1, 0) (see for instance Lemma 26.4 in [15]). Without this condition the gain in the expectations in (1.2) is infinity on the event {τ = σ = ∞}.
Main results.
Below, in Theorems 1-4 we give a qualitative and quantitative exposition of the solution to (1.3). Before doing so, we need to give a brief reminder of a class of special functions which appear commonly in connection with the study of spectrally positive Lévy processes. For each p ≥ 0 we introduce the functions W (p) : IR → [0, ∞) which are known to satisfy for all x, y ≥ 0, where τ + y := inf{t > 0 : X t > y} and τ − −x := inf{t > 0 : X t < −x} (cf. Chapter 8 of Kyprianou [10]). In particular W (p) (x) = 0 for all x < 0 and further, it is known that on (0, ∞) W (p) is almost everywhere differentiable, there is right continuity at zero and We make the very mild assumption that Π has no atoms when X has paths of bounded variation. This suffices to deduce (cf. [7]) that W (p) ∈ C 1 (0, ∞) and hence Z (p) ∈ C 2 (0, ∞) and further, if X has a Gaussian component they both belong to C 2 (0, ∞). It is also known that if X has bounded variation with drift d, then W (p) (0+) = 1/d and otherwise W (p) (0+) = 0. (Here and in the sequel we take the canonical representation of a bounded variation spectrally positive Lévy processes X t = S t − dt for t ≥ 0 where (S t , t ≥ 0) is a driftless subordinator and d is a strictly positive constant which is referred to as the drift). Further, when X has unbounded variation, which is understood to be +∞ when b 2 = 0. Consider the exponential change of measure Under P (λ) , the process X is still a spectrally positive Lévy process and we mark its Laplace exponent and scale functions with the subscript λ. It holds that for θ ≥ 0 and, by taking Laplace transforms, we find for p ≥ 0. The reader is otherwise referred to Chapter VII of Bertoin [5] or Chapter 8 of Kyprianou [10] for a general overview of one-sided Lévy processes and scale functions. It turns out that the solution to the stochastic game can fall in four different regimes, depending on the value of the discount factor q. We remind the reader of the standing assumption (A). Theorem 1. Suppose q ≤ α/K. Then a saddle point for the stochastic game (1.3) is given by .
In particular, , there is smooth fit at log a * (q) if and only if X has paths of unbounded variation and otherwise there is continuous fit.
is an interval whose infimum we denote by q 1 . In particular q 1 > α/K.
(ii) When b 2 > 0 and q ∈ [q 1 , q 0 ], a saddle point for the stochastic game (1.3) is given by In particular, (iii) When b 2 > 0 and q ∈ [q 1 , q 0 ] there is smooth fit at log K if and only if q = q 0 or q = q 1 .
has a unique solution in (−∞, log K) which we denote by c * (q).
(ii) A stochastic saddle point is given by the pair
In particular, for x < c * (q) and for x ≥ c * (q) V (x) = e x ∨ K.
(iii) There is smooth fit at c * (q) if and only if X has paths of unbounded variation and otherwise there is continuous fit.
The order in which we present these statements above (first q ≤ α/K, followed by q ≥ q 0 , q ∈ [q 1 , q 0 ] when b 2 > 0, and finally q ∈ (α/K, q 1 )) is convenient with regard to the dependency between their proofs. We also note that with the exception of Theorem 3, the conclusions with regard to smooth versus continuous fit are consistent with existing results in the literature which generally exhibit smooth fit at boundary points of the stopping region if and only if that point is regular for the interior of the stopping region (see for example [1] and [13]). In our case, thanks to stationary and independent increments of X, this boils down to the point 0 being regular for (0, ∞), which also corresponds to the case that X has unbounded variation for the special case of spectrally positive Lévy processes.
The remainder of this paper is dedicated to proving these theorems and is structured as follows. In the next section we state and prove a Lemma which will be repeatedly used to implement proofs on the basis of 'guess and verify' such as is common with solving optimal stopping problems. Thereafter we prove the four main theorems above in the order that they are stated.
Guess and verify
Following classical ideas in optimal stopping, we verify that a candidate solution solves the stochastic game (1.3) by checking certain associated bounds and martingales properties. Specifically, we use the following verification lemma which is of a similar form to Lemma 5 in [3].
Lemma 1 (Verification Lemma). Fix x ∈ R. Suppose that τ * ∈ T 0,∞ and σ * ∈ T 0,∞ are candidate optimal strategies for the stochastic game (1.4) such that has finite mean under P x . Let is a right continuous supermartingale.
Proof: Define H q τ,σ := L τ 1I {τ <σ} + U σ 1I {σ≤τ } , where τ and σ are stopping times. Since we have assumed that q > ψ(−1) ∨ 0, we have that From the supermartingale property (vi), Doob's optional sampling theorem, (iv) and (i) we know that for any stopping time τ and t ≥ 0, It follows from Fatou's lemma by taking t to ∞, that Now using (v), Doob's optional sampling theorem, (iii) and (ii) we have for any stopping time σ and t ≥ 0, Taking limits as t goes to ∞ and applying the monotone convergence theorem for the first two terms on the right-hand side and the dominated convergence theorem for the last term on the right-hand side (see (3.9)), we have and hence (τ * , σ * ) is a saddle point to (1.3).
Proof of Theorem 1
Suppose q ≤ α/K. We claim that the process (Z t , t ≥ 0) defined by showing that, as β > 0, Z is an adapted, strictly increasing process, i.e. a submartingale.
We may now invoke the Verification Lemma, since the other properties are automatically satisfied by taking σ * = 0. Note in particular that the condition (3.9) is automatically satisfied since
Proof of Theorem 2
The basis of the proof of Theorem 2 is the assumption that the optimal strategies take the form σ * = inf{t > 0 : X t > log K} and τ * = inf{t > 0 : X t > y * } for some optimally chosen y * . On this basis, establishing the value function in the Stackelberg equilibrium, V , would boil down to computing H y * where for any −∞ < y ≤ log K, H y (x) := E x L τ + y , that is to say, We thus proceed by evaluating the above expression in terms of scale functions, then we choose the value of y * by blindly applying the principle of smooth and continuous fit respectively to the cases that X has paths of unbounded and bounded variation and finally we verify that the established strategy is indeed optimal with the help of the Verification Lemma.
With the help of the exponential change of measure, (2.8), and (2.6), the first term of the right-hand side of the above expression for H y satisfies On the other hand from Theorem 8.7 in [10], the second term of the right-hand side of (5.10) satisfies where P denotes the law of the dual process X = −X. Finally noting that We also see in particular, making use of the fact that W (q) (0−) = 0 and Z (q) (0) = 1, that for all x > y.
Having expressed H y in terms of scale functions, we now turn our attention to making the choice of y * using the principle of smooth and continuous fit.
Bounded variation and continuous fit: In this case it is known that W (q) (0+) > 1/d where d > 0 is the drift term of the process X. It follows that In order to avoid a discontinuity at a we choose it equal to the value a * which satisfies Note that this is equivalent to requiring that providing q > ψ(−1) + β. In order to respect the requirement that a * ≤ K we also need to check how the function a * = a * (q) varies with q. To this end, note that hence a * (·) is strictly decreasing. Note also that lim q↓β+ψ(−1) a * (q) = ∞ and lim q→∞ a * (q) = 0, which implies the existence of a unique q 0 > β + ψ(−1) such that a * (q 0 ) = K. Note that it also turns out that q 0 > α/K on account of the fact that for q ≤ α/K where in the equality we have appealed to the well-known identity for one of the Wiener-Hopf factors of X (cf. Chapter 8 of [10]).
Unbounded variation and smooth fit: In this case it is known that W (q) (0+) = 0 and hence in the above analysis one sees that H log a (log a−) = a = H log a (log a+). In that case, the principle of smooth fit can be implemented and we insist on there being no discontinuity in H ′ log a at log a. We have Recall that W (q) (0+) = 0 and that W (q)′ (0+) = 2/b 2 which should be interpreted as +∞ in the case that the Gaussian coefficient b 2 = 0. We find In order to obtain the smooth fit H ′ log a (log a+) = a we must thus have that which, after a simple integration on the right hand side, gives the same expression of a * as in the bounded variation case. The same bounds on q 0 are thus still applicable in this case too.
In both cases, we obtain our candidate value function We now proceed to verify our candidate solution when q ≥ q 0 . That is to say, we shall verify that fulfill the conditions of the Verification Lemma. Note in particular that τ * ≤ σ * .
Submartingale and supermartingale properties: To this end, note that (5.10) together with an application of the Markov property gives us for all t ≥ 0, That is to say, Λ = (Λ t : t ≥ 0) is a martingale. This confirms the submartingale property (v) in the Verification Lemma. An easy computation shows that and hence V * belongs to C 2 (−∞, log a * ). Moreover, the latter conclusion is sufficient to show that ΓV * (x) is continuous on (−∞, log a * ) where Γ is the infinitesimal generator of X, and in particular, (See for example the argument in Lemma 4.1 of [11]). For any −n ≤ x ≤ a < log a * , where n ∈ N, the aforementioned facts concerning smoothness and continuity allow us to apply Itô's formula to Λ, but stopped at τ − −n , where τ − −n = inf{t > 0 : X t < −n}, and deduce that is a local martingale such that B is the Gaussian component in X and X (1) is the martingale part of X consisting of compensated jumps of size strictly less than unity. In fact, thanks to the boundedness of V * ′ and ΓV * on [−n, a], the process {m t : t ≥ 0} is a martingale. The latter, together with the fact that Λ is a martingale, implies that the drift term in (5.15) must almost surely be equal to zero. Taking expectations and writing R (q) (x, dy; a, −n) for the q-resolvent measure of the process X when issued from x and killed on first entry into (−∞, −n) ∪ (a, ∞) we have for all −n ≤ x ≤ a < log a * , [(Γ − q)V * (y) + (α + βe y )]R (q) (x, dy; a, −n) = 0.
As R (q) (x, dy; a, −n) is absolutely continuous with respect to Lebesgue measure with a strictly positive density in (−∞, 0) (cf. Chapter 8 of Kyprianou [10]) it follows that for Lebesgue almost every x < log a * . The latter can be upgraded to every x < log a * as the left hand side of (5.16) is continuous. It is also trivial to check that V * (x) = e x on (log a * , ∞) and hence it follows from q > ψ(−1) + β that Next, note that it is straightforward to see that V * is twice continuously differentiable on (−∞, log a * ) ∪ (log a * , ∞) with the existence of a left and right derivative at log a * . We may thus apply the Meyer-Itô formula (cf. Theorem 70 of Protter [14]) to the process V (X t∧τ + log K ) and then integrate by parts to obtain, in a similar vein to (5.15), that where M := (M t : t ≥ 0) is a local martingale and ℓ := (ℓ t : t ≥ 0) is the semi-martingale local of X at log a * . Note that when b 2 = 0, the final integral is identically zero owing to the fact that the local time process ℓ is also identically zero and otherwise, when b 2 > 0, the final integral is still identically zero thanks to smooth pasting. Note also that although the quantity (Γ − q)V * (x) + (α + βe x ) is not defined at x = log a * , this is not a problem in the context of the above calculus as the Lebesgue measure of the time that the process X spends at log a * is zero.
Recalling that (Γ−q)V * (x)+(α+βe x ) ≤ 0 on (−∞, log a * )∪(log a * , ∞), by taking expectations with the help of a suitable localizing sequence of stopping times {T n : n ≥ 1} for M, Fatou's lemma and monotone convergence, we obtain The last inequality above together with the Markov property is sufficient to deduce the supermartingale property (vi) in the Verification Lemma. Note that right continuity follows immediately from the continuity of V * and the fact that X has càdlàg paths.
Lower and Upper bounds: The bounds (i) and (ii) in the Verification Lemma can be deduced directly from the expression for V * . To this end, write Note that g(0) = 0. Since V * (x) = e x for all x ≥ log a * , we have the required lower bound for V * if we can prove that g ′ (z) > 0 for all z > 0. To this end we differentiate and find that Finally, to show that g ′ (z) > 0 we note from (8.20) of Kyprianou (2006 where e p is an exponentially distributed random variable which is independent of X and has parameter p. For the upper bound on V * it suffices to show in a similar vein to the lower bound that V * ′ (x) ≥ 0. Calculations in the spirit of the ones above show that where we have made use of (5.12).
Stopped values: Note that since V * (x) = e x for x ≥ log a * both conditions (iii) and (iv) are automatically satisfied.
Having now checked properties (i)-(vi) of the Verification Lemma, and noting that the justification for (3.9) is the same as in the proof of Theorem 1, we may conclude that the proposed triple (τ * , σ * , V * ) is a stochastic saddle point.
Proof of Theorem 3
The proof of Theorem 3 relies on the following optimal stopping problem. Recall that for q > 0 U t = e −qt (e Xt ∨ K) + t 0 e −qs (α + βe Xs )ds. (6.17) Then w has the following properties,
is a right continuous submartingale and (ix) the process
is a right continuous supermartingale.
Then the process Z := (Z t , t ≥ 0) given by is Markovian and starts from (0, 0, x) under the measure P x . For (t, i, x) ∈ IR 2 + × IR denote by P (t,i,x) the law of Z when it started at (t, i, x). Thus the optimal stopping problem (6.17) reads as follows where F (t, i, x) = e −qt (e x ∨ K) + i. Since F : IR 2 + × IR → IR + is continuous and X * is quasi-left continuous we can deduce that w is upper semicontinuous. Furthermore, we have so we can apply a variant of Theorem 3.3 on p.127 of Shiryaev [16] (see also Corollary 2.9 on p.46 of Peskir and Shiryaev [13]) to conclude that , is an optimal stopping time. Note that for all (t, i, x) ∈ IR 2 + × IR, the following identity holds and thus we deduce that D = {x ∈ IR : w(x) = e x ∨ K} and τ D = τ + log K ∧ inf{t ≥ 0 : X t ∈ D}. In what follows, if ς is a stopping time for X we shall write ς(x) to show the dependence of the stopping time on the value of X 0 = x. Similarly, we denote for all t ≥ 0 and thus, also appealing to the definition of w as an infimum, which implies that w non-decreasing. (ii) This property follows directly from the definition of w as an infimum and taking for instance the stopping time σ = 0. (iii) Recall that w is upper semicontinuous. Thus the set is open. From (ii), we deduce that C = D c and therefore D is a closed set. The fact that w is non-decreasing and that D is a closed set implies that there exists a c * ≤ K such that D = [c * , ∞).
In that case τ D = τ + c * . (iv) We first note that from the definition of w as an infimum, we have for some constant K(c * , β) > 0 which depends on c * and β. Therefore, using part (i), we deduce that w is continuous and moreover that w(c * ) = K.
(v) In what follows, for q > 0, it is convenient to denote the function w by w(x, q) and U t = U t (q) for all t ≥ 0. Note that for any t ≥ 0, U t (q) is non-increasing in q. Hence, On the other hand, recall from Theorem 2 that when q = q 0 , a saddle point for the stochastic game (1.3) is given by τ * = σ * = τ + log K , and in particular the value function satisfies Therefore, appealing to the definition of V as an infimum and using the lower bound on the solution to Theorem 2, we have (vi) and (vii) These are trivial statements.
(viii) and (ix) These are standard results from the theory of optimal stopping. See for example Theorem 2.2 on p.29 or Theorem 2.4 p.37 of Peskir and Shiryaev [13].
According to the previous Lemma and the Verification Lemma, a stochastic saddle point of the Kühn-Gapeev game exists and is given by τ * = τ + log K and σ * = τ + c * , for a given c * ≤ log K. (Note that the condition (3.9) is dealt with in the same way as before). Therefore the associated value function is given by The proof of Theorem 3 is thus complete as soon as we can characterize c * as given in the statement of the theorem. Suppose that b 2 > 0. Our objective is to show that τ * = σ * = τ + log K is the stochastic saddle point provided q is smaller than q 0 but not too small (to be made precise below). We again do this with the help of the Verification Lemma.
We show that c * = log K if and only if H ′ log K (log K−) ≥ 0. Note that from (5.13) we find that where we have used the fact that W (q) (0+) = 0 and W (q)′ (0+) = 2/b 2 when b 2 > 0 (cf. Chapter 8 of Kyprianou [10]). Taking account of the monotonicity of H log K (x, q) in q this implies that those q ∈ (0, q 0 ) for which form an interval the left end point of which we shall denote by q 1 . First consider q > q 1 . It then holds that H ′ log K (log K−) > 0 and hence H log K (x, q) < H log K (log K, q) = K for x ∈ [log K − ε, log K) for some ε > 0. Now any choice of c * < log K would imply H log K (x, q) < w(x) for some x < log K, since w(x) = K for all x ∈ [c * , log K]. This leads to an immediate contradiction due to the fact that τ + log K is a feasible strategy for the optimal stopping problem (6.17). We conclude that for q > q 1 we have that c * = log K.
Next, we show that c * = log K also in the case when q = q 1 . For any q > q 1 it holds that H log K (x, q) ≤ K ∨ e x for all x and thus we find that H log K (x, q 1 ) ≤ K ∨ e x due to continuity of H log K (x, q) in q.
Furthermore, note that is a martingale for q ∈ (q 1 , q 0 ), as it is both a submartingale and a supermartingale due to items (viii) and (ix) of Lemma 2. From monotone convergence it follows that (6.19) is also a martingale when q = q 1 . Next, we show that q 1 > α/K. It seems unclear how to prove this inequality directly using the definition of q 1 and instead we argue by contradiction, hence suppose that q 1 ≤ α/K. Due to monotonicity in q in the definition of w and Theorem 1 it would then follow that K ∨ e x ≥ w(x, q 1 ) ≥ V (x, α/K) = K ∨ e x for all x. Hence in this case is a martingale. Recall from the proof of Theorem 1 however that when x < log K, the process above is strictly increasing. We get a contradiction with the martingale property and thus conclude that q 1 > α/K. From (6.18) it is clear that smooth pasting can only occur when H ′ log K (log K−) = 0 or K. This occurs precisely at the end points of the interval [q 1 , q 0 ]. We conclude the proof by noting that when b 2 = 0, by considering (5.11) and (5.13) with a = log K and recalling that W (q) (0+) > 0 if X has bounded variation and W (q)′ (0+) = +∞ if X has unbounded variation, the strategies τ * = σ * = inf{t ≥ 0 : X t > log K} do not constitute a stochastic saddle point when q < q 0 as otherwise the necessary upper bound, K ∨ e x on the value function V will not be respected.
Proof of Theorem 4
The proof of Theorem 4 again relies on the optimal stopping problem introduced in the previous Section. Assume that α/K < q < q 1 , which is possible thanks to Theorem 3.
Let us first address the issue of continuous and smooth fit. We know from Lemma 2 that the value function V is always continuous and hence in particular there is always continuous fit at the point c * . Note that necessarily c * < log K as otherwise c * = log K and then from the previous theorem, q = q 1 which is a contradiction. As we shall see, this will be sufficient to uniquely characterize the value c * in the case that X has paths of bounded variation. When X has paths of unbounded variation, consistently with prior experience, continuous fit is not enough and the following lemma will be needed instead. Proof: Thanks to monotonicity of the value function we know that V (x) ≤ V (c * ) for all x ≤ c * and hence lim inf The proof is thus complete as soon as we show that lim sup which is o(ǫ) on account of the fact that W (q) is monotone increasing with W (q) (0+) = 0 (the latter is due to the assumption that X has paths of unbounded variation). Taking this into account and combining the inequalities (7.21) and (7.22) we get Lemma 11 in Baurdoux and Kyprianou [3] states that lim sup ǫ↓0 W (q) (2ǫ)/W (q) (ǫ) ≤ 2 and hence the expression in the brackets on the right-hand side above is o(ǫ). This in turn implies (7.20) and hence the proof is complete. (7.23) and note that for x ≥ c, we have G c (x) = e x ∨ K.
We may now put the features of continuous and smooth fit to use and characterize the value of c * . Our immediate aim is to give and explicit form of G(x), for x < c, in terms of scale functions and the characteristics of X. We first note that the integral on the right-hand side of (7.23) has been computed before and is equal to The first term on the right-hand side of (7.23) satisfies Recall that P denotes the law of X = −X. By Theorem 8.1 in [10], we get that the first term on the right-hand side of (7.24) satisfies Now using the exponential change of measure (2.8) with λ = Φ(q), we write the second term in the right-hand side of (7.24) as follows Let f (y) = e Φ(q)y e x+y − K 1I {y+x>log K} . From Theorem 4.4 in [10] and since x < c ≤ log K, we deduce where R Φ(q) (z, dy; 0) plays the role of R(z, dy; 0) but under the measure P Φ(q) z . Therefore, by Corollary 8.8 in Kyprianou [10] we get Finally, putting the pieces together, using in particular that Π Φ(q) (dx) = e −Φ(q)x Π(dx) and W Φ(q) (x) = e −Φ(q)x W (q) (x), we obtain the following formula for G c (x), when x < c, Now that we have an expression for G c we may find the one which corresponds to the optimal solution by choosing c = c * so that there is smooth or continuous fit accordingly with the path variation of X.
Bounded variation case: In this case we know that W (q) (0+) = 1/d > 0. Hence, checking for a discontinuity at c we find that It is important to note that f (z) ∼ z 2 as z → 0, and f (z) ∼ 1 Φ(q) + 1 e z as z → ∞, thus from the hypothesis (A) and the fact that Π is a Lévy measure, we have So, we take In order to show that this expression has a unique solution, it is more convenient to note from (7.25) that on account of the assumption that q > α/K. Moreover, as in the case of bounded variation paths, and ψ(Φ(q)) = q, we may compute from (7.25) where the strict inequality follows from the fact q < q 1 = q 0 . Thus, we get the existence of the unique solution if we prove that G · (·−) is continuous and increasing in (−∞, log K]. The continuity of G . (.−) follows from (7.25) and the fact that when the measure Π has an atom at log K − c, the integrand on the right-hand side of (7.25) is equal to 0 at z = 0. Now, note that f ′ (z) = 1 Φ(q) + 1 (e z − e −Φ(q)z ) > 0 for all z > 0, which implies that f is positive and increasing. Then from (7.25) it is clear that G . (.−) is increasing in (−∞, log K]. Unbounded variation case: In this case W (q) (0+) = 0 and hence in the above analysis one sees that G c (c−) = K = G c (c+). In that case, the principle of smooth fit can be implemented and we insist on choosing c such that there is no discontinuity in G ′ c (c−).
Recall that W (p) ∈ C 1 (0, ∞) and let x < c. Therefore, using a standard argument involving dominated convergence to differentiate through the integral in the last term of G c (x), we have × e Φ(q)(u+y) e c+u+y − K 1I {u+y+c>log K} .
Hence from (7.29) where the strict inequality follows from the fact q < q 1 (recall that q 1 = q 0 when b 2 = 0). The existence of the unique solution now follows from the continuity and the monotonicity of F which can be proved as in the bounded variation case. | 2009-04-24T13:41:50.000Z | 2009-04-24T00:00:00.000 | {
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118896348 | pes2o/s2orc | v3-fos-license | Reactive Hall constant of Strongly Correlated Electrons
The zero-temperature Hall response within tight-binding models of correlated electrons is studied. Using the linear response theory and a linearization in the magnetic field B, a general relation for the reactive (zero frequency) Hall constant in the fast (transport) limit is derived, involving only matrix elements between the lowest excited states at B=0; for noninteracting fermions, the Boltzmann expression is reproduced. For a Fermi liquid with a well defined Fermi surface and linear gapless excitations an analogous expression is found more generally. In the specific case of quasi-one-dimensional correlated systems a relation of $R^0_H$ to the charge stiffness D is recovered. Similar analysis is performed and discussed for D and the compressibility.
I. INTRODUCTION
Properties of metals with strongly correlated electrons can be strikingly inconsistent with the usual picture of ordinary Fermi liquid. The most intensively studied example are the superconducting cuprates [1,2], which behave in the normal state as hole doped magnetic (or Mott-Hubbard) insulators. Here an evident and challenging theoretical problem is to reconcile the Hall constant R 0 H , following approximately a simple semiclassical behavior R 0 H ∼ 1/n h e 0 consistent with an (semiconductor-like) interpretation of low concentration n h of holes in an insulator, with the evidence for large Fermi surface as it emerges e.g. from photoemission experiments.
The theoretical analysis of the Hall constant R 0 H and more generally of the dynamical Hall response R H (ω) in systems with correlated electrons has proved to be very difficult. Within the general linear response theory the procedure for the calculation of Hall response is in principle well established [3] and requires the introduction of a modulated magnetic field B exp(iqr) and consequently a modulated vector potential A in order to formally allow a linearization of the offdiagonal conductivity σ βα in B = 0 and to derive an expression for R H (q, ω) not involving B. At T = 0 the relevant case for usual transport measurements of d.c. R 0 H is the limit: q → 0 first, and then ω → 0 [3,4]. The formulation originally designed for nearly free lectrons has been extended to tight-binding models for strongly correlated electrons [5]. Nevertheless there have been so far rather few results for strongly correlated models obtained following this framework. The Hall mobility of a single carrier at large T has been evaluated [6]. R H (ω) has been calculated in the high-ω, T * Electronic address: peter.prelovsek@ijs.si † Electronic address: Xenophon.Zotos@epfl.ch expansion [5], indicating on the change of sign of R H in the vicinity of the Mott-Hubbard insulator. One of the present authors [7] also showed a plausible but nontrivial result that a single carrier doped into a magnetic insulator at T = 0 indeed follows the semiclassical formula for R 0 H . More direct numerical evaluation of R 0 H within the linear response approach (at finite B > 0) is also quite delicate for prototype models of correlated electrons. Namely, small-system studies give even some controversial conclusions regarding the sign of R 0 H close to the magnetic insulator [8,9,10]. It has been shown [11] that a better controlled approach at T = 0 can be obtained for a system with open boundary conditions in one direction, i.e. on a ladder geometry, where R 0 H can be expressed in terms of derivatives of the ground state energy with respect to external fields.
Recently, the present authors [12] derived at T = 0 a quite general relation between the reactive Hall constant R 0 H and the derivative of the charge stiffness D with respect to the electron density n, It should be pointed out that the derivation uses, in the direction transverse to the driving field, the non-standard (slow) limit ω → 0 first, then q → 0. It is a question and also our aim to find out whether or under which conditions this relation applies to the more relevant fast limit: q → 0, then ω → 0. Eq.(1) has some very attractive properties for the analysis of strongly correlated electrons: a) D is at T = 0 the central quantity distinguishing the Mott-Hubbard insulator from a conductor (metal), b) close to the Mott-Hubbard insulator where the stiffness is expected to be proportional to hole doping, i.e. D ∝ n h = 1 − n, Eq.(1) directly implies the plausible semiclassical result R 0 H = 1/e 0 n h , which has been hard to establish by any other analytical method so far.
A qualitatively similar relation to Eq.(1) was also proposed in [4], where σ xy was related to the variation of the kinetic energy, i.e. ∂ T /∂n. Note that in a tight binding system T and D are interrelated through the optical sum rule.
Our goal is to express the T = 0 reactive Hall constant R 0 H in terms of eigenstates of correlated electrons in the absense of B and the fast limit, q → 0 first and then ω → 0. The present approach is the extention of the previous analysis for a single carrier [7] to the general electron concentration n. As first formulated by Kohn [13], the diagonal conductivity in a metal is singular (reactive) at T = 0 and low frequencies, i.e. σ αα ∝ D αα /ω, defining the charge stiffness D αα . The concept of charge stiffness has been essential in the studies of correlated systems and is quite well established both by analytical and numerical methods in a number of relevant models [14].
On the other hand, in the same system at B 0 one also expects a singular off-diagonal conductivity σ α =β ∝ BΛ αβ /ω 2 , leading to a finite reactive R 0 H . Λ αβ (independent of B) is thus a central quantity of interest, playing the role of the off-diagonal charge stiffness. We find a general expression for Λ αβ in terms of low lying states at B = 0, and a more specific one for Fermi liquids with a well defined Fermi surface and charge excitations with linear dispersion. In the latter case we discuss the relation of the new formulation to Eq.(1) as well as to the standard Boltzmann theory for R 0 H in metals within the single relaxation-time approximation [15,16].
The paper is organized as follows: In Sec. II we introduce the linear response formalism for R 0 H . We perform the linearization in B and derive a general expression for Λ in terms of electron eigenstates. In Sec. III we treat in analogous way as a limit q → 0 the stiffness D 0 αα and the compressibility κ in order to demonstrate that in these quantities appear similar matrix elements as in Λ. We also discuss the Hall conductivity σ yx in the slow limit (ω → 0 first), thus relating it to the generalized D. In Sec. IV we deal with two specific cases: a single carrier in a Mott-Hubbard insulator and 1D systems. Sec. V is devoted to a derivation assuming a Fermi liquid system, obtaining an expression analogous to the one in the relaxation-time approximation. As the application of the analysis we discuss noninteracting fermions, an isotropic Fermi liquid, and in particular the quasi-1D system where we recover the relation (1).
II. DYNAMICAL HALL RESPONSE
For simplicity we consider only a two-dimensional system in the x − y plane, with a magnetic field perpendicular to the plane, i.e. B =Be z . To calculate the dynamical Hall constant R H (ω) we are looking on the response to a uniform electric current J = J x e x . We follow the standard linear response analysis [3], introducing a modulated magnetic field. Here we choose the modu-lation in the y direction with q = qe y (the final result in the limit q → 0 should be independent of the q direction) requiring also a modulated electric field with the same q, while the vector potential in the Landau gauge is The dynamic Hall response is given by [5] whereσ αβ , σ αβ denote the conductivities at B = 0 and at B = 0, respectively, higher order terms in B have already been neglected in Eq.(4); the hat will denote quantities in magnetic field from here on and we are interested in the limit q → 0, B → 0. Models for strongly correlated electrons are usually analysed within the tight binding framework H = T + H int where the magnetic field (flux) enters through the kinetic energy T via the Peierls phase, i.e.
Here it is meaningful to distinguish the phase due to a constant magnetic field B (which in principle can be large), i.e. θ ij = er ij · A(r = R ij ), and the small driving (time dependent field) φ ij (t) = er ij · φ(r = R ij , t) where r ij = r j −r i and R ij = (r i +r j )/2; the sum (ij) runs over pairs of sites. The (particle) current J can be defined as, whereĵ p α andτ p αβ refer to the paramagnetic current and stress tensor (diamagnetic contribution), respectively, both in the presence of finite A, The conductivity tensor at B = 0, as a linear response to φ 0 β (t), can be expressed as [3,5], where N is the number of cells (volume of the unit cell is assumed unity) and B denote averages at B = 0.
A. Linearization in B
In order to calculate R H (ω) we have to evaluateσ yx up to the linear term in B. Since τ q yx = 0, we need In a conductor at T = 0 the conductivities σ 0 αα are singular at ω = 0 [13] defining the charge stiffness D 0 αα , where σ reg αα (ω) is the regular part of the conductivity. In the same limit K xy (ω) is also expected to be singular leading to a finite R 0 so that we can express the reactive Hall constant as Performing now the linearization in the static vector potential A q we take into account the analogy to Eq. (6) and the coupling to the field, where j p α =ĵ p α (B = 0) and τ p αβ =τ p αβ (B = 0). Instead of a general formalism at T > 0 [3] we assume here explicitly T = 0 and we can expresŝ where |0 B ,Ê 0 ,Ĥ refer to B = 0. We consider only linear terms in A q thereforeÊ 0 = E 0 . Taking into account that we obtain with an analogous expression holding for K II yx .
B. Fast limit
It is helpful to express K yx (ω) in terms of eigenstates. Assuming that the ground state |0 has momentum Q it is convenient to separate excited states into sectors: |m with momentum Q, |m with Q − q, and |m with Q + q. Denoting ǫ = E − E 0 and using we can write [7] where and and analogous expressions forγm andδ m . Since we are interested in the limit q → 0 it follows anyway fromσ αβ (−ω) =σ * αβ (ω) thatγm = γm = real and δ m = δ m = real.
The fast limit corresponds taking first q → 0 and then ω → 0. In Eq.(18) the singular part ∝ 1/ω in this case emerges from the class of excited statesM which exhibit ǫm → 0 in the q → 0 limit. On the other hand δ m ,δ m terms are not contributing since ǫ m > 0 do not depend on q and remain finite in the limit q → 0. Λ yx can therefore be expressed as We note that (d −q αα )m n can also be represented as the matrix element of the stiffness operator d −q αα , provided that (j 0 α ) nn = 0. It is quite evident that the operatord −q is closely related to the charge stiffness since it follows from Eq.(8),(10) that D 0 αα = 0|d 0 αα |0 /N . Moreover, there are other more compact representations of (d −q xx )m 0 . We first note that it just the matrix element at B > 0, as follows directly from Eq.(14) by using the representation of |m B states and extracting the singular term at q → 0, The latter emerges from m B corresponding tom and the only term linear in B involves Eq.(23).
(d −q xx )m 0 can be as well interpreted as a derivative of the current matrix elements with respect to a uniform vector potential (fictitious flux) θ [13], coupled to the current as H ′′ = θ · j. By taking a derivative with respect to θ x , we obtain In the limit q → 0 we can replace ǫ l → ǫ l − ǫm for l > 0, and ǫl − ǫm → ǫl forl =m. Since (j 0 x ) 00 = 0 we can identify Eq.(25) with Eq.(19) except the terml =m, For a general correlated system we have thus expressed Λ xy and consequently R 0 H in terms of matrix elements involving only lowest excited statesm ∈M (in the absense of B) and their derivatives to a homogenenous flux. In addition we note that the second term in Eq.(26) does not contribute to Λ xy in several nontrivial cases as shown later: a) for a single carrier in a correlated system, b) for noninteracting fermions on a bipartite lattice with only nearest neighbor hopping.
III. CHARGE STIFFNESS AND COMPRESSIBILITY
Before proceeding with the approximations to Λ xy let us stress that also other quantities like the charge stiffness and the compressibility can be expressed solely in terms of the same excited statesm ∈M .
We first consider σ 0 αα for B = 0. D 0 αα can be expressed as hence D 0 αα is evaluated from the ground state energy E 0 (θ α ), which is the usual procedure.
Alternatively we can consider σ 0 αα as the limit q → 0 of σ q αα (direction of q here is arbitrary), In the case of q = 0 it follows τ 0 αα = χ q αα (0) (optical sum rule), so there is no (strictly) reactive term which would correpond to the singularity in Eq. (10). As in Sec. II we can then represent σ q αα in terms of eigenstates, We can nevertheless extract the reactive part as the singular part which behaves as 1/ω for q → 0. This again emerges from statesm ∈M and consistent with Eq.(10) we get Eqs. (27) and (30) define two alternative approaches to evaluate D 0 αα , where the first is the standard one. The equivalence of both is expected to give more insight into the excited states and matrix elements (j q α ) 0m . On the other hand we note that D 0 αα , Eq.(30), contains the same matrix elements as Λ yx indicating that both quantities as well as R 0 H are related. Closely related is also the generalized compressibility κ q . Let us consider a perturbation induced by the modulated chemical potential so that we deal with a hamiltonian H µ = H + µ q ρ q . Then we can express in analogy to D q αα , We also note the relation following from the conservation law for q → 0, so (j q ) 0m and (ρ q ) 0m are evidently related. From Eq.(32) it follows that (ρ q ) 0m ∝ q form / ∈M , so only states m ∈M contribute in the limit q → 0, In order to find a closer relation between Λ yx and the stiffness D αα as e.g. manifested in Eq.(1) let us consider now the stiffness D −q xx in a perturbed ground state [12], We can again evaluate Eq.(34) within the first order of the perturbation theory in H ′ = µ q ρ q and recognize the correspondence withσ yx as explicitly expressed in Eqs. (8), (9), (17) - (20). In fact it has been already shown [12] that Using Eqs. (8), (18) and decomposing 1/[ω(ω − ǫ m )] as in Eq. (17), we can rewritẽ taking into account that at q > 0 there is no singularity strictly at ω = 0, hence terms 1/ω should cancel. For ω = 0 we get It seems plausible that in the limit q → 0 in Eq.(38) only states with ǫm ∝ q contribute, i.e.m ∈M , so that The relation to Λ yx in Eq.(21) is evident and will be exploited later on.
IV. SPECIFIC CASES
A. Single charge carrier A nontrivial example of the above formalism is a single charge carrier i.e. a hole or an electron doped into a Mott-Hubbard insulator [7]. We have to assume only that the carrier behaves as a quasiparticle. I.e., excited states have a well defined effective mass, and The semiclassical result follows finally from Eq. (12), by inserting e = −e 0 . There remains to determine the sign of R 0 H , which should be plausibly positive for a holedoped insulator although this is not trivial to show analytically [7]. For a single carrier we also get for q → 0 (ρ q ) 00 = 1 which is an alternative requirement for a well defined quasiparticle. We note also that the second term in Eq.(26) does not contribute since (j 0 x )00 = 0 if x and y are symmetry directions of the D tensor.
B. 1D systems
Naturally one cannot discuss Hall effect and R 0 H in a strictly 1D system but it is instructive to consider relations which follow from our analysis for D 0 and κ 0 . We assume here that the correlated electron system behaves as a Luttinger liquid with gapless charge excitations characterized by a linear dispersion for q → 0, The counting of states |m is then as for electron-hole excitations in the normal Fermi liquid. Assuming that Eqs.(30),(33) behave regularly as q → 0, we can replace (j q ) 0m → j c and (ρ q ) 0m → r c . From Eq.(32) it follows also that j c = v c r c and we get (taking into account also the spin degeneracy), These expressions are in agreement with the phenomenology of the Luttinger liquids [17] where we can identify the renormalization factor r c with the density exponent K ρ = r 2 c . Although there is another gapless branch of spin excitations, we note that this does not enter the quantities as the charge stiffness D 0 and the charge compressibility κ 0 . Our analysis is at T = 0, it is easy to argue that for low T specific-heat coefficient we get
V. FERMI LIQUID
Let us now consider as an illustration of the above formalism a Fermi system characterized by gapless charge excitations with a linear dispersion (for q → 0), corresponding to electron-hole excitations and a Fermi surface k F . The states |m are then determined as electron-hole excitations in the normal Fermi liquid so at given q they are characterized by k ∈ FS q . For the general direction e β = q/q the states are given by k = k F +ke β and −q <k < 0, where the latter condition is satisfied only along half of the Fermi surface.
Assuming the fermionic character of such excitations (with spin), we can write the sums over excited states explicitly for the thermodynamic limit taking into account spin degeneracy and q → 0, again restricting our analysis to 2D systems.
Here we note that in general the operators j q α , τ q αβ , ρ q (at B = 0) can be represented as where v k = ∂ǫ k /∂k and τ k = ∂ 2 ǫ k /∂k∂k. In the following we can consider as a test noninteracting electrons with dispersion ǫ k , where the relevant excited states are |m = c † k−q/2,s c k+q/2,s |0 .
Let us first treat D 0 αα , Eq.(30), For q → 0 the result must be independent of q so it is plausible that depends only on k ∈ k F , and For noninteracting fermions the expression (52) is straightforward since we know from Eqs.(48),(49) that j α (k) = v α (k) = v α k . In the same way we can also argue that (ρ q ) 0m → r(k), which can be concluded from Eq.(31), Furthermore for noninteracting fermions we get (ρ q ) 0m = r(k) = 1.
Let us turn to the discussion of Λ yx . Matrix elements (d −q xx )m 0 within a Fermi liquid are (for chosen q direction) expected to depend only on k along the Fermi surface, so we can replace (d −q xx )m 0 → d xx (k) and Taking into account Eq.(26) and that the role of θ is to shift k we can also relate, Eq.(21) can therefore be written as At this stage we are unable to put also the second term in analogous form as the first one. Still the expression is very similar to the symmetric one, which is formally equivalent to the Boltzmann expression within the relaxation-time approximation [15,16]. A clear advantage of the symmetric expression is that the required xy symmetry Λ yx = −Λ xy is evident, since Eq.(57) can be represented as where S j is the area spanned by the vector j(k F ).
Again testing with the case of noninteracting fermions we note that in Eq.(26) the second term is therefore we reproduce the usual semiclassical expression [16] forΛ yx , Eqs.(57),(58), up to a constant (relaxation time) which anyhow cancels out in R 0 H , Eq. (12). It should be also reminded that for noninteracting fermions on a bipartite lattices with nearest neighbor hopping the term (59) vanishes since we have v α (k α ).
A. Quasi-1D systems
Let us assume a very anisotropic Fermi liquid with a dispersion large only in the x direction and consequently also a nearly flat Fermi surface with |k F | ∼ k 0 . It is plausible that for large anisotropy Eq.(54) can be decoupled as It follows also that Now we can use relations (36),(39) for a Fermi liquid in the limit q → 0, For a nearly flat Fermi surface we can replace d xx (k) ∼ d xx (k F ) and we get where we have also assumed that taking the derivative ∂D/∂µ is regular for q → 0. So finally we can express R 0 H as (e = −e 0 ), For a quasi-1D system we expect that r(k F ) ∼ r F , v(k F ) ∼ v F and therefore j(k F ) = r F v(k F ) and consequently A ∼ 1. Hence we have reproduced in this case the desired expression (1).
B. Isotropic Fermi liquid
Although the tight binding model, as introduced initially in Eq.(5), does not lead to an isotropic Fermi surface, an isotropic Fermi liquid can still be of interest for illustration and can also emerge in specific cases. We assume here that j(k) = j(k)e k and v(k) = v(k)e k , so that Eqs.(52),(58) lead to and where n F = k 2 F /2π is the effective density of electrons as determined by the volume of the Fermi surface.
VI. DISCUSSION
The theory of the Hall constant in systems with strongly correlated electrons is evidently a difficult subject. In spite of its relevance for the intensively investigated anomalous properties of cuprates, there has been so far no consensus on the behavior and even less in the appropriate formalism for an analytical evaluation of the R H (ω).
It is clearly an advantage to deal with the system at T = 0 since here the transport (reactive) Hall constant R 0 H is well defined but does not involve any scattering or dissipation. One could hope that such R 0 H remains a reasonable approximation for R H (T ) at finite but small T > 0. This is for example the case for normal metals and semiconductors where within the approximation of uniform (in k) but T dependent relaxation rate τ (T ) the latter cancels out and finally R H (T ) ∼ R 0 H . The central quantity in our approach for R H (ω) is the off-diagonal stiffness Λ yx which plays analogous role as the charge stiffness D αα in the diagonal optical conductivity σ αα (ω). We show in Sec. II that Λ yx can be expressed in terms of matrix elements involving solely lowest excited state (at B = 0) which is a conceptual and technical simplification, which is also well adapted for application to a broader class of Fermi liquid systems. For a Fermi system with a well defined Fermi surface and gapless electron-hole excitations we also find a formal correspondence (apart from some ambiguities with the second term in Eq.(56)) of the expression for Λ yx with the one in the relaxation-time approximation. The main difference in correlated system is that effective quantities v(k) and j(k) are not directly related.
From the general formalism in Sec. II it is clear that there is intimate relation between Λ yx and the matrix elements of the stiffness operator, Eqs.(22),(23). More directly we can express R 0 H with the D(n) itself in the case of quasi-1D correlated system, where we recover (under certain restrictions) the expression (1) derived quite generally in the slow limit (first ω = 0, then q → 0). The Hall response of such quasi-1D systems is of direct experimental interest, in particular recently in connection with the apparent controversies in 1D conductors [18] as well as with the striking vanishing of the Hall constant in the stripe phase of cuprates [19,20]. Since strongly correlated quasi-1D systems are not expected to be singular one can expect that the relation (1) remains qualitatively valid even for a broader class of strongly correlated electrons. | 2019-04-14T01:57:56.982Z | 2001-07-12T00:00:00.000 | {
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246478086 | pes2o/s2orc | v3-fos-license | Developmental Origins of Metaflammation; A Bridge to the Future Between the DOHaD Theory and Evolutionary Biology
Metabolic syndrome refers to obesity-associated metabolic disorders that increase the risk of type 2 diabetes, coronary diseases, stroke, and other disabilities. Environmental imbalance during the early developmental period affects health and increases susceptibility to non-communicable diseases, including metabolic syndrome, in later life; therefore, the Developmental Origins of Health and Disease (DOHaD) theory was established. According to the DOHaD theory, the hypothesis of the energy-saving ‘Thrifty Phenotype’ in undernourished fetuses is one of the well-accepted schemes as a risk of developing metabolic syndrome. This phenotype is evolutionarily advantageous for survival of the fittest in a hangry environment after birth, a strong selection pressure, but increases the risk of developing metabolic syndrome under an obesogenic diet according to the ‘Mismatch’ hypothesis. Increasing evidences support that chronic inflammation pathophysiologically connects obesity to metabolic disorders in metabolic syndrome, leading to the concept of ‘Metaflammation’. ‘Metaflammation’ in humans is proposed to originate from the evolutionary conservation of crosstalk between immune and metabolic pathways; however, few studies have investigated the contribution of evolutionary maladaptation to the pathophysiology of ‘Metaflammation’. Therefore, it is promising to investigate ‘Metaflammation’ from the viewpoint of selective advantages and its ‘Mismatch’ to an unexpected environment in contemporary lifestyles, in consideration of the principal concept of evolutionarily conserved nutrient sensing and immune signaling systems.
INTRODUCTION
Metabolic syndrome refers to the co-occurrence of cardiovascular risk factors, including obesityassociated metabolic disorders, such as insulin resistance, atherogenic dyslipidemia, and hypertension, and is now a global public health issue despite being initially reported in Western countries (1). The prevalence of metabolic syndrome was recently reported to be higher in the urban populations of some developing countries than in Western countries, which has, in turn, increased the prevalence of type 2 diabetes, coronary diseases, stroke, and other disabilities (2).
Numerous epidemiological and animal studies demonstrated that environmental disturbances in the early critical period have an impact on health and increase susceptibility to noncommunicable diseases, such as metabolic syndrome, in later life; therefore, the theory of Developmental Origins of Health and Disease (DOHaD) was established (3)(4)(5)(6). According to the DOHaD theory, one of the well-accepted proposals for the risk of developing metabolic syndrome is the hypothesis of the energysaving 'Thrifty Phenotype' in undernourished fetuses, which is evolutionarily advantageous for survival of the fittest in a starved environment after birth, a strong selection pressure, but increases the risk of metabolic syndrome under an obesogenic diet according to the 'Mismatch' hypothesis (7). This concept connects metabolic syndrome to the maladaptation of the evolutionarily acquired plasticity of metabolic regulation against the selection pressure of starvation.
Recent studies reported that chronic inflammation is pathophysiologically associated with obesity and metabolic disorders in metabolic syndrome; therefore, the concept of "Metaflammation" has been established (8,9). 'Metaflammation' in humans is proposed to originate from the evolutionary conservation of crosstalk between immune and metabolic pathways, for example, based on the composition of the fat body of Drosophila melanogaster (9). In the evolutionary history of humans, immune and metabolic crosstalk appeared to be associated, at least partly, with responses and/or adaptation to the selection pressures of infection and/or starvation; however, to the best of our knowledge, few studies have focused on its contribution to the pathophysiology of 'Metaflammation' in metabolic syndrome. On the other hand, according to the DOHaD hypothesis, offspring with the energy-saving 'Thrifty Phenotype' (10) are predisposed to metabolic syndrome under an obesogenic diet according to the 'Mismatch' hypothesis (7).
In this mini review, we introduce the relationship between the 'Thrifty Phenotype' and metabolic syndrome in the DOHaD scheme as well as that between 'Metaflammation' and evolutionarily conserved nutrient sensing and immune signaling systems. We also discuss the importance of investigating 'Metaflammation' from the viewpoint of selective advantages and its 'Mismatch' to unexpected modern environments in consideration of the DOHaD concept, in addition to the principal concept of evolutionarily conserved nutrient sensing and immune signaling systems.
THE 'THRIFTY PHENOTYPE' HYPOTHESIS IN THE DOHaD THEORY; STARVATION AS A SELECTION PRESSURE
Initial evidence to support the concept of DOHaD was the deterioration of health in adulthood of British small babies with low birth weight (11,12) and Dutch fetuses with undernourishment due to maternal starvation in World War II (13,14). Rapid infantile growth, presumably indicative of an abundant nutrient supply after birth, particularly with low birth weight, is also causatively associated with obesity and/or metabolic syndrome in later life (15)(16)(17)(18)(19). These findings suggest that the continuous trajectory from an undernourished environment during the fetal period to an excessive nutrient supply after birth specifically leads to metabolic disruptions in later life. Hales and Barker proposed the 'Thrifty Phenotype' hypothesis, in which the body size of fetuses is reduced as an adaption to an insufficient energy supply in utero through the acquisition of the permanent energy-saving phenotype, resulting in a low birth weight (10,20,21). The 'Thrifty Phenotype' in offspring is hypothesized to be advantageous for survival of the fittest in a starved environment because of low energy demands, but increases the risk of diabetes and/or obesity under an obesogenic diet (10,21) due to reduced insulin sensitivity, a predisposition to authentic and ectopic fat accumulation, and a lower respiratory oxygen quotient, which are risk factors for metabolic syndrome (20)(21)(22)(23).
Starvation is one of strongest selection pressures from an evolutionary viewpoint not only in humans, but also in animals (24). The 'Thrifty Phenotype' is acquired phenotypic plasticity that changes offspring into energy-saving individuals in response to the presence or absence of a starved environment after birth as an adaptation to the selection pressure of repeated starvation waves (25). Based on the long evolutionary history of humans with repeating periods of starvation, the recent era of an overwhelming food supply in developed and some rapidly developing countries is an evolutionary exception. Therefore, it is plausible that the developmentally acquired plasticity of the 'Thrifty Phenotype' with energy-saving metabolic regulation mismatches an environment with an excess energy supply, thereby increasing the risk of obesity and diabetes, the socalled state of metabolic syndrome.
Gluckman and Hanson proposed not only the 'Mismatch' hypothesis (7), but also the 'Predictive Adaptive Responses (PARs)' hypothesis (26,27). In the intrauterine setting, PARs primarily function to improve future fitness to expected conditions after birth, such as starvation, through evolutionarily acquired phenotypic plasticity for adaptation (26). The 'Mismatch' hypothesis indicates maladaptation to the unexpected environment of the new era, which increases susceptibility to non-communicable diseases in adulthood (7). These concepts indicate that the upstream risk of metabolic disorders is connected, at least partly, to the maladaptation of developmentally modified phenotypes by evolutionarily acquired plasticity, particularly in response to the expectation of starvation, a strong selection pressure (27,28).
EVOLUTIONARY ASPECT OF 'METAFLAMMATION'; EVOLUTIONARY CONSERVATION OF CROSSTALK BETWEEN IMMUNE AND METABOLIC PATHWAYS
The pathogenesis of obesity with various metabolic disorders is based on a close relationship between nutrient excess and the activation of the innate immune system in the majority of organs involved in energy homeostasis (8,9,(29)(30)(31). Increasing evidence indicates that inflammation occurs with obesity and may play a causative role not only in the development of insulin resistance and disruption of other aspects of energy homeostasis, but also in the augmentation of fat accumulation (9,29). The characteristics of obesity-associated chronic inflammation differ from other general inflammatory paradigms in that it involves tonic activation of the innate immune system, which has an impact on metabolic homeostasis, generally for a lifetime, and affects multiple organs, such as adipose tissue, the pancreas, liver, muscle, and brain (9,29). This led to the establishment of the concept of 'Metaflammation' (8,9). In addition to starvation, infection is a strong selection pressure in animals (32). The avoidance of these two major selection pressures through adjustments to nutrient and immune conditions has been the most important task for animals to survive for hundreds of millions of years (32,33). The strong relationship between nutrient sensing and immune signaling is rooted in their common evolutionary origins. For example, the hematopoietic system, adipose tissue, and liver are all organized in one functional unit in the fat body of D. melanogaster (8). This developmental heritage is responsible for the highly overlapping biological repertoire of these organs, their effects on metabolic and immune cells, and the close relationship between immune and metabolic response systems, which supports the concept of 'Metaflammation' from an evolutionary viewpoint (8,9). The fat body of Drosophila is capable of sensing both infectious and metabolic disturbances, and studies on Drosophila have provided important insights into highly conserved immuno-metabolic pathways in mammals (8,9). Accumulated evidence has also highlighted the crucial role of metabolic reprogramming in macrophage activation not only in immuno-metabolic pathways, but also in the pathophysiological concept of 'Metaflammation' (34)(35)(36). The infiltration of macrophages and also its associated immune cells into metabolic organs, such as the liver, brain, pancreas, and adipose tissue, is an important factor influencing the maintenance of tissue homeostasis as well as the pathogenesis of metabolic disorders (9). Tissue macrophages function as direct modulators of metabolism, for example, by inducing the polarization of macrophages towards a pro-inflammatory (M1-polarized) phenotype that blocks the effects of insulin (37), to which the contribution of epigenomic alterations (38) and macrophage-secreted products (39) has been demonstrated. It is important to note that many other immune cell types, including dendritic cells, mast cells, eosinophils, and lymphoid cells, may also be involved in metabolic tissue homeostasis and the control of glucose metabolism (9). Therefore, the evolutionary preservation of crosstalk between immune and metabolic pathways is one of the principal concepts of metabolic syndrome.
'METAFLAMMATION' IN THE DOHaD SCHEME
To the best of our knowledge, limited evidence is currently available to support a direct relationship between 'Metaflammation' and the scheme of DOHaD for the pathophysiology of metabolic syndrome. The liver and adipose tissues are representative organs of 'Metaflammation', where infiltration of immune cells as well as fat accumulation is frequently observed in metabolic syndrome (8).
We developed mice animal model of fetal undernutrition by maternal energy restriction, the offspring of which showed deterioration of fat deposit in the adipose tissue (40) and liver (41) on a high fat diet. Interestingly, the offspring also showed the significant infiltration of macrophages into the adipose tissue (40) and liver (41) (Figure 1); thus, we proposed this as a model of 'Metaflammation' in the DOHaD scheme. We also demonstrated that intrauterine undernutrition induced significant increases in endoplasmic reticulum (ER) stress markers in the fatty liver of adult pups (41), while the oral administration of the ER stress alleviator, tauroursodeoxycholic acid (TUDCA), markedly ameliorated macrophage infiltration and hepatic steatosis only in pups that experienced undernourishment in utero (41, 42) (Figure 1). Based on these findings, we propose the involvement of ER stress programming in the developmental origins of 'Metaflammation' (Figure 2). This speculation is consistent with recent findings showing the critical involvement of ER stress in the co-regulation of chronic inflammation and metabolic disorders (43)(44)(45).
Fetal-derived immune cells have been implicated in the development of immune diseases. Mass et al. proposed fetalderived immune cells as prime transmitters of the long-term consequences of prenatal adversity, namely, inflammatory, degenerative, and metabolic disorders, and, thus, are potential contributors to the DOHaD theory (46). Previous studies suggested the commitment of erythro-myeloid progenitors produced in the extra-embryonic yolk sac to the establishment of long-lasting immunological memory (36,(46)(47)(48). Yahara et al. proposed the involvement of erythro-myeloid progenitors in bone regeneration after birth (49). Wu et al. also reported the potential contribution of erythro-myeloid progenitors to homeostasis after birth (50). Nevertheless, the mechanisms by which the memory of tissue-resident macrophages, if actually present, is transferred to mature macrophages remain unclear.
DISCUSSION
The DOHaD theory is partly derived from retrospective epidemiological observations of susceptibilities to metabolic disorders in offspring that experienced maternal starvation during gestation, such as in the Dutch Famine in World War II (13,51) and the Great Chinese Famine (52,53). Since the basic structures of all organs are formed and basic cross-talk between organs is constituted during the embryonic and fetal stages, the 'Thrifty Phenotype' hypothesis of acquiring a permanent constitution of low energy consumption in order to adapt to the low nutrient supply in utero is plausible (10). The 'Thrifty Phenotype' is a type of evolutionarily acquired plasticity in metabolic regulation for humans to survive against the powerful selection pressure of cyclically repeating periods of starvation. However, the 'Thrifty Phenotype' mismatches an obesogenic diet and is causatively associated with diabetes, obesity, and associated metabolic disorders in developed and some rapidly developing countries; therefore, the 'Mismatch' hypothesis was proposed (7). The essential concepts of the 'Thrifty Phenotype' and 'Mismatch' hypothesis of the DOHaD theory involve the evolutionary acquisition of plasticity in nutrient sensing against starvation and its maladaptation to an unexpected modern environment ( Figure 2). Chronic low-grade inflammation has recently been proposed as a bridge between augmented fat accumulation and metabolic disorders, such as insulin resistance (29); therefore, the concept of 'Metaflammation' is now widely accepted (8,9). The concept of 'Metaflammation' is also based on evolutionary adaptation against the selection pressures of starvation and infection, i.e. nutrient sensing and immune signaling (8,9). The fat body of Drosophila is capable of sensing both infectious and metabolic disturbances and evolutionarily differentiated into adipose tissue, the liver, and immune cells in mammals; therefore, this mutual functional control mechanism has been preserved between immune cells and the representative organs of adipose tissue and the liver (8,9). A similar mutual regulatory mechanism with immune cells has also been proposed in other organs, such as the pancreas and brain, in the concept of 'Metaflammation' (9). Therefore, a similar evolutionary trajectory of nutrient sensing and immune signaling underlies the 'Metaflammation' concept ( Figure 2).
The 'Thrifty Phenotype' is hypothesized to be advantageous for survival of the fittest in a starved environment; however, to the best of our knowledge, there is limited evidence to support the contribution of 'Metaflammation' to survival against infection and/or starvation. The contribution of the potentially long-lasting memory of erythro-myeloid progenitors to the risk of specific diseases in later life, but not to overall survival, has been investigated (36,(46)(47)(48). However, we cannot deny the possibility of some unidentified host survival advantage to chronic inflammation or low-grade inflammatory responses incapable of pathogen elimination due to its preservation throughout evolution (Figure 2). Although the involvement of crosstalk between immune and metabolic pathways in the acquisition of the 'Thrifty Phenotype' in the DOHaD scheme has not yet been elucidated, the evolutionary conservation of this crosstalk has been suggested to contribute to the maintenance of homeostasis in individual organs and is presumably associated with the significant accumulation of fat deposits concomitant with metabolic disruption in metabolic syndrome ( Figure 2).
Our mouse model revealed that undernourishment in utero significantly enhanced the infiltration of macrophages into adipose tissue (40) and the liver (41) (Figure 1) only in mice fed a high-fat diet, and this was concurrent with the deterioration of metabolic disorders. We previously reported that this mouse model of undernourishment in utero partly represented the 'Thrifty Phenotype' due to low levels of diet-induced thermogenesis and a predisposition to obesity (54). The findings of these animal studies strongly suggest that a 'Mismatch' to an obesogenic diet in 'Thrifty Phenotype' offspring is causatively associated with a malfunction or imbalance in immunometabolic crosstalk, namely, 'Metaflammation', particularly under an obesogenic diet ( Figure 2). Therefore, a more detailed understanding of the fundamental pathophysiology of 'Metaflammation' is needed to clarify plasticity in the memory of tissue-resident immune cells, such as macrophages, from the viewpoint of selective advantages and its mismatch to an unexpected new environment, in addition to the principal concept of evolutionarily conserved nutrient and immune sensing systems. ER is a major site in cells for protein folding and trafficking and ER malfunctions, such as ER stress, promote the unfolded protein response and activate various stress signaling pathways (43,45). Previous studies proposed roles for ER stress in the common upstream regulators of immune and metabolic functions in 'Metaflammation' (43,45). In our mouse model of the 'Thrifty Phenotype', the oral administration of the ER stress alleviator, TUDCA, to pups significantly ameliorated the infiltration of macrophages in the liver only if they experienced undernourishment in utero (41) (Figure 1). These findings suggest the importance of the regulation of ER stress as a promising research target upstream of developmentally induced 'Metaflammation' (Figure 2).
On the other hand, functional 'Trade-off' for adapting to the environmental disruption is also an important concept of the DOHaD theory (6). It is known that the immune function of hibernating animals is suppressed during the hibernation period when a large amount of fat is stored (55), suggesting a possible presence of a kind of 'Trade-off' between fat accumulation and immune activation for the purpose of adapting to the cyclical transitions between hibernation and activity periods. Since coordinate regulation of nutrient and immune functions is a key concept of 'Metaflammation', it might be a clue for understanding the pathogenesis of 'Metaflammation' from DOHaD theory, to investigate a possible 'Trade-off' in 'Metaflammation' between nutrient sensing and immune signaling systems in response to the environmental diversity.
In conclusion, in consideration of the 'Thrifty Phenotype' and 'Mismatch' hypothesis in the DOHaD theory, a promising research target is 'Metaflammation' from the viewpoint of selective advantages and its mismatch to an unexpected modern environment, in addition to the principal concept of evolutionarily conserved nutrient sensing and immune signaling systems.
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. | 2022-02-03T14:20:49.087Z | 2022-02-03T00:00:00.000 | {
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220265443 | pes2o/s2orc | v3-fos-license | Filtering of stationary Gaussian statistical experiments
This article proposes a new filtering model for stationary Gaussian Markov statistical experiments, given by diffusion-type difference stochastic equations.
Stationary statistical experiments
The statistical experiment (SE) is defined as the averaged sums: in which the random variables δ r (k), 1 ≤ r ≤ N, k ≥ 0, are equally distributed and independent for each fixed k ≥ 0, which take binary values 0 or 1.
In this case, the random amount S N (k), k ≥ 0, describes the relative frequencies of presence of the attribute A in a sample of fixed volume N at each time instant k ≥ 0. Introducing the normalized fluctuations ζ N (k) := √ N(S N (k) − ρ), where ρ be the equilibrium of SE [1,2], one gets an important representation of SEs.
The next theorem [5] gives the necessary and sufficient conditions of the stationarity, in wide sense, of the DMD (2). Theorem 1. (Theorem on stationarity). The DMD (2) is a stationary random sequence in wide sense if and only if the following relations take place: Now consider a stationary, in wide sense, two-component random sequence ζ(k), ∆ζ(k + 1) , k ≥ 0, with the following joint covariances: In filtering problem of Gaussian stationary DMD, the equivalence formulated in the following theorem (see [5]) is essentially used.
Proof. By Theorem on normal correlation [6, Th. 13.1], one has: Hence Considering the martingale-differences Let's calculate its first two moments.
E ∆W (k + 1) Now it remains to prove that, the stochastic part covariations are: Suppose for determination, that r < k. Using the Markov property of the sequence (ζ(k), k ≥ 0, and the relation (9), one obtains: Theorem 2 is proved.
Filtering of discrete Markov diffusion
The filtering and extrapolation problem is considered by many authors (for ex., [7,8]. In our constructions of the new filter, we proceed from the following basic principle: the presence of two normally distributed random sequences implies the presence of their covariances, which contain information about the filtering. The task is to estimate the unknown parameters of a stationary Gaussian Markov signal process α(k) by using the trajectories of the signal (α(k), ∆α(k +1)), and a stationary Gaussian Markov filtering process (β(k), ∆β(k + 1)), k ≥ 0.
The signal with unknown parameters is determined by the next equation: The filtering process -by the equation: It is known that the best estimate (in the mean square sense) of the signal (α(k), ∆α(k + 1)), by observing the filtering proccess (β(k), ∆β(k + 1)), coincides with the conditional expectation The next calculation of filtering matrix Φ Φ β , determined by the conditional expectation (15), essentially uses Theorem 2 on equivalence and is based on Theorem on normal correlation by Liptser and Shiryaev.
Theorem 3. The estimate (15) is determined by the filtering equation with the filtering matrix where Corollary 1. The interpolation of the signal α(k) by observing the filtering process β(k) is Corollary 2. One has the following statistical parameter estimation: Corollary 3. One has the following statistical parameter estimation: Proposition 1. Under the assumption of mutual uncorrelatedness (24), the statistical estimates (19) 1 -(20) are unbiased and strongly consistent, as T → ∞.
Proof of Theorem 3. By the Theorem on normal correlation [6, Th. 13.1], the filtering matrix introduced in (16) has the following form: where and the covariances are defined as follows: Under the assumption of mutual uncorrelatedness the equations (13) -(14) imply the following representations: So the matrix has the following inversion: Now let's calculate the elements of the filtering matrix By (21), taking into account (27), one has: 1 For the filtering parameter estimation see the Section 4.
The filtering error
Let's denote the filtering mean square estimation error By stationarity of the processes α(k) and β(k), k ≥ 0, we shall skip the parameter k where it is considered possible and convenient. By the normal correlation theorem [6, Theorem 13.3], the mean square error of the filtering is expressed as the trace of the following error matrix: Let's denote F β k the natural increasing sequence of σ-algebras of events, generated by the trajectories of the filtering DMD β(k), k ≥ 0. Then the elements of error matrix Γ Γ are defined as: the covariation matrix R αβ is defined in (20) and the term Φ Φ β is defined in formula (33). Let's calculate the term Φ Φ α .
The filtering empirical estimation
In real physical observations, the condition of mutual uncorrelatedness (24) is practically not satisfied. Therefore, the covariance characteristics (24) should be taken into account in the covariance analysis of filtering, if they are nonzero. The corresponding correction terms are subject to estimates, based on the filtering equation (16).
(51) Now our task is to express the filtering matrix in the terms of empirical covariances. By the definition (43) one has Taking into account the relation d T β = E(R T β ) 2 = σ · R T β , one obtains: which one can rewrite in the matrix form as The empirical matrix representation (54) contains two terms. The first addendum defines the filtering matrix under conditions of uncorrelatedness (24) of the stochastic components of signal and filter. The second addendum defines additional statistical estimates, generated by the correlation of the stochastic components of signal and filter. | 2020-02-27T09:26:34.211Z | 2019-10-21T00:00:00.000 | {
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257000922 | pes2o/s2orc | v3-fos-license | Oral Manifestations Associated with COVID-19 Infection: A Cross-Sectional Study of Recovered Iraqi Patients
Aims The aim of this study was to determine prevalence of oral manifestations related to COVID-19 infection among a sample of recovered patients in the Basrah province of Iraq. Methodology. This cross-sectional study included a total of 574 individuals from Basrah city, Iraq (196 males and 378 females), who had been previously infected with COVID-19. A questionnaire was developed and used to record the demographic data, medical history, severity of respiratory infection followed by hospitalization along with oral signs and symptoms that occurred during the COVID-19 infection and their persistence after recovery. Results Oral manifestations were reported in 88.3% of the studied sample. The most common oral manifestation was ageusia (66.8%), followed by dry mouth (59%), gustatory changes (46%), dysphagia (40.5%), burning sensation (20.8%), oral ulceration (14.5%), and gingival bleeding (3.3%). The findings suggested that ageusia was the only symptom that persisted following recovery from the COVID-19 infection. The results showed a significant statistical correlation between the incidence of oral manifestations and the severity of COVID-19 infection followed by hospitalization. A significant correlation was also found between the age groups and COVID-19 oral manifestations, whereas no significant statistical relationship was observed between gender, smoking, and systemic diseases. Conclusions COVID-19 infection has considerable impacts on the oral cavity and salivary glands and after recovery from the infection, some patients continue to complain of ageusia for several months. There is a positive correlation between the incidence of oral signs and symptoms associated with COVID-19 infection and the severity of the infection.
Introduction
It has been more than two years since the recording of the frst case of the novel coronavirus disease in Wuhan City, China, and the declaration by the World Health Organization (WHO) of pandemic disease [1] but we could not identify the Wuhan City case as the truly frst case all over the world. Since that time, approximately 643 million infection cases worldwide with more than 6 million deaths had been reported [2]. Fever, sore throat, severe headache, loss of smell and taste, shortness of breathing, dry cough, and tiredness are considered as the most common symptoms associated with the COVID-19 infection [3]. Tis viral disease is transmitted from human to human through respiratory droplets [4]. Previous studies have established that the SARS-CoV-2 virus invades human cells through an interaction with the host angiotensin-converting enzyme 2 receptors (ACE2) [5]. Tese receptors are expressed by a variety of human tissues such as lung, gastrointestinal tract, liver, kidney, skin, and the oral cavity [1,3]. Furthermore, the oral cavity has a crucial role in the dissemination of SARS-CoV-2 virus since it serves as a gateway to the internal environment of the human body. It has been suggested that oral manifestations associated with COVID-19 may be either related to the direct efect of the SARS-CoV-2 on the tissues of the oral cavity or an indirect efect due to an impairment of the immune system by the virus [6,7]. Oral manifestations related to COVID-19 infection have important negative impacts on nutrition, quality of life, and the psychological status of patients. In addition, they have an important role in the clinical diagnosis of disease [8].
Terefore, the aim of this cross-sectional study is to examine the types and prevalence of oral manifestations associated with COVID-19 and evaluates their persistence. In addition, any correlation between the incidence of these oral manifestations and several factors such as: gender, age, systemic disease, smoking, and severity of the infection have been investigated.
Methodology
2.1. Survey Procedure. Tis cross-sectional study was conducted on patients attending dental clinics of the College of Dentistry, University of Basrah, between October 2021 and April 2022. All the participants had been previously infected with COVID-19 and their infection was confrmed by a positive reverse transcription-polymerase chain reaction (RT-PCR) test.
Ethical approval was obtained by the Research Ethics Committee, College of Dentistry, Basrah University (BDC/ 07/09/2021), and the study was performed in accordance with Helsinki Declaration. All the participants were volunteers and had completed and signed a consent form.
Te study sample consisted of a total 574 patients from Basrah province (196 males and 378 females), with an age range between 18 and 78 years.
A questionnaire was flled out to collect information from each patient. Te patients were asked about their sex, age, occupation, address, smoking habits, the presence of any systemic diseases such as cardiovascular, respiratory, diabetes, cancer, kidney, and autoimmune disease and whether the respiratory infection was severe and required hospitalization. Details of any oral manifestations that occurred during the COVID-19 infection were recorded along with whether these persisted following the recovery.
Statistical
Analysis. Data were statistically analyzed using Microsoft Excel software. Te frequency and percentage were used to calculate the numerical data, whereas the Chi-square test was used to test signifcant statistical correlations. A p value of <0.05 was considered signifcant.
Results
Te results showed that 507 patients (88.3% of the total number of COVID-19 patients studies), had at least one manifestation related to the oral cavity and salivary glands. Whereas only 67 (11.7%) did not report any sign or symptom associated with oral cavity.
Te fndings showed that ageusia was the only symptom that persisted following recovery in 31% of the total patients who complained from ageusia for approximately one month, 4.6% for 3 months and 2.6% after 6 months ( Figure 2).
Regarding the gender of the patients, 172 (87.7%) males and 335 (88.6%) females had at least one oral sign and symptom associated with the COVID-19 infection. Tere was no statistically signifcant diference in the prevalence between the males and females (p � 0.7) as shown in Table 1.
With regards to the presence of systemic diseases, the results showed that 128 patients in the study sample had systemic diseases and 112 (87.5%) of them complained of at least one oral manifestation during their COVID-19 infection. On the other hand, 446 participants had no systemic diseases, and 395 (88.6%) of them showed oral signs or symptoms during the period of infection. No statistically signifcant diference was detected between the patients with systemic diseases and healthy patients (p � 0.7, Table 3).
Among 67 smokers in the study sample, 61 (91.1%) of them had at least one oral sign and symptom. Whereas the number of nonsmoker patients who were afected by oral manifestations was 446 forming 87.9% of the total number of nonsmoker patients. Tere was no statistically signifcant diference recorded between smoking patients and nonsmokers (p � 0.4, Table 4).
Finally, the fndings showed that 69 patients in the study sample was hospitalized during their COVID-19 infection due to the severe respiratory infection with 67 (97.1%) of them complaining of oral manifestations. 505 patients did not require admission to hospital and 440 (87.1%) of them had oral manifestations, a signifcant statistical diference between both groups (p � 0.01, Table 5).
Discussion
COVID-19 infection has efects on multiple organs and sites including oral cavity [1]. Although many studies have been published to investigate its general impacts, oral manifestations are still underestimated and unclarifed [9]. Terefore, the rationale of the current study was to investigate the most common oral manifestations that are accompanied by the disease, their prevalence as well as the presence of any correlations with demographic factors and the severity of disease in an Iraqi cohort.
Te results of the study showed that 66.8% of the examined patients had ageusia (complete loss of taste sensation) which is higher than that found by Ganesan et al. [11]. Two hypotheses could explain the high incidence of ageusia and gustatory changes during COVID-19. First, high expression of ACE2 receptors in epithelial cells in taste buds of the tongue that binds SARS-CoV-2 virus results in inactivation of these receptors and as a consequence, the taste perception is alter [12][13][14]. Te second hypothesis suggests that taste loss might be attributed to side efects of medications used in the treatment of COVID-19 infection [15,16]. However, the later hypothesis is considered questionable and remains unsupported due to the occurrence of ageusia and gustatory changes in drug-free COVID-19 patients with mild and moderate infection [12]. Dry mouth (xerostomia) is the second most common oral fnding found in 59% of the total study sample which is comparable to that found by Ameen et al. (56%) but higher than that revealed by El Kady et al. (39.7%) [11] and Ganesan et al. (28%) [8]. Recent literature attributed the incidence of xerostomia during COVID-19 infection to prolong antibiotic treatment and poor hydration as well as the presence of Candida infection [8].
Te results of the current study showed dysphagia in 40.5% of the participants which is higher than that reported by El Kady [17].
In this study, the prevalence of burning sensation is 22.8% which is similar to that found by El Kady et al. (22.4%) [11]. Several studies suggested that burning sensation is a nonspecifc symptom that might occur during COVID-19 infection as a consequence of other conditions such as dry mouth, candidal infection, oral ulceration, and any secondary infection [1,18].
Our fndings showed that 14.5% of the studied sample complained of oral ulceration following COVID-19 infection which is comparable to that found by El Kady et al. (17.2%) [11] and higher than that found by Natto et al. (6.4%) [10]. Previous studies have considered oral ulceration to be a nonspecifc symptom and indirectly related to COVID-19. Tis could be due to several reasons such as blister as a result of high fever, aphthous ulcers triggered by stress, and recurrent herpes type 1 infection due to a compromised immune system during COVID-19 infection [19][20][21][22].
Te last oral manifestation investigated in this study was gingival bleeding which is found in 3.3% of the study sample, which is less than that reported by El Kady et al. (7%) [11]. Gingival bleeding could be explained by the fact that COVID-19, like other debilitating diseases leads to reduced measures of maintaining oral hygiene. Tis leads to the International Journal of Dentistry accumulation of dental bioflm which is associated with an increased infammatory reaction and accompanied by clinical signs of gingivitis [23].
To the authors' best knowledge, no previous study has examined the persistence of oral signs and symptoms following recovery from the infection. Our results demonstrated that only ageusia continued in some patients for up to 6 months following the recovery. Tere is no specifc explanation for this as the exact pathogenesis of loss of smell and taste in COVID-19 is unknown [24]. Furthermore, many studies established that loss of taste accompanied olfactory dysfunction and most individuals considered both symptoms as a single entity rather than two [25].
In this study, the relationships between the incidence of oral manifestations with gender, age, smoking, systemic diseases, and the severity of infection were evaluated. Te results showed a signifcant statistical correlation between oral manifestations and age of patients as well as the severity of infection. Tese fndings are in consistent with the study by Ganesan et al. [8] who found no correlation between oral manifestations and gender and smoking, whereas a signifcant correlation was found with severity of the infection. In addition, the fndings are in accordance with that reported by El Kady et al. [11] who showed that there is no correlation between genders with oral manifestations.
It is obvious in the current study that the high frequency of the oral manifestations reported by hospitalized patients. Tis could be attributed to the fact that hospitalized patients had severe acute respiratory infection, and most of them were intubated and are likely to have an impaired immune response. Furthermore, those patients were debilitated and had a long hospital course. All these factors lead to compromised oral hygiene and a subsequent increased incidence of oral signs and symptoms [8].
Conclusions
It is evident that COVID-19 infection has signifcant efects on the oral cavity and salivary glands. Tese efects increased with severity of the infection and patient hospitalization. In addition, some patients continue to complain of loss of taste for several months after recovery from the infection. Terefore, it is recommended that the dentists should have the adequate knowledge of the patterns of presentation and management of common oral manifestations associated with COVID-19. Furthermore, it is also suggested that dentists should be part of the COVID-19 therapy team so as to improve the process of recovery and the quality of life for the afected patients.
Data Availability
Te data that support the fndings of this study are available from the corresponding author upon reasonable request.
Conflicts of Interest
Te authors declare that they have no conficts of interest.
Authors' Contributions
Mohanad JN Al-Magsoosi conceptualized the study, performed investigation, performed methodology, performed project administration, performed supervision, and wrote and edited the original draft. Oras KB Al-Asadi conceptualized the study, performed methodology, performed data curation, and wrote the original draft. Nibrass T Al-Quraine performed data curation and data analysis. Suha M Sami conceptualized the study and performed formal analysis. Julfkar Haider performed supervision; performed formal analysis; and wrote, reviewed, and edited the manuscript. | 2023-02-19T16:15:33.024Z | 2023-02-17T00:00:00.000 | {
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251479139 | pes2o/s2orc | v3-fos-license | Renewal MI Dental Composite Etch and Seal Properties
This study’s aim was to assess whether the Renewal MI composite can self-etch enamel, seal sound cavities, and stabilize demineralized dentine. Etching was assessed using scanning electron microscopy (SEM). Cavity sealing was quantified using the ISO-11405 dye microleakage test. Demineralized dentine stabilization was evaluated by visualizing resin tag formation, enzyme activity and mineral precipitation at the adhesion interface. Renewal MI provided a mild etching of sound enamel in comparison with 37% phosphoric acid. It provided a comparable seal of sound cavities to Z250/Scotchbond Universal adhesive and a superior seal to Activa, Fuji IX and Fuji II LC. With demineralized dentine, Renewal MI formed 300–400 µm resin tags covering 63% of the adhesion interface compared with 55 and 39% for Z250/Scotchbond and Activa. Fuji IX and Fuji II LC formed no resin tags. A higher tag percentage correlated with lower surface enzyme activity. Unlike Activa and Fuji II LC, Renewal MI promoted mineral precipitation from simulated body fluid, occluding adjacent dentinal tubules within 6 months. These novel etching and sealing properties may facilitate Renewal MI’s application in minimally invasive dentistry.
Introduction
Dental caries is caused by specific oral bacteria (e.g., Streptococcus mutans and lactobacillus) producing organic acids that demineralize the enamel and then the underlying dentine [1]. Enzyme-catalysed hydrolysis and solubilization of demineralized dentine by matrix metalloproteinases (MMPs) subsequently occurs [2]. Caries is one of the most common human diseases worldwide, with disadvantaged groups disproportionately affected [3]. In 2019, the average percentage of 5-year-old children with caries was 23% in England (up to 34% in more deprived areas). Only 10% of teeth with caries in dentine were restored [4,5]. Untreated caries can progress to pulpitis, periapical pathology, and the need for tooth extraction. In the UK, this is currently the main reason for a child's hospital admission [4].
The difficulty of providing dental care to anxious or pre-cooperative children is one of the most common reasons for them not to have their teeth restored. Silver-mercury amalgam fillings were the mainstay of treatment for many years. They are strong, simple to place and antibacterial [6,7]. Following the global Minamata agreement (Minamata convention on mercury, UN, 2013), however, all mercury-containing products are being phased out globally. Consequently, the use of amalgam in children was banned in July 2018 across the EU [8].
The main alternative filling material is the dental composite. Unlike amalgam, composites are not antibacterial. Instead, they rely on an effective cavity seal, restricting bacterial growth. For conventional composites to work, multiple complicated steps must be followed to ensure effective adhesion to the tooth structure [9,10]. Prior to composite placement (as with amalgam), the total removal of dental caries is necessary [11]. This generally requires local anaesthesia and high-speed drilling. Acid-etching and rinsing are then required to provide a surface to which the composite adhesive can bond. Ideally, placement 1. sealing sound drilled cavities. 2.
forming resin tags and inhibiting enzyme activity with demineralized dentine.
Teeth
Teeth were obtained from the Eastman Dental Institute biobank. The study has been approved by the UCL Eastman Biobank Ethics Committee under a generic project ethical approval number 1304 (first dated 29 April 2014). Primary molars were used in drilled cavity studies, as this was the population and tooth type of main interest. Permanent teeth, however, were used in etching and demineralized dentine investigations. This was because extracted primary teeth are usually deeply decayed. Conversely, sound adult premolars and molars may be extracted for orthodontic reasons. They therefore provided sufficiently large depth of sound dentine required for the demineralized dentine studies.
Enamel Etching
To compare the effect of Renewal MI versus phosphoric acid gel on enamel etching, flat enamel surfaces were obtained by grinding down the buccal surface of human permanent teeth using 500-grit abrasive paper. Teeth were then shaken in water for 5 min (Jintai ® JT-14 Dental Lab Round Shaker Oscillator, Jentai, Zhejiang, China) to ensure the removal of any debris. Enamel surfaces were blot dried and Renewal MI or phosphoric acid gel applied for 20, 40, 60, 90 or 120 s. Phosphoric acid was rinsed off with water. Renewal MI Materials 2022, 15, 5438 4 of 16 was dissolved using acetone and removed by vortexing for 1 min followed by vigorous washing with water for a further minute. The etched enamel surfaces were blow-dried, sputter-coated with gold/palladium and visualized through scanning electron microscopy (SEM, Phillip XL-30, Eindhoven, The Netherlands).
Tooth Restoration
Microleakage at the drilled tooth-restoration interface was investigated following the instructions of ISO/TS 11405:2015 [22] using primary molars. As access to primary teeth was limited, to maximize sample numbers, multiple cylindrical cavities were drilled in each tooth. Cavities were 2 mm in diameter and 2 mm deep for enamel leakage assessment. For dentine leakage assessment (with the surface enamel removed), the depth was increased to 3 mm. Seventy teeth were used, with 3 or 4 cavities per tooth. Only non-carious buccal, lingual, or proximal surfaces at the middle third were used. Where possible, each tooth had one cavity restored with each of the different restorative materials. Cavities were restored with the different materials after the following tooth preparation steps: Restorations were carefully polished (using composite polishing bur) to ensure the removal of any excess material on the outer enamel surface. For the assessment of leakage at the dentine/restoration interface with no enamel, cavity depth was increased to 3 mm. The restoration was then polished down to remove the top~1 mm-thick surface of both enamel and material and expose the dentine/material interface. Restored teeth were then incubated in water at 37 • C for 24 h prior to dye leakage assessment.
Dye Microleakage Evaluation
Before immersion in dye, apices of the above teeth were sealed with adhesive wax. The rest of the tooth surface was covered with 2 layers of nail varnish (except for the top surface of the fillings and 1 mm around them). Specimens were subsequently placed in aqueous methylene blue (1%) for 4 h to allow dye penetration through any gaps at the adhesion interface. Following rinsing and drying, teeth were carefully cross sectioned longitudinally to expose each restoration/tooth interface, one at a time. These were examined under a light microscope and dye microleakage scored as follows: • 0: None • 1: Within enamel (only used for cavities with enamel cavosurface) • 2: In dentine but did not reach the pulpal wall • 3: Reached the pulpal wall When the results were more variable (e.g., for the RMGICs), sample numbers were increased to enhance confidence in any differences with those for Renewal MI. Sample numbers were: for Renewal MI, 18 and 26, Z250, 11 and 11, Activa, 30 and 31, Fuji IX, 16 and 16 and for Fuji II LC, 27 and 37 for enamel and dentine, respectively. Data are reported as both the number with each score and this number as a percentage of the group number. For example, for Renewal MI, numbers are provided as a percentage of 18 or 26 for enamel versus dentine, respectively. tems, Milton Keynes, UK) and then obtaining the top 2 mm-thick slice of coronal dentine. These discs were immersed in 15 mL of formic acid (4M) for 2 days to achieve total demineralization. Complete demineralization without collapse of the dentine tubule structure was demonstrated through SEM with EDX (Inca X-Sight 6650 detector, Oxford Instrument, Abingdon, UK). The final discs were flexible, with tubules from the top to the lower surfaces providing a porous mesh-like structure composed largely of collagen [24].
Resin Tag Formation
For material/demineralized dentine penetration assessment (resin tag formation), pastes were confined within circlips of 10 mm internal diameter and 1 mm thickness. Circlips were placed on top of an acetate sheet. Pastes included Renewal MI, Z250, Activa, Fuji IX and Fuji II LC. The above demineralized dentine discs were then pressed on the top surfaces of the uncured material pastes. For Z250, Scotchbond was pre-applied to the dentine surface prior to dentine placement in contact with the composite. Materials requiring light exposure were then exposed to the above LED light for 40 s on each side. The light tip was placed in direct contact with acetate or demineralized dentine. To visualize the paste's ability to penetrate the demineralized dentine, the collagen was completely dissolved by immersion in sodium hypochlorite (15%) for 2 days. This exposed any polymerized resin tags formed by material penetration into the demineralized dentine mesh.
Relatively high-magnification SEM images (×100-500) were used to enable visualization of localized tags density and length. Additionally, lower-magnification images (×40) of the whole collagen imprint area were used to quantify total surface coverage percentage. Lower-magnification images demonstrated that regions containing tags seen at a high magnification could be patchy. Imprint areas were significantly darker in regions of high resin tag density. ImageJ software was used to determine the percentage of the collagen surface that had been sealed by tags. This was achieved through first determining the total adhesion area (dentine/material contact area with the central pulp horn region subtracted). The remaining imprint dark and lighter regions were converted to black and white, respectively (n = 3 per material). Tag coverage was defined as the percentage of the adhesion area that was black.
Additionally, restored demineralized dentine discs (n = 3) were polished using 500-grit abrasive paper to expose the adhesion interface. The exposed interface was then stained with Rhodamine B (0.2% in isopropanol, Sigma, Welwyn Garden City, UK) for 2 min followed by a gentle rinse. This enabled the visualization of resin tags within tubules using confocal laser scanning microscopy (CLSM Bio-Rad Confocal Microscope/Olympus BX51 Upright Microscope, Olympus Corporation, Tokyo, Japan). An oil objective lens (×60) and the red fluorescence channel were used. The laser microscope settings for rhodamine B were 568 nm excitation and emission through 600-630 nm filter. The Z motor was used to acquire 35 scans of the same field up to 18 µm deep at 2 µm intervals. These scans were compiled using ImageJ software to enable visualising an 18 µm depth of the interface in a 2D image.
Enzyme Activity
To assess enzyme activity at the demineralized dentine/restorative material interface, a Molecular Probe (EnzChek Collagenase Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA) was employed. This kit contains a denatured collagen/fluorescein conjugate that, when degraded, produces strong green fluorescence. In this study, 5 µL of the kit collagen substrate was diluted 1:4 with buffer to obtain a collagen concentration of 200 µg/mL. The diluted collagen probe was applied to the blot dried collagen disc surfaces and left for 5 min to soak in. The probe-soaked surface was then restored, as in the resin tag test above, using Renewal MI and commercial restorative material pastes (n = 3). A negative control of non-restored demineralized dentine was used. Polymerization, when required for setting, was initiated through exposure of the lower surfaces to LED light for 40 s. Set restorations were stored in a humid sealed pot with a moist tissue in a 37 • C incubator.
Samples were polished down to expose the interface after 1 day or 2 weeks to enable scanning through CLSM. Samples were orientated to scan a representative cross-section region of the adhesion interface. The selected field was scanned using a ×60 lens and focused to gain the highest fluorescence possible without detecting the weak collagen green auto fluorescence. Images were analysed using ImageJ software v1.8 (NIH, LOCI, University of Wisconsin, Madison, WI, USA). Relative MMP activity was taken as the area percentage of the scanned field which had strong green fluorescence.
Mineral Precipitation at Set Material/Demineralized Dentine Interfaces
The above demineralized dentine discs were also used on set materials to determine their tooth remineralization potential. Renewal MI, Fuji II LC and Activa pastes were placed in 10 mm-internal diameter and 1 mm-thick circlips sandwiched between 2 acetate sheets. Set discs were obtained by the 40 s exposure of the top and bottom surfaces to the above LED light placed in direct contact with the acetate sheet. Following set disc removal from the mould, demineralized dentine discs, prepared as above, were gently clamped against the material surfaces (n = 3). Samples were then fully immersed in 10 mL of simulated body fluid (SBF) prepared according to ISO 23317:2014. Samples were incubated at 37 • C, with the SBF being replaced every 2 weeks. After 6 months, discs were gently detached, and the dentine interface imaged by SEM.
Statistical Analysis
All values and error bars reported were the mean with 95% confidence intervals (95% CI). SPSS Statistics v24 for Windows (IBM, Armonk, NY, USA) was used for statistical analysis. For dye leakage, ordinal regression and Kruskal-Wallis pairwise comparisons were undertaken. For tag coverage and enzyme activity area, Levene's test was used to assess the homogeneity of variance. When variances were equal, data were analysed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons when needed. The Kruskal-Wallis test was used if the variances were not equal, followed by pairwise comparisons if needed [25]. The significance value was p = 0.05.
Enamel Etching
Enamel exposed to Renewal MI showed a roughened uneven surface in comparison to untreated ground enamel ( Figure 1). The etching pattern observed, however, was different from that seen with phosphoric acid etching. It also required longer exposure times (60 instead of 20 s) to become well-defined.
Dye Microleakage in Sound Cavities
Blue dye microleakage results with enamel or dentine cavosurfaces are shown in Figure 2. The darker the column, the deeper the dye penetration. Statistical analysis showed that microleakage with enamel or dentine cavities restored with Z250 (with etch and bond) and Renewal MI were comparable to each other. Both were significantly lower compared with all other commercial comparators (p = 0.01 to 0.007).
Tags Formation
Example SEM images of resin tags formed within demineralized dentine after collagen dissolution are provided in Figure 3. Some tag-free areas were detected often around the edge of the collagen imprint area or surrounding the central pulp horn areas. Percentages of the collagen imprint area covered by tags are provided in Figure 4.
Dye Microleakage in Sound Cavities
Blue dye microleakage results with enamel or dentine cavosurfaces are shown in Figure 2. The darker the column, the deeper the dye penetration. Statistical analysis showed that microleakage with enamel or dentine cavities restored with Z250 (with etch and bond) and Renewal MI were comparable to each other. Both were significantly lower compared with all other commercial comparators (p = 0.01 to 0.007).
Dye Microleakage in Sound Cavities
Blue dye microleakage results with enamel or dentine cavosurfaces are shown in Figure 2. The darker the column, the deeper the dye penetration. Statistical analysis showed that microleakage with enamel or dentine cavities restored with Z250 (with etch and bond) and Renewal MI were comparable to each other. Both were significantly lower compared with all other commercial comparators (p = 0.01 to 0.007). Renewal MI showed an extensive network of 300-400 µm-long tags ( Figure 3) covering 62% (Figure 4) of the demineralized dentine adhesion surface. Whilst imprint surface area covered by tags by the Z250 bonding agent (55%) was not significantly different, the tags within these areas were clearly shorter and less densely packed. In comparison, Activa tags were of intermediate length but covered significantly less (39%) of the surface (p = 0.034 to 0.001). Fuji II LC and Fuji IX provided no detectable resin tags. In the latter case, the use of sodium hypochlorite was questionable, as it caused Fuji IX to soften, as well as dissolve collagen.
The CLSM scans ( Figure 5), however, could help address this concern. As seen with SEM, tags formed using Renewal MI were much longer in comparison to those of the Z250 Scotchbond adhesive. With Z250, a few microns-thick adhesive layer was detectable beneath the resin tags. Fuji IX and Fuji II LC, however, showed minimal evidence for material tubule penetration and a less well-defined interface. group sample number. The cavosurface was either enamel (E) or dentine (D). The darker the column, the more extensive the dye penetration, and the worse the cavity sealing.
Tags Formation
Example SEM images of resin tags formed within demineralized dentine after collagen dissolution are provided in Figure 3. Some tag-free areas were detected often around the edge of the collagen imprint area or surrounding the central pulp horn areas. Percentages of the collagen imprint area covered by tags are provided in Figure 4. Renewal MI showed an extensive network of 300-400 µm-long tags (Figure 3) covering 62% (Figure 4) of the demineralized dentine adhesion surface. Whilst imprint surface area covered by tags by the Z250 bonding agent (55%) was not significantly different, the tags within these areas were clearly shorter and less densely packed. In comparison, Activa tags were of intermediate length but covered significantly less (39%) of the surface (p = 0.034 to 0.001). Fuji II LC and Fuji IX provided no detectable resin tags. In the latter case, the use of sodium hypochlorite was questionable, as it caused Fuji IX to soften, as well as dissolve collagen.
The CLSM scans ( Figure 5), however, could help address this concern. As seen with SEM, tags formed using Renewal MI were much longer in comparison to those of the Z250 Scotchbond adhesive. With Z250, a few microns-thick adhesive layer was detectable beneath the resin tags. Fuji IX and Fuji II LC, however, showed minimal evidence for material tubule penetration and a less well-defined interface.
Enzyme Activity in Restored Demineralized Dentine
Representative example CLSM images of green fluorescence due to the collagenase molecular probe at the interface between materials and demineralized dentine discs are provided in Figure 6. Renewal MI showed the least fluorescence at the adhesion interface, which disappeared almost totally after 14 days. For other materials, the fluorescence was
Enzyme Activity in Restored Demineralized Dentine
Representative example CLSM images of green fluorescence due to the collagenase molecular probe at the interface between materials and demineralized dentine discs are provided in Figure 6. Renewal MI showed the least fluorescence at the adhesion interface, which disappeared almost totally after 14 days. For other materials, the fluorescence was mostly seen as a band at the interface, although in Fuji II LC and Activa it could additionally be observed penetrating down into the dentine tubules. Figure 7 provides green fluorescence area percentages obtained using ImageJ. With Renewal MI, less than 1% of the image areas were highly fluorescent at either 1 or 14 days. This was significantly lower in comparison to all of the other commercial groups (p = 0.01 to 8.3 × 10 −8 ). Activa and Z250 gave intermediate results. These were significantly lower than Fuji IX and Fuji II LC, and significantly higher than Renewal MI at day 14. mostly seen as a band at the interface, although in Fuji II LC and Activa it could additionally be observed penetrating down into the dentine tubules.
Mineral Precipitation at Demineralized Dentine/Set Restoration Interfaces
Collagen discs, incubated with discs of Activa or Fuji II LC for 6 months in SBF, showed no sign of minerals at the interface. Instead, widely opened dentinal tubules were observed. Collagen discs incubated with Renewal MI, however, showed an interface totally covered with minerals, and the dentinal tubules were fully occluded (Figure 8).
Mineral Precipitation at Demineralized Dentine/Set Restoration Interfaces
Collagen discs, incubated with discs of Activa or Fuji II LC for 6 months in SBF, showed no sign of minerals at the interface. Instead, widely opened dentinal tubules were observed. Collagen discs incubated with Renewal MI, however, showed an interface totally covered with minerals, and the dentinal tubules were fully occluded (Figure 8).
Figure 8.
Example SEM images of the collagen mesh discs following their contact with pre-cured Activa, Fuji II LC and Renewal MI discs (n = 3 per material). Samples were incubated at 37 °C in SBF for 6 months. Activa and Fuji II LC shows widely opened dentinal tubules. Conversely, Renewal MI caused formation of a layer of minerals that covered the whole surface (n = 3).
Discussion
This study employed a standard technique for assessing the sealing of sound drilled cavities. It also introduced new methods of investigating fully demineralized dentine stabilization, through penetration and sealing, enzyme deactivation and remineralization. Renewal MI properties were compared with different restorative material classes that have been in clinical use for many years and the newer "bioactive" material, Activa.
Composition of Renewal MI
The experimental composite Renewal MI is urethane dimethacrylate (UDMA)-rather than BISGMA-based, which removes the phthalate (a potential BISGMA impurity) leaching risk [26]. The high molecular weight of its diluent monomer, polypropylene glycol dimethacrylate (PPGDMA), helps to keep shrinkage low. The Renewal MI liquid phase also contains 3 wt% of the adhesion promoting monomer, 4-methacryoyloxy trimellitate anhydride (4META) [24]. Its total filler content is 75 wt% (~55 vol%). It is a hybrid composite containing a mixture of 7 µm-and 0.7 µm-average diameter, radiopaque, silanetreated, barium aluminosilicate glass particles and a lower level of silica nanoparticles.
The food preservative polylysine (PLS) is also included in Renewal MI filler phase at 4 wt% [24]. This component is crucial for enabling composite penetration into dentine tubules [24]. The PLS particles are too large (20-50 µm) to penetrate the tubules themselves, but may act through absorbing water. This could allow the hydrophobic resin phase to penetrate and interact with tooth structure, but additionally lowers strength.
The filler phase of Renewal MI also contains 8 wt% monocalcium phosphate (MCP). In water, MCP can disproportionately form dicalcium phosphate (DCP) and phosphoric acid. The phosphoric acid may, in the presence of surface water, provide self-etch of apatite in tooth structure. The DCP rapidly precipitates as brushite. This process requires water and produces crystals of lower density that occupy greater volume [27]. This can provide expansion of Renewal MI to compensate for polymerization shrinkage or fill gaps at the tooth restoration interface [24,28].
Renewal MI has a high conversion rate and sufficient level and depth of cure (72% at 3 mm depth) for bulk filling without layering [24]. Flow characteristics and polymerization shrinkage (3%) are between those of packable (e.g., Z250) and flowable composites [24,29,30]. The biaxial flexural strength is 120 MPa at 1 day, but declines to 75 MPa at 3-6 months due to water sorption [24]. For comparison, that of Z250 is greater (>150 MPa), those of Activa and Fuji II LC are comparable (100-70 MPa) and that of Fuji IX is weaker (<40 MPa).
Enamel Etching
Enamel prisms can demonstrate one of three etching patterns: demineralized edges, centres of the prisms or a mix of both. Clinically, the pattern has been found to be unimportant, provided there is sufficient roughening to increase the surface area and enable an effective seal [31][32][33].
Discussion
This study employed a standard technique for assessing the sealing of sound drilled cavities. It also introduced new methods of investigating fully demineralized dentine stabilization, through penetration and sealing, enzyme deactivation and remineralization. Renewal MI properties were compared with different restorative material classes that have been in clinical use for many years and the newer "bioactive" material, Activa.
Composition of Renewal MI
The experimental composite Renewal MI is urethane dimethacrylate (UDMA)-rather than BISGMA-based, which removes the phthalate (a potential BISGMA impurity) leaching risk [26]. The high molecular weight of its diluent monomer, polypropylene glycol dimethacrylate (PPGDMA), helps to keep shrinkage low. The Renewal MI liquid phase also contains 3 wt% of the adhesion promoting monomer, 4-methacryoyloxy trimellitate anhydride (4META) [24]. Its total filler content is 75 wt% (~55 vol%). It is a hybrid composite containing a mixture of 7 µmand 0.7 µm-average diameter, radiopaque, silane-treated, barium aluminosilicate glass particles and a lower level of silica nanoparticles.
The food preservative polylysine (PLS) is also included in Renewal MI filler phase at 4 wt% [24]. This component is crucial for enabling composite penetration into dentine tubules [24]. The PLS particles are too large (20-50 µm) to penetrate the tubules themselves, but may act through absorbing water. This could allow the hydrophobic resin phase to penetrate and interact with tooth structure, but additionally lowers strength.
The filler phase of Renewal MI also contains 8 wt% monocalcium phosphate (MCP). In water, MCP can disproportionately form dicalcium phosphate (DCP) and phosphoric acid. The phosphoric acid may, in the presence of surface water, provide self-etch of apatite in tooth structure. The DCP rapidly precipitates as brushite. This process requires water and produces crystals of lower density that occupy greater volume [27]. This can provide expansion of Renewal MI to compensate for polymerization shrinkage or fill gaps at the tooth restoration interface [24,28].
Renewal MI has a high conversion rate and sufficient level and depth of cure (72% at 3 mm depth) for bulk filling without layering [24]. Flow characteristics and polymerization shrinkage (3%) are between those of packable (e.g., Z250) and flowable composites [24,29,30]. The biaxial flexural strength is 120 MPa at 1 day, but declines to 75 MPa at 3-6 months due to water sorption [24]. For comparison, that of Z250 is greater (>150 MPa), those of Activa and Fuji II LC are comparable (100-70 MPa) and that of Fuji IX is weaker (<40 MPa).
Enamel Etching
Enamel prisms can demonstrate one of three etching patterns: demineralized edges, centres of the prisms or a mix of both. Clinically, the pattern has been found to be unimportant, provided there is sufficient roughening to increase the surface area and enable an effective seal [31][32][33].
This study showed that Renewal MI created different etching patterns that also required longer application time to form. MCP in Renewal MI can form phosphoric acid in contact with water as explained above. This acid, however, might provide localized, inhomogeneous hydroxyapatite dissolution primarily near the MCP particles thereby leading to the different enamel roughening patterns. The concentrations of acid produced will be dependent upon levels of moisture and MCP particles at the tooth/composite interface and, as seen in this study, application time. Further work is required to assess whether increasing the time between placement and light exposure can enable improvements in both etch and then bond strengths.
Dye Microleakage in Sound Cavities
Dye microleakage is one of the methods of testing adhesion to permanent teeth detailed in ISO/TS 11405:2015 [22]. In this study, however, primary teeth were employed, since Renewal MI is proposed for paediatric patients. Samples were aged in water at 37 • C for just 24 h to determine early sealing properties. Further work with longer aging and/or thermocycling will be required to assess the effects of long-term interface degradation or mineral precipitation.
In this study, Renewal MI was cured soon after placement, providing limited time for the self-etching properties to enhance interlocking and bond strengths. Furthermore, Renewal MI's moderate shrinkage did not result in higher microleakage. This contradicts what was expected based on previous studies with other materials [34]. A new early sealing mechanism is possible, maybe due to Renewal MI's flowability. Additionally, the surface water sorption by PLS and the subsequent absorbed water reaction with MCP and 4META could be important. Tooth surface water removal may enable improved contact and interlocking of the hydrophobic resin phase with the tooth surfaces. Furthermore, the water sorption may promote localized composite swelling to compensate for polymerization shrinkage as the material sets. This sealing could be further improved by water sorption during the 24 h of restoration storage prior to immersion in dye [24]. Further studies will be required to assess if this seal can be maintained in the long term through additional swelling and mineral precipitation to compensate for any loading damage.
The null hypothesis that Renewal MI microleakage is not significantly different from commercial materials can be rejected with the GIC and RMGICs. It cannot, however, be rejected when comparing Renewal MI to Z250 with adhesive. For Z250 (with etch and Scotchbond), microleakage was comparable to Renewal MI and similar to previous observations [35,36]. The high variability in the results with Activa Kids and Fuji II LC suggests the materials may bond to one surface but not the other. The more consistently high leakage with Fuji IX may be due to a more constant gap. The manufacturer, GC, recommends tooth preconditioning with a polyacrylic acid solution and rinsing for both Fuji IX and Fuji II LC. This step, however, was excluded in this study as it is not always employed. Despite this deviation, the dye leakage observed with glass ionomers in this study was similar to previous work in which a conditioner and thermocycling were employed [37]. This suggests that gaps are formed from the start. The variable results with Activa are consistent with clinical study variability. In 1-year clinical studies, Activa (with etch only) gave good results in one study [38], but unacceptably high failure in a different study [39].
Interactions with Demineralized Dentine
In carious dentine, the top surface is infected, degraded, soft, and readily removed. Beneath this, there is an affected, leathery layer which is partially demineralized but repairable [40]. This may cover harder, translucent dentine with tubules occluded by excess minerals. These minerals provide a natural mechanism to protect the underlying pulp. In this study, fully demineralized collagen discs were used to mimic the structure of the intermediate affected dentine layer. Using originally sound teeth, controlling disc thickness and the time of immersion in formic acid enabled the complete removal of the minerals. This provided reproducible standardized model discs that consisted of non-collapsed collagen [24]. These had sound dentinal tubules that could be used to assess collagen tubule sealing by tags or occlusion through precipitation of minerals.
The ability of a restorative material to penetrate and seal caries-affected dentine might improve the interlocking with and strengthening of the diseased tissue. Furthermore, it should reduce water that may promote enzyme catalysed hydrolysis of collagen and consequently limit microleakage and recurrent disease. Additionally, the prevention of nutrient ingress should also cause remnant caries to arrest and provide a favourable environment for remineralization. These features could enable the use of the material without the need for total caries removal or complicated etch and bond procedures.
Resin Tags within Demineralized Dentine
The null hypothesis that Renewal MI tags coverage is not significantly different from commercial materials can be rejected with the GIC and RMGICs. It cannot, however, be rejected when comparing Renewal MI to Z250 with adhesive, despite the considerable difference in tags lengths. While resin tag formation is not by itself a true indicator of the ability of the material to bond to tooth structure, it is an important property to stabilize caries. Renewal MI could penetrate demineralized dentine without any surface pre-treatment and form a dense network of long resin tags. The hydrophilic PLS and MCP particles, together with 4META, have been shown to be primarily responsible for the high-dentine surface coverage [24]. These components may absorb water from the dentinal tubules, creating space for the fluid paste to penetrate. The patchy tag-free areas may be due to remnant water blocking tubules. The length of the tags is mainly controlled by the fluidity of the paste rather than hydrophilic component levels. Lower filler content enables faster and thereby greater movement into the tubules [24]. The resin tags formed by Renewal MI within demineralized dentine were much longer (>200 µm) than those reported in the literature. Typically, adhesive resin tags in acid-treated dentine are 20-30 µm [41,42]. Further work is needed, however, to analyse the chemical composition of these resin tags.
Preliminary studies showed that the conventional composite Z250 formed no tags on its own due to its high viscosity and hydrophobicity [24]. The observation of tags when Z250 was used with Scotchbond could be a consequence of the hydrophilic adhesive. The high fluidity of Scotchbond should encourage fast flow into the tubules. The small amount of adhesive applied combined with its BISGMA content, however, might limit the amount of water that could be absorbed. This might explain the shorter, less dense resin tags of Scotchbond compared with Renewal MI.
The flowability of Activa in combination with hydrophilic components might explain its ability to form resin tags with demineralized dentine. The lower surface coverage than that seen with Renewal MI, however, could be a consequence of Activa's lesser water sorption ability.
Whilst Fuji II LC and Fuji IX can both absorb water, their high viscosities might restrict their ability to penetrate the demineralized dentine. Furthermore, the large size of the polyacids in their liquid phase and interactions with positively charged amino acids in the dentine might further slow the rate of flow. Instead, a distinct few micron-thick water insoluble layer is known to form at the tooth interface [43].
Enzyme Activity in Restored Demineralized Dentine
The molecular probe used in this study provided a layer of high fluorescence on the surface of the control sample. This fluorescence will have been due to hydrolysis of collagen particles catalysed by enzymes activated in the demineralized dentine surface. The limited depth of fluorescence could be a consequence of the probe failing to diffuse into the dentine bulk. Alternatively, enzymes may only be activated near the dentine surface. Reduction in the fluorescence after 2 weeks might have arisen through the surface layer collapsing following enzyme-catalysed collagen hydrolysis.
The null hypothesis that Renewal MI enzyme inhibition is not significantly different from all commercial comparators can be rejected at day 1 and 14. For restored discs, the fluorescence area at the adhesion interface correlated with level of tags formation.
The reduction in fluorescence area at 2 weeks with Renewal MI might be a consequence of it absorbing the remaining water and improving the seal by forming resin tags.
Similarly, the Scotchbond adhesive used with Z250 might have provided an effective seal. With the other restorative materials, larger interfacial gaps containing water and the molecular probe might have caused both the higher area of interface fluorescence and lesser area reduction with time.
The importance of good dentine sealing on inhibiting MMP activity has previously been addressed in the literature [44,45]. Low pH caused by the natural caries process (bacterial acids) or acid-etching can result in activation of the MMP enzymes. This could be responsible for future degradation and nanoleakage at the interface, leading to recurrence of the disease and failure of the restoration. Research has shown that host-derived MMPs combined with cysteine cathepsins are the most abundant and active enzymes in carious dentine. Furthermore, MMP release from collagen is a key feature for caries progression [40,[46][47][48][49].
Mineral Precipitation in Demineralized Dentine
The above observed ability of Renewal MI to precipitate minerals, at the demineralized dentine interface, can be explained by the MCP content. The hydrophilic MCP can interact with water as explained above to form phosphoric acid and dicalcium phosphate. The release of these from the composite will provide calcium and phosphate ions. These may supersaturate the storage solution (SBF), resulting in the precipitation of various calcium phosphate minerals. The amount of remineralization thickness and occluding dentinal tubules looks more promising in comparison to a casein phosphopeptide-amorphous calcium phosphate (CPP-ACP)-containing paste [50]. The lack of mineral precipitation with Activa and Fuji II LC suggests that their release of ions is insufficient to supersaturate the SBF. Further analysis of the chemical composition of the precipitated layer at the interface between Renewal MI and dentine is needed.
Conclusions
Renewal MI can self-etch enamel. Its early ability to seal drilled cavities is comparable with Z250 (with Scotchbond) and superior to that of Activa, Fuji II LC and Fuji IX. With demineralized dentine, Renewal MI gave denser and longer resin tags and reduced enzyme activity. Unlike Activa or Fuji II LC, it promoted the precipitation of minerals.
Patents
A.Y has two patents on the use of MCP and PLS in dental composites licensed to a dental company (Davis Schottlander and Davis Ltd., Letchworth Garden City, UK). Informed Consent Statement: Informed consent was obtained from all subjects whose tissues were used in the study. Samples were anonymized and patients cannot be identified.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author.
Conflicts of Interest:
A.Y. has two patents on the use of MCP and PLS in dental composites licensed to a dental company (Davis Schottlander and Davis Ltd., Letchworth Garden City, UK). N.A., W.X. and P.A. declare no competing interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. | 2022-08-11T15:20:34.740Z | 2022-08-01T00:00:00.000 | {
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211126682 | pes2o/s2orc | v3-fos-license | Degrees of Freedom of Cache-Aided Wireless Cellular Networks
This work investigates the degrees of freedom (DoF) of a downlink cache-aided cellular network where the locations of base stations (BSs) are modeled as a grid topology and users within a grid cell can only communicate with four nearby BSs. We adopt a cache placement method with uncoded prefetching tailored for the network with partial connectivity. According to the overlapped degree of cached contents among BSs, we propose transmission schemes with no BS cooperation, partial BS cooperation, and full BS cooperation, respectively, for different cache sizes. In specific, the common cached contents among BSs are utilized to cancel some undesired signals by interference neutralization while interference alignment is used to coordinate signals of distinct cached contents. Our achievable results indicate that the reciprocal of per-user DoF of the cellular network decreases piecewise linearly with the normalized cache size $\mu$ at each BS, and the gain of BS caching is more significant for the small cache region. Under the given cache placement scheme, we also provide an upper bound of per-user DoF and show that our achievable DoF is optimal when $\mu\in\left[\frac{1}{2},1\right]$, and within an additive gap of $\frac{4}{39}$ to the optimum when $\mu\in\left[\frac{1}{4},\frac{1}{2}\right)$.
I. INTRODUCTION
Content delivery applications such as video streaming have occupied a significant proportion in mobile wireless data traffic. It amounts for more than 63% of the total mobile data in 2019 and is foreseen to contribute 76% in 2025 [2]. Wireless caching [3]- [5] is considered as one of the most effective techniques to cope with this increasing content oriented traffic. Its main idea is to exploit the under-utilized network resources during the off-peak hours by prefetching the popular contents in the local memory of edge nodes in order to accelerate the content delivery. Caching is first studied from an information-theoretic framework, known as coded caching [6] where a server communicates with multiple cache-enabled receivers over a shared error-free link. Receiver caching has been shown as an effective way to reduce the traffic load by creating coded multicast opportunities for the memory-equipped networks [6]- [8]. Then the authors in [9] investigate the role of transmitter caching in a 3 × 3 interference channel, and show that transmitter caching can bring transmit cooperation gain and hence provide an opportunity to increase the degrees of freedom (DoF) of the interference channel. The works [10]- [12] consider caching in a general cacheaided interference network with arbitrary number of transmitters and arbitrary number of receivers. It is found in [10] that with a novel cooperative transmitter and receiver caching strategy, the interference network can be turned opportunistically into a new class of channels, namely, cooperative X-multicast channels. The works [13]- [16] study the caching gain by considering multiple antennas deployed at both transmit and receive nodes and show that the spatial multiplexing gain induced by multiple antennas and the caching gain are cumulative. Later on, the impact of caching has been studied in various wireless network models, such as combination networks [17], [18], device-to-device networks [19], [20], and fog radio access networks (Fog-RANs) [21]- [23].
Note that many of the previous works on wireless caching assume a fully connected network topology, i.e., all the transmitters can communicate with all the receivers over independent and identically distributed (i.i.d.) fading channels. This assumption largely simplifies the theoretical analysis but also limits the applicability of these analytical results in practical wireless environment with partial connectivity (due to path loss, shadowing, signal blocking, etc). In [24], the authors attempt to address this issue by studying the storage-latency tradeoff in a partially connected interference network. They find that coded multicast gain and transmitter coordination gain, which are previously obtained in fully connected networks, can also be exploited in the partially connected network. The work [25] focuses on a partially connected Fog-RAN, where each user is served by some locally connected edge nodes, which are not equipped with cache and connected to a cloud server via dedicated fronthaul links. The above works [24], [25], however, are limited to the one-dimensional linear network only. Later on, the authors in [26] consider a two-dimensional cache-aided cellular network modeled by a hexagonal topology where each user can only be connected to three nearby BSs. They obtain an order-optimal per-cell DoF result and show that the per-cell DoF scales linearly with the total amount of cache available in the cell. The delivery scheme in [26] is restricted to linear one-shot processing, and only exploits zero-forcing gain by activating the BSs caching the same requested subfiles at each time. The authors in [27] formulate a joint file placement and delivery optimization problem to investigate the storage-latency tradeoff in a network with given and arbitrary partial connectivity. They adopt a non-splitting cache placement scheme, and thus cannot exploit the traffic load balancing gain by file splitting. Moreover, there is no information-theoretical analysis for the optimality of their results.
In this work, we consider a downlink cache-aided cellular network where the locations of cache-aided BSs are modeled as a grid topology with each grid regarded as a cell 1 . Considering path loss and shadowing effect, we assume that each user within a grid cell can only communicate with the nearby four BSs located at the corners of the cell. To ensure that the whole database is entirely cached at any four BSs around one user, in this paper, we focus on the cache size region µ ≥ 1 4 with µ denoting the fraction of the database that each EN can store locally. The goal of this paper is to investigate how much capacity gain can be achieved by caching in the cellular network. Towards this end, we characterize the DoF, the asymptotic capacity measure in high signal-to-noise (SNR) regime, of the cache-aided cellular network. The main contributions and findings of this paper are summarized as follows: • Achievable DoF: We adopt a file splitting and placement scheme tailored for the cellular network with partial connectivity. According to the overlapped degree of cached contents among BSs, caching brings different levels of BS cooperation for content delivery. We propose transmission schemes with no BS cooperation, partial BS cooperation, and full BS cooperation, respectively, for three typical cache sizes. In specific, for cache size µ = 1 4 where there is no BS cooperation, each BS only emits message to one nearby user at each time and interference alignment is used to coordinate signals of distinct cached contents among BSs. For cache size µ = 1 2 where there exists partial BS cooperation, the main idea is to utilize the common cached contents to cancel part of interference via interference neutralization and align the rest by interference alignment. For cache size µ = 1 where the BSs can fully cooperate, the whole cellular network can be regarded as a virtual multiple-input single-output (MISO) broadcast channel with partial connectivity and all the interference can be neutralized at each user by using a zero-forcing precoding. By applying memory sharing, we obtain the achievable DoF results for the whole cache size region µ ∈ [ 1 4 , 1]. Our achievable results reveal that the reciprocal of per-user DoF of the cellular network decreases piecewise linearly with the cache size µ, and the gain of BS caching is more significant for the small cache size region.
• Converse: We first derive an outer bound on the DoF region of the cache-aided cellular network for any given uncoded prefetching scheme by using a genie-aided approach. Note that this outer bound is applicable not only for the cellular network with grid topology considered in this paper, but also for the Then, under the adopted cache placement scheme, we obtain an upper bound of per-user DoF for the considered cache-aided cellular network. It is shown that the obtained per-user DoF is optimal when µ ∈ 1 2 , 1 , and within an additive gap of 4 39 to the optimum when µ ∈ 1 4 , 1 2 . The remainder of the paper is organized as follows. Section II provides the system model of the cacheaided wireless cellular network and the adopted cache placement scheme. In Section III, we present the achievable DoF results and the corresponding proofs. The converse is given in Section IV. Section V concludes this paper. The proofs of some lemmas and theorems are relegated to the appendices. Notations: x, x, X and X denote scalar, vector, matrix and set, respectively. X † denotes the Moore-Penrose inverse of matrix X. span(X) stands for the column span space of the matrix X. |X | denotes the size of the set X .
II. SYSTEM MODEL A. Cellular Network Model
We consider a cellular network where the BSs are located evenly on a two-dimensional plane, modeled as a square grid shown in Fig. 1. We use coordinate (p, q), with p, q ∈ E, to denote a BS. Each square formed by every set of four neighboring BSs is regarded as a cell. The users are arbitrarily distributed. The user density is assumed to be large enough so that we can always schedule one (and at most one) user in each cell to serve in each resource block. The resource blocks can be defined in either time domain by using TDMA or frequency domain by using OFDMA. Without loss of generality, we consider the transmission in one resource block during one scheduling interval. We use coordinate (i, j), with i, j ∈ O, to denote the scheduled user within each cell. Considering the shadowing effect and distancedependent path loss, we assume that the user within each cell can only be connected to the four nearby BSs located at the corners of the cell without interference from the other BSs. This assumption is valid since each mobile user only reports channel quality indicator (CQI) feedback from at most 3 ∼ 4 dominant nearby BSs in 3GPP standards [26]. Let M (i,j) denote the set of the four nearby BSs around user (i, j), i.e. Define Φ and Ψ as the sets of all BSs and all users respectively. 2 We further divide the set of all BSs Φ into four disjoint sets according to their geometrical coordinates as follows: which are demonstrated with different colors as shown in Fig. 1. These BS sets shall be used to design cache placement as discussed shortly later. At each time slot t, the received signal at user (i, j) can be represented as Here h (i,j),(p,q) (t) denotes the channel coefficient from BS (p, q) to user (i, j), which is time-variant and i.i.d. from a continuous distribution, x (p,q) (t) is the transmitted signal from BS (p, q) subject to an average power constraint P , and n (i,j) (t) denotes the additive white Gaussian noise (AWGN) at user (i, j) with zero mean and unit variance. Following the convention in [9]- [11], [24]- [26], we assume that the perfect channel state information is available at all BSs and all users.
B. Cache Model
Consider a database consisting of L files, denoted by {W 1 , W 2 , · · · , W L }, each of size F bits. Each BS is equipped with a local cache and can cache up to QF bits with Q ≤ L. The normalized cache size is defined as µ Q L , which represents the fraction of the database that each BS can store locally. Considering that in practical scenario, the memory equipped at a mobile user is negligible compared to the size of the whole database, we do not take the user-side cache into consideration. To ensure that the database can be entirely cached at any four BSs around one user, in this paper, we focus on cache size region µ ∈ 1 4 , 1 . The system operates in two phases, a cache placement phase and a content delivery phase. In the cache placement phase, each BS (p, q) maps the L files in the database to its local cached contents Ω (p,q) subject to its cache size constraint without knowing the future user demands. In this paper, we do not allow coding during the cache placement phase. A set of BSs A satisfying (p,q)∈A is defined as a BS cache group, which indicates the set of BSs caching some common contents that are not available in any other BSs. Denote the set of all BS cache groups as Θ {A}. We apply the cache placement scheme proposed in [6] by taking the partial connectivity of the network into consideration. Based on the BS sets {B k } k∈ [4] defined in (2) , is evenly split into six subfiles {W k ℓ } k∈ [6] with size 1 6 F bits, and each is cached in a unique collection of two out of the four BS sets {B k } k∈ [4] . More specifically, the BS cache group of subfiles {W k ℓ } ℓ∈ [L] , for k ∈ [6], is given by respectively. There exists partial overlapping in cached contents among BSs M (i,j) . • µ = 1: In this case, the cache size of each BS is large enough to store the whole database. There is only one BS cache group, i.e., the set of all BSs Φ. In the content delivery phase, each user (i, j) requests a file W r (i,j) from the database, where r (i,j) ∈ [L]. We define r (r (i,j) ) (i,j)∈Ψ as the vector of all user demands. Upon receiving the user demands r, BSs encode the requested files into signal sequences by using a codeword with length T , and transmit them to satisfy user demands. The error probability of the system is defined as P e = max whereŴ (i,j) is the estimated file of user (i, j) from the received signal sequence. Since each user receives a file with size F bits during codeword length T , we define R(µ, P ) F T as the per-user rate at cache size µ and transmit power P . The per-user rate R(µ, P ) of the system is said to be achievable if and only if there exists a coding scheme that all users can decode their requested files with vanishing error probability, i.e., P e → 0 as F → ∞.
C. Performance Metric
We define capacity C(µ, P ) as the supremum of all achievable per-user rates for the given cache size and the power constraint. The per-user DoF of the system is defined as Since the reciprocal of d(µ) is a convex function of cache size µ [9], we will use 1/d to show our results. (0,0) (2,0) III. ACHIEVABLE DOF This section presents the achievable per-user DoF results for the cache-aided cellular network. Theorem 1: For the considered cache-aided cellular network where each BS has a cache of normalized size µ, the reciprocal of the per-user DoF d is upper bounded as Remark 1: It can be seen that the reciprocal of the obtained per-user DoF d decreases piecewise linearly with the normalized cache size µ. In addition, the line segment at the small cache size region µ < 1 2 is steeper than that at µ ≥ 1 2 , indicating that the gain of BS caching is more significant at the small cache region.
In the delivery phase, we consider the worst-case scenario where all user demands are distinct. When user demands are not all distinct, the same delivery strategy can be applied by treating the demands as being different. In the following three subsections, we present the achievable schemes of three corner points (µ = 1/4, 1/d = 2), (µ = 1/2, 1/d = 7/6) and (µ = 1, 1/d = 1) 3 . The achievability of other points can be proved by using memory sharing [6].
A. µ = 1 4 (No BS Cooperation) By the cache placement scheme in Section II-B, the four requested subfiles of each user (i, j) are cached in the four BS cache groups {A k } k∈ [4] , denoted as {W r (i,j) ,A k } k∈ [4] respectively. Since there is no overlapping in the cached contents, no transmission cooperation can be exploited among the BS set M (i,j) . The main idea of the achievable scheme is to divide the whole transmission into different phases so that each BS only serves one user in each phase, and asymptotic interference alignment is used to align the interference for each user.
We choose a part of the cellular network consisting of some specific BSs and users as an example shown in Fig. 2 to present the achievable scheme, and the proof for the entire cellular network is placed in Appendix A. Here, the four BS cache groups are Note that only the four BSs around user 5 are entirely given in this example network. In the rest of this subsection, we only present the specific transmission design associated with user 5 and show that it can achieve the target DoF. The achievable scheme is based on the asymptotic interference alignment [31]. In specific, we use [31, Lemma 2] to design precoding matrices and the proof for the decodability of desired 3 The proposed achievable schemes of µ = 1 4 and 1 are still applicable if the connectivity degree of each user is larger than 4. For instance, consider that each user can be connected to nearby 12 BSs, i.e., M ′ (i,j) In this case, the delivery scheme of µ = 1 4 is very similar to Section III-A by taking M ′ (i,j) into the construction of precoding matrices, and the achievable per-user DoF is d = 1 2 . The delivery of µ = 1 in this scenario also forms a virtual MISO broadcast channel, and per-user DoF d = 1 is achieved by a zero-forcing precoding. signals relies on [31, Lemma 1]. However, due to the partial connectivity of the network, the space design of interference and the specific precoding construction in this paper are different from those in [31] which focuses on a fully connected interference network.
Each BS employs a random Gaussian coding scheme to encode the four subfiles requested by its serving users into signals, i.e.
BS a : where each s (i,j),(p,q) = [s 1 (i,j),(p,q) , · · · , s M (i,j),(p,q) ] T ∈ C M is the signal vector transmitted from BS (p, q) and intended to user (i, j). The whole transmission contains four phases and each phase takes N symbol extensions. In each phase, each BS transmits one of the above four signals to serve one user as illustrated in Fig. 3. Without loss of generality, we take phase 1 as an example and show that the DoF of 1 2 is achievable for user 5. Denote v m (i,j),(p,q) ∈ C N ×1 as the precoding vector of signal s m The choice of the signal dimension M and symbol extension number N is given shortly later in Remark 2. The received signal (ignoring noise) of user 5, denoted by y 5 ∈ C N , can be represented as where each . We aim to align the directions of the three interference signals into a common space, i.e.
where V ′ is an N × (N − M) matrix. Based on [31, Lemma 2], we shall design every element in the η-th row of V (i,j),(p,q) as a multivariate monomial function of entries in the η-th rows of all interference channel matrices, which are denoted by a set I. In this example, I is given by We construct the following set of column vectors where 1 N is an N × 1 vector with each element being one. The column vectors in V(n) can occur in any order to form each of matrices V 5,b V 4,a , V 7,c and V 8,d . For example, the m-th column of where the subscripts of each exponent ι m (i,j),(p,q) ∈ [n] are associated with corresponding base H (i,j),(p,q) , and its superscript m denotes the column index of v m 5,b in V 5,b . The received signal at user 5 can be rewritten as Note that each precoding vector of interference after multiplying channel matrix has a similar monomial structure as that in V(n), except that the range of each exponent is changed from [n] to [n + 1]. This indicates that these received precoding vectors of interference belong to the set V(n + 1). Thus, by letting the matrix V ′ consist of all the vectors in set V(n + 1), the interference alignment conditions (8) are satisfied. Remark 2: Since each user is interfered by three signals in the delivery, the total number of interference channels I is |I| = 3|Ψ| (in this example, |I| = 3 due to that we only consider three interference channels for user 5). Note that the columns of each precoding matrix V (i,j),(p,q) are formed by all the vectors in set V(n) with any order. Thus, the signal dimension M is equal to the cardinality of set V(n), i.e., M = |V(n)| = n 3|Ψ| . On the other hand, the dimension of all received interference vectors is equal to the cardinality of set V(n + 1) and given by (n + 1) 3|Ψ| . To ensure the decodability, the symbol extension number should be N = n 3|Ψ| + (n + 1) 3|Ψ| .
Next, we show that for each user, the received vectors of all desired signals are linearly independent from those of interference. For user 5, we shall prove that the matrix has full rank almost surely. To apply [31, Lemma 1], we need to verify that the elements in the same row of Λ 5 are different monomials. We have the following observations: 1) In the η-th row of Λ 5 , the products in H 5,b V 5,b are different from each other because each column v m 5,b with m ∈ [M] is a unique vector in set V(n); Similarly, the terms in the η-th row of V ′ are also different from each other since each column of V ′ is unique in set V(n + 1).
2) Since H 5,b is not in the construction of V(n), the terms in the η-th row of H 5,b V 5,b and V ′ differ in channel factor h 5,b (η). Therefore, the condition of [31, Lemma 1] is met and the matrix Λ 5 has a full rank almost surely. Since user 5 can decode n |3Ψ| desired signals over n 3|Ψ| + (n + 1) 3|Ψ| symbol extensions, the achievable per-user DoF can be expressed as Note that the above per-user d increases monotonically with n. Taking n → ∞, we can achieve the maximum value 1 2 of the per-user DoF asymptotically.
B. µ = 1 2 (Partial BS Cooperation) By the cache placement in Section II-B, the six requested subfiles of user (i, j) are cached in {A k } k∈ [6] , respectively. There exists partial overlapping in the cached contents among BSs M (i,j) . Thus, partial transmission cooperation can be exploited among BSs M (i,j) . In this subsection, the main idea of the achievable scheme for the cellular network with partial BS cooperation is to first cancel part of the undesired signals and then align the rest of interference for each user by using asymptotic interference alignment. In this case, BSs in each BS cache group A k use a random Gaussian coding scheme to encode subfile W r (i,j) ,A k requested by user (The signal dimension M and symbol extension number N will be determined shortly in Remark 3.) Each precoding matrix V where U (p,q) The above product form of V (p,q) (i,j),A k is inspired by the previous work [10] on cooperative X-multicast channels. The first matrix U (p,q) (i,j),A k is designed to cancel part of the interference while the second matrix W (i,j),A k is used to apply asymptotic interference alignment. Similar to Section III-A, we use the example network shown in Fig. 2 to illustrate the design of matrices U (p,q) (i,j),A k and W (i,j),A k . The specific design methods of U (p,q) (i,j),A k and W (i,j),A k for the entire cellular network are given in Appendix B. In this case, the BS cache groups in Fig. 2 We first elaborate the design of U (p,q) (i,j),A k . For notation convenience, we define as the effective channel of interference at user (i, j), which is caused by the signal transmitted from BS cache group A k to user (i ′ , j ′ ). The design of U (p,q) (i,j),A k contains the following three forms: • Case I: U (p,q) (i,j),A k is designed as a function of channel matrices. Since some adjacent BSs are connected to two common users, e.g., both BSs a and b can simultaneously serve users 2 and 5, we can use zero-forcing precoding to neutralize interference. We take signal s 2,A 1 for an example, which is transmitted cooperatively from BSs a, b ∈ A 1 and interferes user 5. By designing U a 2,A 1 = H 5,b and U b 2,A 1 = −H 5,a , for user 5, the interference caused by signal s 2,A 1 is neutralized as Similarly, for other adjacent BSs connected to two common users, the corresponding precoding matrices can also be designed by zero-forcing. For user 5, the effective channels of interference that can be neutralized as zeros are given by G 2 5,A 1 , G 8 5,A 2 , G 4 5,A 3 and G 6 5,A 4 .
• Case II: U (p,q) (i,j),A k is designed as a zero matrix. Due to the partial connectivity of the network, we can force some U (p,q) (i,j),A k with (p, q) / ∈ M (i,j) to be zeros in order to avoid interference. For example, noticing that both BSs a and b cannot be connected to user 8, i.e., a, b / ∈ M 8 , we can design U a 8,A 1 = U b 8,A 1 = 0 and the interference caused by signal s 8,A 1 at user 5 can be cancelled as Similarly, for user 5, we can obtain the following effective channels of interference as zeros: (i,j),A k linearly independent with those of the effective channels experienced by the desired signals. This condition will be used to prove the decodability of the desired signals and please see Appendix B for more details. In summary, we give the nonzero effective channels of interference at user 5 from each BS cache group A k with k ∈ [6] as follows J 5,A 6 = G 1 5,A 6 , G 2 5,A 6 , G 3 5,A 6 , G 4 5,A 6 , G 6 5,A 6 , G 7 5,A 6 , G 8 5,A 6 , G 9 5,A 6 .
Then we present the design of W (i,j),A k . We aim to align the directions of interference at each user into a common space. For user 5, the interference alignment conditions can be expressed as where W ′ is an N × (N − 6M) matrix. Similar to Section III-A, we shall design every entry in the η-th row of W (i,j),A k as a multivariate monomial function of entries in the η-th rows of effective channels of interference in J 5 . Towards this end, we construct the following set of vectors where the subscripts of each exponent ι (i ′ ,j ′ ),k are associated with corresponding base G (i ′ ,j ′ ) 5,A k . The column vectors in W(n) can occur in any order to form each of {W (i ′ ,j ′ ),A k : i ′ , j ′ , k satisfy G (i ′ ,j ′ ) 5,A k ∈ J 5 }. In this way, the precoding vectors of the interference after multiplying channel matrices belong to the set W(n + 1). The interference alignment conditions (21) are satisfied by letting the matrix W ′ consist of all the vectors in set W(n + 1). The received signal at user 5 can be expressed as where the former six items are desired signals and the remaining items in the summation are interference. Fig. 4 illustrates the signal space of the received signal at user 5. so as to achieve interference alignment at every user. Thus, the signal dimension is M = |W(n)| = n 8|Ψ|( √ |Ψ|−1)+2|Ψ|(|Ψ|−1) . Considering that the total dimension of six desired signals is 6M and the dimension of all interference vectors in set W(n + 1) is (n + 1) 8|Ψ|( √ |Ψ|−1)+2|Ψ|(|Ψ|−1) , the symbol extension number should be N = 6n 8|Ψ|( √ |Ψ|−1)+2|Ψ|(|Ψ|−1) + (n + 1) 8|Ψ|( √ |Ψ|−1)+2|Ψ|(|Ψ|−1) . Next, we show that for each user, the received vectors of desired signals are linearly independent from those of interference to ensure the decodability. Taking user 5 as an example, we denote and shall prove the full rankness of matrix The proof of the decodability for the entire cellular network is placed in Appendix B.
Remark 4: It is worth pointing out that the above delivery scheme of the cellular network differs from the cooperative transmission schemes of fully connected networks [9]- [11], [13], [32], [33]. In the proposed delivery scheme with partial BS cooperation (also the scheme with full BS cooperation introduced in the next subsection), we not only let the nearby BSs M (i,j) cooperatively serve user (i, j), but also let other BSs transmit the common messages desired by user (i, j) to increase per-user DoF of the network by interference neutralization and interference alignment.
C. µ = 1 (Full BS Cooperation)
In this case, all BSs can fully cooperate to serve users. The system thus can be regarded as a partially connected virtual MISO broadcast channel. We denote all the channel coefficients h (i,j),(p,q) as a |Ψ| × |Φ| matrix H Ψ,Φ , with each row denoting the channel coefficients from all BSs to each user. Since each user can only be connected to the four nearby BSs, there are four nonzero elements in each row of H Ψ,Φ . Note that the positions of nonzero elements in each row of H Ψ,Φ are different and all the channel coefficients are independent of each other. Therefore, the matrix H Ψ,Φ has full rank with probability one. A zero-forcing precoding matrix H † Ψ,Φ can be used to neutralize all the interference. Thus, we can achieve per-user DoF d = 1 and the proof of corner point (µ = 1, 1/d = 1) is completed. [26] In this subsection, we compare the proposed scheme with the existing scheme [26] 4 on cache-aided cellular networks. The main idea of our proposed delivery scheme is to utilize the common cached contents among BSs for interference neutralization (at µ = 1 2 and 1), and exploit the distinct cached contents among them to achieve interference alignment gain (at µ = 1 4 and 1 2 ). However, the transmission scheme in [26] is to only activate the BSs caching the same requested file segment at each time and use a zero-forcing precoding to do interference neutralization among different data streams. If applying the scheme [26] in our considered network model, we would obtain worse per-user DoF results. In specific, for µ = 1 4 , the BSs in B 1 , B 2 , B 3 and B 4 are activated alternately and the per-user DoFd = 1 4 can be obtained; for µ = 1 2 , the BSs in 4 and B 3 ∪ B 4 are activated alternately and the per-user DoFd = 1 2 can be obtained; for µ = 1, all the BSs in Φ are activated and the per-user DoFd = 1 can be obtained. By using memory sharing, the per-user DoF achieved by the scheme in [26] satisfies Fig. 5 shows the curves of the reciprocal of per-user DoF versus normalized cache size µ. It can be seen that our proposed scheme is superior in the whole cache size region. IV. CONVERSE In this section, we present an upper bound of the per-user DoF for the considered network under the given cache placement scheme in Section II-B. Based on this upper bound, we show that our achievable per-user DoF in Theorem 1 is optimal in certain cache size region and within a constant additive gap to the optimum in the remaining cache size region. Note that since each user is equipped with one antenna, the cut-set upper bound of the per-user DoF is d ≤ 1 for all possible caching and delivery strategies. This means that our achievable per-user DoF is within a multiplicative factor of 2 to the optimum, which, however is too loose to demonstrate the effectiveness of our proposed delivery scheme. To obtain the new upper bound tighter than the cut-set bound, we first derive an outer bound on the DoF region of the considered network for any given uncoded prefetching scheme. This outer bound is not restricted to the cellular network with grid topology and can be applied to networks with other general topologies. We then obtain an upper bound of the per-user DoF and show the order-optimality of the proposed delivery scheme with a constant additive gap.
A. Outer Bound on DoF Region
Consider a cellular network for any given uncoded prefetching scheme with BS cache groups Θ = {A k }. We define R (i,j),A k (µ, P ) |W r (i,j) ,A k | T as the rate of message W r (i,j) ,A k which is transmitted from BS cache group A k to user (i, j). Define capacity region C(µ, P ) as the set of all achievable rate tuples R = {R (i,j),A k (µ, P )} (i,j)∈Ψ,A k ∈Θ . The DoF region of the cache-aided cellular network is defined as Theorem 2: Consider a cache-aided cellular network with general topology. For each arbitrary user set R, we define a corresponding BS set T which satisfies the following two conditions: 1) |T | = |R|.
2) The matrix H R,T , with each row denoting the channel coefficients from all the BSs in T to each user in R, has full rank almost surely. Note that the channel coefficient from a BS to a user will be zero if there is no connection between them. Denote all the pairs (R, T ) as a set S. Then, an outer bound on the DoF region of the considered network for any given uncoded prefetching scheme with BS cache groups Θ = {A k } is given by whereR denotes the set of the remaining users connected to the BSs in T except the users in R. Proof: Please see Appendix C. To illustrate how to apply Theorem 2, we consider an example network with a given uncoded prefetching scheme as shown in Fig. 6. Here, the set Θ contains two BS cache groups, i.e., (29), we obtain Via adding (31b) and (31c), we can obtain a tighter bound than (32) and thus (32) is redundant. The outer bound D out of the network in Fig. 6 for the cache placement scheme with BS cache groups Θ = {A 1 , A 2 } is given by B. Upper Bound on Per-User DoF Next, we use Theorem 2 to derive an upper bound on the per-user DoF of the considered cellular network for the given cache placement scheme in Section II-B. Recalling that Θ is the set of all BS cache groups {A k }, the first item (29) is equal to |R| · d with d being the per-user DoF.
Since each user requests the same number of subfiles from the BS cache groups in the adopted caching scheme, the second item (29) can be expressed as |R| · A k ⊆T ,A k ∈Θ d (i,j),A k . By dividing both sides of the inequality (29) with |R|, we have By defining a function Based on the specific file splitting and placement strategy in Section II-B, we can determine the optimal T * as shown in the following lemma. Lemma 1: For the considered cache-aided cellular network with the given prefetching scheme in Section II-B, the optimal T * in problem P satisfies one of the following forms: Proof: Please see Appendix D. Since each T * in Lemma 1 is connected to all users Ψ, the corresponding R * satisfies R * ∪R * = Ψ. For T * with size |T * | = |Φ| 4 , |Φ| 2 , 3|Φ| 4 and |Φ|, the value |R * | |R * | is determined and given by 3, 1, 1 3 and 0, respectively. In other words, the optimal value λ * only depends on set T * and the cardinality |R * | (which is equal to |T * |), but not on the specific users in R * . One optimal form of R * is (1) R * = U 1 for [4] are defined as By searching for all possible (R * , T * ), we can obtain the upper bound on the per-user DoF. Theorem 3: For the considered cache-aided cellular network with each BS having a cache of normalized size µ and the given prefetching scheme in Section II-B, the reciprocal of the per-user DoF d is lower bounded as Proof: Please see Appendix E. Remark 5: Comparing Theorem 1 with Theorem 3, we can see that the upper and lower bounds of the per-user DoF coincide and thus the proposed delivery scheme is optimal for cache size µ ∈ 1 2 , 1 , and their additive gap for cache size µ ∈ 1 4 , 1 2 is given by V. CONCLUSION In this work, we characterized the per-user DoF of the cache-aided cellular network where the locations of BSs are modeled as a grid form and users within a grid cell can only communicate with four nearby BSs. By adopting a file splitting and placement scheme tailored for the network with partial connectivity, there exist different levels of BS cooperation in the delivery. We proposed the transmission schemes with no BS cooperation, partial BS cooperation and full BS cooperation, respectively, for different cache sizes. The achievable DoF results reveal that the reciprocal of per-user DoF for the cache-aided cellular network decreases piecewise linearly with the cache size and the gain of BS caching is more significant for the small cache region. Besides, we showed that under the given cache placement scheme, the obtained peruser DoF of the cache-aided cellular network is optimal when µ ∈ 1 2 , 1 , and within an additive gap of 4 39 to the optimum when µ ∈ 1 4 , 1 2 .
The received signal (ignoring noise) of user (i, j) can be represented as where the first item is the desired signal and the remaining three items are interference signals. For each user (i, j), we aim to align the directions of three interference signals into a common space and the interference alignment conditions are given by We collect the channel matrices experienced by interference for all users as and construct Recall that the cellular network is assumed as a square grid with the number of users in each row (or column) being |Ψ|. Thus, we can obtain |J (i,j),A k | = 2( |Ψ| − 1) with k ∈ [4], and |J (i,j),A k | = |Ψ| − 1 with k = 5, 6. We define J (i,j)∈Ψ [6] . For each user (i, j), we aim to align the directions of all the interference signals into a common space and the interference alignment conditions are expressed as span G Towards this end, we construct Here, we still use the simplified notations in Fig. 2 for those BSs and users and show that the decodability for user 5 without loss of generality. According to [33,Lemma 3], we shall prove the linear independence of the following polynomial functions where each w 5,k with k ∈ [6] is given by and each w ′ is given by Denote the sum of the elements in ι r (i,j),k as s(ι r (i,j),k ). The polynomial functions in (54), which have different sum exponent Here, each E 1 5,k denotes a specific constant value of s(ι 1 5,k ) with k ∈ [6]. Considering the grid network topology, it can be found that the polynomial structures of (57a) (57b) (57c) and (57d) are symmetric, and the polynomial structures of (57e) and (57f) are also symmetric. Thus, we shall only prove the linear independence of (57a) (57e) and (57g).
The ( |Ψ| 4 ) × ( |Ψ| 2 + 2) Jacobian matrix of the above polynomial functions has the similar structure as the one in Fig. 8, and thus it also has full row rank with probability one. Due to [33,Lemma 1] and [34,Theorem 3,p. 135 ], the polynomial functions in (59) are algebraically independent.
3) Linear independence of the polynomial functions in (57g): We divide all the sets in (57g) into two parts, i.e, {(g r (i,j),k ) ι r (i,j),k } with k ∈ [4], and {(g r (i,j),k ) ι r (i,j),k } with k ∈ {5, 6}. Since the Jacobian matrix of polynomial functions {g (i ′ ,j ′ ) (i,j),k : (i ′ , j ′ ) ∈ U r \ {(i, j)}}, with k ∈ [4], has the similar structure as the one of {g : (i ′ , j ′ ) ∈ U 1 \ {5}}, the polynomial functions {g (i ′ ,j ′ ) (i,j),k : (i ′ , j ′ ) ∈ U r \ {(i, j)}}, with k ∈ {5, 6}, are also algebraically independent. Thus, we can prove the linear independence of the polynomial functions within each set in (57g). APPENDIX C PROOF OF THEOREM 2 Define the following subfile sets and defineW as the set of the remaining subfiles to be sent in the network. For a user set R, we use y R to denote the vector of the signals received by the users in R, with a similar notation used for the transmitted signals and noise. In the following, we show that given y R , the subfile sets W R,Φ and WR ,T can be recovered withW as side information, i.e.
H(W R,Φ , WR ,T |y R ,W) ≤ ǫ Based on the converse assumption that each user can obtain the desired file from its received signal, W R,Φ can be decoded from y R almost surely. The signals y R and yR can be expressed as respectively. From W R,Φ andW, we can obtain those requested subfiles which are cached at BSs in set Φ \ T and further reconstruct the transmitted signal x Φ\T . Utilizing x Φ\T and y R , we can obtain a degraded version of yR as yR = HR ,T H † R,T (y R − H R,Φ\T x Φ\T ) + HR ,Φ\T x Φ\T = HR ,T x T + HR ,Φ\T x Φ\T + HR ,T H † R,T n R .
SinceỹR and yR only differ in noise, the subfile set WR ,T can be recovered fromỹR almost surely. By using Fano's inequality, we have where (64b) comes from the fact thatW is independent from W R,Φ and WR ,T ; (64f) is due to [13,Lemma 5]. By rearranging (64) and taking T → ∞ and ǫ → 0, we have For all the pairs (R, T ) in S, the above condition must be satisfied, which completes the proof.
APPENDIX D PROOF OF LEMMA 1 We need to prove λ(R, T ) ≤ λ(R * , T * ), where T * is given in Lemma 1 and R * is the corresponding user set for T * . First, we consider T not entirely including any B k . According to the adopted cache placement scheme, each BS cache group A k contains at least one B k . Thus, we have It is obvious that λ(R, T ) ≤ λ(R * , T * ). Then, we consider T including {B k } k∈Q and some other BSs, where Q ⊆ [4], e.g., T = B 1 ∪ {(0, 2)}. We shall prove λ(R, T ) ≤ λ(R * , T * ) with T * = {B k } k∈Q . Since in the adopted caching scheme each BS cache group is a union of some B k 's, we have Noticing that both T and T * are connected to all users Ψ, we have |R| + |R| = |R * | + |R * |. Since |R * | ≥ |R|, we can obtain | 2020-02-17T02:00:25.438Z | 2020-02-14T00:00:00.000 | {
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251333222 | pes2o/s2orc | v3-fos-license | Association between tennis training experience and executive function in children aged 8–12
Cognitively engaging activities have been shown to facilitate the improvement of executive functions in children. However, a limited number of studies have investigated whether the relationship between dose parameters of physical activities and executive functions, and heterogeneity exists. In the present study, we aim to explore the association between tennis training experience and executive functions in children. Sixty children between the ages of 8 and 12 were recruited in this study and were allocated to the short-term (ST) group (<12 months, n = 30) and the long-term (LT) group (more than 12 months, n = 30). The abilities of inhibitory control, cognitive flexibility, and working memory were measured by the Stop-signal task, Switching task, and N-back task, respectively. There was no significant group difference in either the accuracy or reaction time of the Stop-signal task. No significant difference between the groups' accuracy in the Switching task was observed. However, the LT group presented a shorter reaction time than the ST group (731.69 ± 149.23 ms vs. 857.15 ± 157.99 ms, P < 0.01) in the Switching task. Additionally, training experience was positively associated with the reaction time of the Switching task. As for the N-back task, in comparison with the LT group, the ST group showed a longer reaction time (711.37 ± 168.14 ms vs. 164.75 ± 635.88 ms, P < 0.05). Moreover, training experience was also positively associated with the reaction time of the N-back task. But there was no significant group difference in the accuracy of the N-back task. In conclusion, children trained for over 1 year have better performance in cognitive flexibility and working memory than those trained in <1 year; thus, tennis experience is positively associated with executive functions.
Cognitively engaging activities have been shown to facilitate the improvement of executive functions in children. However, a limited number of studies have investigated whether the relationship between dose parameters of physical activities and executive functions, and heterogeneity exists. In the present study, we aim to explore the association between tennis training experience and executive functions in children. Sixty children between the ages of and were recruited in this study and were allocated to the short-term (ST) group (< months, n = ) and the long-term (LT) group (more than months, n = ). The abilities of inhibitory control, cognitive flexibility, and working memory were measured by the Stop-signal task, Switching task, and N-back task, respectively. There was no significant group di erence in either the accuracy or reaction time of the Stop-signal task. No significant di erence between the groups' accuracy in the Switching task was observed. However, the LT group presented a shorter reaction time than the ST group ( . ± . ms vs. . ± . ms, P < . ) in the Switching task. Additionally, training experience was positively associated with the reaction time of the Switching task. As for the N-back task, in comparison with the LT group, the ST group showed a longer reaction time ( . ± . ms vs. . ± . ms, P < . ). Moreover, training experience was also positively associated with the reaction time of the N-back task. But there was no significant group di erence in the accuracy of the N-back task. In conclusion, children trained for over year have better performance in cognitive flexibility and working memory than those trained in < year; thus, tennis experience is positively associated with executive functions. KEYWORDS tennis, physical activity, training experience, executive function, children
Introduction
As an important component of cognitive functions and behaviors, executive functions (EFs) are easy to observe and are often used to measure the gain of the sports on brain and cognition. Executive function is an umbrella term referring to a higher-order cognitive process that is responsible for problem-solving, self-regulation, and goal-directed behavior control (Wickel, 2017;Morgan et al., 2019). EFs are critical in our life since it allows us to think before we act (Diamond and Ling, 2016). Three distinct but interrelated components constitute executive function: inhibitory control (the ability to inhibit, downregulate, or delay the dominant, automatic, or prepotent responses, or to stay focused instead of being interrupted by temptations or distractions), working memory (the ability to keep the information in mind and further process the information), and cognitive flexibility (the ability to shift attention adaptively among multiple tasks, rules, mindsets, and perspectives and to deal with a sudden, unexpected situation in real-time) (Michel et al., 2019;Willoughby et al., 2019). Well-developed executive function contributes to academic achievement (reading, mathematics, and science) (Rhodes et al., 2016;Gerst et al., 2017;Willoughby et al., 2019) and greater behavioral self-regulation (Morgan et al., 2019). In the school setting, students with EFs deficits often present with academic difficulties and behavioral problems (Otero et al., 2014).
Childhood is a highly sensitive and critical period for executive function development due to development of the key cortex, the prefrontal cortex and late maturation (Michel et al., 2018;Chen et al., 2020). The developmental curve of gray matter for the frontal lobe peaks at around age 12 (Giedd et al., 1999). Accordingly, the executive function undergoes a protracted development. During the period from 7 to 12, all sub-components of the executive function experience significant development, consistent with increased gray matter density in the brain (Bidzan-Bluma and Lipowska, 2018). During this time, the development of executive function is more sensitive to environmental and external stimuli, such as sports activity (Ludyga et al., 2016;Takacs and Kassai, 2019). As common sense, physical activity improves physiological indicators, namely, metabolic biomarkers, cardio-respiratory functions, bone health, and muscular strength, which promotes physical fitness, decrease the risk of metabolic syndrome and cardiovascular disease, and reduces psycho-social problems. Recent evidence showed that there was a positive association between regular physical activity and brain functions (Ludyga et al., 2016;Poitras et al., 2016;Chen et al., 2020). Therefore, physical activity may elicit greater benefits for children.
As surmised above, the effects of various types of chronic exercises on cognitive functions in children have been studied extensively. Cognitively engaging activities (e.g., football, basketball, etc.) have been shown to benefit executive functions in children undergoing developmental changes (Ludyga et al., 2016;de Greeff et al., 2018). Compared with physically demanding intervention alone (simple repetitive aerobic exercises), cognitively engaging activities are more efficacious in facilitating executive functions in children between the ages of 6 and 12 years (Schmidt et al., 2015;de Greeff et al., 2018).
Tennis is a technical and tactical racquet sport asking for a complex combination of physical components, namely, strength, power, speed, agility, aerobic and anaerobic capacities, and neuromuscular coordination (Zagatto et al., 2018). Although studies have demonstrated that 12-month high frequency (four times per week) tennis play resulted in a greater improvement in working memory than low frequency (once a week) play (Ishihara and Mizuno, 2018) and tennis experience were positively related to cognitive flexibility (Ishihara et al., 2017a). They only compared differences between the genders and measured only one type of difficulty in working memory, set-shifting task in cognitive flexibility, and interference control in inhibitory control. As heterogeneity exists across studies, more studies are needed to conclude the dose parameters of different physical activities to achieve optimal cognitive improvement (Erickson et al., 2019). The purpose of this cross-sectional study was to investigate whether training experience will influence the promoting effect of tennis training on three sub-components of EFs (i.e., inhibitory control, working memory, and cognitive flexibility). It is hypothesized that longer tennis training experience is associated with better improvement in EFs.
Participant
PASS 11.0 (NCSS, LLC, USA) was used to perform the sample size calculation. The sample size was calculated to be at least 10 in each group, with power was 0.9, alpha was 0.05, and a lost-to-follow-up rate of 10% (Ishihara et al., 2017b). Finally, the proposed sample size for our study was 30 in each group. In total, 60 healthy children from a tennis club were recruited to participate in this study. Those subjects were included only if they fulfilled the following criteria: (1) between the ages of 8 and 12, (2) right-handed, (3) with normal intelligence and hearing, (4) with a normal or corrected vision, (5) without any color blindness, serious somatic disease, or nervous system injury, (6) had never participated in any similar psychological test as this study before, and (7) did not participate in any physical activity moderate-intensity or above other than tennis training.
Tennis training lessons were technique-based, comprising of three parts: (1) warm-up, (2) tennis training or competition, and (3) cool-down. Participants were categorized into two different groups based on training experience, which was measured by training records: (1) short-term group (ST): participants' . /fnhum. .
training experience of fewer than 12 months and (2) longterm group (LT): participants' training experience more than 12 months. The study was conducted following the standards of the Declaration of Helsinki and was approved by the Ethics Committee of the Beijing Sport University. Written informed consent was given to participants and their parents before participation.
Procedure
The present investigation was performed from May 2019 to September 2019. All participants and their parents were informed of the experimental procedure before the test and signed an informed consent form. Information on demographics, namely, age, gender, height, weight, and training parameters (frequency and experience) were collected before the tests.
Three computers were used to perform cognitive tasks. Stimulus test programs were coded by E-Prime (Psychology Software Tools Inc., Pittsburgh, United States). To avoid experimental errors caused by different physical sensations, all tests were conducted from 0:00 to 13:00 in a quiet room at a constant temperature of 23 • C. Only one tester and one participant were allowed to be present in the room each time to avoid interference. Participants were asked to perform 10 trials identical to the formal test first. Formal tests were started when participants comprehend the experimental procedure and performed the task with no <90% accuracy (Wu et al., 2015). The experiments were conducted in the order of Stop-signal task, Switching task, and N-back task with appropriate rest between each test.
Inhibitory control
The Stop-signal task was used to assess inhibitory control (Luo et al., 2021). A black prompt "+" was displayed for 500 ms in the center of the screen to remind participants to start the test. One of the four black geometrical figures (i.e., circle, triangle, rectangle, and diamond) was presented randomly soon afterward, sometimes followed by a STOP symbol with stimulus onset asynchronies (SOAs) of either 200, 400, 600, or 800 ms. Participants were instructed to press the left button of the mouse in the absence the of STOP symbol (Go trial), otherwise, inhibit the motor response (Stop trial). Each figure displayed for no more than 2,000 ms. If the reaction was too slow, the figure would disappear and the trial would be regarded as an error. There were 133 trials in total for each participant. Reaction time (RT, interval between stimuli and response) and accuracy (ACC, percentage of correct response) of the Go trial and the ACC of Stop trial were recorded in real-time.
Working memory
The N-back task was used to assess working memory (Wolf et al., 2015). The task progressed in the order of difficulty level. One of the four black geometric figures (i.e., circle, triangle, rectangle, and diamond) was presented randomly on the screen. In this study, three blocks (the 0-back, 1-back, and 2-back tests) were administered. In the 0-back test, participants need to respond selectively according to the stimuli. Participants were instructed to press the left button of the mouse as soon as the triangle appears on the screen, or otherwise press the right button. In the 1-back test, participants were required to compare the current figure with the previous one and press the left button if the figures were identical, or otherwise press the right button. In the 2-back test, the subjects had to compare the current figure with the two figures presented previously and press the left button if they were identical, or otherwise press the right button. Each figure was presented for 500 ms, and the subjects had to respond within 2,000 ms. There were 46 trials in the 0back test, 60 trials in the 1-back test, and 74 trials in the 2-back test altogether. RT and ACC of each participant were recorded in real-time.
Cognitive flexibility
The Switching task was used to assess cognitive flexibility (Luo et al., 2021). The task began with the appearance of a prompt "+" in the center of the screen for 500 ms in either red or green color, followed by a pair of geometric figures (i.e., circle, triangle, rectangle, and diamond) placed horizontally in red or green, respectively. The figure with the same color as "+" was regarded as the target. The participants were asked to press the left button of the mouse if the target was the triangle, whereas the right button if not. If the participant was not able to respond within 4,000 ms, the figure would disappear and the trial would be considered an error. The participant would wait for another 500 ms and start the next trail. There were 96 trials in total for each participant, which were divided into three blocks. The first was the sustained condition, which contained two consecutive trails and two cues of the same color. The second was the switching condition, which contained two consecutive trails but the two cues were of different colors. The third was the sustained condition between the switching conditions, which had three or more consecutive trails and the cues are in two colors. RT and ACC of each participant were recorded in real-time. . For the mean RT and ACC data obtained from the GO trail of the Stopsignal task, one-way ANCOVA was performed. The mean RT and accuracy data obtained during the other two EFs tasks were analyzed using 2 (group: ST, LT) × 3 (blocks) twoway ANCOVAs. Bonferroni post-hoc techniques were used if a significant main effect or interaction was discovered. The effect size in ANCOVAs was given as η 2 p and was interpreted as follows: η 2 p ≥ 0.01 = a small effect, η 2 p ≥ 0.06 = a medium effect, and η 2 p ≥ 0.14 = a large effect. Correlations between the mean RT or ACC data of the EFs tasks and groups or training experience were performed by calculating Pearson's correlation or Spearman's rank correlation coefficients (r). Correlation coefficient strength was classified as negligible< 0.30, weak 0.31-0.50, moderate 0.51-0.70, or strong >0.71 (Sonesson et al., 2021). Statistical significance was set to P < 0.05 for all tests.
Result Demographics
Participants' demographic characteristics are shown in Table 1. The demographic profile was found to be comparable, Values are presented as mean (SD). ST, the short-term group; LT, the long-term group. *** statistically significant with P < 0.001. a Compared with the long-term group.
with no significant differences in gender, age, height, and weight between groups (P > 0.05). ST had significantly shorter training experience in comparison with LT (Z = −6.732, P < 0.001).
As for training frequency, there was no significant difference between ST and LT.
Inhibitory control
For the GO trail of the Stop-signal task, there were no significant differences in the ACC and RT between groups (Table 2; Figures 1A,B), meanwhile, no significant correlation was apparent between the groups and the ACC and RT of GO trail (Table 2) Table 3; Figure 1C]. The significant main effect of SOAs was observed [F (3,240) = 49.315, P < 0.001, η 2 p = 0.391], and ACC was significantly lower with the increase of SOAs (P < 0.001). No significant correlation was apparent between the groups and SOAs (Table 3).
Working memory
In the case of the RT, there was no significant interaction effect between group and block [F (2,180) = 0.287, P = 0.751, η 2 p = 0.003] but there was a significant main effect of group [F (1,180) = 10.808, P = 0.001, η 2 p = 0.059] and block [F (2,180) = 92.118, P < 0.001, η 2 p = 0.517]. An intergroup analysis showed that the RT of LT was significantly shorter than ST in the 0-back test (P = 0.001, Figure 2D), 1-back test (P = 0.015, Figure 2E), and 2back test (P = 0.014, Table 4; Figure 2F). Furthermore, the RT increased with the increase of blocks (P < 0.001). In addition, a weak negative correlation between the groups and the RT of the 0-back test, 1-back test, and 2-back test (Table 4) The ACC decreased with the increase of blocks (P < 0.001). No significant correlation was apparent between the groups and the ACC of the N-back task (Table 4). The RT of LT was significantly shorter than that of ST in the switching condition (P = 0.002), the sustained condition (P = 0.021), and the sustained condition between the switching conditions (P < 0.001, Table 5; Figure 3B). Meanwhile, there was a weak negative correlation between groups and the RT in the different conditions (Table 5). For the ACC of the Switching task ( Figure 3A), there were no Group×Block interaction effect . /fnhum. .
Correlation between training experience and EFs
In terms of inhibitory control (Figure 4), the ACC in the 400 ms SOAs (r = 0.257, P = 0.047) was positively correlated with training experience, but the strength was negligible. As for working memory (Figure 4), there was a negligible positive correlation between the ACC of the 2back test (r = 0.265, P = 0.04) and training experience. Meanwhile, there was a weak negative correlation between the RT in the 0-back test (r = −0.471, P < 0.001), 1-back test (r = -0.355, P = 0.005), and training experience. In the case of cognitive flexibility (Figure 4), a weak negative correlation between training experience and the RT in the switching condition (r = -0.391, P = 0.002), the sustained condition (r = -0.326, P = 0.011), and the sustained condition between the switching conditions (r = -0.413, P = 0.001) was observed. LT, the long-term group. *Statistically significant with P < . , **statistically significant with P < . .
Frontiers in Human Neuroscience
frontiersin.org . /fnhum. . Values are presented as mean (SD). ST, the short-term group; LT, the long-term group. * Statistically significant with P < 0.05. ** Statistically significant with P < 0.01. a Compared with the long-term group. Values are presented as mean (SD). ST, the short-term group; LT, the long-term group. * Statistically significant with P < 0.05. ** Statistically significant with P < 0.01. *** Statistically significant with P < 0.001. a Compared with the long-term group.
Discussion
In the present study, we examined the three domains of executive functions in children between the ages of 8 and 12 who participate in tennis training for more than 12 months and <12 months. We found that children between the ages of 8 and 12 with long-term training experience performed better than those with short-term training experience in cognitive flexibility and working memory. In addition, longer tennis training experience is associated with better performance in cognitive flexibility and working memory but not inhibitory control.
Our study found that longer training experiences were not associated with accuracy but with reaction time, regardless of the working memory loads. Children between the ages of 8 and 12 with long-term tennis training experience responded faster than children with short-term tennis training experience and had better performance in working memory. This may be since prolonged tennis training enhances the decision-making skills of the players and reduces their response delay times (Grigore et al., 2015). Previous studies have shown that people who regularly perform open-ended sports such as tennis and fencing exhibit faster reaction times, which is consistent with our study (Taddei et al., 2012;Šlosar et al., 2021). Working memory is the capacity to retain knowledge while making it accessible, and it is essential for processing all conscious information (Bergman and Söderqvist, 2017). Two reasons may explain why increased tennis training experiences improve children's working memory. First, working memory is likely to be activated during a child's engagement in tennis training because they must continuously recall tennis rules and techniques. Second, effective information processing, however, necessitates the use of working memory because children must maintain, update, and extract information that is pertinent to the task goal during tennis practice while ignoring or suppressing competing information that is irrelevant to the current context (Ishihara and Mizuno, 2018;Ishihara et al., 2018a,b). However, a recent study found that tennis training experience was not associated with working memory performance, which is inconsistent with our findings. The reason for this may be that they only used the difficult 2-back test, thus suggesting that future studies use the simple 1-back test to measure children's working memory (Ishihara et al., 2018b). Our study used three tasks with different memory loads to test the working memory performance of people with long-and short-term tennis training experience, yielding the same results as other studies that tested with only 2-back tests (Ishihara et al., 2017a). This study fills a gap in tennis research and further confirms the benefits of tennis training on working memory.
Results of the Switching task. (A)
The accuracy of three blocks and (B) the reaction time of three blocks. ST, the short-term group; LT, the long-term group; S between S, Sustained between Switching block, *statistically significant with P < . , **statistically significant with P < . , ***statistically significant with P < . .
Another important finding was that increasing the training experience of tennis can enhance the cognitive flexibility of children. Additionally, longer tennis training experience is associated with better performance in cognitive flexibility tasks. Cognitive flexibility is the capacity to change one's attention and focus to pursue an internal objective or meet task demands (Garon et al., 2008). It is frequently assessed using a variety of task-switching and set-shifting tasks (Diamond, 2013). We obtained results consistent with previous studies without using a set-shifting task, reinforcing the benefits of increased tennis training experience to improve cognitive flexibility. The reason could be that children with longer tennis training experiences have more opportunities to switch between different types of tasks and problem-solving strategies, which are necessary to enhance cognitive flexibility (Ishihara et al., 2018b(Ishihara et al., , 2019. Also, tennis places great demand on attention-shifting ability due to the need for tennis players to focus attention on the opponent's actions and balls, shift attention among different objectives, and make accurate and fast corresponding accordingly in a dynamically changing, unpredictable, and externally paced environment (Carlson et al., 1998;Shangguan and Che, 2018). During tennis play, for instance, players are required to memorize complex movement sequences, focus on the ball and opponent's position, as well .
FIGURE
The heat map of the correlation coe cient (r) matrix. TE, training experience; ACC, the accuracy of each executive functions task; RT, the reaction time of each executive functions task; SOAs, stimulus onset asynchronies; S between S, Sustained between Switching block. *Statistically significant with P < . , **statistically significant with P < . , ***statistically significant with P < . .
as have attention-shifting capacities under time pressure. It is assumed that these activities activate the similar brain regions used to control the higher level of cognitive processes (e.g., cognitive flexibility) (Schmidt et al., 2015;Egger et al., 2019). We were unable to determine the exact mechanisms behind the association between playing tennis and improved working memory and cognitive flexibility. Increased physical activity leads to physiological changes in the brain that might explain the current results. According to the studies, engaging in physical activity has a positive impact on brain structure and volume, namely, an increase in the white matter, parietal lobe gray matter, hippocampus, and basal ganglia volume (Chaddock et al., 2010;Niemann et al., 2014;Chaddock-Heyman et al., 2018). Additionally, physical activity is thought to affect brain neuroplasticity because it boosts the hippocampus's production of brain-derived neurotrophic factor (BDNF), encourages neuronal and synaptic growth and differentiation, and safeguards neuronal and synaptic transmission (Zhao et al., 2017). Moreover, recent research showed that exercise can increase blood flow to the brain, and clustering in exercise plasma reduced inflammation in the brain and enhances memory (De Miguel et al., 2021). Furthermore, another mechanism that may explain the improvement of cognitive performance might be that brain connectivity is enhanced following cognitive demanding exercise by increasing cell density and arborizing axons between brain structures engaged in motor and cognitive functions (Meijer et al., 2020).
However, we did not find a correlation between tennis training experience and inhibitory control. The correlation between tennis training experience and inhibitory control is not clear because (Ishihara et al., 2017c(Ishihara et al., , 2018a obtained different results using the same Stroop Color and Word task. Prepotent response inhibition and interference control are two commonly utilized distinctions in inhibitory control (Diamond, 2013). Tasks like the Go/no-go and Stop-signal are frequently used to test prepotent response inhibition, whereas the Flanker task and the Stroop task are commonly used to measure interference control (van der Bij et al., 2020). Unlike previous studies, in the present study, we used a Stop-signal task to explore the relationship between tennis training experience and prepotent response inhibition. However, we did not find a difference in prepotent response inhibition between the two groups. This phenomenon can be attributed to the fact that, unlike conflict tasks, the Stop-signal task does not require the execution of an alternative response and was not sensitive enough to detect any difference (Best and Miller, 2010). Moreover, because response inhibition grows better with age and our subjects were children between the ages of 8 and 12, the difficulty of the prepotent response inhibition task was insufficient to make a difference (Davidson et al., 2006). Ultimately, because the participants were all experienced in tennis training, the impact of tennis play on this population's inhibitory control may have had a ceiling effect (Ishihara and Mizuno, 2018).
In this study, we sought to assess differences in EFs in children with different tennis training experiences using a different type/difficulty of tasks than in previous studies and to explore the relationship between tennis training experience and three core components of EFs. However, several limitations should be acknowledged. Several potential moderators may influence executive function, namely, physical fitness level (Khan and Hillman, 2014;Borkertiene et al., 2019;Mora-Gonzalez et al., 2019), social-economic status (Vrantsidis et al., 2020), and peer and teacher-child relationship (van Lier and Deater-Deckard, 2016). Since we did not obtain this information, it is unknown if the aforementioned parameters account for variations in results. Given the cross-sectional nature of the study design, another limitation of the present study is that we were not able to infer a causal link between cognitively engaging physical activity and improvement of executive function. Therefore, longitudinal designs are needed in future research to make causal inferences. In addition, we only investigated whether training experience will influence the promoting effect of tennis training on three sub-components of EFs. Furthermore, since only behavior outcomes were assessed, the underlying mechanism of the promotion effect of cognitively engaging sports activities on executive functions was not obtained. Future studies should investigate brain biomarkers or structures to have a better understanding of biological or physiological pathways contributing to cognitive improvement. We only investigated whether training experience will influence the promoting effect of tennis training on three sub-components of EFs. In the future, it is indeed important to investigate whether closed-skill physical activity alone could affect EFs.
It is concluded that longer tennis experience was associated with better performance in working memory and cognitive flexibility in children between the ages of 8 and 12. Furthermore, tennis experience is positively associated with executive functions. The results of the present study may be of great practical importance for parents and educational settings to design physical activity programs that target the improvement of cognitive function for children.
Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.
Ethics statement
The studies involving human participants were reviewed and approved by the Ethics Committee of Beijing Sport University (2019112H). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.
Author contributions
YX, WZ, and LW wrote the manuscript. HZ and YX analyzed the data. HZ recruited subjects and carried out the experiments to collect the data. YL conceived and planned the experiments. GN made critical revisions related to the important intellectual content of the manuscript. All authors contributed to the article and approved the submitted version.
Funding
This study was supported by the Special Fund for Fundamental Scientific Research Expenses of Central Universities (20211003).
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. | 2022-08-05T15:06:07.533Z | 2022-08-03T00:00:00.000 | {
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244580837 | pes2o/s2orc | v3-fos-license | Extrinsic Anisotropy of Two‐Phase Newtonian Aggregates: Fabric Characterization and Parameterization
Abstract Rocks of the Earth's crust and mantle commonly consist of different minerals with contrasting mechanical properties. During progressive, high‐temperature (ductile) deformation, these rocks develop extrinsic mechanical anisotropy linked to strain partitioning between different minerals, amount of accumulated strain, and bulk strain geometry. Extrinsic anisotropy plays an important role in a wide range of geodynamic processes up to the scale of mantle convection. However, the evolution of grain‐ and rock‐scale fabrics causing this anisotropy cannot be directly simulated in large‐scale numerical simulations. For two‐phase aggregates–a good rheological approximation of most Earth's rocks–we propose a method to indirectly approximate the extrinsic viscous anisotropy by combining (a) 3D mechanical models of rock fabrics, and (b) analytical effective medium theories. Our results confirm that weak inclusions induce substantial weakening by forming a network of weak thin layers with limited lateral connectivity. Consequently, even when the inclusion phase is extremely weak, structural weakening is not larger than 30–60%, less than in previous estimates. On the other hand, the presence of strong inclusions does not have a profound impact on the effective strength of the aggregate, and lineated fabrics only develop at relatively low viscosity contrasts. When rigid inclusions become clogged, however, the aggregate viscosity can increase over the theoretical upper bound. We show that the modeled grain‐scale fabrics can be parameterized as a function of the bulk deformation and material phase properties and combined with analytical solutions to approximate the anisotropic viscous tensor.
convection patterns does not yet exist. Previous numerical studies considered compositionally layered media with simplified rheology, and the extrinsic anisotropy has been estimated for a strain-insensitive fabric by the Voigt and Reuss upper and lower bounds (Christensen, 1987;Lev & Hager, 2008;Mühlhaus et al., 2002;Perry-Houts & Karlstrom, 2019). Recent lab experiments at low finite strain have revealed that the effective strength of composites is strongly related to the initial geometry of the weak phase inclusion (Girard et al., 2016), which tends to form a network of layers of strain localization as strain increases. Dabrowski et al. (2012) and Thielmann et al. (2020) numerically studied the strength evolution of 2D, two-phase aggregates at larger deformation, reporting effective strength drops of about 80%. These 2D simulations likely overestimate the degree of weakening as they implicitly assume ideal lateral interconnection of the weak layers.
The goal of this paper is to combine numerical and semi-analytical methods to predict 3D strain-induced fabrics of two-phase (inclusion-matrix) aggregates (representative of a wide spectrum of geological materials) and associated extrinsic viscous anisotropy. First, numerical tools are employed to reproduce 3D fabrics under simple shear deformation to quantify the relationship between the extrinsic viscous anisotropy, amount of strain and volume ratio between inclusion and matrix. Then, we demonstrate that the anisotropy of a two-phase composite is well-predicted by semi-analytical solutions based on the Differential Effective Medium (DEM) theory. Finally, we parameterize the resulting 3D fabrics as a function of strain and strength contrast between the coexisting phases. For an arbitrary deformation state, the fabric can then be approximated, and the associated anisotropic viscous tensor can be calculated by the DEM.
Methods
We employ a Finite Differences solver for the 3D Stokes equations (T. Gerya, 2019) as the main tool to study the fabric development and associated evolution of the bulk viscosity in two-phase aggregates in simple shear. Numerical models are complemented with solutions derived from the DEM theory.
Numerical Methods
Deformation is described by the Stokes equations for incompressible viscous flow: where ∇ is the nabla operator, τ is the deviatoric stress tensor, p is pressure, and v is velocity. Equations 1 and 2 are solved employing a finite differences scheme combined with a particle-in-cell method (T. V. Gerya & Yuen, 2003) in the 3D Cartesian space. Tri-linear interpolation resulting in a weighted arithmetic mean is employed to map the viscosity back and forth between Eulerian nodes and Lagrangian particles. Throughout this paper, we use (a) bold upper case Latin and lower case Greek letters to denote fourth and second order tensors, respectively; (b) bold lower case Latin letters to denote vectors; and (c) regular symbols to denote scalar values.
For simplicity we adopt an isotropic Newtonian rheology for each material phase, so that the relationship between stress and strain is given by the linear constitutive equation 2 , where η is the shear viscosity and is the deviatoric strain rate tensor. This simplified rheology does not account for the effects of dislocation creep, brittle failure, pressure-solution, surface tension or other mechanisms that could affect the deformational behavior of a natural rock. Nonetheless, a Newtonian rheology is expected to be representative of the deformation behavior at mantle conditions where diffusion creep dominates (Ranalli, 1995).
Stokes equation are non-dimensionalized using the characteristic scales of viscosity η c , length l c , and time t c : where the superscripts i and m stand respectively for inclusion and matrix, L is the length of the cubic aggregate, and bg is the background strain rate. The choice of η c is purely out of convenience, so that both normalized viscosities are integers.
Model Setup
For a wide range of P-T conditions, rock-forming polymineral aggregates can be approximated by a population of dispersed particles (inclusions) within a matrix of distinct rheology where, in the simplest case, the initial rock geometry is isotropic with no shape preferred orientation of inclusions. This geometry is a good proxy of the fabric of many magmatic rocks, including mantle and oceanic rocks. Assuming a pyrolitic or the more depleted harzburgitic composition, above 410 km depth mantle rocks are made mainly of olivine and pyroxene (respectively, 60:40 and 70:30, Stixrude & Lithgow-Bertelloni, 2012). In the mantle transition zone this proportion remains roughly constant as olivine crystals transform into the high pressure polyhmorphs wadsleytie and ringwoodite, and pyroxene transforms into majoritic garnet. The lower mantle is mainly composed by bridgmanite and ferropericlase (80:20; ignoring minor presence of Ca-perovskite). After eclogitization the oceanic crust is made by omphacitic pyroxene and pyrope garnet, plus minor quartz (10%). In the transition zone, basalts are made by garnet and 10% stishovite. In the lower mantle, the subducted crust is formed by a four-phase aggregate with about similar volume fractions and unknown relative strength, such that it is not yet possible to predict potential grain-scale fabrics.
A simplified, but representative dimensionless model for such a composite consists of spherical inclusions (here with equal radius r = 0.1) randomly distributed within a cubic matrix of unit volume. This geometry avoids the complex crystal-like shapes of natural aggregates that pose a computational challenge for numerical codes, particularly at large inclusion-matrix viscosity contrast, as the stiness matrix resulting from the Stokes equations becomes more ill-posed. In 3D high-resolution models, this causes a signicant slow-down in the convergence rate of the iterative method (Geometric Multigrid Method, or GMG) used to solve the linear system of equations (see next section 2.1.2). In addition, spherical inclusions generally deform into pseudo-ellipsoids, so that tracking and quantifying the evolution of their shape is easier than for irregular shapes. Aggregates of (Mg,Fe) SiO 3 perovskite and ferropericlase, synthesized at uppermost lower mantle equivalent conditions (Yamazaki et al., 2009) consist of clusters of ferropericlase equant grains of comparable grain sizes and shapes resulting in near-isotropic samples prior to deformation. To a first approximation, the crystal shape ferropericlase grains is reasonably well approximated by spheres within a (Mg,Fe)SiO 3 matrix. In other geological scenarios, porphyroclasts-matrix aggregates in mylonites and bubble-bearing magmatic rocks are well approximated by composites containing pseudo-spherical inclusions in many cases.
The domain of the model is spatially discretized in an immutable and regular grid of cubic cells with 245 × 245 × 277 nodes. To simulate simple shear a kinematic reference frame is set where: the X-axis is the shear direction; the Y-axis is the vorticity axis, and the X-Y plane is the shear plane orthogonal to Z. Two rigid plates with thickness 0.08 We explore the development of fabrics at different viscosity contrasts between the inclusion and matrix phases by fixing the viscosity of the weak phase to 1 and varying the viscosity of the strong phase, so that strong phase [ , , ] 10 10 10 2 3 . The viscosity contrast between both phases is then defined as Δη = η i /η m . We further study the cases of composites with different volume fractions of the least abundant phase [ , , ]% 10 20 30 .
Model Convergence
3D Stokes problems with large discontinuous viscosity contrasts, such as those presented here, result in a linear system of equations with millions of degrees of freedom (∼66.5 millions in the models presented here) and highly spatially heterogeneous coefficients within the discretized elliptic operator. The linearized Stokes equations are solved iteratively via GMG, with Gauss-Seidel smoothing operating on every V-cycle level. This solution scheme works well for models with low and moderate inclusion-matrix viscosity contrast, producing well resolved flow solutions. However, at high viscosity contrast the GMG does not converge to a well-resolved flow solution after small finite strains when inclusion aspect ratio is different than one. May et al. (2015) employed matrix-free operators and a combination of GMG and Algebraic Multigrid Methods (AMG) to solve a sinker problem with viscosity contrasts of up to six orders of magnitude. However, the problem was solved only for instantaneous flow and with spherical sinkers. Although promising, such solution schemes are yet to be tested 10.1029/2021JB022232 4 of 25 for model set-ups and complex geometries comparable to the ones in this paper. Here we employ the following strategy: (a) at the first time step we employ the PARDISO direct solver (De Coninck et al., 2016;Verbosio et al., 2017;Kourounis et al., 2018) at some intermediate multigrid level to obtain the exact solution; (b) the solution is prolonged to the finest multigrid level; (c) multigrid V-cycles are performed until the desired tolerance is reached; (d) when the solution starts to diverge, the solution from the previous time step (initial guess) is reloaded and the V-cycles restarted with the pressure and velocity relaxation parameters halved. The latter step ensures a safer updating of the unknowns and a more stable, although slower, convergence toward a well-resolved solution.
Deformation History of the Inclusions
An isolated, spherical, isotropic inclusion suspended in a viscous matrix transforms into an ellipsoid (the inclusion finite strain ellipsoid, FSE) by homogeneous deformation of the matrix. In aggregates of spatially dense inclusions, particle interactions result in more complex deformed inclusion shapes. For these complex shapes the amount of accumulated strain is evaluated by computing the finite strain equivalent ellipsoid (FSEE), that is, the average FSE of the inclusions. The FSEE is given by eigenvalues a i and eigenvectors λ i , with i = 1, 2, 3, of the left stretch tensor (e.g., Becker et al., 2003), which define the length and orientation, respectively, of the FSEE semiaxes in the Cartesian space. The FSE is computed a posteriori in a set of selected Lagrangian particles sampling homogeneously the inclusions. We use the routines included in the software D-Rex (Kaminski et al., 2004) to compute the velocity gradient tensor, and update the deformation gradient tensor and the FSE. Arithmetic averaging of the Lagrangian particle FSE sampling a given inclusion is used to compute its FSEE. D-Rex does not inject particles when the cells of the discretized domain become empty, yielding artifacts on FSE(E) at large strain when weak inclusions are extremely stretched and/or heterogeneously deformed.
Analytical and Semi-Analytical Solutions for Viscous Tensors of Multi-Phase Aggregates
The Voigt and Reuss bounds define the upper and lower limits, respectively, for any bulk material property of composites with continuous fibers. For a given field or mechanical property Ψ, the Voigt and Reuss (e.g., Handy, 1990) bounds are given by: where i is a material phase and n the total number of material phases. In our case, Equation 4 predict the orthogonal and parallel viscosities with respect to the fabric of a laminar material, respectively. Natural multi-phase rocks rarely display a perfect laminar fabric and the Voigt and Reuss limits fail to provide accurate estimates of the mechanical properties. However, these limits still represent the upper and lower bounds of the mechanical properties of an aggregate. To overcome these limitations, alternatives methods in the field of micro-mechanics (Mura, 1987;Nemat-Nasser & Hori, 2013;J. Qu & Cherkaoui, 2006) based on Eshelby's work Eshelby (1957Eshelby ( , 1959 have been developed and applied to geology in recent years (e.g., Dabrowski et al., 2012;Jiang, 2014;Jiang & Bhandari, 2018;Yang et al., 2019). These methods however work under the assumption of ellipsoidal inclusions. Here we employ the DEM, which additionally assumes aligned inclusions, to draw comparisons with the rheology of 3D models and explore its capability to quantify the viscous tensor in aggregates with realistic rock-like fabrics. The DEM was first introduced by Roscoe (1952) to calculate the viscosity of suspensions of rigid particles and has been widely employed (e.g., Boucher, 1976;Dabrowski et al., 2012;Mainprice, 1997). The DEM tensorial formulation was developed by McLaughlin (1977): where η D is the viscosity tensor of the aggregate and A is the inclusion shape-dependent interaction fourth order tensor (e.g., Jiang, 2014;Mainprice, 1997): where the symmetric fourth order identity tensor is defined as J J J ijkl s ijkl jikl 1 2 ( ) , with J ijkl = δ ik δ jl being the fourth order identity tensor and δ ij the Kroenecker's delta. For a general viscous material, the Eshelby tensor S is given by: where T is the fourth order Green interaction tensor (Lebensohn et al., 1998): where ξ = [ sin(θ) cos(ϕ), sin(θ) sin(ϕ), cos(θ)] T , 〈⋅, ⋅〉 indicates inner product, and with ij ikjn D k n . The ordinary differential Equation 5 is solved by iteratively increasing ϕ (taking ϕ 0 = 0) until reaching the desired volume fraction, and integrated employing a fourth order Runge-Kutta scheme. In the first iteration, the viscous tensor of the aggregate is defined by the viscosity of the matrix, that is, η D (ϕ = 0) = 2J s η m . In Appendix B we discuss the numerical implementation of Equation 5 and the associated computational cost.
The main downside of the DEM is that all the inclusions are assumed of equal shape and orientation. However, stress and strain are not evenly distributed among all the inclusions during shear deformation due to inclusion interactions. This results in a heterogeneous distribution of inclusion shapes and orientations. Therefore, to compare the numerical results with the DEM, we average the FSE of all the inclusions to obtain the FSEE.
Evolution of Two-Phase Aggregates in Simple Shear Deformation
We first describe the evolution of a single spherical inclusion with Δη = 10 ±1 and dimensionless radius 0.2. Then, we describe the development of the fabric and anisotropic viscosity for multiple inclusions. The fabric is quantied by the aspect ratio of the FSEE principal axes and the inclination (α) of the FSEE a 1 -axis with respect to the horizontal shear plane.
Two-Phase Aggregate With a Single Inclusion
During simple shear, a spherical weak inclusion transforms into an FSE with the maximum a 1 -axis initially inclined at 45° with respect to the X-axis. With increasing strain (a) the FSE a 1 : a 3 ratio increases with the intermediate a 2 -axis (parallel to Y) rapidly growing initially of about 15% and then remaining constant in length for larger strain ( Figure 1a); and (b) the maximum a 1 -axis of the FSE progressively rotates in the XZ plane toward the X-axis to achieve a nearly stable position at ca. α = 1.5° for γ > 15 ( Figure 1b). As illustrated by 2D analytical solutions (Moulas et al., 2014), the pressure inside the inclusion depends on the viscosity contrast with the matrix and the maximum axis inclination α. In the case of a weak inclusion, the internal pressure increases to a maximum value at about α = 20°, and then decreases as the inclusion further rotates toward the Cartesian X-axis. The pressure eventually becomes negative at α < 7°, and then remains roughly constant at higher strain ( Figure 1c). The presence of a single weak inclusion weakens the bulk strength of the composite, inducing a decrease of the effective viscosity of about 10% with respect to the matrix viscosity γ > 5 ( Figure 1d).
In simple shear, a single spherical inclusion stiffer than the matrix permanently rotates (Figure 1e and 1f). During a rotation cycle, the inclusion transforms into a 3-axes ellipsoid having the a 1 -and a 3 -axis at their maximum and minimum, respectively, when the a 1 -axis is aligned with the shear plane. As rotation continues, the inclusion recovers its spherical shape. As predicted by analytical expressions (Moulas et al., 2014), the pressure inside the inclusion oscillates depending on the inclusion orientation and is (a) negative when the long inclusion axis is oriented in the range of 0° < α < 45°; and (b) positive for 45° < α < 90°. The presence of the rigid inclusion results in a 10% increase in the composite strength, which remains relatively constant during the whole deformation history ( Figure 1h).
Weak Inclusions
Similar to the case of a composite with a single weak inclusion, both the a 1 -axis and a 2 -axis of the FSEE increase with increasing γ (with an almost linear trend for the former up to γ = 7 − 8), while the a 3 -axis shows an initial rapid decrease with γ (Figures 3a and 3c). Given the much larger increase rate of the a 1 -axis and a 2 -axis, deformation results in the development of a strip-like shape of the individual particles aligned to form an L-S type fabric (Figures 2 and S1, and Movies S1 and S2). The a 2 -axis of the FSEE increases up to ca. 20% depending on the viscosity contrast (Figure 3a-3c), reaching a near-steady state length after ca. 600% of shear strain. The growth of the a 2 -axis indicates inclusion flattening.
The shape of individual inclusions is a function of the viscosity contrast and volume fraction between the two phases. The former determines how much the weak phase can deform within the strong matrix; the latter determines the amount of inter-crystal deformation. At low viscosity contrasts and low volume fractions of the weak phase (Figure 2g), the matrix opposes little resistance to deformation, and the inclusions, quite spaced apart, are relatively free to grow laterally without getting in contact. In this case, the inclusions are well defined ellipsoids (see Flinn diagram in Figure S3) that match with the associated FSEE.
At low viscosity contrast and high volume fractions ( Figure 2i and Movie S1), the weak phase evolves into a dense network of thin, weak surfaces. The close spacing of inclusions promotes interactions, which results in gently convex/concave pseudo-ellipsoidal inclusion shapes at high γ. An increase in the matrix rigidity affects To summarize, the models show that relatively low viscosity contrasts are needed to develop very well-defined networks of weak thin layers, while the foliated (S-type) fabric is less well-defined at large viscosity contrasts.
The evolution of the average inclusion inclination (α) is shown in Figure 4, top row. At the onset of deformation, the inclusions are inclined between 40° and 45°, subparallel to the third principal stress. With increasing γ, the angle α decreases exponentially to reach a near steady state value of 5° at γ > 10. The observed alignment of the inclusions with the flow direction is consistent with natural observations, 2D numerical models (e.g., Dabrowski et al., 2012;Thielmann et al., 2020), and micromechanic approaches with linear and non-linear rheology (e.g., Jiang, 2012Jiang, , 2014Yang et al., 2019). As previously stated, some inclusions experience rotation along the X-axis at large viscosity contrasts; however, rotations along Y-axis largely dominate. Our results further show that α is effectively independent of the viscosity contrast and matches the inclination of the FSEE a 1 -axis of the bulk composite.
Due to severe flattening and lateral growth in models with ϕ = 10% and Δη ∈ [10 −2 , 10 −3 ], during the calculation of the FSE the distance between Lagrangian particles of the inclusions becomes large enough so that some matrix particles fill empty grid cells. This results in the artificial segmentation of the inclusion, and causes the oscillations present in some of the panels in Figures 3 and 4. The resulting flattening, elongation, and alignment of the inclusions with the flow direction increases the amount of weak surface in the shear plane. This has positive feedback with the relative amount of strain accommodated by the weak phase and strain progressively localizes in the weak phase as the S-type fabric matures ( Figure 5). The strain rate accommodated by the "flat" S-type fabric is always larger than the bulk, and the fraction of strain accommodated by the inclusions rapidly increases before stabilizing after a few per cent deformation. For a given volume fraction, the final strain partitioning depends on the viscosity contrast, with larger viscosity contrasts yielding larger amounts of strain being accommodated by the weak inclusion phase.
For ϕ > 10% the amount of deformation accommodated by the inclusion phase is equal or larger than the strain absorbed by the matrix. This phenomenon is not observed for Δη = 10 −1 , although we cannot discard that it may occur at higher volume fractions than those considered here.
Strong Inclusions
Strain is mostly accommodated by the weak matrix and two types of fabrics are observed depending on the viscosity contrast. The first fabric type occurs at relatively low viscosity contrast (Δη < 10 2 ), where the inclusions a 1 -axis grows in the direction of the flow, while the a 3 -axis shrinks considerably (Figures 3d-3f). The a 2 -axis decreases up to 10% at high packing numbers, indicating constructional deformation. The inclusions thus deform into a prolate (cigar-like) shape and gradually rotate toward the shear plane (Figures 4k and 4l and Movie S3).
The reduced inter-crystal spacing at ϕ > 10% enhances the development of the L-type fabric (Figures 6h, 6i, S2h and S2i), because strain localizes at the contact between neighbor inclusions, enhancing the inclusions stretching. At lower ϕ, the L-type fabric is less pronounced and depends on the initial spatial distribution of the inclusions, since isolated inclusions barely deform in any direction and mainly rotate along the Y-axis. The second fabric type is observed for Δη ≥ 10 2 , where relative large viscosity of the inclusions inhibits large intra-crystal deformation and the inclusions constantly rotate around the Y-axis (Figure 4, bottom row), and small deviations from the initial shape are the result of interactions between inclusions. For ϕ < 30%, strain localizes just at the boundary with the rigid plates and the rigid inclusions concentrate toward the domain center. This phenomenon can be explained with the tendency of the system to maintain a balance in the grain dispersive pressure, which is caused by the interactions between rigid particles (Bagnold effect, Komar, 1972). The dispersive pressure increases with both the particle concentration and the velocity gradients. Thus, near the rigid boundaries the high velocity gradients must be compensated by a low rigid particle concentration, and vice versa toward the center of the composite. Clustering of the inclusion phase in a tight band is not observed at ϕ = 30%, where high strain localizes at the contact of the clogged inclusions ( Figures S2c and S2f). For all the different combinations of Δη and ϕ, strain is mainly accommodated by the weak matrix and remains more or less constant with increasing shear, absorbing more than 90% of the total deformation (Figures 5c and 5d).
Viscosity Evolution
Two-phase aggregates are mechanically heterogeneous materials where the stiffness tensor of the composite is no longer isotropic. It is not possible to infer all the components of the anisotropic viscous tensor of the composite directly from the 3D models, but only an effective viscosity (e.g., Jiang & Bhandari, 2018): where 〈⋅〉 indicates the volume average of a given field, and the subscript II indicates the square root of the second invariant of an arbitrary second-order tensor C, defined as C II 1 2 C C : . In simple shear, the effective viscosity is equivalent to the shear viscosity component in the plane parallel to the shear direction, i.e., η eff ≡ η xzxz , or η eff ≡ η xz in the reduced Voigt notation. To retrieve the remaining shear viscosities η xyxy and η yzyz , the model is rotated along the X-and Z-axes, respectively, at different stages of deformation to solve the instantaneous flow and compute the effective viscosity. The same approach was used by Hansen et al. (2012) to measure the normal viscosity under uniaxial extension on olivine aggregates after a certain amount of simple shear deformation. The normal viscosity components η xxxx , η yyyy and η zzzz are estimated here by imposing pure shear boundary conditions, with v = (1, 0, 0) and v = (0, 0, −1) prescribed at the boundary on the plane x = 1 and at the top of the domain, respectively, and free-slip boundary condition at the remaining boundaries of the domain. In this latter case, the rigid plates located at the top and bottom of the model are removed. The inclusions do not align perfectly with the horizontal plane and, therefore, the anisotropic viscous tensor contains non-zero values in the off-diagonal blocks, as well as in the off-diagonal indices of the lower diagonal block. However, direct retrieval of these components of the viscous tensor from the numerical models is not possible.
The numerical convergence of the rotated models is difficult to achieve as the result of large viscosity discontinuities, pre-existing complex morphology, and lack of a good estimate of the flow solution. Furthermore, some inclusions may split in two across the bottom and top non-periodic boundaries after rotation. In particular, pure shear models fail to converge in models with viscosity jumps larger than one order of magnitude, and even in some cases with low viscosity contrast. Only converged models are shown in this section. In Appendix A we demonstrate that the normal and shear components of the anisotropic tensor can be recovered from 3D models with a simple morphology where a fully converged flow solution is achieved.
Weak Inclusions
The shear-parallel viscosity exhibits the same trend in all simulations (Figure 7), with a short initial stage of hardening followed by rapid weakening and finally a new stage of gentle hardening at large deformation. The first hardening occurs at 0.5 < γ < 1 and is related to the transition of the initial spherical shape of the weak inclusions to σ 3 -parallel ellipsoids. This produces a disturbance in the otherwise quasi shear-parallel flow and results in a 3%-5% increase of the bulk viscosity. This initial hardening has been documented in 2D forward models with inclusion with spherical (Dabrowski et al., 2012) and random (Thielmann et al., 2020) shapes, in self-consistent micro-mechanical models with varying-shape ellipsoids and power-law rheology and flow fields more complex than simple shear (Jiang, 2014).
After the viscosity peak is reached, the models experience a phase of intense weakening, where the weakening rate is controlled principally by, and increases with, viscosity contrast. For example, models with Δη = 10 −3 and 10 −1 require about 500% and 1,600% of shear deformation, respectively, to reach the maximum weakening. After this point, the composite does not reach a steady-state of constant viscosity, but a stage of slight hardening. We infer that this last hardening stage is a combination of three mechanisms. First, even though our models do not exhibit a general loss of resolution, the edges of the inclusions may become thinner than the vertical spacing of the grid at large strain and introduce some numerical artifacts (Thielmann et al., 2020). Second, given an ellipsoidal inclusion with angle β < 45° between their second principal semi-axes and the horizontal plane, any rotation so that β ≤ β new ≤ 45° yields a stronger aggregate. At large strain, the inclusions develop different degrees of non-homogeneous internal rotation with the vorticity axes given by the shear-parallel direction (X-axis) that creates planes within the inclusion at a higher inclination with respect to the horizontal plane (Figure 2), thus arguably hardening the aggregate. And third, segmentation of severely sheared inclusions at large strain. Quantifying the contribution of these mechanisms to the total hardening would require running the models with considerably denser particle density and finer grid. In the latter case, adaptive meshes should be used, as the problem soon becomes computationally prohibitive with regular grids. On the other hand, normal stress is mainly supported by the matrix and, therefore, the normal viscosity components harden as the inclusions flatten, tending toward the Voigt upper bound (Figure 7). Figure 7 represent the shear-parallel viscosity calculated from the DEM Equation 5 by employing the average inclusion shape (FSEE) and subsequent correction of the average inclination of the fabric. The DEM matches well both initial and peak shear viscosity components, as well as the weakening stage of η xz and η yz , but overestimates the maximum amount of weakening. The η xy component is also well predicted by the DEM at low viscosity contrast (Δη = 10 −1 ). At larger viscosity contrasts, the DEM fails to predict the observed softening behavior and predicts a phase of hardening for η xy . We hypothesize that the observed weakening is caused by lateral distortions of the ellipsoidal shape (Figures 2b, 2c, 2e, and 2f) that are not captured by the DEM. As in Figures 3 and 4, the wiggles in the DEM curves in the middle and bottom panels of the left-hand-side column in Figure 7 are caused by poorly resolved FSE related to the segmentation of weak inclusions.
It is important to note that ω differs from the structural weakening ω struct , where the latter refers to weakening of the bulk aggregate relative to the initial, undeformed state (i.e., ing (ω struct ), always lower than the weakening normalized against the matrix viscosity (ω), oscillates between 30% (for Δη = 10) and up to 60% (for larger Δη).
The dashed blue line in Figure 8 represents the reduction in the bulk shear viscosity obtained by solving the DEM Equation 5. As previously discussed in Section 3.3, this semi-analytical equation predicts a stronger weakening of the composite in comparison to our 3D models. The misfit between the weakening from the DEM and the numerical models increases with Δη and ϕ, since the characterization of the average fabric becomes more difficult.
Strong Inclusions
The presence of rigid inclusions only moderately affects the effective strength of composites. A viscosity contrast of 3 orders of magnitude results in an increase of no more than 5-6 times the viscosity of the matrix, while the impact of the rigid inclusions is considerably less pronounced at lower Δη. Shear viscosity of composites with L-type fabrics (Figure 9, second row) exhibit slight strain softening, related to the rotation of the cigar-shaped inclusions from a σ 3 -parallel orientation to a stable position at few degrees off the flow direction. The resulting fabric is well characterized by the FSEE and, consequently, the DEM predicts well the shear-parallel viscosity with great accuracy. The L-type fabric no longer develops at Δη > 10, where particle rotations and small inclusion interactions are reflected in the oscillatory evolution of the effective viscosity (Figure 9, third and fourth rows). This is consistent with previous studies based on a multi-scale self-consistent micro-mechanical approach with power-law rheology (Yang et al., 2019), with the difference that the upper bound of viscosity contrast at which L-type fabrics develop was set at Δη = 5. These models show a clustering of the inclusions in a tight band at the center of the model domain that reduces the space between inclusions, resulting in a jammed aggregate where high stress localizes inside the inclusions and yields a viscosity that exceeds the Voigt upper bound. The DEM does not accurately predict the strength of jammed configurations and significantly underestimates the anisotropic viscosity.
Up-Scaling to Large Scales: Fabric Parameterization
The scale of the fabrics developing in multi-phase composites, aimed at simulating rock fabrics, is several orders of magnitude smaller than geological structures at the regional-to-plate tectonics scale. Therefore, it is computationally not feasible to include realistic multi-phase materials in numerical codes given the currently available computational power. As a consequence, mechanical anisotropy linked to the development of SPO cannot be incorporated in numerical models and materials are typically approximated as isotropic bodies.
Given the good match between the extrinsic viscous tensor obtained from forward numerical simulations and the DEM, the latter could be used to approximate the SPO-related viscous anisotropy of two-phase aggregates with a given fabric shape and orientation. In this section we propose a parameterization for the simulated 3D fabric as a function of parameters that might be either known or easily computed by geodynamic codes. Once the average composite morphology and orientation are estimated, the viscous tensor can be approximated by first solving the DEM Equation 5, and then rotating the anisotropic tensor with the angle α around the Y-axis. The black solid line correspond to the 3D models, the dashed black line is the Reuss bound, the dashed blue lines are the predictions from the differential effective medium (DEM), the dashed green line is the fit to the 3D models, and the solid red line with red circles is for 3D models with cylindrical inclusions. Figure 9. Evolution of the anisotropic viscosity tensor of a two-phase aggregate with a strong inclusion phase. Solid lines represent the viscosity retrieved from forward numerical simulations. Dashed lines are the η xz obtained from the differential effective medium (DEM) using the average shape of the weak phase as input and corrected for the average inclusion inclination. Gray dashed lines are the Reuss and Voigt lower and upper bounds, respectively. The normal components of the viscous tensor (top panels) are shown only for converged models.
Weak Inclusions
In the simplest case where the matrix and the inclusions have equal viscosity, the latter are perfectly aligned and their shape is given by the bulk FSE, which in simple shear and plane strain implies that a a a a 1 2 2 3 This expression is linear in the Flinn diagram and plots over the diagonal. For aggregates with weak inclusions we observe that the path of the FSEE is also quasi-linear in the Flinn space, and is approximately linearly proportional to the diagonal (i.e., bulk deformation), at least for the strain range in our models. Thus the fabric can be approximated as: where r i = log 10 (a i /a i+1 ) and A a a , with i = 1, 2, are the respective ratios of the FSEE and bulk FSE semi-axis, and ζ i is the proportionality constant. The evolution of the ratio between the intermediate and shortest semi-axis of both FSEE and bulk FSE is approximately the same (i.e., r 2 ≈ A) and only the parameterization of r 1 is necessary. The fitting coefficient ζ i = ζ(ϕ, Δη) slightly depends on the morphology and rheology of the aggregate (Table 1) and decreases as the inclusion volume fraction and viscosity contrast increase, reflecting that inclusion flattening is enhanced by inclusion interactions in composites with strongly varying mechanical properties. Note. The fitting parameters found in this table correspond to composites with weak inclusions. Combining Equation 11 with the DEM to estimate the anisotropic viscosity has to be done with caution, as the latter yields a considerably weaker aggregate at large strain compared to the forward models (Figures 7 and 8).
As a work-around, the maximum weakening observed in the 3D models can parameterized and used as a lower cut-off for the viscosity predicted by the DEM. The maximum weakening shows a non-linear relationship with the bulk deformation and physical parameters of the aggregate, which we find to be well-estimated (R 2 > 0.98) as: where the fitting coefficients ζ, ξ, χ, θ, ψ, λ depend on the volume fraction of the weak phase and are shown in Table 1. Extrapolations to larger Δη must be taken with caution, as the hardening behavior of ω is not well-reproduced by Equation 12 and additional data is needed to further understand this effect.
Strong Inclusions
The deformation path of the FSEE of hard inclusions is complex and it is no longer proportional to bulk deformation (Flinn diagram diagonal). Additionally, r 2 ≉ A and two parameterizations are needed to fully estimate the fabric. After testing several expressions, we find the following third order polynomial to reproduce well (R 2 > 0.97; Figure 10) the fabric evolution: where the fitting coefficients ζ, ξ, χ, θ (Table 2) are found via linear regression. We found difficult to produce an accurate parametrization of the fabric for Δη > 10, as small inter-crystal interactions yield a highly non-linear deformation path. Nonetheless, these inclusions barely deform and maintain a near-spherical shape at large strain ( Figure 6). Therefore, they can be safely considered as such.
Average Fabric Orientation
The components of the viscosity tensor vary with the relative orientation of the fabric with respect to the coordinate system of choice. Thus a good prediction of the fabrics must be completed with the orientation of the fabric with respect to the reference coordinate system. Our models show that fabric rotations off the Y-axis are negligible, and therefore only the angle α between the longest axis of the FSEE and the horizontal (XY) plane is relevant.
Weak Inclusions
The orientation of the fabric is linearly proportional to the orientation of longest axis of the bulk FSE: yielding R 2 > 0.95. The values of the proportionality constant (Table 1) are ζ ≈ 1, exemplifying that as already discussed in Section 3.2, the mismatch between the fabric orientation and the orientation of the longest axis of the bulk FSE is minimum (Figure 4), so that α ≈ α bulk is a good approximation of the inclusions orientation. Alternatively, in plane strain deformation and again assuming the alignment of the fabric with the bulk FSE, the analytical solution from McKenzie (1979) can be used: where / is the vorticity number, with Ω being the magnitude of the rotational component about the Y-axis.
Strong Inclusions
Hard inclusions exhibit a constant rotational behavior where the small amount of inter-crystal deformation introduces negligible disturbances in the evolution of the viscosity tensor. Only at ϕ > 10% and low Δη (about one order of magnitude), rigid inclusions develop an L-type fabric whose orientation is predicted by Equation 14 Figure 10. Flinn diagrams. Scattered symbols describe the average inclusion shape of the 3D models at different γ. The solid lines are the average inclusion shape predicted by Equations 11 and 13.
with the fitting coefficients in Table 2. Because of heterogeneous particle interactions at ϕ = 10% and Δη = 10, some elongate, while others adopt a rotational behavior (Figures 4 and 6g). To produce an accurate estimate of the orientation for this case, the forward model should run to larger deformation to study whether the aggregate fully develops an L-type fabric or reaches an stationary average rotational state.
Discussion
The kinematics of porphyroclasts are known to be sensitive to the rheology of the surrounding matrix (e.g., Passchier & Sokoutis, 1993). Additionally, rock-analogue experiments of isolated ellipsoidal and rhomboidal rigid inclusions suggest that a slipping boundary between the inclusion and the matrix is essential to attain a stable configuration (Mancktelow et al., 2002), otherwise the inclusion continuously rotates. These observations are in agreement with our models, with perfect coherence between the inclusions and the matrix, for (a) an isolated rigid inclusion, and (b) rigid inclusions of at least two orders of magnitude stiffer than the matrix, where a stable configuration is not reached. At moderately low viscosity contrast (Δη < 10 2 ), instead, we observe the development of a strong SPO. The fabric development rate is strongly related to the volume fraction of the rigid phase: in densely populated aggregates, channels of high-strain-rate, weak matrix form in between nearby inclusions, inhibiting the rotation and accelerating the inclusion elongation. Large finite strain (γ ≫ 20) is required to reach a stable configuration at low volume fractions. The inclination of the SPO increases with increasing Δη (e.g., about 8° at Δη = 10, and 25° at Δη = 50, both with ϕ = 30%). Therefore, the angle α may be used as a proxy for the aggregate rheology. We note that rheological non-linearities, such as dislocation creep or clast fracturing, and the degree of coherence between matrix and inclusions (Ceriani et al., 2003;Mancktelow et al., 2002) may significantly influence the kinematic behavior of the inclusions. These parameters should be included in future numerical simulations to better constrain the dynamics of matrix-inclusions systems.
When ϕ < 30%, rigid particles tend to concentrate away from the sliding rigid plates and toward the center of the model domain due to the Bagnold effect (Komar, 1972;Figures 6a, 6b, 6d, and 6e). This is consistent with the progressively larger concentration of phenocrystals and clasts observed toward the center of, respectively, magmatic dikes (Komar, 1972) and pseudotachylite veins (Di Toro & Pennacchioni, 2004). However, the latter result is not applicable to deforming viscous rocks where rigid walls are absent and a more homogeneous distribution of the harder inclusions is expected (as in models with ϕ = 30%; Figures 6c, 6f-6i). In contrast, weak inclusions are not affected substantially by the model boundary conditions and setup, and the modeled structures are likely representative of real composites. For instance, the L-S fabrics obtained in models with weak inclusions and low ϕ (Figures 2a, 2d, and 2g) are strikingly consistent with those of bubble-bearing magma sheared at large strains (Caricchi et al., 2011).
More generally, the modeled fabrics display several similarities with those observed in natural and experimentally deformed samples, which suggests the 3D models are capable of capturing, at least to a first order, the mechanical behavior of these composites. For example, intensively sheared gneiss rocks of the continental crust are frequently characterized by elongated ribbons of harder feldspar grains surrounded by flattened and laterally (Figures 6h and 6i) and the extrinsic viscous anisotropy is small (Figures 9a, and 9b). The numerical models reproduce the fabrics, strain weakening and strain partitioning in sheared synthetic samples representative of two-phase mantle aggregates (e.g., Girard et al., 2016), but a direct comparison to mantle rocks samples is difficult, as outcrops of the latter are generally part of the exhumed lithosphere, and hence they are not entirely representative of the deep and hot mantle where a more diffused and long-lasting deformation accommodated by high-T creep takes place.
Recent numerical studies demonstrated that anisotropy related to lattice preferred orientation (LPO) in olivine crystals can yield a weakening of about ω = 30% in the shear direction, and up to one order of magnitude viscosity variations depending on the dominant slip system (Király et al., 2020). The weakening is thus about a half of the predicted by SPO in our models, while the directional variations linked to LPO claimed by Király et al. (2020) can be up to two larger than what observed in our two-phase aggregates and DEM models (Figure 7). This implies that an aggregate with mechanically anisotropic crystals should be more susceptible to changes in flow directions than when only isotropic SPO-related fabrics are present.
2D numerical simulations (Dabrowski et al., 2012;Thielmann et al., 2020) show that strain progressively localizes in the weak inclusions as they elongate under simple shear deformation, considerably weakening the bulk composite before developing a network of fully interconnected weak planes. This implies a fabric maturity-dependent transition from a load-bearing framework to a network of interconnected weak layers. The weakening resulting from the compositional layer has been invoked to inhibit the mixing of material in the lowermost mantle (Ballmer et al., 2017) and enhance the connection between the upper and lower mantle through narrow conduits of rapidly ascending hot material (Christensen, 1987). The layering and strain-softening behavior of composites is well-reproduced by 3D models. However, our results suggest that weakening related to compositional layering is considerably less than reported by 2D plane-strain simulations.
Plane strain implies that the model is infinitely continuous along the direction orthogonal to the 2D cross-section. In other words, the inclusions in 2D representations of two-phase aggregates are continuous fibers of infinite length. The maximum reduction in normalized bulk viscosity, the evolution of the shear-parallel viscosity and the inclusion morphology are compared in Figures 8b and 11 for an aggregate with initially spherical inclusions and an aggregate with full-width cylindrical inclusions (ϕ = 20% and different Δη). Both model set-ups yield comparable cross-section morphologies (Figures 11c and 11d), which are comparable to the 2D morphologies in Dabrowski et al. (2012) and Thielmann et al. (2020).
The weakening observed in the model with cylindrical inclusions (Figure 11a) is also within the range of weakening of 2D models, and amounts to max. 60-80%. However, perfect inter-connectivity in the Y-direction has a strong influence on the effective viscosity of the composite and the strength of models with a more realistic 3D set-up quickly diverges from the plane-strain approximation at γ > 2. Plane strain models overestimate the amount of weakening by about 25% at Δη of one order of magnitude. The overestimation of weakening increases exponentially with increasing Δη between coexisting phases, predicting a three-times weaker composite for Δη of three orders of magnitude. Structural weakening ω struct (i.e., ratio between the bulk minimum and initial viscosities) is also significantly exaggerated by plane strain, which yields between 60% and 85% of ω struct at Δη = 10 −1 and Δη = 10 −3 , respectively. In contrast, only about 30% and 60% of ω struct is predicted by models with spherical inclusions. This comparison shows that while 2D models are a valid tool to provide first-order insights on highly 3D problems, the quantitative results should be taken with caution and further 3D studies with increasing degrees of complexity are required to better constrain the dynamics of multi-phase aggregates.
In this work, we considered only two-phase aggregates with isolated spherical inclusions of equal dimensionless radius 0.1. As discussed in Section 3.3, although this geometry is a good proxy for many relevant cases, two-phase aggregates may be comprised by overlapping inclusions forming heterogeneous clusters of random shape, such as the synthesized ferropericlase samples from Yamazaki et al. (2009). To assess the effect of overlapping inclusions and inclusion size, we ran two additional sets of experiments with: (a) spherical inclusions of equal radius 0.05 and 0.025; and, (b) random heterogeneous random media with a morphology such that the aggregate is statistically isotropic ( Figure S5). To generate the latter models, we use the statistical approach developed by Thielmann et al. (2020), and the average inclusion radii is 0.05 and 0.025. These models are run only with Δη = 10 −1 and 10 −2 , and ϕ = 20%. The evolution of the normalized effective viscosity (Figure 12) shows that the size and shape distribution of the inclusion phase does not dominate the aggregate rheology, which is mainly determined by the volume fraction of the weak phase. The early onset of post-weakening hardening in aggregates with inclusions of r = 0.025 is triggered by inclusion segmentation and/or loss of vertical resolution ( Figure S5d).
The extrinsic anisotropy of two-phase composites with weak inclusions, and strong inclusions with low viscosity variations between coexisting phases, is well-predicted by the DEM. The estimated viscous tensor given by the DEM is particularly accurate at low strain. The DEM overestimates the amount of weakening by a factor of 0.1-0.15 at large strain because deformation is not equally partitioned among individual inclusions and the average fabric defined by the FSEE does not perfectly represent the morphology of the composite. The fabrics developed in our models are well predicted as a function of bulk deformation. This, combined with the DEM, provides a simple framework to forecast the extrinsic anisotropy for a wide range of geological applications. The set of parameterizations proposed here are calibrated for a linear Newtonian rheology, simple shear deformation, and relatively low finite deformation. The validity of the proposed parameterization under different conditions (e.g., for composites deforming by power-law creep such as dislocation creep, or deforming during different bulk strain geometry) is to be explored. On top of this, the deformation fabric may be destroyed, causing an increase in the bulk viscosity, by different microstructural processes: (a) post kinematic annealing at low strain; (b) phase mixing related to dissolution/precipitation, nucleation and cavitation processes (e.g., Skemer et al., 2010;Kilian et al., 2011) at high strain; and, (c) as a result of partial melting of the aggregate.
Conclusions
We present a method to predict the viscous anisotropy of two-phase aggregates in simple shear deformation by combining numerical simulations and analytical solutions. Numerical models are used to simulate the development of 3D fabrics and quantify viscous anisotropy linked to SPO of two-phase (matrix and inclusion) aggregates. A range of geologically relevant inclusion volume fractions and viscosity contrasts have been considered.
Weak inclusions quickly flatten with increasing bulk strain and grow laterally forming a complex network of weak thin layers where most strain localizes. The aggregate is progressively weakened in the flow direction, reducing the strength of the aggregates by up to 80% relative to that of the matrix at moderate-to-high volume fractions of the weak inclusions and large viscosity contrasts. The structural weakening of the aggregate is lower, reaching a maximum value of about 60% with respect to the undeformed aggregate effective viscosity. The models suggest that this maximum weakening occurs at viscosity contrasts of two orders of magnitude. With the matrix-inclusion distributions considered here, the resulting system of equations becomes ill-posed at higher viscosity contrasts. Further improvement on the stability of linear solvers is necessary to confirm the latter observation from a numerical point of view.
In aggregates with strong inclusions, a linear fabric develops at low viscosity contrasts, and the inclusions remain largely undeformed at moderate-to-large viscosity contrasts. The strength of the aggregate remains roughly constant even at high strain.
The anisotropic viscosity of two-phase aggregates with weak inclusions, as well as aggregates with inclusions slightly stiffer than the surrounding matrix, is in good agreement with the solution of the DEM using an average shape and orientation of the fabric. Quantification of the anisotropic viscous tensor is of paramount importance for large scale geodynamic simulations since numerical limitations do not allow for a direct representation of multi-phase aggregates.
The fabric shape and orientation of weak laminar fabrics is approximately proportional to bulk deformation in a linear manner, while high-order polynomials are needed to approximate the development of L-type fabrics. A combination of this parameterization with the DEM might be used to retrieve the anisotropic viscosity of the aggregate. Future work will implement this methodology to obtain the viscous tensor in modern geodynamic codes to explore the effects of viscous anisotropy on global mantle convection patterns and other large scale geodynamic processes.
of inclusion shapes found in our models. As a computationally viable alternative, one can build a database of viscous tensors solving Equation 5 for a given range of physical parameters. Figure B1. Computational cost of differential effective medium (DEM) solution for different numbers of threads. The volume fraction in this test is ϕ = 30% with Δϕ = 5 ⋅ 10 −3 . The Green interaction tensor is numerically integrated by discretizing the integration space in 100 nodes in both dimensions. Parallelization is done at the coarsest level and all the calculations were run on an AMD Ryzen 5 3600. | 2021-10-16T15:18:29.142Z | 2021-10-13T00:00:00.000 | {
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20397759 | pes2o/s2orc | v3-fos-license | A molecular signature of preclinical rheumatoid arthritis triggered by dysregulated PTPN22
1Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA. 2Harvard Medical School, Boston, Massachusetts, USA. 3Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan. 4Division of Allergy, Immunology and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan. 5Division of Rheumatology, University of Colorado School of Medicine, Aurora, Colorado, USA. 6Department of Epidemiology, Colorado School of Public Health, Aurora, Colorado, USA. 7Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. 8Department of Life Sciences and 9Agricultural Biotechnology Center and Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan.
Introduction
The presence of anti-citrullinated protein antibodies (ACPA) is a unique feature of rheumatoid arthritis (RA) (1,2). Citrullinated proteins are generated by deimination catalyzed by peptidyl arginine deiminases (PADs), including PAD1-4 and PAD6 (3). Although the etiology of RA is still not fully understood, alterations in protein citrullination probably contribute to certain risk factors in RA. For example, smoking, a major risk factor of RA, can increase the level of extracellular PAD2 and intracellular citrullinated proteins in lung lavage (4,5). Porphyromonas gingivalis, the most common pathogen of periodontitis, expresses a PAD-like enzyme that is capable of citrullinating host proteins (6,7). Several epidemiological studies have suggested an association between periodontitis and risk of developing ACPA or RA (8)(9)(10). In addition, a functional haplotype stabilizing the mRNA from padi4, the gene encoding PAD4, is associated with a higher risk of RA and the presence of ACPA in RA patients (11)(12)(13)(14). Hematopoietic cells express mainly PAD2 and PAD4. These two PADs have overlapping but not identical substrate spectra (15,16). However, it is still unknown if either PAD has a unique contribution to the pathogenesis of RA.
A unique feature of rheumatoid arthritis (RA) is the presence of anti-citrullinated protein antibodies (ACPA). Several risk factors for RA are known to increase the expression or activity of peptidyl arginine deiminases (PADs), which catalyze citrullination and, when dysregulated, can result in hypercitrullination. However, the consequence of hypercitrullination is unknown and the function of each PAD has yet to be defined. Th cells of RA patients are hypoglycolytic and hyperproliferative due to impaired expression of PFKFB3 and ATM, respectively. Here, we report that these features are also observed in peripheral blood mononuclear cells (PBMCs) from healthy at-risk individuals (ARIs). PBMCs of ARIs are also hypercitrullinated and produce more IL-2 and Th17 cytokines but fewer Th2 cytokines. These abnormal features are due to impaired induction of PTPN22, a phosphatase that also suppresses citrullination independently of its phosphatase activity. Attenuated phosphatase activity of PTPN22 results in aberrant expression of IL-2, ATM, and PFKFB3, whereas diminished nonphosphatase activity of PTPN22 leads to hypercitrullination mediated by PADs. PAD2-or PAD4-mediated hypercitrullination reduces the expression of Th2 cytokines. By contrast, only PAD2-mediated hypercitrullination can increase the expression of Th17 cytokines. Taken together, our data depict a molecular signature of preclinical RA that is triggered by impaired induction of PTPN22.
It was recently demonstrated that Th cells of ACPA + RA patients expressed less PFKFB3, a rate-limiting enzyme of glycolysis, and ATM, a cell cycle checkpoint kinase, but a higher level of G6PD, which catalyzes the pentose phosphate pathway and promotes the generation of NADPH and glutathione (17,18). Therefore, RA Th cells are hypoglycolytic, hyperproliferative, and under reductive stress, but the cause of these features is still unknown. In addition, it is unclear whether these features appear before or after the development of ACPA or prior to the onset of clinical symptoms. In this regard, it is notable that ACPA are found in the serum of asymptomatic individuals for an average of 3-5 years prior to the onset of clinically apparent arthritis and the classification of subjects as having RA (19,20).
Citrullination converts positively charged arginines to neutral citrullines and is expected to alter protein folding and function. Indeed, citrullination critically regulates embryogenesis (21,22), epithelial-to-mesenchymal transformation (23), and pluripotency of embryonic stem cells (24). In addition, PAD4-mediated citrullination of histones is essential for the formation of neutrophil extracellular traps (NETs) (25)(26)(27), which can induce the expression of inflammatory cytokines, such as IL-6 and IL-18, as well as CCL20 and ICAM-1 from fibroblast-like synoviocytes (28). NETs are also a rich source of citrullinated antigens. Thus, PADs can contribute to RA pathogenesis by promoting the generation of citrullinated antigens and aggravating inflammation through the formation of NETs. However, it is still unclear if citrullination and PADs have other regulatory roles in immune cells in addition to promoting NETosis.
We recently discovered that PTPN22, a nonreceptor protein tyrosine phosphatase, can interact with and inhibit the activity of PAD4 (29). This function of PTPN22 is independent of its phosphatase activity. A C-to-T SNP, located at position 1,858 of human PTPN22 cDNA and replacing an arginine (R620) with a tryptophan (W620), carries the highest risk among all non-HLA genetic variations that are associated with RA (30)(31)(32)(33). This R-to-W conversion renders PTPN22 unable to interact with PAD4 and suppress citrullination. Accordingly, the C1858T SNP is associated with hypercitrullination in peripheral blood mononuclear cells (PBMCs) and heightened propensity for forming NETs even in healthy donors. Whether PTPN22 also inhibits the activity of PAD2 remains to be determined.
The frequency of the C1858T SNP in individuals of European descent is approximately 10%, highest among of all ethnicities. It is unclear if hypercitrullination is also present in PBMCs of other at-risk individuals (ARIs) who do not carry the C1858T SNP. If it is, what is the cause and functional consequence of hypercitrullination? To answer these questions, we studied PBMCs obtained from ARIs, including unaffected RA first-degree relatives (FDRs) and ACPA + individuals without RA. Positive family history and positive ACPA status each carries an odds ratio of approximately 4 to 5 or higher (34)(35)(36), which is higher than that of the C1858T SNP. Thus, these individuals are at high risk of developing RA. We found in two independent cohorts of ARIs that hypercitrullination in PBMCs is common. Their PBMCs, regardless of ACPA status, have a defect in the induction of PTPN22, display an aberrant Th cytokine profile, and exhibit a phenotype very similar but not identical to that of RA Th cells. We establish the chronological and causal relationships of these abnormal features and demonstrate that attenuation of both phosphatase and nonphosphatase activities of PTPN22 caused by impaired induction of this protein is likely to be at the root of these abnormal features. , and early/untreated rheumatoid arthritis (eRA) patients (C) were directly analyzed by Western blots for the level of citrullinated histone H3 (cit-H3) or total histone H3 (H3). The identity of each donor within each group is denoted with Arabic numerals. The level of cit-H3 was quantified with densitometry and normalized against that of H3. The normalized density of CT #4 in A was arbitrarily set as 1. The normalized density of cit-H3 from all donors is shown in D. The normalized cit-H3 levels of FDR of anti-citrullinated protein antibody (ACPA + ) or ACPAprobands are shown in E. Statistical analysis was performed with 1-way ANOVA followed by multiple comparison tests (D) or with 2-tailed Student's t test (E). The CC group was used as the control group. ***P < 0.001; ****P < 0.0001. The bars shown in D and E represent mean ± SD.
the FDRs of the BWH cohort, its frequency is approximately 20% in RA patients in North America, so there should be no more than 5 CT FDRs in the BWH cohort. All ARIs had no systemic inflammation at the time of blood draw. Therefore, the elevated level of cit-H3 in ARIs is unlikely to be due to smoking, the C1858T SNP, or systemic inflammation. If hypercitrullination in PBMCs is a precondition of RA, then we should expect to see hypercitrullination in early RA patients, particularly before any treatment. Consistent with our hypothesis, PBMCs from early RA patients also displayed hypercitrullination compared with controls ( Figure 1, C and D, and Figure 2, A and B). Interestingly, no hypercitrullination was detected in 10 treated RA patients (Figure 1, B and D). These treated RA patients had a mean CDAI of 2.6. This observation strongly suggests that effective treatment reduces the level of cit-H3. In the following experiments, ARI samples from the BWH and UC cohorts were used indistinguishably. Early RA patients were not analyzed because they were treated soon after diagnosis.
Cellular sources of cit-H3 in PBMCs. The hypercitrullination seen in ARI PBMCs can come from a single population or multiple populations of blood cells. To identify the source of cit-H3 in PBMCs, we decided to use intracellular staining to quantify the level of cit-H3 on a single-cell basis. We stimulated splenocytes collected from WT mice or mice deficient in PAD4 (PAD4KO), the only PAD bearing a nuclear localization signal, with PMA and then subjected the cells to intracellular staining of cit-H3. PMA increased the staining of cit-H3 in T, B, and non-T/non-B cells (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/jci.insight.90045DS1). This increase was almost completed abrogated in the absence of PAD4. There was still trace cit-H3 staining in PAD4KO splenic T cells. This residual staining is most likely caused by PAD2, which is also expressed in mouse T cells and can also be found in the nucleus (39). We then stained PBMCs from ARIs and control donors with anti-cit-H3 or control IgG. We excluded dead cells and used CD3 and CD20 to identify T, B, and non-T/non-B cells ( Figure 3A). Despite interexperimental variations in the level of cit-H3 staining, we reproducibly detected more cit-H3 in T cells from ARIs compared with those from control donors ( Figure 3, B and C). No difference in the level of cit-H3 between ARIs and control donors was observed in B and non-T/non-B cells, indicating that T cells are the major contributors of cit-H3 in PBMCs of ARIs.
Impaired induction of PTPN22 in ARI PBMCs. As T cells are the major source of cit-H3, it is possible that ARI PBMCs have a higher percentage of T cells, which causes the higher level of cit-H3. However, in the 3 pairs of donors analyzed in Figure 3A and 5 additional pairs of donors, we did not see any major difference in the distribution of T or B cells, even though the percentage of non-T/non-B cells was slightly lower in ARIs ( Figure 3D). We then examined the expression of PAD2 and PAD4, two dominant PADs in hematopoietic cells (40). Interestingly, anti-CD3 stimulation led to a reduction in the transcript level of PAD2 and PAD4 ( Figure 3E). However, the levels of PAD2 and PAD4 in either resting or stimulated PBMCs were very comparable between controls and ARIs.
An alternative explanation for the hypercitrullination seen in ARI PBMCs is impaired expression of PTPN22. We found that the level of PTPN22 transcript in resting PBMCs was comparable between controls and ARIs ( Figure 3F). Anti-CD3 stimulation expectedly increased the level of PTPN22 transcript by almost 2-fold in control PBMCs. Surprisingly, no such induction was detected in ARIs PBMCs ( Figure 3F). The level of PTPN22 transcript in half of the ARIs was actually reduced by anti-CD3 Figure 2. Hypercitrullination in PBMCs from healthy at-risk individuals recruited at the University of Colorado. PBMCs from first-degree relatives (FDR), individuals positive for anti-citrullinated protein antibodies (ACPA + )/non-FDR (ACPA + ), individuals with early rheumatoid arthritis (eRA), and controls were analyzed by Western blots for the level of citrullinated histone H3 (cit-H3) and total histone H3 (H3) (A). The density of cit-H3 was measured and normalized against that of H3. The normalized density of FDR donor #8 was arbitrarily set as 1. The normalized density of cit-H3 of all donors is shown in B. The normalized cit-H3 density from ACPA + and ACPAat-risk individuals (ARIs) is shown in C. Statistical analysis was performed with 1-way ANOVA followed by multiple comparison tests (B) and with 2-tailed Student's t test (C). *P < 0.05. The bars shown in B and C represent mean ± SD.
stimulation. There was no difference in the induction of PTPN22 between ACPA + and ACPA -ARIs (Supplemental Figure 2). We were able to examine the level of PTPN22 protein after anti-CD3 stimulation in 9 ARIs and 8 controls ( Figure 3G). Indeed, the level of PTPN22 protein was significantly lower in stimulated ARI PBMCs ( Figure 3H). Contrarily, the level of cit-H3 in stimulated ARI PBMCs was higher than that in stimulated control PBMCs ( Figure 3H). There was a reverse correlation between the level of PTPN22 and cit-H3 when ARIs and controls were analyzed together ( Figure 3I). Those with a higher level of PTPN22 tends to have a lower level of cit-H3, suggesting that the impaired induction of PTPN22 contributes to the hypercitrullination.
Abnormal phenotype of ARI PBMCs. Th cytokines, such as IL-17, IL-4, and IFN-γ, play a critical role in the pathogenesis of RA and many other autoimmune diseases. To determine if ARI PBMCs have any defect in the expression of Th cytokines, the PBMCs were stimulated with anti-CD3 for 24 hours and the production of Th cytokines was measured with ELISA or real-time PCR. We found no difference in the level of IFN-γ between ARIs and controls ( Figure 4A). Interestingly, ARI PBMCs produced more IL-2 Anti-CD3 stimulated PBMCs from 9 ARIs and 8 control donors were analyzed with Western blotting for PTPN22, cit-H3, and total histone H3 (H3). The density of PTPN22 and cit-H3 was normalized against that of H3 and shown in H. The normalized PTPN22 values were plotted against normalized cit-H3 values and shown in I. Statistical analysis was performed with Student's 2-tailed t test in C, D, the right panel of F, and H; 1-way ANOVA in E and the left panel of F; and Spearman's test in I. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The bars shown in H represent mean ± SD. and Th17 cytokines, including IL-17A and IL-17F, but almost 50% less Th2 cytokines, such as IL-4 and IL-13. There was no difference in cytokine production between ACPA + and ACPA -ARIs ( Figure 4B).
A recent study indicates that naive Th cells from ACPA + RA patients express less PFKFB3 and ATM but more G6PD in response to stimulation (18). We found that PBMCs of ARIs also had a reduced level of PFKFB3 and ATM after stimulation ( Figure 4C). Accordingly, PBMCs of ARIs generated less lactic acid upon stimulation ( Figure 4D). The levels of PFKFB3, ATM, and lactate were comparable between ACPA + and ACPA -ARIs (Figure 4E and data not shown), suggesting that these changes do not require the development of ACPA. Interestingly, the expression of G6PD was normal in ARIs ( Figure 4C). This observation indicates that the aberrant expression of G6PD is not coupled to the attenuated expression of PFKFB3 and ATM and occurs independently of ACPA.
Distinct effects of phosphatase and nonphosphatase activities of PTPN22. When we pooled ARI and control samples together, we found that the levels of IL-2, IL-17A, and IL-17F reversely correlated with the induction of PTPN22 ( Figure 5A). There appeared to be a threshold effect for IL-2 and IL-17F. When the induction of PTPN22 dropped to below 1, the level of IL-2 and IL-17F started to rise. Contrarily, there was a positive correlation between the induction of PTPN22 and the level of ATM or PFKFB3. There was also a trend of positive correlation between Th2 cytokines and the induction of PTPN22, but this trend did not reach statistical significance.
These observations prompted us to investigate if impaired induction of PTPN22 was responsible for the phenotype of ARI PBMCs. We transfected PBMCs from 6 randomly selected ARIs with PTPN22 expression vector ( Figure 5, B and C). The transfected cells were then stimulated with anti-CD3. In agreement with our hypothesis, forced expression of PTPN22 reduced the level of cit-H3 ( Figure 5B); increased the level of Th2 cytokines, PFKFB3, ATM, and lactate; and reduced the level of IL-2 and Th17 cytokines ( Figure 5C).
PTPN22 is a phosphatase and is known to attenuate activation signals in lymphocytes. Thus, impaired induction of PTPN22 is expected to augment activation signals in T cells. However, the reciprocal changes in Th2 and Th17 cytokines observed in ARI PBMCs cannot be explained by stronger activation signals in T cells. In addition to acting as a phosphatase, PTPN22 also has nonphosphatase activities, including suppressing citrullination and promoting TLR-induced expression of type I interferon (29,41). To determine if PTPN22 shaped the phenotype of ARI PBMCs through its phosphatase or nonphosphatase activity, we also expressed W620-PTPN22 or a catalytic dead PTPN22 (CD-PTPN22) in ARI PBMCs. CD-PTPN22 carries two point mutations at the protein tyrosine phosphatase domain of PTPN22 and has little phosphatase activity (42). However, it is still fully capable of suppressing citrullination (29), whereas W620-PTPN22 retains the phosphatase activity but no longer has the nonphosphatase activities (29,41). In agreement with our published data (29), CD-PTPN22 but not W620-PTPN22 reduced the level of cit-H3 in ARI PBMCs as efficiently as WT-PTPN22 ( Figure 5B). In addition, CD-PTPN22 was able to normalize the level of Th2 and Th17 cytokines but not IL-2, ATM, PFKFB3, or lactate ( Figure 5C). Although the effect of CD-PTPN22 on IL-17F did not reach statistical significance in 1-way ANOVA analysis, it carried a P value of 0.0487 when directly compared with empty vector control in Student's t test analysis. By contrast, W620-PTPN22 was able to normalize the level of IL-2, ATM, Figure 4. Abnormal phenotype of at-risk individual PBMCs. PBMCs from control donors (C) and at-risk individuals (ARIs) (14-17 per group) were stimulated with anti-CD3 for 24 hours. The expression of indicated cytokines was quantified with ELISA or real-time PCR (A). The levels of cytokines from ARIs who are ACPA + and ARIs who are ACPAare compared in B. In addition, the transcript levels of PFKFB3, ATM, and G6PD measured with real-time PCR are shown in C, and the concentration of lactate in supernatant is shown in D. The transcript levels of PFKFB3 and ATM in ACPA + and ACPA -ARIs are compared in E. Statistical analyses were performed with Student's 2-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The bars shown represent mean ± SD.
PFKFB3, and lactate but not Th2 or Th17 cytokines (Figure 5C). These results indicate that the aberrant expression of IL-2, ATM, and PFKFB3 is caused by attenuated phosphatase activity of PTPN22, whereas the abnormal Th2 and Th17 cytokine profile is due to attenuated nonphosphatase activity of PTPN22.
PTPN22 regulates the Th2/Th17 cytokine profile by suppressing citrullination. Attenuation of nonphosphatase activities of PTPN22 is expected to lead to hypercitrullination and impaired TLR-induced expression of type I interferon. The observation that normalization of Th2 and Th17 cytokines correlated with a reduction in the level of cit-H3 prompted us to postulate that hypercitrullination, but not dysregulated expression of type I interferon, is the cause of the abnormal Th2/Th17 cytokine profile of ARI PBMCs. To test this hypothesis, we randomly selected 9 ARIs and examined the Th2 and Th17 cytokine profile of their PBMCs after stimulation with anti-CD3 in the absence or presence of Cl-amidine, a pan-PAD inhibitor. Expectedly, Cl-amidine reduced the level of cit-H3 ( Figure 6A). It also increased the level of IL-4 and IL-13 and decreased the level of IL-17F ( Figure 6B). There was also a trend toward reducing the level of IL-17A. However, the effect of Cl-amidine treatment is rather modest. This modest effect may be expected considering that hypercitrullination already existed in ARI PBMCs before stimulation.
We therefore took two additional approaches to further examine the impact of hypercitrullination on the expression Th cytokines. We generated a line of Jurkat cells that express an exogenous PAD2 in an inducible manner (43). We found that inducing the expression of PAD2 increased the expression of Th17 cytokines but inhibited the expression of Th2 cytokines ( Figure 6C). In addition to PAD2, human PBMCs also express PAD4. Attenuated nonphosphatase activity of PTPN22 may lead to hypercitrullination through PAD2 and/or PAD4. To recapitulate the cytokine phenotype of ARI PBMCs and to distinguish between the effects of PAD2-mediated and PAD4-mediated hypercitrullination, we overexpressed PAD2 or PAD4 in control PBMCs, which were then stimulated with anti-CD3. Exogenous PAD2 and PAD4 equally increased the level of cit-H3 ( Figure 6D) and comparably suppressed the expression of IL-4 and IL-13 ( Figure 6E). However, only PAD2, but not PAD4, was able to enhance the expression of Th17 cytokines. Figure 4 were plotted against the fold induction of PTPN22 shown in the right panel of Figure 3F. Statistical analysis was performed with Spearman's correlation test.
(B and C) PBMCs from at-risk individuals were transfected with plasmid vector expressing PTPN22, W620-PTPN22, CD-PTPN22, or the empty vector (-), and then stimulated with anti-CD3 for 24 hours. The levels of PTPN22, citrullinated histone H3 (cit-H3), and total histone H3 (H3) in transfected/stimulated cells were quantified by Western blotting (B). Representative blots of 6 independent experiments are shown. The density of PTPN22 and cit-H3 was quantified with densitometry and normalized against that of H3. The normalized levels of PTPN22 and cit-H3 are shown. The expression of indicated cytokines and genes was measured with ELISA or real-time PCR (C). The concentration of lactate in supernatant was quantified with a colorimetric assay (C). The data values from the same donors were connected with lines. Statistical analysis for B and C was performed with 1-way ANOVA followed by multiple comparison tests using the empty vector-transfected groups as controls. *P < 0.05; **P < 0.01; ***P < 0.001.
Discussion
A molecular signature of preclinical RA has emerged from our data (Supplemental Figure 3). Impaired induction of PTPN22 leads to attenuated phosphatase and nonphosphatase activities of PTPN22. Attenuated phosphatase activity of PTPN22 results in augmented expression of IL-2 but diminished expression of ATM and PFK-FB3, which subsequently leads to hypoglycolysis. By contrast, attenuated nonphosphatase activity of PTPN22 causes hypercitrullination, which is responsible for aberrant production of Th2 and Th17 cytokines. PAD2-or PAD4-mediated hypercitrullination suppresses the expression of Th2 cytokines, whereas only PAD2-mediated hypercitrullination is able to augment the production of Th17 cytokines. All these molecular events very likely take place before the heightened expression of G6PD and independently of ACPA.
Unfortunately, there are only 4 ACPA + ARIs in our study, a number that is probably too small to show any difference between ACPA + and ACPA -ARIs. In addition, we were unable to examine the molecular signature in purified ARI Th cells, given the limited amount of blood that we were allowed to collect from each donor. It is possible that the molecular signature is influenced by non-T cells in PBMCs. This possible scenario may explain the normal expression of G6PD in ARIs. Recruiting more ACPA + ARIs and collecting more blood from each ARI will be needed to address these issues.
The observation that PBMCs of ARIs contain more citrullinated proteins further strengthens the notion that hypercitrullination is a precondition of RA, regardless of the status of ACPA. Our data indicate that impaired induction of PTPN22, but not smoking, systemic inflammation, abnormal expression of PADs, or the C1858T SNP, is the cause of hypercitrullination in the ARIs of this study. Then, what is the cause of the impaired induction of PTPN22? PTPN22 is expressed mainly in hematopoietic cells, and its expression in T cells is induced after anti-CD3 stimulation in vitro. There is no other SNP, in addition to the C1858T within the PTPN22 gene, that carries a significant risk of RA. Thus, it is unlikely that ARIs share a SNP in the PTPN22 gene that prevents its induction by anti-CD3. We propose that yet-to-be identified test (A and B) or 1-way ANOVA using empty vector-transfected groups as controls (D and E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. environmental factors play a key role in regulating the expression of PTPN22. Recently, it was reported that gut and oral microbiomes are different between RA patients and healthy controls (44,45). Specifically, Prevotella copri and Lactobacillus salivarius were overrepresented in RA patients, whereas Haemophilus spp. were depleted. These observations not only highlight the critical role of the environment in the pathogenesis of RA, but also raise the possibility that the microbiome may influence the expression of PTPN22. Thorough investigation into the molecular mechanism regulating the expression of PTPN22 will be the first step to test this hypothesis.
Our data indicate that phosphatase and nonphosphatase activities of PTPN22 are equally important in shaping the phenotype of PBMCs. However, the detailed mechanism of action of PTPN22 is still poorly understood. Attenuated phosphatase activity of PTPN22 is expected to strengthen the activation signals in lymphocytes (42,(46)(47)(48). This scenario can explain the higher level of IL-2. However, the impaired expression of ATM and PFKFB3, which are also induced by anti-CD3 stimulation, in ARI PBMCs cannot be explained by stronger activation signals and suggests a novel mechanism. The phosphatase activity of PTPN22 also inhibits the signals induced by type I interferon (49), modulates macrophage polarization (50), and activates the inflammasome by dephosphorylating NLRP3 (51). A role of NLRP3 in Th cells was recently discovered (52). It is possible that PTPN22 promotes the expression of PFKFB3 through the inflammasome in T cells. This hypothesis remains to be tested. Interestingly, several SNPs located between pfkfb3 and prkcq are associated with a higher risk of RA in genome-wide association studies (53,54). Our finding that PTPN22 directly or indirectly regulates the expression of PFKFB3 further provides a molecular link between these two RA-associated genes.
Thus far, two nonphosphatase activities of PTPN22 have been identified: suppressing citrullination and promoting LPS-induced production of type I interferon by myeloid cells (29,41). The observation that the abnormal Th2/Th17 cytokine profile of ARI PBMCs was partly normalized by a pan-PAD inhibitor and was recapitulated by overexpression of PADs strongly suggests that failure to suppress citrullination, but not failure to produce type I interferon, is the cause of the abnormal cytokine profile. In addition, the Th2/Th17 cytokine profile of ARI PBMCs is almost a mirror image of the ex vivo cytokine profile of mice treated with another pan-PAD inhibitor, BB-Cl-amidine (55). While it is still unclear how hypercitrullination causes the aberrant expression of Th2 and Th17 cytokines, our data have expanded the role of hypercitrullination in RA pathogenesis. Hypercitrullination not only enlarges the pool of citrullinated antigens, but also actively modulates the expression of Th cytokines.
We demonstrate again that PTPN22 is an inhibitor of citrullination. The C1858T SNP ablates this function of PTPN22, resulting in hypercitrullination and excessive production of citrullinated antigens. This mechanism can explain the synergy between the C1858T SNP and HLA shared epitopes in ACPA + RA (56). Through hypercitrullination, this SNP also increases the propensity of the formation of NETs, which have been shown to play a pathogenic role in SLE and other autoimmune diseases (57,58). This latter mechanism satisfactorily explains the association between the C1858T SNP and a higher risk of several other autoimmune diseases (59,60), which do not have ACPA. It will be very interesting to examine the phenotype of PBMCs from healthy donors carrying this SNP. As the conversion of R620 to W620 alters mainly nonphosphatase activities of PTPN22, one would expect that those PBMCs should have abnormal expression of Th2 and Th17 cytokines but normal levels of IL-2, ATM, and PFKFB3. Impaired induction of PTPN22 leads to augmented expression of Th17 cytokines, and only PAD2-mediated, but not PAD4-mediated, hypercitrullination recapitulates this feature. These data strongly suggest that PTPN22 also inhibits the activity of PAD2. This scenario remains to be confirmed. The latter observation also demonstrates that PAD2 and PAD4 have different roles in regulating the differentiation and function of Th cells. It is also consistent with previous reports showing that these two PAD enzymes have overlapping but not identical substrate spectra (15,16). Accordingly, PAD2 deficiency should preferentially attenuate Th17 response. However, PAD2-deficient mice are still sensitive to experimental autoimmune encephalomyelitis (61), a model of multiple sclerosis that is heavily dependent on Th17 cells. This discrepancy may originate from the fundamental difference between gain-of-function and loss-of-function approaches or from the intrinsic difference between mice and humans. Identifying the substrates of PAD2 and PAD4 in mouse and human Th cells will clarify this issue.
Approximately 50% of the ARIs in our study had impaired induction of PTPN22. This finding is reminiscent of our recent observation showing that nearly 40% of ACPA -FDRs already had detectable levels of APCA in their sputum (62). It will be of great interest to examine if the presence of APCA in sputum correlates with the impaired induction of PTPN22 in PBMCs of ARIs. While the Th cytokine profile of ARIs is different from that of controls, the difference is modest overall. This is not surprising, given that all ARIs are free of systemic inflammation. However, the functional consequence of this modest difference is still unclear. The odds ratio for developing RA in our ARIs is 4 to 5. Accordingly, only approximately 5%-10% of ARIs are expected to develop RA. This discrepancy strongly suggests that the molecular signature we discovered in this study is necessary but not sufficient for the development of RA and that additional "hits" are needed. One potential candidate is heightened expression of G6PD. It is a feature of Th cells from ACPA + RA patients and positively correlated with disease activity. However, the level of G6PD was normal in the ARIs of our study, suggesting that the heightened expression of G6PD is a late event that appears after the development of the molecular signature of preclinical RA. It remains to be determined if this feature occurs before or after the onset of clinical symptoms. Longitudinally studies following ARIs will be needed to test this hypothesis.
Methods
Human subjects. PBMCs were obtained from the following sources: (a) BWH PhenoGenetic Project (38); (b) Partners HealthCare Biobank, an enterprise biobank of consented patient samples at Partners HealthCare (Massachusetts General Hospital and BWH), according to IRB-approved protocols; (c) Personalized Risk Estimator for Rheumatoid Arthritis Family Study, an NIH-funded prospective, randomized, controlled trial designed to evaluate whether personalized RA risk education affects willingness to change RA-related behaviors among unaffected FDRs of RA patients (37); (d) Profiling of Cell Subsets in Human Diseases, a research initiative of BWH comparing immune cells in blood from patients with or without inflammatory diseases; and (e) Studies of the Etiology of Rheumatoid Arthritis, a multicenter study designed to examine the role of environmental and genetic factors in the development and progression of RA-related autoimmunity (63).
Plasmid. The expression vectors of WT and W620 PTPN22 were described previously (29). CD-PTPN22 (D195A/C227S) was generated through site-specific mutagenesis. The expression vectors for PAD2 and PAD4 were provided by Hyejeong Lee at Vanderbilt University (Nashville, Tennessee, USA) and Anthony Rosen at The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) (64, 65), respectively.
Intracellular cit-H3 staining. PBMCs were washed twice with 1% BSA in PBS, resuspended in 100 μl of 1% BSA in PBS, and incubated with anti-CD3 and anti-CD20 at 4°C for 30 minutes. The cells were washed twice with cold PBS, fixed with 100 μl of fixation buffer (eBioscience IC Fixation Buffer) at room temperature for 20 minutes in the dark, mixed with 2 ml permeabilization buffer (0.5% Triton X-100 in 1% BSA/PBS), centrifuged at 300 to 500 g and washed again with 2 ml permeabilization buffer. The washed cells were resuspended in 100 μl permeabilization buffer containing 1:300 rabbit IgG or anticit-H3 (ab5103, Abcam) at 4°C in the dark for 30 to 60 minutes, washed twice with 2 ml permeabilization buffer, and resuspended in 100 μl permeabilization buffer containing 1:400 goat anti-rabbit IgG-PE (sc-3739, Santa Cruz Biotechnology) at 4°C in the dark for 30 to 60 minutes. The stained cells were washed twice with 2 ml permeabilization buffer at 4°C and resuspended in PBS for flow cytometry.
Measurement of lactate concentration. Lactate concentration was measured with the Lactate Assay Kit (K607-100, BioVision) according to the manufacturer's instructions. Briefly, 50 μl of supernatant was mixed with 50 μl of the Reaction Mix (BioVision) for 30 minutes, and OD 570 nm was measured for colorimetric assay.
Statistics. Statistical analyses were performed with 1-way ANOVA followed by multiple comparison tests, 2-tailed Student's t test, or Spearman's correlation test, as indicated in the figure legends. A P value less than 0.05 is considered significant.
Study approval. This study has been approved by the Partners Human Research Committee, Boston, Massachusetts, USA, and the Colorado Multiple Institution Review Board, UC Denver, Anschutz Medical Campus, Aurora, Colorado, USA. Informed consent was obtained from participants prior to inclusion in the studies. | 2018-04-03T00:44:50.498Z | 2016-10-20T00:00:00.000 | {
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147753055 | pes2o/s2orc | v3-fos-license | Photojournalism: Journalistic Reality and Necessity
Aylan Kurdi, a three year old Syrian boy’s image carried on the front pages of newspapers and magazines in September 2015 was enough to stop the world in its tracks. It embodied the ravages of the Syrian war which has made headlines in newspapers and in the mass media in the past few years. Photo journalism is “Journalism in which written copy is subordinate to pictorial presentation of news stories or in which a high proportion of pictorial presentation is used, is broadly news photography” according to Miriam Webster’s dictionary. News photography sears, it captures reality. It is a necessity in this world which requires evidence and substantiation. This paper aims to study the photos related to the war in Syria; especially photos of Aylan Kurdi a three year old boy washed ashore while escaping with his family from Syria. The impact of these photographs on readers has been made through a qualitative study with in-depth interviews. The disturbing nature of the photographs, the knowledge about the war in Syria, the need and necessity of using of such photographs in media, feelings evoked, and the impact of the photographs by being shown on social media was gauged through a questionnaire and in-depth interviews.
Introduction
The Syrian war has been chaotic to say the least; with a number of different organizations from inside and outside the organization playing a role in the war.This war in Syria is being fought over the past four years.More than 200,000 persons have been killed in the war in the last four years and millions of persons have been displaced.A number of contenders have joined the fray to make the situation more chaotic.
President Al-Assad has ruled Syria with an "iron hand" according to rebel forces: "President Assad's counter-insurgency strategy has appeared to involve targeting the civilian population and medical facilities in rebel areas, in order to deprive the armed opposition of its support" says Glass, chief Middle East correspondent for ABC news and a veteran of the Lebanon war.He has provided a record of sorts with his book "Syria Burning: ISIS and the Death of the Arab Spring" published by OR Books, 2015.
IRA International Journal of Education and Multidisciplinary Studies
ISSN 2455-2526 Vol.03 Issue 01 (April, 2016) Paper DOI: https://dx.doi.org/10.21013/jems.v3.n1.p9 Glass further distributes the blame into what is now a seemingly intractable war: "Armed men were a minority among dissidents" at the start of the conflict he feels and they "gained ascendancy by the force of their actions and the international support they gained for their choice of the rifle over the banner."And in came the rifles and the war spread."Saudi Arabia and Qatar…poured in weapons and money.Turkey opened its border to arms, rebels and refugees.Clandestine training and logistical help came from US, Britain, France.Protests turned to civil war." This was a deadly game.Britain and France initially and then USA contributed to the machinations in the Middle east and resulted in the expelling of over 100,000 Syrians from their homes when Israel seized the Syrian Golan.Hezbollah joined the already chaotic fray.Aylan Kurdi's family was one such family which had to flee Syria due to this war.They were part of the terrible exodus resulting from the civil war in Syria against its President.
The Role of Photojournalism:
Photojournalism has been termed as "Visual truth".David Finklestein says: "It participates in the maintenance and reinforcement of cultural narratives-the images embedded in daily, weekly, monthly newspapers, magazines and journals create and sustain social constructions of self, identity and nationhood and challenge and confirm world views imbibed by consumers of our media."Further he clarifies the role of important photo journalists: "Photojournalism is most closely identified with the active adrenaline-driven dash to record war and human conflict, embodied in the work of such war photography veterans as Robert Capa (214)".Sontag to explain Photojournalism: "To take a photograph is to participate in another person's mortality, vulnerability, mutability."And to take photos of war is even more lethal according to Ron Haviv, the journalist in Darfur: For years, the new media have displayed photos of Syrian refugees: images of the dead wounded and displaced.But few of them seem to have made much of an impression-until last week, when people around the world saw photos of a 3-year-old boy named Aylan Kurdi, whose lifeless body washed up on a Turkish beach.
The boy had drowned, along with his brother and mother, while trying to get from Turkey to the Greek island of Kos.The Wall Street Journal published an image of a Turkish officer carrying Aylan's dead body, with the boy's face not quite visible and the man looking away, as if not able to bring himself to look at the child.Other news organizations published an even starker photo of the dead little boy, face down in the surf.(Ref. 1) This was an unusual image to say the least.It was a heart rending picture and most unusual and therefore it attracted immense attention."Once in a while, an image breaks through the noisy, cluttered global culture and hits people in the heart and not the head," says Douglas Brinkley, a professor of history at Rice University.This was one such extraordinary picture which struck out from the rest.
The documentary filmmaker Ken Burns admitted that he once worried that the still image had been devalued, "that a picture was no longer worth a thousand words because there were so many of them."But the photos of Ayland Kurdi are a reminder, he says, that "the power of the single image to convey complex information is still there.It has that power to shock and arrest us.To make us stop for just a second and interrupt the flow." The photograph appeared on the cover of The Independent in the U.K. among other titles, and in Le Monde, the only French newspaper to publish it quickly.Nicholas Jimenez director of photography at le Monde says: "I'm convinced that until you've shown this photograph, you havn't shown the reality of this crisis.We'd written about it in the past, but we hadn't shown it in such a hard way.I feel that to show like this is an important step." The photograph was also widely circulated on Twitter and Face book and other social media sites.Pinney further adds that viewers and readers may forget the conflict but when "they realize that this is about real people and about the fact that people will risk everything-the lives of their children to cross open waters" the pathos and tragic element of the migration becomes evident.
In fact this photograph and the others of the little boy Aylan Kurdi which followed highlighted the tragedy of little children in the migration from Syria.The pathos and heart rending tragedy was clearly evident.
Methodology:
Five photographs of the Aylan Kurdi stories were chosen from international newspapers who published the story on their front pages: These newspapers were: The www.intentblog.com,The Guardian, Reuters and www.express.co.uk as given in Reference 1.The choice of photographs was based on the fact that these were published on the front page of well-known newspapers, read by millions of readers.With the help of a questionnaire and in-depth interview of 10 persons, the impact of the photographs was gauged.
Findings:
The questionnaire and in-depth interviews were taken between 3 rd December, 2015 and 14 th December, 2015.Reactions of readers were recorded and tabulated Findings were charted and compiled.The photographs shown to 10 respondents elicited several interesting facets.The follow up intensive interviews conducted were to gauge the impact of the photographs and their effect on the viewers.
Demographics:
There were three respondents over the age of 60 years, two respondents aged 29-32 years and 5 respondents were between the ages of 35 and 40 years.There were 5 male respondents and 5 female respondents.
Regarding occupation, 5 respondents are university lecturers, 1 a retired medical doctor, one respondent is a regional marketing executive, one a freelance translator and one respondent is a chief finance officer.
Findings and Discussion:
Seven respondents had seen the photographs before while 3 respondents had not seen the photographs before the interview.Seven respondents knew about the baby in the photograph while 3 respondents did not know about the child in the photograph and were not familiar with the child's name or background.
Regarding awareness of the war going on in Syria all respondents said that they were aware about this war which was going on in Syria.Most of the respondents seemed well aware of the war which was going on in Syria for the past four years though the intricacies were not too clear.However they were aware that this was a war which had led to the migration of many people.
Regarding their knowledge about the war in Syria, two respondents had very little knowledge; another respondent had deep knowledge while the rest 7 respondents had only satisfactory knowledge about the war in Syria.
When questioned whether such images should be shown in the media (newspapers, magazines, television and internet) only one respondent said that images such as these should not be shown on media, 8 respondents said that this image should be shown on the media.One respondent from these 7 respondents also said that though very disturbing these images should be shown in the media.
The last and most important question was regarding the feelings which these images evoked: Respondents were asked to express their feelings openly and to be eloquent regarding them.One respondent said: "Very disturbing and show the effects of religious fanaticism on innocent lives.War leads to war!" "Touching, heart rending photographs, reality of the situation where such innocent lives are lost," said one respondent."It is pathetic where such innocent lives are lost and a situation is created by warring factions resulting in such misery and terror and difficulties which lead to such an exodus of people fleeing from the atrocities of war." Another respondent opined that -He had great sympathy for the people affected by this war.War was useless and not a solution to the problem.Peace should be kept and problems solved.He also said that it was heart rending that races should be affected in this manner and this issue was humanitarian and should be addressed at once.
A young respondent said "I feel sorry for the boy".Another respondent who was vocal and vociferous said "War is sad and leads to suffering.It should be stopped since kids and families separate.Institutions like the United Nations, UNICEF, and European Union should find ways to stop the war.Innocent children do not know about the war and they suffer the most.This photograph has been shared many times on social media especially Twitter and Face book.It made us think that war should be stopped and there should be peace and kids who are innocent should not be affected by this terrible war." A senior respondent felt that this was a terrible situation where innocent children suffered due to no fault of their own.War could happen anywhere at any time and kids were the victims.However this situation where kids were victimized should never be tolerated and kids should never be subject to this terrible fate and to such sadness.War is terrible and should stop immediately, said the respondent.The apparent terror which war evoked and the resultant violence were abhorred by the respondent and the feeling was that children became innocent victims who were torn due to no fault of their own.
Another respondent said that it was extremely disturbing to see these images on social media.The respondent added: "Not only disturbing but sad as well.Media should be used to raise awareness, not to raise bias.I can only imagine the distress it must have caused for the family of the victim." Another respondent intensely felt that though Syrians were welcomed by Saudi Arabia they prefer to migrate to Europe.He felt that millions of Muslims have died due to infighting among Muslims and interference of Western countries.Turkey has added to the fighting in Syria and so has ISIS a militant group which he feels is funded by western countries.If peace is to prevail in the Middle East such migration should stop and western forces should stop buying oil from ISIS and leave the area.
A respondent opined that the images made her sad and realize how terrible the situation was.Syrians were risking their lives and the lives of their families by escaping.The photos were the photos of a single parent but there must be several other people in a similar situation and she hopes that media can help raise awareness to help children like the one shown in the pictures.
Another saddened respondent at the state of affairs in Syria said that he felt grateful as well to be brought up in an affluent country which gave all opportunities to succeed in life.Though these pictures are alarming he said that "I think it's important to educate society as to these kinds of atrocities around the world.Although these pictures may be alarming, for better or worse, it's these types of pictures and stories that will make others realize what is happening in other parts of the world…I do believe however, that the more media exposure it receives, the more people will hopefully make an effort to put a stop to it and to offer assistance to those in need of it."
Conclusion
The cataclysmic war in Syria has caused untold damage to millions of people in the country and led to mass migration.This has been reflected in the photographs chosen for this study.The photo graphs evoked a mixed response: Barring two respondents all others found the images disturbing.However they all agreed that such images should be shown in the media like newspapers, television, internet and social media.
A number of respondents focused on the futility of war and its terrible aftermath especially on children.The victimization of young kids as a result of war was a focus of discussion and response.Innocent children who are unaware of disturbing events in the world around them yet died: They aroused the ire and sympathy of viewers and media consumers like the respondents of this study.
The role of important world organizations like United Nations or European Union was pointed out.The responsibility for dissipating war or stopping it was also the responsibility of these bodies.However, the withdrawal of western powers from the region was opined giving a holistic view to the entire problem.
The repeated focus of most respondents was the circulation of these images on social media.Not only did the social media display the images, they promoted debate, censure and discussion about the war and the resultant migration of innocent people.The images were disturbing and distressing.According to Hugh Pinney, Vice President at Getty Images, a distributor of news images, says: "The reason we're talking about this photograph is not because it's been taken or not because it's been circulated, but it's because it's been published by mainstream media.And the reason we're talking about it after it's been published is because it breaks a social taboo that has been in place in the press for decades: a picture of a dead child is one of the golden rules of what you never publish." The photograph has appeared on the cover of "The Independent" in the U.K., among other titles, and in Le Monde in France.Nicholas Jimenez, director of photography at Le Monde says: We'd written about it in the past, but we hadn't shown it in such a hard way.I feel that to show like this is an important step.This is an image people have to see.This is an image that can galvanize attention around a crisis that has been ignored for too long." The idea behind showing the image was not to shock said many editors of leading media from UK and Europe; it was to focus on stopping senseless deaths in the Mediterranean.If it succeeds in doing that, the image will have contributed a lot to the world.
Maxwell McCombs has discussed the role of media as setting the agenda or agenda setting model of mass communication.This model enumerates that the media sets an agenda or focus which help the general public to focus on issues and form opinions about what they see in the media in the long run.The model is applicable to the present study-The media has indeed set an agenda by displaying the images of Aylan Kurdi and his family.
The possible effects will permeate down to all sections of society since they have been picked up by internet and other social media.The power and influence of photographs is thus reiterated and re-emphasized.Its permeating role is brought to the fore and the enormous responsibility of media personnel also requires being emphasized.Which image is "right" or which is "wrong" is a debate which needs to be studied in the future.What can be said at this juncture is that mass media has an important responsibility and that is to focus and sustain interest on issues which matter and which require the limelight. | 2018-01-25T22:17:53.006Z | 2016-05-10T00:00:00.000 | {
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13535766 | pes2o/s2orc | v3-fos-license | Nondestructive tribochemistry-assisted nanofabrication on GaAs surface
A tribochemistry-assisted method has been developed for nondestructive surface nanofabrication on GaAs. Without any applied electric field and post etching, hollow nanostructures can be directly fabricated on GaAs surfaces by sliding a SiO2 microsphere under an ultralow contact pressure in humid air. TEM observation on the cross-section of the fabricated area shows that there is no appreciable plastic deformation under a 4 nm groove, confirming that GaAs can be removed without destruction. Further analysis suggests that the fabrication relies on the tribochemistry with the participation of vapor in humid air. It is proposed that the formation and breakage of GaAs-O-Si bonding bridges are responsible for the removal of GaAs material during the sliding process. As a nondestructive and conductivity-independent method, it will open up new opportunities to fabricate defect-free and well-ordered nucleation positions for quantum dots on GaAs surfaces.
Q uantum dots (QDs) are three-dimensionally confined semiconductor nanocrystal with quantum effect 1,2 . Due to their unique optical and electronic properties, such as photoionization 3 , cavity quantum electrodynamics effect 4 and quantum size-effect tunability 5 , QDs have attracted extensive research and can be widely used in photodetector 3 , nano-lasers 6 and 3 rd generation solar cells 5 , etc. However, the realization of these applications relies on the ability to manage ordering and positioning of high-quality quantum dot arrays 2 . A popular solution is growing QDs on nanopatterned GaAs substrate by epitaxial processes, where the hollow nanopatterns on GaAs surface serve as nucleation positions 7 . Nevertheless, the defects on nucleation positions can degrade the optical and electrical properties of quantum devices 8 . Therefore, the nondestructive and sitecontrolled nanopatterning of GaAs surface is a fundamental issue for the quantum technologies.
Many efforts have been devoted to fabricate nucleation positions on GaAs surfaces. For example, photolithography is an efficient method for nanofabrication of GaAs, but the limited resolution, the contaminants induced by wet etching and the complex processes still are tough challenges 2 . Mechanical stamping by indenting diamond tips can directly produce well-ordered nanoholes on GaAs surface 9 . However, since the required Hertzian contact pressure is quite high, plenty of defects may form in the substrate and it is difficult to grow high-quality quantum dots on these defective positions [9][10][11][12] . Recently, anodic oxidation nanolithography based on atomic force microscopy (AFM) has been successfully utilized for nanofabrication on GaAs surface 13,14 . This method depends strongly on the conductivity of sample and the endurance of Pt coating on the tip 15,16 , so that it is not fit for fabrication on semi-insulating GaAs samples, such as undoped GaAs. In addition, a post-etching process is necessary to remove the oxidation products on the fabrication area, which may cause complex processes and low fabrication efficiency. Therefore, it is essential to develop a nondestructive and straightforward method for fabrication of nucleation sites on GaAs surface.
In the present study, a tribochemistry-assisted nanofabrication method has been developed by directly sliding a SiO 2 microsphere on GaAs surface in humid air. The capability of this new method is demonstrated by various nanostructures including nanoholes, nanolines, nanoplanes and so on. Cross-sectional transmission electron microscope (XTEM) observation revealed no lattice distortion beneath the fabricated area, which implies that the involved fabrication mechanism should be greatly different from traditional mechanical scratching or cutting. Further analysis suggests that the tribochemistry may have facilitated the removal of GaAs material during the sliding process, resulting in defect-free hollow structures. It is also found that the potential of this one-step nanofabrication method could be realized for GaAs surfaces in different plane orientations and doping types. It is thus expected that this nondestructive tribochemistry-assisted fabrication method will shed new light on the fabrication of high-quality quantum dots on GaAs surfaces.
Results and discussion
Tribochemistry-assisted removal of GaAs by SiO 2 microsphere. It is well-known that grooves could be formed on a surface when the yield of material occurred 17,18 . When a diamond tip was used, the lowest limit of the contact pressure for the fabrication of grooves on GaAs surface was about 4.9 GPa, under which the contact area of GaAs yielded (Supplementary Section 1). Since the severe lattice distortion induced by the plastic yield will degrade the properties of the QDs 9,10,12 , the mechanical cutting is unsuitable for the defect-free nanofabrication on GaAs.
Previous work suggests that material can be removed by sliding SiO 2 tip under much lower contact pressure because of the tribochemical wear 19 . It is speculated that the tribochemistry may provide a nondestructive nanofabrication method on GaAs. To verify it, SiO 2 microspherical probe was selected as fabrication tool to remove GaAs material. As shown in Figure 1, it was found that the effective contact pressure P c for the fabrication by a SiO 2 tip could be as low as 0.54 GPa, which was much less than P y 5 4.8 GPa for the initial yield of GaAs (Supplementary Section 1). When the contact pressure P c increased from 0.54 GPa to 0.92 GPa, the fabrication depth increased from 1.8 nm to 3.3 nm. Compared with the mechanical cutting of sharp diamond tip, the scratching by blunt SiO 2 tip can make deeper grooves on GaAs surface even under a lower contact pressure. Therefore, the removal mechanism by SiO 2 tip should be quite different from that by diamond tip. It is thus speculated that the tribochemistry should account for the formation of grooves during sliding SiO 2 tip 20,21 .
XTEM characterization on the fabrication area. If the tribochemistry dominates the removal of GaAs by SiO 2 probe, the fabrication destruction should be avoided since the fabrication is independent of the plastic yield. To demonstrate whether it is a nondestructive method, the lattice of fabrication area was characterized by XTEM. From the AFM image in Figure Figure S2a), although there were few slight distortions on the fabricated surface, no appreciable plastic deformation was observed underneath this 4 nm deep groove. Such distortions can be further avoided when the nanolines are fabricated under a contact pressure P c below 0.85 GPa (Supplementary Figure S2b). Therefore, the XTEM results indicate that the defect-free nanofabrication of GaAs can be realized by using the tribochemistry-assisted method with a SiO 2 probe under a low pressure.
Mechanism for the nondestructive tribochemistry-assisted nanofabrication. Because the mechanical interaction between a SiO 2 tip and GaAs substrate is difficult, if not impossible, to produce grooves under a contact pressure far below P y 5 4.8 GPa, the tribochemistry should be an important mechanism responsible for the removal of materials during sliding. It has been reported that both oxygen and vapor in humid air can affect the tribochemical reaction 19,22 . To clarify the respective role of the two gases, the scratching tests were conducted on the same n-type GaAs(100) surface by a SiO 2 tip under low contact pressures (P c 5 0.54 , 0.85 GPa) in various atmospheres, namely vacuum with a pressure lower than 2.7 3 10 24 Pa, dry air (relative humidity RH , 1.5%) and dry nitrogen (RH , 1.5%). As shown in Figure 3, the fabrication depth increased with the contact pressure under four atmosphere conditions. However, under the same loading condition P c 5 0.85 GPa, the fabrication depth decreased from 2.9 nm in humid air to 0.7 nm in dry gases, and further decreased to 0.3 nm in vacuum. According to the chemical kinetics, the oxidation reaction between GaAs and O 2 was related to the striking number per second between the reactants. Assuming that the average fabrication area on GaAs was 10 4 nm 2 , the striking number of O 2 on the fabrication area could be estimated as 5.7 3 10 12 times per second both in humid and dry air because the content of oxygen remained unchanged 23 . Thus, the dramatic reduction of the depth can be mainly attributed to the decrease of the RH. However, there were still some shallow nanolines formed in dry air. To further understand the mechanism, dry nitrogen (RH , 1.5%) atmosphere was prepared in a renewed vacuum. Although the content of oxygen was down to less than 0.1% in dry N 2 and the striking number decreased by at least 200 times, the depth of nanolines formed in dry nitrogen was almost the same as that formed in dry air. These results implied that instead of the oxygen, it was the residual vapor that induced the tribochemical reaction and thus produced the nanolines on GaAs surface in dry gases. When the residual vapor was further reduced in vacuum www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9020 | DOI: 10.1038/srep09020 (2.7 3 10 24 Pa), the depth of nanoline was 43% of that in dry gases or 10% of that in humid air. Clearly, the vapor in air should have played a key role in the tribochemical reaction during the fabrication process. Figure 4 schematically depicts the possible tribochemical reaction between a SiO 2 tip and a GaAs surface during sliding. It is known that water molecules could be adsorbed on the SiO 2 and GaAs surfaces in experimental humid ambience (RH 5 50%) [24][25][26] . When the tip contacted the sample, water meniscus could form on the contact surfaces 27 . With the participation of the vapor and the adsorbed water film, both the SiO 2 tip and the GaAs surface could be chemically modified by hydroxylation, resulting in hydroxy termination on the contacting surfaces (Figure 4a) [28][29][30] . Upon sliding contact, the opposite hydroxyls on the contacting surfaces could come in close proximity and collide each others. As shown in Figure 4b, with the help of frictional energy, interfacial bonding bridges between GaAs and SiO 2 (GaAs-O-Si) might be formed by dehydration reaction [31][32][33] . During the sliding, GaAs-O-Si bonding bridges would be stretched and the energy was stored in the interfacial bonds. These high energy bonds might readily be broken by adsorbed water through hydrolysis reac-tions, probably resulting in the formation of oxides such as Ga(As)O x on GaAs and following hydroxylation on other bonds (Figure 4c) 28,34 . Such hydrolysis reactions should preferentially occur on GaAs side because the bond strength of Ga-O (353.5 kJ/mol), As-O (481 kJ/mol) or Ga-As (209.6 kJ/mol) was much weaker than that of Si-O (799.6 kJ/mol) (Supplementary Section 3) 35 . As the neighboring Ga-As bonds were hydrolyzed, the oxides products were detached from GaAs substrate, resulting in the wear debris with high oxidation degree (Supplementary Section 4). Finally, such tribo-oxidation products on GaAs can be cleaned easily by ultrasonic water washing 36 . Through this mechanism, the tribochemical reaction and removal of GaAs took place during the continuous tip sliding.
It was reported that only patchy water islands were occasionally observed on mica surfaces at 2% RH 37 . Therefore, when the fabrication was conducted in dry gases (RH , 1.5%), there should be no reliably water adsorption between the SiO 2 /GaAs contact pair since the GaAs surface was more hydrophobic than mica surface. The slight wear in dry gases might result from the partial water adsorption induced by the residual vapor. While in the vacuum (RH 5 0), the tribochemical reaction was further restricted due to the absence of the vapor. Since the fabrication depends on the tribochemical reaction during sliding, this method can be named as tribochemistry-assisted nanofabrication.
Site-controlled nanofabrication on GaAs. Finally, site-controlled and defect-free nanofabrication could be realized on a GaAs surface. Based on the controllable scanning of AFM, various types of hollow nanostructures including holes, lines and planes can be designed and produced by this one-step nanofabrication method. As shown in Figure 5a, a nanohole can be fabricated into a GaAs surface by scanning 2 cycles over a 70 nm 3 70 nm area under a contact pressure P c 5 0.92 GPa (F n 5 2.5 mN). The depth was about 5.9 nm and the diameter was about 150 nm. The letters ''QDs'' in www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9020 | DOI: 10.1038/srep09020 by adjusting the fabrication parameters (such as contact pressure and scratching cycles) and the dimension of SiO 2 microsphere (Supplementary Section 5). When a smaller SiO 2 microsphere is used, a narrower nanoline is supposed to be fabricated.
Just like the electrochemical reaction during local anode oxidation, tribochemical reaction provides additional energy loading pathway for the material removal 17 . However, the contact pressures P c during anodic oxidation by Pt-coated AFM tip was estimated to be 1 , 2 GPa when the tip radius was about 50 nm 38 , which was still higher than the contact pressure during the tribochemistry-assisted nanofabrication in this study. This means that the extent of lattice damage induced by tribochemistry-assisted method should not be worse than that by local anodic oxidation. In fact, no appreciable crystal distortion was observed in the tribochemistry-assisted fabrication areas on GaAs surface by a SiO 2 tip under such low contact pressure in present paper. Moreover, post-etching process can be omitted because hollow structures are formed directly after tribochemistry-assisted nanofabrication. Furthermore, the tribochemistry-assisted method does not rely on the electrical conductivity of sample and AFM probe, thus the fabrication can also be realized on GaAs in different plane orientations and doping types (Supplementary Section 6). The proposed method can provide new chances for the fabrication of defect-free and well-ordered nanostructures on GaAs and other chemical reactive surfaces such as Si 19,20 . Moreover, such hollow structures can also be used to define quantum structures underneath GaAs surface through charge depletion 39 .
Conclusion
Tribochemistry-assisted nanofabrication has been realized on GaAs by sliding a SiO 2 tip under ultralow contact pressures in humid air. Various hollow nanostructures can be directly fabricated on different types of GaAs surfaces. XTEM results show that the tribochemical reaction during scanning enables us to locally remove the GaAs materials though a nondestructive way. Comparison of the scratching tests in different ambient conditions suggests that the vapor plays a key role in the tribochemical reaction during the fabrication process. As a nondestructive and conductivity-independent method, it will stimulate the new development of the quantum technology.
Experimental
Materials. GaAs wafers in different plane orientations and doping types, including ntype GaAs(100), n-type GaAs(111)A, n-type GaAs(111)B and undoped GaAs(100), were purchased from Tianjin Jingming Electronic Materials Co., Ltd., China. By using an atomic force microscopy (AFM, SPI3800N, Seiko, Japan), the root-mean-square roughness of GaAs wafer was measured to be 0.4 nm over a 1 mm 3 1 mm area. Before the fabrication, samples were ultrasonically cleaned in methanol, ethanol and deionized water for 10 min. The water contact angle on the samples was measured to be 89u by a contact angle goniometer (DSA100, Krüss, Germany). Then, the samples were placed in AFM chamber with a vacuum capability. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9020 | DOI: 10.1038/srep09020 Fabrication method. Spherical SiO 2 probes (Novascan Technologies, USA) with a radius of 1200 nm were used as fabrication tools. The spring constant of the cantilever was measured as 14 N/m by using a standard cantilever (CLFC-NOBO, Veeco, USA) 40 . Figure 6 schematically shows the nondestructive tribochemistry-assisted nanofabrication process. When a spherical SiO 2 tip slid on a GaAs surface under a low contact pressure in humid air, a groove could be easily fabricated without destruction. Unlike anodic oxidation nanolithography, the tribochemistry-assisted fabrication process does not need any applied electric field and the surface is not electrically necessary conductive. The upper right inset picture in Figure 6 briefly illustrates the fabrication mechanism involved. Instead of plastic deformation, the water-assisted tribochemistry facilitated the removal of the GaAs. Therefore, the fabricated area could keep its original single crystal lattice. During the fabrication, the sliding speed of the tips was set as 2 mm/s, the temperature was controlled at 20 6 3uC and the relative humidity RH was ranged between 50 6 5% in humid air, if not specially mentioned. After the fabrication, the topograph of the nanostructure was scanned by Si 3 N 4 tips with a spring constant of 0.1 N/m (MLCT, Veeco, USA).
XTEM Characterization.
To verify whether the crystal distortion occurs during the tribochemistry-assisted fabrication process on GaAs surface by SiO 2 tip under a low contact pressure, the cross-sectional transmission electron microscopy (XTEM, JEOL JEM-2100 LaB6, JEOL Ltd., Japan) was used to study the microscopic structure and the potential deformation of fabrication area. The XTEM sample was prepared by using a Quanta 3D FEG focused ion beam miller (FIB, FEI Company, USA). In order to facilitate the FIB cutting across the fabrication area, a mechanical scratch in the depth of about 20 nm was produced by a diamond tip on the GaAs samples as a marker. Beside the marker, a series of nanogrooves in depth of about 4 nm and width of about 200 nm were produced by SiO 2 tip in humid air. | 2018-04-03T04:59:15.669Z | 2015-03-12T00:00:00.000 | {
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244150163 | pes2o/s2orc | v3-fos-license | DPP4 rs16822665 and rs2268694 had Protective Effect on Osteonecrosis of the Femoral Head
Background: It is reported that DPP4 is associated with bone metabolism, osteoporosis and other orthopedic diseases, but the correlation between DPP4 and osteonecrosis of the femoral head (ONFH) is not clear. It was the purpose of this study that was to explore the relationship between DPP4 gene and ONFH. Methods: We genotyped four single nucleotide polymorphisms (SNPs) from DPP4 gene using the Agena MassARRAY platform. The association between DPP4 variants and ONFH susceptibility was assessed using odds ratio (OR) and 95% condence intervals (CIs) via logistic regression. Results: The results showed that the allele C of rs16822665 was related to a lower risk of ONFH (OR = 0.76, 95%CI = 0.63-0.92, p = 0.006). In the case of stratied analysis, we found that rs16822665 could reduce the incidence of ONFH risk in four genetic models (dominant, codominant, log-additive, and recessive models) in drinkers and people age ≤ 51 years (p < 0.05). In gender stratication analysis, both rs2268694 and rs16822665 were contributed to bring down the risk of disease, which were mainly reected in the codominant, dominant and log-additive models in female (p < 0.05). The subgroup analysis was conducted based on smokers revealing that rs2268894 was vitally correlated with a decreased risk of ONFH in the codominant (C vs. T: OR = 0.51, 95% CI: 0.34-0.76, p = 0.001), dominant (TC-CC vs. TT: OR = 0.53, 95% CI: 0.36-0.77, p = 0.001), and log-additive (OR = 0.65, 95% CI: 0.48-0.88, p = 0.006) models, while it was not found in the non-smokers. Conclusions: This nding provide evidence that DPP4 variants play a key role in the occurrence of ONFH among the Chinese Han population. a major impact on glucose metabolism, immune regulation, signal transduction, cell migration differentiation 23 . Study have shown that the expression of CD26 mRNA in patients with rheumatoid arthritis is related to disease activity and bone erosion, suggesting that this molecule may play a role in the immunopathology of rheumatoid arthritis and bone erosion 21 . Some researches 24 found that higher plasma DPP4 levels were signicantly associated with higher bone turnover and a higher incidence of osteoporotic fracture. These results showed that DPP4 may be related with osteoporotic fracture by mediating bone turnover rate. Carbone et al found that in elderly group,there was no relationship between DPP4 and osteoporosis 25 . These lines of evidence have demonstrated that DPP4 gene played a crucial role in bone-related disease. However, the association between DPP4 gene and osteonecrosis susceptibility has not been reported. Rs2268694 and rs16822665 polymorphisms, located in the DPP4 gene protected susceptibility to ONFH was indicated in our results, but had not been previously reported in other diseases.
Introduction
Osteonecrosis of the femoral head (ONFH) is a common disease in orthopedics, which is di cult to treat 1 Femoral head necrosis has a high disability rate, which has a strong impact on the quality of life of patients. In recent years, some progresses have been performed in its treatment, but its speci c pathogenesis needs to be further studied.
The human DPP4/CD26 gene, located at 2q 24 , is a transmembrane glycoprotein with a molecular weight of 110ku and contains 26 exons encoding 766 amino peptides 6, 7 . Its product is a proteolytic enzyme, which has a unique proteolytic effect and belongs to the serine protease family 8, 9 . In addition, scholars found that DPP4/CD26 was involved in physiological processes such as invasion, apoptosis, migration, adhesion, and immune modulation 10 . As a marker of T-cell activation, DPP4/CD26 also plays a key role in immune regulation and activates broblast proteins and participate in signal pathways [11][12][13] .
It is well known that DPP4 and some of its substrates interact with adipokines, playing a pivotal role in the energy metabolism 14 . In recent years, DPP4 not only plays a vital role in protein regulation in the context of energy metabolism in the literature, but also has a direct and indirect effects on bone metabolism. Many substrates of DPP4 have played role in bone metabolism, and that include incretins, neuropeptides and gastrointestinal peptides. Overall, although some effects are conducive to bone formation, others are intricate and have not been absolutely expounded.
The activity of DPP4 is related to diabetes hyperglycemia, osteoporosis bone loss 15 and general osteoporosis, which indicates that the regulation of DPP4 is not directly involved in pathophysiology of these bone diseases, is also in uenced by its development.
Experimental studies have clearly con rmed some effects of DPP4 matrix on bone 16, 17 . In addition, some meta-analyses of clinical studies on DPP4 inhibitors and GLP-1-based bone therapy have been published 18 and have indicated that DPP4 inhibitors are relevant to a reduced fracture risk 18 . DPP4 inhibitor can improve vertebral bone mineral density and trabecular structure in vivo 19 , suggesting a harmful effect of DPP4 on bone metabolism. Studies have shown that DPP4 can digest several molecules playing an important role in bone metabolism. It was found that inhibition of DPP4 reduced serum reabsorption markers and trabecular bone volume and increased cortical and bone strength in rats with diabetes 20 . Yeganeh1 et al. 21 showed that DPP4 may be directly involved in bone resorption. In recent years, studies have displayed that the development of human osteoclasts is hindered when DPP4 signaling is blocked 22 .
So far, DPP4 has been reported to be associated with bone formation, bone loss, and osteoporosis. However, the mechanism of DPP4 gene effect on osteonecrosis remains unclear. In this study, we discussed the association of DPP4/CD26 genetic variations with ONFH susceptibility among Han individuals in China, which will help understand the function of DPP4/CD26 in the development of ONFH and provide new insights into its pathogenesis.
Study population
In this study, we enrolled 936 subjects, including 468 cases and 468 health controls. All cases were newly diagnosed by the following criteria: 1) diagnosed and selected by anteroposterior and bilateral hip X-ray lms and /or magnetic resonance imaging; 2) No other direct trauma, cardiovascular disease, rheumatoid arthritis, ankylosing spondylitis, hip joint disease (such as dysplasia of the hip), diabetes, renal insu ciency, cancer, glucocorticoid, alcohol and family genetic diseases were included. The selection criteria of all healthy people were: 1) they are from the same hospital during the same period; 2) People who are not long-term users of alcohol and steroids; 3) no pain in the buttocks; 4) no lesions were found in the pelvic anteroposterior lm and frog leg lateral lm.
DPP4 genotyping
Four SNPs (rs2268894, rs16822665, rs6741949, and rs67399148) in DPP4 gene were selected from 1000 Genomes Chinese Han Beijing population, with minor allele frequency (MAF) >5%. 5ml of fasting peripheral venous blood was collected from all subjects, and dispensed in ethylene diamine tetraacetic acid (EDTA)-containing tubes. Genomic DNA was puri ed with a commercially available DNA extraction kits (GoldMag Co. Ltd, Xi′an, China). Genotype analysis of DPP4 gene polymorphism was performed according to the protocol using Agena MassARRAY platform (Agena Bioscience, San Diego, CA, USA). The genotyping results were managed and analyzed using Agena Bioscience TYPER software (version 4.0).
Statistical analysis
Student's t-test was used to assess the difference of age, while χ 2 test was to compare the distributions of gender between the two groups, assess the frequency of SNP allele between the case and control groups, and gure up the Hardy-Weinberg equilibrium (HWE) of the control group. Odds ratio (OR) and 95% con dence intervals (CIs) were using for evaluating the relationship between DPP4 polymorphisms and ONFH risk by logistic regression analysis. Then, the interaction of SNP-SNP in the risk of ONFH was detected by multifactor dimensionality reduction (MDR).
Basic information of research objects
As rendered in Table 1
As shown in
After strati ed analysis (Table 4), we found that rs16822665 could reduce the incidence of ONFH risk in the four genetic models (dominant, codominant, log-additive, and recessive models) in drinkers and patients age ≤ 51 years (p < 0.05). In gender strati cation analysis, both rs2268694 and rs16822665 were contribute to bring down the risk of disease, which were mainly re ected in the dominant, codominant, and log-additive models in female (p < 0.05).
The subgroup analysis, which was conducted based on smokers revealed that rs2268894 was vitally related with a decreased risk of To sum up, we realized that two SNPs (rs2268894 and rs16822665) were not associated with patients at age > 51 years, non-smokers, and non-drinkers, suggesting that age, gender, smoking, and drinking are signi cant factors that affect the risk of ONFH.
SNP-SNP interactions
We analyzed the SNP-SNP interactions on the risk of ONFH by using the MDR. Table 5 represents that the best four-locus model including rs2268894, rs16822665, rs6741949, and rs67399148 was the best model to predict ONFH risk (training accuracy: 0.569; testing accuracy: 0.534; CVC = 10/10, p < 0.001).
Discussion
To the best of our knowledge that genetic studies have offered insights into a great number of diseases, including ONFH. In this study, it was expounded that DPP4 genetic polymorphisms were associated with ONFH risk among Han individuals in China. Allele, genotype of four loci between patients with ONFH and healthy samples were compared, and strati cation analysis were marched. We obtained that the allele C of rs16822665 was related to a decreased risk of ONFH in the Chinese Han individuals. In addition, the results showed that rs2268894 and rs13822665 have a protective effect on the risk of ONFH in patients age ≤ 51 years, or females, or smokers, or drinkers. Therefore, all of those date underscore the importance of DPP4 in ONFH development, and those two SNPs may become new biomarker for the early treatment and prevention of ONFH.
Dipeptide peptidase-4 (DPP4), also known as CD26, is an exopeptidase that is widely expressed on diverse types of cell surface 23 . By controlling the activity of its substrate or by physical binding with other proteins, the activity of this transmembrane exopeptidase has a major impact on glucose metabolism, immune regulation, signal transduction, cell migration differentiation 23 . Study have shown that the expression of CD26 mRNA in patients with rheumatoid arthritis is related to disease activity and bone erosion, suggesting that this molecule may play a role in the immunopathology of rheumatoid arthritis and bone erosion 21 . Some researches 24 found that higher plasma DPP4 levels were signi cantly associated with higher bone turnover and a higher incidence of osteoporotic fracture. These results showed that DPP4 may be related with osteoporotic fracture by mediating bone turnover rate. Carbone et al found that in elderly group,there was no relationship between DPP4 and osteoporosis 25 . These lines of evidence have demonstrated that DPP4 gene played a crucial role in bone-related disease. However, the association between DPP4 gene and osteonecrosis susceptibility has not been reported. Rs2268694 and rs16822665 polymorphisms, located in the DPP4 gene protected susceptibility to ONFH was indicated in our results, but had not been previously reported in other diseases.
Although our study found that the relevant loci of DPP4 gene have a protective effect on ONFH disease, which can offer a new theoretical basis for disease treatment and prevention, several potential limitations are unavoidable in our study. First, all participants were Han ethnicity, so we need more different ethnic populations to con rm our ndings. Second, the sample size is too small to con rm our results abundantly. Third, only four polymorphisms in DPP4 gene were studied, more polymorphisms are needed to be investigated.
Conclusions
All in all, we con rmed that DPP4 variants play a key role in the occurrence of ONFH among the Han population in China. This nding may provide new sights for the prevention and diagnosis of ONFH.
Declarations
Ethics approval and consent to participate The ethical approval of this study is in line with the ethical principles of the Helsinki declaration on human medical research. Our study has been approved by the ethics committee of Hong Hui hospital in Xi 'an, China, and all participants have signed informed consent before participating in the study.
Consent for publication
Not applicable.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Competing interests
The authors declare that they have no competing interests.
Funding
There was no funding support for this work.
Author's contributions Chang Liu designed this study protocol and supervised the study; Xuan Liu drafted the manuscript and performed the DNA extraction and genotyping; Xiaowei Li performed the data analysis and performed the sample collection and information recording. All authors have read and approved the nal manuscript. Bold values indicate statistical signi cance (p < 0.05). | 2021-11-17T16:23:10.815Z | 2021-11-15T00:00:00.000 | {
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261431568 | pes2o/s2orc | v3-fos-license | Study on mechanical efficiency of 65 ml/r fuel pump and its piston optimization
The 65 ml/r fuel pump proposed in this paper is a new type of large-displacement piston pump. The pump is of the roller plunger type construction. This structure enables the pump to have good performance even under high and variable speed conditions. Firstly, force analysis of this pump and establishment of mathematical model of pump mechanical efficiency. The influence of load pressure and speed on mechanical efficiency was obtained by numerical simulation through matlab. Then the prototype was designed and manufactured. The pump outlet pressure, outlet flow rate, and torque were measured on the test bench under various load pressure and speed, and mechanical efficiency of pump at various load pressure and speed was got. The experimental data and simulation are close. 65 ml/r fuel pump has a volumetric efficiency of 99% and a mechanical efficiency of 58.6% at a load pressure of 3 MPa and a speed of 1500 rpm. From experimental results, 65 ml/r fuel pump volumetric efficiency is high, but there is a problem of low mechanical efficiency. The piston of pump will be optimized for improved mechanical efficiency of pump. Then mechanical efficiency of the optimized pump is calculated and compared with mechanical efficiency before optimization. Results show that piston optimization will increase mechanical efficiency of pump.
Introduction
2][3] And regardless of the changes in the control system of the power unit, fuel pumps are necessary in the control system. 4Fuel pumps are used in a wide range of fields, such as aerospace, marine, and automotive. 5The positive displacement fuel pumps used in ships can be classified as gear pumps and axial piston pumps. 6The advantages of gear pumps are high flow rate and simple structure.However, the gears will make contact between the top of the teeth and the pump casing due to the unbalanced radial force, which will making the pump work less well.Gear pumps with high leakage and low volumetric efficiency. 7,8The piston fuel pump has a small leakage and high volumetric efficiency because the piston and the cylinder bore are cylindrical surfaces with high precision sliding surface fit.][11][12][13][14][15] The mechanical efficiency of the axial piston pump is affected by three frictional pairs.These three frictional pairs are the flow distribution pairs, the piston pairs, and the slide shoe pairs. 16,17he following are the studies made by domestic and foreign scholars on the three friction pairs and mechanical efficiency of the piston pump.Professor Manring 18 proposed a new mathematical model for piston sub friction based on lubrication conditions, which agreed well with experiment, but did not consider the effect of piston state on the results.Bergada et al. 19,20 solved the Reynolds equation for the oil film under piston tilt conditions, and the experimental results were close to the theoretical solution, but did not consider the effect produced by temperature.Gao et al. 21used deviation analysis to select suitable test points, simplify and improve the model, and predict the efficiency of a piston pump quickly and accurately with an error within 1%, but the method fails at low load rates.Costa and Sepehri 22 obtained the overall efficiency of the pump and motor from the perspective of energy balance, which was previously obtained by separate construction of mechanical and volumetric efficiencies, which is a new way of building the overall efficiency and verifying it experimentally.Xue and Du 23 considered the effect of operating pressure, temperature, and air mixed in the oil on the volumetric efficiency in the mathematical model built, but have not yet verified its accuracy experimentally.
To balance the axial force of the piston pump as well as to eliminate the structural flow pulsation and increase the reliability of the pump.And to make the pump perform well under variable speed conditions and when starting with load.A new structure of roller piston pump was proposed by the team of Professor Ruan J at Zhejiang University of Technology. 24This pump uses the reciprocating motion of the piston driven by the guide rail to change the size of the piston chamber to achieve oil suction and discharge, and the flow distribution function is achieved by the circumferential rotation of the flow distribution shaft.Eliminates the need for a distribution plate structure.Then, the pump was simplified in its mechanism.The pump replaces the sliding support of the traditional piston pump distributor with the cylinder block by rolling support of the guide rail to the roller.Rolling support makes the pump less dependent on the oil film, which can make pump still have good performance in high and variable speed conditions.
The 65 ml/r fuel pump is a large displacement piston pump with a roller piston structure.In this paper, a mathematical model of mechanical efficiency of 65 ml/r fuel pump will be built.The mechanical efficiency of the 65 ml/r fuel pump will be compared through experiments and simulations.The reasons of theoretical and experimental errors will be analyzed.And by optimizing the piston, we get the purpose of improving the mechanical efficiency of 65 ml/r fuel pump.
Pump structure and working principle
The interior of 65 ml/r fuel pump mainly consists of four parts: flow distribution shaft, cylinder block, piston, and guide rail, as shown in Figure 1.Inside distribution shaft, there are four through-holes for flow distribution; on the outside of the distribution shaft, there are eight flow distribution windows, four windows at the front and rear, and each through-hole communicates two flow distribution windows, one side is the high pressure flow distribution window and the other side is the low pressure flow distribution window.The small window is the low pressure distribution window.The big window is the high pressure distribution window, as shown in Figure 2. The curved surface of guideway is a law of motion with equal acceleration and deceleration.Eight pistons are evenly installed on the guide rail in the circumferential direction.Since the piston roller is close to guide rail, the motion law of the piston is the same as that of the guide rail, which is equal acceleration and deceleration motion.The motion trajectory between adjacent pistons is 45°apart.Meanwhile, the two cylinders are installed in opposite directions with the left and right sides of the pistons, and the inner side has an oil passage hole to communicate with piston chamber and shaft, and the outer side is equipped with a plug for sealing.The roller of 65 ml/ r fuel pump is made of 14 cones arranged circumferentially along the inner wall of the idler wheel, as shown in Figure 3.During operation, the superimposed rollers fasten the inner wall of the roller shaft and idler pulley under load pressure.
The 65 ml/r fuel pump distributes the flow through the drive shaft.65 ml/r fuel pump compared with the conventional piston pumps, the flow distribution plate structure is omitted.The structure of the pump has been greatly simplified.The guide rail is divided into two pieces with four wedges in the middle.Then the wedge will be under the action of centrifugal force will be propped up guide rail, so that it is close to the piston roller, so as to eliminate the gap and prevent the piston reciprocating movement and guide rail collision.65 ml/ r fuel pump is symmetrical construction, the two pistons with 180°phase difference have the same trajectory, and the two pistons with 90°phase difference have the opposite trajectory.So the inertia force of the pump is balanced.The role of the piston with 45°phase difference is to eliminate structural flow pulsation, but also makes the pump movement more smooth.
The fuel distribution process of 65 ml/r fuel pump is shown in Figure 4, in which the red area indicates high pressure oil and the blue area identifies low pressure oil.The motor drives the shaft and the guide rail for rotational movement through the transmission device.The piston idler wheel is restrained by the left and right sides of the guide rail.The piston starts to do reciprocating motion.The pump's working volume cavity changes periodically to reach the purpose of oil suction and discharge.
The position of the piston, the overlap area between the oil passage port of the cylinder and the flow distribution window of the flow distribution shaft, and the position of the contact point between the guide and the piston idler wheel will change with the rotation of the flow distribution shaft, and the position relationship of each moment is as follows: taking Figure 4(a) as the initial position, the piston idler wheel is in contact with the cam guide at the highest point of the surface, the volume of the right chamber is the minimal, the volume of the left chamber is the biggest, and the oil passage port corresponding to the two chambers and the flow distribution window Zero communication.From right to left, as the contact point between the guide and the idler wheel moves from the highest point to the lowest point, the piston moves from the right end to the left end, the volume of the right chamber increases from minimum to maximum, the flow distribution area between the flow distribution axis and the cylinder increases from zero to maximum and then decreases to zero, the volume of the right chamber of the piston increases and the volume of the left chamber of the piston decreases.Then the contact point of guide rail and idler wheel moves from the lowest point to the highest point, piston is moved to the right end from the left end, the mating area between the flow shaft and the cylinder body goes from zero to the maximum and then decreases to zero, the volume of the right chamber of the piston decreases and the volume of the left chamber of the piston increases and absorbs oil.At this time, piston completes a reciprocating motion, flow distribution shaft rotates 180°, piston unilaterally carries out a suction and discharge oil.So shaft rotates for 1 week, a single piston will finish sucking and discharging oil for four times.65 ml/r fuel pump has eight pistons which can finish sucking and discharging oil for 32 times.
Mathematical model
The 65 ml/r fuel pump is driven by the motor to rotate the spindle and drive the piston through the guide rail for axial reciprocating motion.The motion pattern of the plunger is limited by the guide rail.Starting at the lowest end of rail, and the movement law of guide rail is as follows.
Where s is the displacement trajectory of piston.s# is the movement speed of piston.s$ is the acceleration of piston.h is piston stroke.v is the angular velocity when guide rail rotates.u is the angle of rotation of guide.
The torque that needs to be provided when the 65 ml/r fuel pump works can be divided into three parts, which are the torque T i provided by the guide rail to the piston, the oil shear torque T s between shaft and cylinder block, and the churning loss torque T c caused by the rotation of the guide rail in the oil.
In the figure, L 3 is the width from the edge of the valve orifice to the edge of the cylinder block, L 4 is the width of the flow distribution orifice, L 5 is the width from the valve orifice segment to the high-pressure groove, and L 6 is the width from the high pressure slot to the end of cylinder block.
The 65 ml/r fuel pump's distribution shaft is rotating while cylinder is fixed, and the gap between the distribution shaft and the cylinder is composed of relatively moving walls.Since the relative motion of the two walls, and because of the fluid visibility, shear force is generated between the moving walls and the fluid in the gap.At the same time, the existence of high and low pressure oil cavity makes the differential pressure flow between the distribution shaft and the cylinder body, as shown in Figure 5.
In the axial direction, there is no relative movement between shaft and cylinder, so there is no shear flow in the axial direction, only differential pressure flow.The annular groove on the outlet side is the high pressure groove, oil in high pressure groove will flow to the outside of cylinder and low pressure chamber.The oil in the high pressure chamber will also flow to the outside of the cylinder through the gap.
In the circumferential direction, the oil flows from the high pressure chamber to the low pressure chamber through the clearance between the cylinder block and the distribution shaft.However, due to the circumferential arrangement of the high and low pressure chamber, the forces generated by the differential pressure flow can cancel each other numerically.Consider only the shear flow formed by the relative motion of the distribution shaft and the cylinder block.
Guide rail rotation 0°-45°, the acceleration direction of the piston is the same as the velocity direction, and piston shows equal acceleration motion.The force situation is shown in Figure 6.F a is the inertial force received by piston during movement.F N is the support force of guide rail on idler wheel.F Nf is the frictional force between guideway and idler wheel.F p is the pressure of high pressure chamber on piston.F f is the resistance received by piston.
To determine the number of contact surfaces between both sides of the plunger and the cylinder bore wall.Create a simplified model of the plunger force in ansys.
The established equivalent model is in Figure 7.
Where A is the pressure on the piston coming from the oil.C is the compression support simulating the support of the cylinder on the side of the piston.B, D is the compression support simulating the support of the rail on the idler wheel.E is the support force of the rail on the idler wheel.F is the fixed support simulating the fixed state of the cylinder.
The interaction force between the cylinder bore wall and the piston is expressed by the deformation amount using the principle of Hertz contact.The distortion of cylinder bore wall is shown in Figure 8.It can be seen that the left side of the cylinder bore wall has deformation on both sides, which means that the plunger has two contact surfaces.The right side of cylinder bore wall only has deformation on the lower side, which means that plunger has only one contact surface.This is because the guide rail has support force to the left idler wheel, while high pressure chamber has pressure to the left piston, and the deformation is not well transferred to the other side, resulting in the deformation of the left side of piston in Figure 8 is larger than the deformation of the right side.
The force analysis of the piston in this state is performed to solve the magnitude of the frictional force.As shown in Figure 9, piston is subjected to inertia force, gravity, support force of the cylinder on the piston and friction force, support force of the guide rail on the piston and friction force.The forces on the piston are analyzed to establish the equilibrium equation of the force system.The x-axis is the vertical side.yaxis is the vertical paper side.z-axis is the horizontal side.
The equation of force balance in the x direction is: The equation of force balance in the y direction is: The force balance equation in the z direction is: The balance equation of torque in x direction is: The equilibrium equation of torque in y direction is: The balance equation of torque in the z direction is: Where, F 1 , F 2 are the lateral forces acting on cylinder on the side of high pressure chamber.F 3 is the supporting force of low-pressure chamber side cylinder on piston.F 1f , F 2f are the lateral force F 1 , the friction force of F 2 .F g is the gravity exerted by piston.L 1 , L 2 is the length of plunger in the left and right end of cylinder respectively.L 7 is the length from piston end and the center of idler wheel.L 8 is half of the length from the roller axes on both sides.h 1 is the thickness from the top side of the plunger to the top of the bracket.R is the radius of idler wheel.r is the radius of piston.R 1 is the radius of the spindle.a is the roller pressure angle.f is the friction coefficient of cylinder wall face.The lengths l 1 and l 2 of the lateral forces F 1 and F 2 acting on the left end of plunger.
The interval between the piston and cylinder bore is much smaller than the piston diameter and the length of the piston in the cylinder, so it can be assumed to be clearance-free sliding.Also assume that the sliding friction will not affect the distribution of the contact pressure between the piston and the cylinder.No relative rotation between plunger and cylinder.Then the contact length between the piston and cylinder bore wall according to the stress triangle similarity principle, l 1 , l 2 length as shown in equations ( 13) and (14).
The action points of F 1 and F 2 are located at l 1 /3 and l 2 / 3 far from the contact edge between the piston and the cylinder block respectively.The lateral forces F 1 , F 2 of the cylinder on the high pressure chamber side on the piston are solved by solving the force system balance equation to obtain the component forces F 1x , F 1y , F 2x , and F 2y on the x-axis, y-axis, and the support force F N of the guide on the piston, expressed by equations ( 15)- (19).
Where F Ny is the component force of the rail-to-piston support force on the y-axis, and F Nfy is the component force of the rail-to-piston friction force on the y-axis.It can be expressed as follows.
The lateral force F 1 , F 2 of the high-pressure chamber side cylinder block against the piston can be calculated by F 1x , F 1y , F 2x , and F 2y .
Assuming that the force at the right end of the piston is not transmitted to the left end of the piston and the left end of the piston only acts as a support, the support force F 3 is given by the following formula.
The pressure F p of the high-pressure chamber on the piston end face can be expressed by equation (25).
p is the pressure.
There is a drain port on the cylinder block.Therefore, the two cylinders are not filled with oil between them.The resistance F pi to the axial movement has three main parts.The first part is the resistance of the piston by the oil when it is moving in the oil.The second part is due to the piston being supported by the guide rail, which has a circumferential force in the rotation process due to the support force of the guide rail on the piston.This force brings the piston side into contact with the cylinder transition section.The third part is the piston side and the cylinder transition section between the existence of clearance there is a shear flow resistance F n .For plungers that are not immersed in oil.Only the plunger is in connection with the transition section generating friction force.
S 1 is the area of the bottom surface of the piston minus the area of the cross-section of the piston.S 2 is the area of piston on the right side of rail.S 3 is the area of piston side in contact with the cylinder.F n is the force of cylinder on the side of plunger.v 1 is the velocity of plunger when it moves axially.r is the fluid density.d 1 is the interval between the side of plunger and wall.m is the dynamic viscosity of the oil.Assume that the pressure of the piston side to the cylinder can be regarded as the idler wheel axis on its upper side of the end of the force on the cylinder.The force acting on the upper side of the idler wheel is equal to half of the support force of the guide to the piston.Therefore, F n generates the frictional force F nf can be indicated by formula 28.
When the guide rail rotates from 45°to 90°, the piston acceleration direction is opposite to the velocity direction, and the piston is in equal deceleration motion.The force analysis diagram is shown in Figure 10.The acceleration produces unilateral inertia force can be provided partly by the frictional force to the piston and the resistance to the axial motion, and partly by the guide rail.At this time, the support force F N of the guide rail on the piston can be expressed as equation ( 29).
Since the situation is the same when the guide is rotated from 0°to 45°and from 90°to 135°, only the force on the piston when the guide is rotated from 0°to 90°can be studied.The value of m f is negligible due to its small size.The inertial forces can be canceled by integrating the solution for rail rotation from 0°to 90°.So the inertia force of two adjacent pistons can be balanced, which is also the reason why 65 ml/r fuel pump can balance the axial inertia force.The average value of the support force on the piston roller during the rotation of the guide rail from 0°to 90°can be approximated by equation (30).
When the guide rail rotates from 0°to 45°, the contact state of both ends of plunger with cylinder block is shown in Figure 11.The presence of the gap between the two generates the oil shear force F sh , as in equation (31).
Frictional force F nf on the side of the plunger.The resistance F pi of the piston in axial motion and the friction between the piston and the bore wall of the cylinder.
The torque T i of the guide rail to the piston subjected to friction.
The torque T s generated by the differential pressure flow F f1 and the shear flow F f2 .
Where, D 1 is the diameter of the circle where the contact point between the idler wheel and the guide rail is located.
The churning loss moment of the guideway can be obtained by fitting Fluent simulation T c . 25 T c =7:964310 À10 Án 2 +2:153310 À6 Án+5:473310 À4 ð35Þ Summing these moments gives the total torque T for the 65 ml/r fuel pump 0°rotation to 90°input.
Where, T# is pump output torque.As a result, the mechanical efficiency of the 65 ml/r fuel pump can be expressed as equation (37).
Where V D is the displacement of 65 ml/r fuel pump and t 90 is the time required to rotate the rail by 90°.
Mechanical efficiency simulation study
The mathematical model listed above is used as the basis.The effect of rotational speed and load pressure on the mechanical efficiency of the pump is studied by matlab.Specific parameters are shown in Table 1.The 65 ml/r fuel pump adopts the structure of a idler wheel piston pump with eight pistons arranged circumferentially.As the installation phase between two adjacent pistons differs by 45°, the movement law of two pistons also differs by 45°.The single plunger is rotated from 0°to 45°by the guide and the torque provided by the guide to it is increased.During the rotation of the guide rail from 45°to 90°, the torque provided by the guide rail to it is decreasing.This is due to the fact that in the process of 0°-45°of rail rotation, the acceleration is provided by the support force of the rail on the idler wheel, and the inertia force plays a hindering role.During the rotation of the rail from 45°to 90°, the acceleration is provided by the resistance of the piston movement and the reduction of the support force of the rail on the idler wheel, and the inertia force plays a role in helping the rotation.
For the whole 65 ml/r fuel pump.Eight pistons in the process of motion four for the speed up four for the speed down, and its output torque changes every 45°r otation for a cycle.The motion state is the same when the rail is rotating 0°and 45°.The plunger has the same number of plugs at the lowest, highest and middle of the rail, and the motion law is identical.Therefore, when studying the 65 ml/r fuel pump torque variation, it is only essential to research the variation in the process of guide rotation from 0°to 45°.
The oil shear resistance, piston friction resistance, and churning resistance of 65 ml/r fuel pump will increase with the speed.Under the same load pressure, rotational speed and torque are proportional.Under the same speed, the values of pressure and torque are proportional, as in Figure 12.The mechanical efficiency of the 65 ml/r fuel pump decreases due to the increase in speed.65 ml/r fuel pump operation resistance also increases.At low pressure, the initial torque is a large percentage of the input torque.The load pressure rises.Mechanical efficiency of 65 ml/r fuel pump raises, as in Figure 13.
Experimental verification
Prototype of 65 ml/r fuel pump was built.Its substance is shown in Figure 14.65 ml/r fuel pump configuration data are shown in Table 2.
The schematic diagram of the designed test bench system is shown in Figure 15, which mainly composed by fuel tank, motor, pressure sensor, torque/speed sensor, flow meter, relief valve, and console.The pump under test is equipped with a torque speed sensor between the motor and the pump, which can measure the input torque and pump speed.The pump inlet and outlet are equipped with pressure sensors to measure the pressure coming out of the pump inlet and outlet.The pump outlet is also equipped with a flow meter to indicate the pump output flow.The test bench relies on the self-priming of the 65 ml/r fuel pump for fuel supply.The values of pressures, flows, speeds, and input torques can be read in the instrumentation on the console.
Since the test stand has two parts of circuits, as shown in Figure 16, one circuit tests the pump performance and the other circuit tests the motor performance.When testing the pump performance it is necessary to cut off the left half of the motor circuit and return it to the tank through the relief valve.Therefore, only one part of the instrumentation on the console is functional, as shown in Figure 17.The system tested the efficiency of 65 ml/r fuel pump at various speeds and load.Records output torque and flow rate at 1, 2, and 3 MPa at 500, 1000, and 1500 rpm of the pump.There was a lot of metal debris generated during the run-in process.This is due to the friction between the piston pairs and the friction between idler wheel and rollers on the lower side of the piston holder.This indicates that the piston and the cylinder bore wall and the idler wheel and the roller are in contact with each other and have a large friction force.
The instantaneous torque and flow rate of the 65 ml/ r fuel pump at different load pressures and speeds were measured experimentally, as shown in Figure 18.
The experimental results of 65 ml/r fuel pump mechanical efficiency are shown in Figure 19.The experimental trend is in accordance with the trend of the theoretical value.However, there is still a gap for theoretical value and experimental value.At constant pressure, the input torque increases as the speed rises.At constant speed, the input torque increases as the pressure increases.It is also clear from the experimental results that the initial torque of 65 ml/r fuel pump is too high, resulting in a low mechanical efficiency of pump at low pressure.Due to the high viscosity of the oil.The pump leakage is low and the flow rate remains almost unchanged.
Three major factors for the gap between theory and experiment exist.One is because the calculation lacks the consideration of the friction between the idler wheel and the roller, the idler wheel and the roller on the lower end of the piston; two is because the piston as a whole in the case of continuous force bending the bottom of the piston leads to an increase in friction between the idler wheel and the roller on the lower end of the piston, and the deformation of the bottom of the piston leads to the hydraulic pressure and the support of the guide on the idler wheel is no longer aligned, will produce a x-axis torsional force, so that the force F 1x , F 2x increase in cylinder; three is due to the machining accuracy of the pump in the assembly there will be an initial torque.
In the theoretical calculation of 65 ml/r fuel pump the shear force of the oil, the friction between piston and cylinder is the most important factor affecting the mechanical efficiency of 65 ml/r fuel pump.The oil selected for the experiment is the common working medium of marine fuel pumps, which is hydraulic oil No. 46.The viscosity of this fluid is larger, so the shear force of the pump fluid is larger.And the plunger is subjected to a higher friction force due to more contact surfaces.The mechanical efficiency is 63.3% at 500 rpm, 60.9% at 1000 rpm, and 58.6% at 1500 rpm under a load pressure of 3 MPa.This is due to the rise in pump speed will lead to shear force of the oil and plunger is subject to increased friction q is the flow rate.n is the speed.V D is the pump displacement.At the same time, the outlet flow rate of 65 ml/r fuel pump is almost equal to the theoretical flow rate, and calculated by equation (38) to obtain the volumetric efficiency of more than 99%, as can be seen from the outlet flow rate Figure 19 of the pump.The pump leakage is very small and the oil flowing from the pump leakage port is much smaller than the pump leakage measured by the test bench.During the experiment, the amount of oil discharged from the pump leakage is very small.The leakage of the pump is mainly internal leak and there is almost no outside leak.Internal leakage mainly occurs between the shaft and the cylinders.There will be external leak between the piston and the cylinder.However, the plunger-to-hole wall clearance is too small as evidenced by the amount of leakage.The oil may not be able to fully lubricate the piston.When the piston is deformed, one end of the plunger will easily form two contact surfaces, and the contact surface increases leading to an rise in friction as well.To sum up, reducing the friction between piston and cylinder block is the most feasible way to improve the mechanical efficiency.
Table 3 shows the comparison of 65 ml/r fuel pumps with conventional piston pumps.Compared with the traditional piston pump, the 65 ml/r fuel pump is more integrated, has higher volumetric efficiency and higher power-to-weight ratio.However, the mechanical efficiency of this pump is currently low.
Piston optimization
The mechanical efficiency of the pump is low, and a large amount of mechanical energy will turn into frictional heat during the operation of the pump, making the temperature of the pump rise rapidly.And the rise of temperature will make the oil viscosity drop.Oil viscosity drop will make the pump leakage increase, volumetric efficiency is reduced.At the same time, the low mechanical efficiency of the pump also indicates that the pump has a large wear, which will have an impact on the life of the pump.
The shear force of the oil and the friction to which the piston is subjected are the most important factors affecting the mechanical efficiency of the 65 ml/r fuel pump.The size of the shear force of the oil is related to the clearance between distribution shaft and cylinder, and the change of the clearance will cause the change of the volumetric efficiency.However, reducing friction can improve mechanical efficiency without affecting volumetric efficiency.
To reduce the friction force on the piston.A novel flexible plunger is proposed.What is expected to be achieved by this flexible piston structure is to reduce the friction force and also to reduce the weight of the 65 ml/ r fuel pump.The mechanical efficiency and the workto-weight ratio of the 65 ml/r fuel pump are further improved without reducing the volumetric efficiency.
Due to the same time only one side of the piston is subjected to the support force of the guide rail.And the original piston middle section thickness is larger, the force on the left end of the piston can not be transferred to the right end, the deformation of the piston in the case of force is concentrated in the left end, while the right end of the piston is less force, such as the left figure in Figure 20.By reducing the thickness of the middle section of the piston, the middle section of the piston can be bent more easily, so that the force can be transferred from one side to the other, increasing the value of F 3 and decreasing the value of F 2 .
Decrease the deformation of the left end of the piston in the left diagram of Figure 20 so that the acting force F 1 is no longer generated.The mass of the piston can be further reduced by adjusting the piston structure.As the strength of the roller shaft in the piston in the ansys static analysis is much greater than the force to be withstood.Therefore, the idler wheel shaft is changed to a hollow structure to reduce the mass of the pump.The thickness of the middle section of the plunger is reduced.The mass of the plunger also decreases greatly.It is meaningful to improve the work-to-weight ratio of the pump.
Build the flexible piston model as shown in Figure 21.
At that moment the force at the end of the plunger exists only F 2 , F 3 .Due to the x-axis direction by the smaller force can be approximated as F 2 , F 3 equal to F 2y , F 3y , so from the formula 38 to solve the value of F 2 , F 3 .
Substituting the calculated results into the above, the mechanical efficiency of the piston pump when using a flexible piston can be obtained by calculating the mechanical efficiency before and after the piston optimization.
Figure 22 shows the variation curve of theoretical mechanical efficiency with load pressure at 500 rpm for 65 ml/r fuel pump with flexible piston and 65 ml/r fuel pump with normal piston.From the above graph.It can be seen that 65 ml/r fuel pump with flexible pistons will increase the mechanical efficiency by 5% over the original pump.Therefore, it is feasible to increase the mechanical efficiency of the 65 ml/r fuel pump by using a flexible piston.
Conclusions
(1) A new type of large-displacement piston pump is proposed.Unlike the traditional axial piston pump whose drive mechanism uses oil film support, this pump uses rolling support.The flow distribution is realized through the rotation of the flow distribution shaft.65 ml/r fuel pump does not need a separate flow dispensing structure.It eases the design of the pump.The roller type pump has a symmetrical design, so that the pump can achieve inertia force balance and no structural flow pulsation.Experimentally measured when the pump load pressure is 3 MPa, the speed is 500-1500 rpm, mechanical efficiency from high to low, varying from 63.3% to 58.5%, volumetric efficiency is almost constant 99%.(2) Develop a mathematical model of the mechanical efficiency of the 65 ml/r fuel pump.Simulation of piston contact state by ansys.On this basis, the mathematical model of mechanical efficiency of 65 ml/r fuel pump is established for 65 ml/r fuel pump and the way of rotating flow distribution of the distribution shaft.Analyze the effect of speed and load pressure on the variation of pump mechanical efficiency.The change curve of mechanical efficiency is obtained by matlab calculation.When the load pressure of the pump is constant, the mechanical efficiency decreases with the increase of the rotational speed; when the rotational speed of the pump is constant, the mechanical efficiency increases with the increase of the load pressure.(3) The experimental results are slightly lower than the simulation results.Reason one is the presence of friction within the piston assembly, friction between the wheel and roller and its upper and lower end surfaces, and friction between the wheel and roller.The second reason is the presence of the initial torque of the pump.Finally, a new type of flexible piston is proposed to improve the mechanical efficiency of the pump.This piston structure can reduce the frictional force on the plunger.A mathematical model of the force on the flexible piston was established, and the mechanical efficiency of the pump at 500 rpm was improved by 5% by calculation.(4) The pump can have better performance in variable speed conditions and with load start, and it can cope with more conditions in practical application.However, the internal structure of the pump is more complex and the machining cost is higher.A potential challenge with this design is that the pump can be difficult to maintain, as the highly integrated structure of the pump requires disassembly of the entire pump in the event of a failure.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Figure 4 .
Figure 4. 65 ml/r fuel pump core flow distribution process: (a) schematic diagram when rotation angle is 0°and (b) schematic diagram when rotation angle is 90°.
Figure 9 .
Figure 9. Force diagram of the piston.
Figure 11 .
Figure 11.The contact state between the two ends of the piston and the bore wall of the cylinder.
Figure 13 .
Figure 13.65 ml/r fuel pump mechanical efficiency as a function of speed and load pressure.
Figure 16 .
Figure 16.65 ml/r fuel pump test stand.
Figure 20 .
Figure 20.Optimize the contact between the front and rear pistons and the cylinder wall.
Figure 22 .
Figure 22.Comparison of theoretical mechanical efficiency before and after piston optimization at 500 rpm.
Table 2 .
Table of main structural parameters of the pump under test.
Table 3 .
Performance comparison with conventional piston pump at 1500 rpm and 3 MPa.Comparison of experimental and theoretical mechanical efficiency at different speeds. | 2023-09-02T13:04:07.378Z | 2023-09-01T00:00:00.000 | {
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117903086 | pes2o/s2orc | v3-fos-license | Kaon Physics: What the Future Holds in Probing the Standard Model and Beyond
The status and prospects of current and future kaon physics experiments is discussed. Both precision measurem ents and the search for and measurement of ultra-rare decays are powerful probes of many models of new physics beyond the Standard Model. The physics reach of these experiments is briefly discussed.
Introduction
Experimental kaon physics remains an active field with the following experiments producing results this year: These experiments are pursuing measurements to attack the "Flavor Problem" that persists in particle physics today.There are strong theoretical arguments for new physics at the TeV scale, such as Super-Symmetry (SUSY).
With generic O(1) couplings this new physics should manifest itself as anomalous quantum corrections to flavor physics phenomena within the Standard Model.The absence of observed anomalous corrections today severely constrains the space of generic couplings…thereby giving rise to the Flavor Problem.
Highlights from the current round of experiments.
The KLOE experiment this year has substantially improved the measurement of |Vus| by extracting this CKM amplitude from a variety of kaon decay channels in order to control experimental systematic biases 1 .This precision measurement of |Vus| enables a an incisive test of first-row CKM unitarity which can probe the presence of 4 th generations and other new physics.In contrast, assuming CKM unitarity, the effective Fermi coupling (G F ) determined from these quark decays can be compared with G F determined from muon, tau, and W/Z decays.This comparison is thereby sensitive to physics beyond the Standard Model as summarized below in Table 1. 1 .The level of agreement in G F extracted from different sources within the Standard Model constrains the contribution from new physics.In models such as SO(10), corresponding gauge boson (Zχ ) masses can be limited 2 to greater than the 10 TeV scale.
Source G F (GeV
The CERN NA62 experiment is pursuing a precision measurement of the ratio R = (K + e + ν e )/(K + μ + ν μ ) which is senstive to new physics.The K + e + ν e decay is helicity suppressed to a branching ratio of 1.1x10 -5 .SUSY processes, in particular charged Higgs amplitudes can modify this branching ratio by up to the 1-2% level 3 .The NA62 experiment has collected a sample of more than 100,000 K + e + ν e decays and has recently report results 3 on the ratio R that is consisitent with the Standard Model expectation at the 0.3% level.This new measurement contributes substantially to furthering constraints on charged Higgs couplings within the context of SUSY.
Expectations for the future program.
The following experiments and initiatives are preparing or considering major upgrades: 3) The certainty with which the Standard Model contribution to K π νν is known permits a 5σ discovery potential for new physics even for enhancements of the branching fraction as small as 25% .
This sensitivity is unique in quark flavor physics and probes essentially all models of new physics that couple to quarks within the reach of the LHC.Further, precision measurement of K π νν The E14 experiment at JPARC in Tokai Japan is pursuing sensitivity of the 0 0 L K π νν → process at the single Standard Model event level, which would be sensitive to several models beyond the Standard Model.This nearly x100 increase in sensitivity is achieved though upgrading the existing E391 detector and an new high intensity beam-line at the JPARC accelerator complex.
Summary
Measurements in the kaon system have contributed greatly to establishing both the framework and the details of the Standard Model.The broad sweep of consistency of all flavor physics phenomena today within the Standard Model has led us to the Flavor Problem, which is a steadily growing tension between the expectations of TeV-scale new physics and the apparent absence of corresponding quantum corrections that should affect quark flavor measurements at the current state of the art.The next round of experiments in kaon physics are aimed squarely at the K π νν + + → and 0 0 L K π νν → processes, which are particularly promising tools to crack the Flavor Problem.Proton beam facilities exist world-wide today to mount these experiments which have the potential to deliver these incisive measureents within the coming decade.
E391a at KEK: Proton beam driven K L decay-in-flight experiment.E949 at BNL: Proton beam driven K + decay-at-rest experiment.KLOE at Frascati: e + e -storage ring φ K + K -/ K L K S experiment.KTeV at Fermilab: Proton beam driven K L decay-in-flight experiment.NA62 at CERN: Proton beam driven K decay-in-flight experiment.
at JPARC: Proton beam driven K L decay-in-flight experiment.NA62 at CERN: Proton beam driven K + decay-in-flight experiment.K-initiative at Fermilab: Proton beam driven K + decay experiment.A high precision measurement of the ultra-rare K and or Fermilab would be one of the most incisive probes of quark flavor physics in the coming decade.The CERN experiment, NA62, is a major upgrade of the existing detector and beam-line systems and proposes a sensitivity of about 100 Standard Model events.An initiative at Fermilab is studying experiment concepts with a sensitivity goal of 1000 Standard Model events.The dramatic physics reach of a precision measurement of K recognized as theoretically robust to the 2-4% level.No other loop-dominated quark process can be predicted with this level of certainty.highly suppressed in the Standard Model with an expected branching fraction of 7x10 -11 .This suppression allows physics beyond the Standard Model to contribute noticeably to the branching fraction with enhancements of up to factors of x3 above the Standard Model level.
many models of new physics far beyond the direct mass reach of the LHC.The experimental challenge of measuring K 10-billion Standard Model rate has been met successfully.Several events of the K by BNL E949 using the stopped K + technique.The NA62 experiment is pursuing a new technique based on in-flight decays.The Fermilab initiative is now studying the optimal concept for a 1000-event experiment.
TABLE 1 .
G F extracted from various sources | 2018-12-15T07:40:00.747Z | 2010-02-01T00:00:00.000 | {
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6911865 | pes2o/s2orc | v3-fos-license | Fertility and Markers of Male Reproductive Function in Inuit and European Populations Spanning Large Contrasts in Blood Levels of Persistent Organochlorines
Objective We synthesized the main findings from an international epidemiologic study on the impact of biopersistent organic pollutants (POPs) on human reproductive function. Data sources and extraction We used a database with interview and biological data from 2,269 women and their spouses, and 18 published core papers. Data synthesis The study did not provide direct evidence of hormone-like activity of the polychlorinated biphenyl (PCB) congener CB-153 and the main dichlorodiphenyltrichloroethane (DDT) metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE), as serum concentrations of these compounds were not consistently related to either endogenous or exogenous hormone activity in serum. Nevertheless several links bewteen POP exposure and biomarkers of male reproductive function were identified. First, an association between high CB-153 serum levels and low sperm counts was detected within a subgroup of men with short androgen receptor CAG repeat length. Second, a relationship between increased CB-153 serum concentrations and decreased sperm motility was seen in all four studied regions, and indications of reduced neutral α-glucosidase activity in seminal plasma point to a post-testicular effect. Third, damage of sperm chromatin integrity was considerably less frequent in Greenlandic Inuits compared with that in European groups, and only in the latter was impairment of sperm chromatin integrity related to POPs. Despite these effects, fertility in terms of time taken to conceive was not related to POPs except in Inuits. A likely explanation of the latter was not identified. Conclusions POPs may interfere with male reproductive function without major impact on fertility. The data do not provide direct evidence for endocrine disruption, hence other mechanisms should also be considered.
Subfertility is encountered among some 15% of all couples trying to conceive (Juul et al. 1999), and sperm counts in the subfertile range are prevalent in some countries (Carlsen et al. 2005). Although advances in understanding of causes and mechanisms have been made during the past few decades (Aitken and Baker 2004;DeMasters 2004;Sharpe 2003), major gaps in knowledge still preclude effective prevention of the majority of infertile cases. Lessons learned from the occupational arena and indications of contemporary declining trends and geographic variation in the occurrence of male reproductive disorders have fueled the search for environment-and lifestyle-related risk factors. The hypothesis that ubiquitous man-made chemicals in the environment may interfere with proper development of reproductive organs through interaction with natural hormonal regulation has achieved much attention (Toppari 2002). Proponents of the hypothesis focus on a) an apparent decline of male reproductive health and on evidence that several male reproductive disorders are of fetal origin and may share common causes (Skakkebaek et al. 2001); b) that similar developmental disorders of male reproductive organs can be induced in experimental studies after exposure to various compounds that interfere with hormonal homeostasis (Toppari 2002); and c) that reproductive anomalies have been documented among sons of mothers treated during pregnancy by the potent synthetic estrogen diethylstilbestrol (Sharpe and Skakkebaek 1993). Opponents argue that the evidence on major temporal shifts of, for example, sperm counts, is circumstantial (Handelsman 2001) and that the large number of xenobiotics detectable in human tissues have low hormonal potencies (Daston et al. 1997) and occur in concentrations far below levels that conceivably could have a major impact on reproductive organ development (Safe 2004). Updated comprehensive reviews conclude that current experimental and epidemiologic evidence does not with sufficient certainty support the view that environmental endocrine disruptors contribute to an increase in male reproductive disorders; neither does it provide sufficient grounds to reject this hypothesis (Storgaard et al. 2005;Vidaeff and Sever 2005). More recently, attention has shifted from direct xenobiotic agonistic or antagonistic actions on the human estrogen or androgen receptor to interference with endogenous hormonal regulation (Sharpe and Irvine 2004).
Epidemiologic studies explicitly designed to corroborate or refute the environmental hormone hypothesis are few, but such studies are important in providing reliable answers as to whether xenobiotics are among important preventable causes of subfertility and other reproductive disorders. From 2002 to 2005 a European Union (EU) Fifth Framework Programme Research and Development project, INUENDO, was carried out explicitly to address this deficit in current knowledge. The main findings have been published in a number of focused original papers (Axmon et al. 2006;Bonefeld-Jorgensen et al. 2006;Elzanaty et al. 2006;Giwercman et al. 2007;Giwercman AH et al. 2006;Jönsson et al. 2005;Long et al. 2006Long et al. , 2007Spano et al. 2005;Stronati et al. 2006;Tiido et al. 2006;Toft et al. 2004Toft et al. , 2005aToft et al. , 2005bToft et al. , 2006Toft et al. , 2007. The purpose of this article is to provide an overview of the main results that emerged from the INUENDO project and to review the findings relative to the overall evidence on persistent organic pollutants (POPs) and human fertility.
Methods
The INUENDO studies combine four interview studies of time to pregnancy (TTP) with four cross-sectional studies of male reproductive hormones and semen quality (Table 1). Three study populations included pregnant women and their male spouses who had antenatal care visits from May 2002 through February 2004 at one of three locations: a) the local hospitals in 19 cities and settlements throughout Greenland, b) a large central hospital in Warsaw, Poland, and c) three hospitals and eight antenatal clinics in Kharkiv, Ukraine. A fourth study population included Swedish fishermen and fishermen's wives. They were enrolled independent of current pregnancy in two separate steps from an existing cohort (Rignell-Hydbom et al. 2004;Rylander et al. 1995). A total of 2,269 women from the four localities were enrolled (participation rates 26-90%; Table 1). In Greenland, Kharkiv, and Sweden, the age distribution and the number of children did not differ between participants, those who declined participation, and nonrespondents. Available data did not allow for nonresponse analysis in the Polish sample (Toft et al. 2005a). Male spouses were consecutively encouraged to participate in a semen study until approximately 200 men at each site had agreed (participation rates 7-79%; Table 1). The low particpation rate among Swedish fishermen (7%) is due to the more elderly study population and the recruitment procedure that involved postal correspondence in several consecutive steps.
Information on TTP for the current (the three pregnancy-based cohorts) or the latest planned pregnancy (the Swedish populationbased cohort) was obtained by in-person interviews with the women at the hospital or residence or by telephone (Sweden) using a structured questionnaire (Toft et al. 2005a). The male partners provided separate interview data on lifestyle factors, urogenital disorders, and infertility that were used to describe reproductive characteristics of couples, with men providing or not providing semen samples (Toft et al. 2005a). Finally, women and men had a venous blood sample drawn (Table 1).
Polychlorinated biphenyls (PCBs) and the dichlorodiphenyltrichloroethane (DDT) metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p´-DDE) were selected as biomarkers of exposures of interest because of documented hormonal actions (Bonefeld-Jorgensen et al. 2001;Kelce et al. 1997;Moore et al. 1997), widespread occurrence worldwide (Bignert et al. 1998), and existence of reliable and relatively inexpensive assays suitable for large-scale epidemiologic studies. 2, 2´,4,4´,5, was selected as a marker of PCB congeners because it correlated well with both total PCB concentration in plasma and serum from Swedish subjects and Inuits from Greenland (Glynn et al. 2000;Grimvall et al. 1997) and with the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalent (TEQ) in plasma from PCB as well as the total POP-derived TEQ in plasma in American Vietnam veterans (Gladen et al. 1999). The antiandrogenic compound p,p´-DDE, the major metabolite of the insecticide DDT was selected as a supplementary marker of POP exposure. Moreover, xenohormone activity in serum cleared for endogenous hormones and dioxin-like activity in the lipid fraction of serum was measured in a subset of the study population by ex vivo chemicalactivated luciferase gene expression (CALUX) assays. Reporter gene constructs and cell culture systems were used to explore whether integrated measures of xenobiotic receptor binding are related to reproductive end points. The luciferase activity was expressed as relative light units (RLUs) per milliter serum; reference levels were calculated to 3.31 RLU/mL serum [estrogen receptor (ER), androgen receptor (AR)] and 6.67 RLU/mL serum [aryl hydrocarbon receptor (AhR)]. For details, the reader is referred to original papers (Bonefeld-Jorgensen et al. 2006;Long et al. 2006). An overview of biological markers of male reproductive function investigated in the present study is given in Table 1.
The fine tuning of the AR function is regulated by two polymorphic sequences in the transactivating part of the receptor, namely, polyglutamine-encoding CAG repeats and polyglycine-encoding GGN triplets (Gao et al. 1996;Tut et al. 1997). Earlier studies have indicated that the number of CAG repeats and the GGN triplets can influence the functional status of the AR. Analyses of interactive effects of AR polymorphisms were performed to provide clues as to causal inference and insight into possible mechanisms . For these analyses CAG repeat numbers were categorized into five groups of almost equal size: < 20, 20/21, 22/23, 24, and > 24. Data analysis. To create an overview of the cross-sectional relations between POP exposure and all reproductive end points and to enable a systematic and coherent assessment of strength, exposure response, and internal consistency of associations, we summarized the data using a simplified, uniform approach building on comprehensive statistical analyses in previous papers (Table 1).
Common risk estimates. We present data at the aggregated level across all regions unless inappropriate because of heterogeneous associations. Heterogeneity was examined by interaction terms in multiple regression models. However, because the frequency of gene Abbreviations: APOPTOSIS, percentage of ejaculated sperm cells expressing the Fas protein, as an indicator of apoptosis, and the Bcl-xL antigen, as an indicator of anti-apoptosis (Stronati et al. 2006); CALUX, chemical-activated luciferase gene expression; CASA, computer-aided sperm analysis; SCSA, sperm chromatin structure assay; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end-labeling; WHO (World Health Organization), sperm count, morphology and motility measured according to the WHO 1999 guidelines (WHO 1999); Y/X SPERM, ratio between ejaculated spermatozoa with Y and X sex chromosome.
polymorphisms regulating male reproduction seems to differ substantially between Inuits and Europeans ), all analyses were stratified accordingly. With this approach, effect modification by European regions may be hidden, but region-specific associations are presented comprehensively in the core articles. Exposure categorization. Central measures of distributions in terms of medians and corresponding adjusted geometric mean values are presented according to exposure levels divided into three intervals. Cutoff values were selected as trade offs between numbers in each category, contrast of exposure, and ranges within each interval for CB-153 (ng/g lipid): 0-100, 101-200, and > 200 (highest value 5,460); and for p,p´-DDE (ng/g lipid): 0-500, 501-1,000, and > 1,000 (highest value 13,200).
Xenobiotic CALUX values were dichotomized into values above and below the average reference value of 3.13 RLU/mL serum for ER and AR assays and 6.67 RLU/mL serum for the AhR assay. For the agonistic assays, higher CALUX values indicate agonistic transactivation, and for the competitive assays, higher values indicate enhanced (synergistically or additively) transactivation of the natural or synthetic ligands and lower values indicate an antagonistic effect on ligand-induced receptor transactivation (Bonefeld-Jorgensen et al. 2006;Long et al. 2006).
Statistical methods. The distribution of hormonal and semen characteristics in each exposure category was compared with the reference category by multiple linear regression. In addition, tests for linear trends across the entire exposure range were performed by similar methods but with exposure entered as a continuous variable. With few exceptions, end points were transformed by the natural logarithm to normalize skewed distributions as described in the core articles. In linear trend analyses, the exposure variables were also transformed by the natural logarithm to account for the higher variability in the high end of the distribution. Adjustments for potential confounding variables included only a few well-established determinants, which were included regardless of effects in the present data set. These determinants are listed in Table 2 (footnotes). Comprehensive confounder analysis according to the change in estimate method is provided in the core articles. The parsimonious approach we use here and the comprehensive approach applied in the core articles resulted essentially in similar findings and, if not, deviances are explicitly addressed. Geometric mean values and their confidence intervals (CIs) were obtained by back transformation, whereas linear regression coefficients in analyses based on continuous exposure variables were not. All summary analyses presented in this article were performed using SAS 9.13 software (SAS Institute Inc, Cary, NC, USA).
Systematic criteria used to help distinguish spurious from causal associations.
A priori hypotheses declared before the execution of the project were held in general terms. Therefore, most of the several hundred comparisons that have been performed should be considered explorative, and the risk of spurious associations regardless of statistical significance testing may be high. Evaluation of consistency of findings across regions including assessment of strength (magnitude) of associations and exposure-response relationships has been performed throughout.
Results
Exposure levels. The blood measurements of POP markers in men and women demonstrated large variations between and within study populations ( Figure 1) (Jönsson et al. 2005). The median serum concentrations of the most abundant PCB congener, CB-153 as well as the DDT metabolite p,p´-DDE varied > 10-fold between regions. The within-region ranges of the 5th and 95th percentiles were of similar magnitude.
The INUENDO project is the first largescale population-based epidemiologic study that has evaluated the integrated xenohormone (ER, AR) and dioxin-like (AhR) activity in serum by CALUX assays (Bonefeld-Jorgensen et al. 2006;Long et al. 2006). These assays demonstrated agonistic as well as competitive receptor interference of serum cleared for endogenous hormones, although the between-and within-region variation was small compared with variations in POP concentrations ( Figure 2). The CALUX activities were only weakly correlated with CB-153 and p,p´-DDE, indicating that these organochlorines are not the important contributors to the measured xenobiotic serum activity.
Reproductive end points according to polychlorinated biphenyls (CB-153) and
p,p´-DDE. Fecundability measured by TTP in couples that conceived was not related to CB-153 among Europeans, but among Inuits the fecundability was reduced among intermediate-and high-level exposed men and women compared with those exposed to low levels of CB-153, although no obvious exposureresponse relations were found and findings were of borderline significance (Axmon et al. 2006). The risk associated with male exposure was most likely not confounded by female exposure and vice versa, but because of the strong correlation between CB-153 and p,p´-DDE among Inuits, it was not possible to determine if the risk was associated with CB-153 or p,p´-DDE or an interaction between the two compounds.
None of the five male reproductive hormones measured in serum varied consistently with CB-153 serum levels among Inuits and Europeans, but luteinizing hormones increased with increasing CB-153 among Inuits and free testosterone decreased, whereas sex hormonebinding globulin levels became higher with increasing CB-153 among European men (Table 2). Comprehensive additional analyses of reproductive hormones within each region indicated several endocrine responses associated with CB-153 blood levels in some regions but not in others (Giwercman AH et al. 2006).
Sperm count and the proportion of morphologically normal sperm was not related to CB-153 in any study group, but progressive sperm motility was inversely related to CB-153 in serum among both Inuits and Europeans, with consistent indications of exposure response relationships (Table 2; Toft et al. 2006). The percentage of progressively motile sperm decreased by 3.6% (95% CI, 1.7-5.6) per 1 U increase in the logarithm of serum CB-153 (nanograms per gram lipid) in the entire data set .
We observed significant interactive effects of CB-153 and AR CAG repeats on sperm counts but not on semen volume, sperm motility, or sperm morphology ). Thus, when total sperm number was compared in men with CB-153 levels above median and those with exposure below median, sperm output was 40% lower in subjects with AR CAG repeat length < 20 but not in those with ≥ 20 repeats.
Two independent indicators of sperm chromatin integrity [% DNA fragmentation index (%DFI) and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)] were strongly related to CB-153 serum levels among Europeans but not among Inuits ( Table 2; Spano et al. 2005;Stronati et al. 2006). Among Europeans the proportion of sperm with impaired sperm chromatin integrity increased with increasing CB-153 blood concentrations to a level almost double that in the high-level-exposed group. This association seemed not to be confounded by concomitant exposure to p,p´-DDE. Our study failed to demonstrate any relation between CB-153 and apoptotic sperm biomarkers (Fas and Bcl-xL) in any region or overall (Stronati et al. 2006). No interactive effects of AR polymorphisms and CB-153 on %DFI were found .
Associations between CB-153 and the proportion of Y spermatozoa exhibited strong heterogeneity across regions (Tiido et al. 2006). In the Swedish fishermen cohort, CB-153 serum levels were positively associated with the proportion of Y chromosomebearing spermatozoa [linear regression coefficient β = 0.53 (95% CI, 0.001 to 1.05)], whereas among Polish men, levels of CB-153 correlated negatively with the proportion of Y sperm [β = -0.54 (95% CI, -0.92 to -0.14)]. The difference in average proportion Y-bearing sperm between these groups was small (51.2% for the fishermen sample, 50.3% for the Polish sample).
None of several seminal markers of epididymal and accessory sexual gland function varied consistently with CB-153 serum levels among Inuits or Europeans across all regions (Table 2; Elzanaty et al. 2006). However, a statistically significant negative association between the levels of CB-153 and the total activity of NAG was seen among men from Greenland and Warsaw and in the entire data set (Elzanaty et al. 2006). Moreover, NAG was also lower in high-level-exposed Swedish fishermen, whereas in Kharkiv, higher levels of CB-153 tended to be related to higher levels of NAG. Associations between serum concentrations of p,p´-DDE and reproductive end points are given in Table 3.
Reproductive end points in relation to ER, AR, and AhR CALUX serum activity. In the subset of couples with valid TTP data and male CALUX activity data (n = 182), no consistent indications were found across regions of deviating fecundability according to ER, AR, or AhR CALUX assay activity. Moreover, sperm count, morphology, and motility were not related to agonistic, competitively enhanced, or antagonistic estrogenic serum activity across all regions or-with few exceptions-in any of the regions (Tables 4 and 5). Similar negative findings were observed for the AR and AhR CALUX measurements (data not shown). Summary findings with respect to other seminal characteristics are given in Tables 4 and 5 for the ER CALUX assays (data for other assays not shown). Comprehensive analyses of correlations between ER as well as AR and AhR xenobiotic activities and semen characteristics, including indicators of sperm DNA damage and abnormal sperm apoptosis, did not reveal consistent patterns of associations across regions. For details, the reader is referred to the original articles Toft et al. 2007).
Discussion
Reports on secular trends in male reproductive health in the early 1990s became a major impetus for research on the hypothesis that male reproductive disorders may be related to xenobiotics with hormone-like properties. Two recent systematic reviews reexamined the evidence on the importance of xenohormone exposure in human reproductive health. Both reviews emphasize the current lack of explicit epidemiologic observations to validate the hypothesis (Storgaard et al. 2005;Vidaeff and Sever 2005). The purpose of the INUENDO project was to contribute additional insight into links between putative xenohormonal dietary exposure, as measured by POP markers in blood, and human fertility, as measured by an array of functional and biological indicators.
To accomplish this objective approximately 5,000 women and spouses were enrolled in four cohorts in Greenland, northern, central, and eastern Europe. Below we discuss the coherence and synthesis of the main findings. Endocrine disruption. Experimental data have shown weak hormonal activity of the PCB congener CB-153 and the main DDT metabolite p,p´-DDE (Bonefeld-Jorgensen et al. 2001;Kelce et al. 1995;Moore et al. 1997), but our study does not provide strong or consistent evidence that these POP markers express hormone-like activity in humans at the prevailing exposure levels. Thus, the serum POP concentrations were not consistently related to either serum concetrations of sex hormones or to exogenous CALUX activities of serum across all study populations. However, an association between the concentration of p,p´-DDE in male serum and sex hormones in blood was observed in the Kharkiv region (Giwercman AH et al. 2006). Moreover, a weak increase in follicle-stimulating hormone with increasing CB-153 was observed in all regions but did not reach statistical significance in the pooled analysis. Whether these results have any bearing on impaired male reproductive function is not clear. One interpretation is that associations between POP and sex hormones are heterogeneous because of heterogeneous xenobiotic exposure that is only partly reflected by the POP markers measured in the present study. This is to some extent supported by the xenobiotic CALUX activities showing regional variation and weak correlations with POP markers. Another possibility is spurious associations emerging from a great number of explorative comparisons that have been carried out or uncontrollable bias inherent in study design and data collection. Because of the lack of strong and consistent associations, it is not possible to distinguish these different interpretations. But it should be kept in mind that cross-sectional associations based on point measurements of plasma concentrations of sex hormones reflect dynamic time-varying interactions and compensatory feedback mechanisms to a limited degree. Thus, no strong negative conclusions on the effect of POPs on hormonal homeostasis can be made from these findings.
Post-testicular effetcs of POPs. In the present study, progressive sperm motility decreased with increasing POP blood levels in all four regions and seemed strongest among Inuits with rather high exposure to both POP markers. This finding is consistent with the results of six of seven cross-sectional studies (Aneck-Hahn et al. 2007;Dallinga et al. 2002;Danadevi et al. 2003;de Jager et al. 2006;Guo et al. 2000;Richthoff et al. 2003; Rozati et al. 2002). The two most recent studies carried out in Mexico and South Africa enrolled men with high environmental exposures and both demonstrated strong associations between serum concentrations of p,p´-DDE and various measures of sperm motility. Additional supportive evidence comes from two adult rat studies reporting reduced sperm motility after treatment with high doses of coplanar as well as noncoplanar PCBs (Hsu et al. 2003(Hsu et al. , 2004. Interestingly, the non-coplanar PCB congeners (CB-132 and CB-149) only affected sperm motility, whereas the coplanar dioxin-like PCB congener CB-77 also reduced sperm counts. The immotile testicular spermatozoa are gaining motility through slow passage through the epididymal tubules. Once ejaculated, secretions from the seminal vesicle and the prostate play a crucial role in energy supplies and composition of the seminal fluid and thereby in the ability of the spermatozoa to move (Elzanaty et al. 2006). Considering the consistent associations between CB-153 and sperm motility, it is of interest that NAG decreased with increasing CB-153 serum levels (but not p,p´-DDE serum levels), which is fairly consistent across the four regions (Elzanaty et al. 2006). NAG is excreted by the caudal part of the epididymis and is widely used as a marker of epididymal function in the clinical setting-low seminal levels, indicating impaired excretion and reduced epididymal function. Little is known about the physiologic role of the enzyme, but our study and an earlier study by Richthoff et al. (2002) show that NAG is weakly correlated with percentage motile sperm. For other seminal markers of epididymal and accessory sex gland function, findings were less consistent across regions. Sex hormones regulate epididymal and accessory sex gland function. These organs express the estrogen, androgen, and the aryl hydrocarbon receptors. Some PCB congeners bind to the ER and AhR and elicit agonistic or antagonistic activity in vitro. Moreover, synthetic estrogens such as diethylstilbestrol and antiestrogens such as tamoxifen reduce epididymal and accessory sex gland weight when administered to adult rats in high doses (Elzanaty et al. 2006). However, as discussed above, this present study does not provide any explicit support for the assumption of endocrine disruption as the basic mechanism of action. Polymorphisms in the AR gene did not modify POP-related effects on sperm motility (Giwercman et al. 2008), and none of the CALUX activities that are assumed to represent the integrated xenohormone action from the mixture of pollutants in the serum extracts were consistently related to sperm motility across study populations. In addition, it can be argued that the estrogenic and androgenic equivalents contributed by xenobiotics in these highly exposed populations only are a few percent of the endogenous hormone activity, even when possible higher bioavailability of the xenobiotics is taken into consideration (Toft et al. 2007). However, in controlled rodent experiments, effects on reproductive function have been observed at very low exposure levels, particularly after exposure during the fetal and postnatal development periods (Anas et al. 2005;Mably et al. 1992)-an issue not explicitly addressed by this project. Thus, the picture with respect to mechanisms at the cellular level is far from clear, and the possibility cannot be excluded that POPs also exert toxic effects independent of hormonal actions. There is therefore a need for further research into the impact of POPs on epididymal function and gene expression.
Gene-environment interaction. There are several earlier human studies on the effects of postnatal POP exposure on testicular function. In the study with subjects with the highest exposure to PCBs and polychlorinated dibenzo-p-dioxins after the Yucheng accident in Taiwan, abnormal sperm morphology and reduced sperm capacity to penetrate hamster eggs were found (Hsu et al. 2003). Two recent large cross-sectional studies of men with high environmental exposure to DDT in Mexico Bonde et al. 274 VOLUME 116 | NUMBER 3 | March 2008 • Environmental Health Perspectives Table 3. Adjusted geometric mean values and linear regression coefficients of male reproductive hormones in serum, semen characteristics, and markers of epididymal and accessory sex gland function by categories of p,p´-DDE.
Inuits Europeans p,p´-DDE (ng/g lipid) p,p´-DDE (ng/g lipid) Inuits Europeans 0-500 501-1,000 > 1,000 0-500 501-1,000 > 1,000 Linear regression coefficient ( and South Africa failed, however, to demonstrate associations between serum concentrations of p,p´-DDE and sperm concentrations, whereas effects on semen volume and total sperm were found in one study (Aneck-Hahn et al. 2007) but not in another (de Jager et al. 2006). The lipid-adjusted serum concentrations were approximately 50-200 times higher in these groups of men than in men in our study. These findings seem consistent with the results in the present study where neither sperm count nor sperm morphology was related to POP exposure in any of the four study groups. However, among the subset of men with short AR CAG repeat length, which makes up about one-fifth of the entire study population, high levels of CB-153 were significantly related to low levels of sperm counts ). This observation is an indication of gene-environment interaction between POPs and AR configurations. We acknowledge, however, that this finding emerges from a pooled analysis of four diverse study groups and calls for independent replication. The mechanism by which the CAG repeat length might modify the effect of POP on semen characteristics is not known. However, as the three-dimensional structure of the receptor is affected by the length of the CAG stretch, one could hypothesize that the strength of the POP binding, or of any necessary co-factor, is regulated by CAG number ). The INUENDO studies also indicate interactive effects on sperm chromatin integrity, although not necessarily gene-exposure interactions. The sperm chromatin structure assay measures two different types of sperm chromatin anomalies, one linked to DNA damage (%DFI) and the other reflecting abnormal condensation of proteins during the tight packaging of the sperm DNA (%HDS). Both types of chromatin anomalies are traced indirectly by in situ acid denaturation of sperm cells. Independent of sperm count, morphology, and motility, %DFI is related to male fertility, which starts decreasing when the percentage of abnormal sperm cells increases above approximately 20% (Spano et al. 2000). The %DFI measure is rather stable within subjects, and a number of studies of several European populations have shown remarkably homogenous distributions (Spano et al. 1998). Therefore, it is interesting that the %DFI levels were lower in Inuit men, indicating more "healthy" sperm chromatin. The sperm TUNEL assay demonstrated even more pronounced low levels of sperm DNA damage in Inuits. Comparable data have to our knowledge not been published previously. The low frequency of chromatin damage among Inuits, as demonstrated by two independent assays, could be genetic in origin, although dietary constituents as polyunsaturated fatty acids and selenium could also play a role. Interestingly, high serum concentrations of CB-153 were strongly related to low sperm integrity among European men, but there was no association among Inuit men. That exposure to POPs interferes with sperm chromatin integrity is further supported by the modifying effects of polymorphisms in the AR antigen Abbreviations: %DFI, percentage of sperm with denaturable DNA, mainly due to DNA damage (Spano et al. 2000); %HDS, percentage of sperm with high levels of green fluorescence, indicating immature sperm (Spano et al. 2000); n, number of men; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end-labeling. a Reference 3.13 RLU, the mean value of solvent control samples. b Samples with spillage excluded. c Adjustment by the logarithm of period of abstinence (day). d Samples with a delay of > 1 hr from collection excluded. e Adjustment by the logarithm of age (years). studies have shown that epigenetic changes in the sperm DNA, evoked by in utero exposure to vinclozoline, may pass to and cause infertility in, at least, three subsequent generations (Anway et al. 2005). Further research is warranted to understand why Inuits have better sperm chromatin integrity than Europeans and why POPs are impairing chromatin integrity in Europeans but not among Inuits. Impact on fertility. Sperm count, morphology, motility, and chromatin integrity are all reliable and independent indicators of male fecundity (Bonde et al. 1998;Spano et al. 2000). The INUENDO studies demonstrated effects related to serum levels of POPs for three of these biological markers-most consistently for sperm motility. Nevertheless, the INUENDO study revealed no indications of delayed conception related to male or female POP serum levels except in Inuits where fecundability was reduced by 30% among the high-level-exposed groups. This finding was of borderline statistical significance and without strong exposure-response relationships but withstood a comprehensive analysis of potential bias and confounding factors including age and self-reported urogenital diseases. It is hard to explain why POPs interfere with fertility in only one of the four study groups. Neither exposure characteristcis nor POP-related effects on male biomarkers of fecundity offer any clear solutions to this enigma. To our knowledge, there are no comparable studies on fertility related to POP exposure among Inuit people, and the evidence from studies of other populations is conflicting (Toft et al. 2004). If not caused by bias inherent in the TTP methodology (Olsen et al. 1998) or residual confounding that escaped recognition, yet unknown gene-environement interactions should be identified in future research.
Internal validity issues. The strengths of the INUENDO study include large exposure contrasts and large sample sizes relative to most end points, adherence to uniform design, protocol and data collection procedures, individual biological exposure markers, indicators of integrated xenohormone and dioxin-like actions, inclusion of a wide range of established and novel reproductive end points, analysis of selected AR polymorphisms, centralized data management and laboratory analyses with internal and external quality control, and an agreed upon protocol for the data analysis.
Several limitations must also be addressed. Few highly specific hypotheses were stated a priori, for example, whether PCBs or p,p´-DDE were to be considered the most important risk factor for the various reproductive end points. The same applies for the three CALUX assays, which represent a large range of possible risk-factor scenarios. It is important to acknowledge that most analyses were explorative. Multiple comparisons may constitute a serious risk for data-induced and post hoc interpretation of random associations (Smith 2001;Smith and Ebrahim 2002). Magnitude and statistical strength of associations, exposureresponse relationships, gene polymorphisms, and biological plausibility has been considered throughout to help distinguish real from spurious associations. Another option to evaluate internal validity was to request consistent associations across the four study regions before assigning high credibility to the findings. Although still important, the obvious heterogeneity among regions with respect to POP and xenobiotic CALUX exposure profiles makes this "consistency-approach" less powerful than first anticipated. Systematic differences among regions in the genetic make-up that regulates metabolism of xenobiotics and reproductive functioning add further difficulties to the interpretation of heterogeneous associations. Even if in many aspects still elusive, environment-environment, gene-environment, and gene-gene interactions can play important roles in determining the relationship between exposure to environmental chemicals such as POPs and associated risks to human health and reproduction. Nevertheless, few disagree that associations that consistently survive background variation and heterogeneity, such as the relation between PCB and sperm motility, are more likely to be real. Furthermore, studies of associations stratified on relevant gene polymorphism may be important tools to advance knowledge in the field. Thus, the analyses of androgen gene polymorphisms in the INUENDO populations have for the first time demonstrated associations compatible with genetic modification of POP-related effects on male reproductive health.
Potential confounding factors were addressed systematically in all analyses according to a uniform protocol. Investigators considered well-established determinants as well as a number of hypothetical risk factors. Important determinants of the various outcomes may still escape adequate control because at present and to our best knowledge, they are unknown. Finally, the strong correlation between CB-153 and p,p´-DDE among Inuits and Swedish fishermen precludes allocation of effects to one or the other POP. Much weaker correlations yet large exposure ranges in the Warsaw and Kharkiv samples can, to some extent, resolve this problem.
External validity issues. The sampling frame in three of the four regions was pregnant women and their partners. This should not be considered an important limitation to the external validity of all core studies. The couples enrolled consecutively also include those in the clinical setting who are labeled infertile, namely, those couples that took > 1 year to conceive. Approximately 15-20% of the "fertile" couples enrolled in this project would have been classified as infertile in the clinical terminology. Only a small minority of sterile couples are not represented in the database. This is not a problem unless the exposures being studied have an on-off effect, which is very unlikely (Baird 1988;Olsen 1999;Olsen et al. 1998). Among the few known human reproductive toxicants, tobacco smoking has been demonstrated with equal efficiency in pregnancy and population-based studies (Bolumar et al. 1996). Thus, the selection of pregnant women and their spouses for this study is not expected to reduce the chance of detecting the effects of POP on reproductive function.
There are several pieces of evidence that indicate reproductive organs are more sensitive to detrimental effects of chemicals during critical periods of fetal and postnatal development than during adult life (e.g,, Anas et al. 2005). It is therefore important to acknowledge that the research reviewed in this article does not explicitly address fetal exposures.
Conclusion
The INUENDO study indicates that POPs may interfere with adult male reproductive function without major impact on fertility and sperm counts. However, subsets of men with specific polymorphisms in the AR gene seem more vulnerable. It is unknown if POP interference with sperm chromatin integrity and sperm DNA may have health consequences in the offspring. PCBs seem more to blame than DDT. No-effect thresholds could not be established. Findings provided limited direct evidence of xenobiotic disruption of the endocrine regulation. Further research is needed into POPrelated effects on sperm chromatin integrity and epididymal function that include modifying effects related to AR polymorphisms. VOLUME 116 | NUMBER 3 | March 2008 • Environmental Health Perspectives Figure 2C was incorrect in the manuscript originally published online and has now been replaced with the corrrect version. | 2014-10-01T00:00:00.000Z | 2007-11-23T00:00:00.000 | {
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245154028 | pes2o/s2orc | v3-fos-license | U NDERSTANDING THE D EMAND D RIVEN M ATERIAL R EQUIREMENTS P LANNING S COPE OF A PPLICATION : A C RITICAL L ITERATURE R EVIEW
The supply chains complexity generated by the dynamic market demand imposes the improvement of the classical production control systems. A recently introduced method, the Demand Driven Material Requirements Planning (DDMRP), is proposed as an upgrade of the Material Requirements Planning (MRP), widely used in industry, capable of overcoming the nervousness of MRP environment and the bullwhip effect affecting supply chains under uncertainties. The DDMRP approach, however, is still not well established since the conditions for its application are still little investigated. Thus, this study aims at reviewing the existing scientific literature concerning DDMRP method in order to critically analyse its main scope of application as well as its real practical performance. From the reviewed literature three main research lines emerged: DDMRP basic principles, comparison with other methodologies, and case studies. The analysis of both research oriented papers and case studies points out some critical issues that are limiting the diffusion of the DDMRP method, including the additional costs necessary to adapt the in use control and planning software. The main criticality of the method is recognized to be the high subjectivity affecting the positioning of the buffers, and the need for classifying the suitable sectors of application.
Introduction
The current evolution of the market, guided by the increasingly volatile needs of the customers, put companies in front of new challenges: the supply chain has become more complex, moving from mainly linear and vertically integrated structures to highly branched ones; greater volumes of material are requested and handled; the heterogeneity and complexity of the available products have significantly increased; technological advancement has contributed to rapidly changing consumer preferences, reducing the life cycles of products and making them more and more customizable.In the context of production planning, these issues translate into the need for improving the "classical" production control systems (PCS) to adjust them to the new market requirements.Many PCS's emerged in the last couple of decades to respond to the market evolution as well as to specific environments of application [1].
One of the most widely used production control and planning systems in the industry is MRP (Material Requirements Planning), and this system is often embodied in the enterprise resource planning (ERP) software [2].MRP techniques use the bill of materials (BOM), the master production schedule (MPS), and the inventory records to schedule the required materials replenishment for the manufacturing of the final product for a given time horizon.It is based on a control system for the planning of stocks and production of the push-type, according to which organizations use forecasts to determine the market demand for their products.The MRP approach works well in a deterministic context, while it shows some weaknesses under uncertainties concerning the demand and lead time [3].The continual adjustments in the scheduling due to even small changes of the codes of the higher levels of the BOM make the MRP unstable, causing the so-called MRP nervousness [4].Moreover, the dependence between the nodes of the supply chain produces the bullwhip effect, transmitting and amplifying the variability that affects all the levels of the BOM to all the nodes in the supply chain [5].
Among the new approaches proposed to overcome MRP criticalities [6], [7], [8], the Demand Driven MRP (DDMRP) was firstly introduced in 2011 by Ptak and Smith [9], as a new PCS.According to the authors [10], the DDMRP is a hybrid push-pull method that combines some of the principles and practices of MRP, Lean, Six Sigma, and the Theory of Constraints, resolving possible conflicts among the different methods through innovative solutions.It uses Lean Manufacturing principles to allow a pull logic with approximation to real demand and the concept of signalling mechanisms to have visibility of stock levels; like TOC it focuses on products that limit the system performance and uses buffers to protect the process; from Six Sigma it considers the strong sensitivity to variations, trying to control them.DDMRP considers a decoupled lead time, keeping some pre-defined BOM components in stock, and uses the Available Stock Equation (ASE) to calculate, daily, the future stock based on actual demand and orders in production.The methodology is implemented through five steps: the strategic inventory positioning, where the positioning of the buffers responds to the need of decoupling the planning and execution horizons of the logistics chain reducing the bullwhip effect; the definition of the buffers profiles and levels, for sizing the three buffer layers (red, yellow and green) calculated by three factors: item type, lead time and variability; the dynamic adjustments of the buffers' layers with changes in demand; the demand-driven planning, for creating the production and purchase orders; the execution phase, to manage the orders once they are generated.For a more detailed analysis of the method see for example [11], [12].
In the last years the DDMRP method, initially developed by practitioners, gained new interest within the academic research that produced both theoretical and empirical studies [1].However, though the method presents some innovative aspects, mainly regarding its ability in overcoming the bullwhip effect typical of supply chains and the MRP nervousness, the conditions for its application and its real practical performance are still little investigated.Only a few literature reviews tried to organise the available studies and analyse some specific issues related to DDMRP implementation.Thus, this study aims at reviewing the existing scientific literature concerning the DDMRP method, in order to investigate the more recent results on the methodology applicability and effectiveness in the industrial environment.It is organised as a critical literature review that updates the few available reviews, providing additional insights.Following the three main research lines that emerged from the literature, the paper discusses the advantages and limits of the methodology in the view of the various authors, analyses the critical issues that can limit its application, and outlines its possible future developments.
Scope of the review
Despite the recent attention to the study and application of the DDMRP and the number of articles available on the website of the Demand Driven Institute (DDI), the consulting company founded by Ptak and Smith operating in the DDMRP field, the method is still under-investigated by the scholars.Therefore, this literature review aims at exploring the academic perspective on the methodology and is driven by three main research questions: 1. Are the basic principles of DDMRP improved by academic research?2. What are its required future evolutions according to the scientific community view?3. Considering the benefits and difficulties encountered by the manufacturing industry applying the DDMRP, in what productive environments did this system prove useful?
Our study aims at updating and extending the three literature reviews published on the DDMRP methodology, which reveal different focuses.Marzougui et al. identify papers containing simulated or real case studies with the aim of comparing the academic results with the improvements achieved by companies implementing the DDMRP method as claimed by the DDI; master's theses and non-English paper are excluded from the review, which is updated to 2019 [11].The systematic literature review of Orue et al. analyses the studies investigating the standardization of the procedure to implement the DDMRP model, to build up a standard application model enabling full exploitation of the methodology potential benefits, through reducing the implementation time and costs, the possible errors, and improving the quality of the process; also this review is updated to 2019 [13].
The more recent systematic review, presented by Azzamouri et al. and updated to April 2020, explores the literature contributions to three main issues providing knowledge on the method: the method validation, the improvement of the original techniques, and the proposal of new tools for its implementation [14].
Publications search and selection
The exploration of the literature was conducted using the online databases Science Direct, Web of Science and Google Scholar and completed on May 14, 2021.An initial set of papers was identified employing the keywords "Demand Driven Material Requirements Planning", "DDMRP", and "Demand Driven"; the saturation point occurred when the same articles kept appearing in search engines.Due to the relatively small number of collected papers and to ensure that our research was as comprehensive as possible, we further conducted a snowball search.At the end of the search process, we collected a total of 140 papers.
Then, we proceeded with the selection of the relevant papers according to the following exclusion criteria: • Duplicates between search engines have been removed, i.e.48 papers.
• Both qualitative and quantitative articles were selected, considering publications in academic journals or conferences, and Master and PhD degree theses; in this review, we included the theses because they made available the knowledge on the DDMRP methodology to the wide practitioners' community in their different national languages.Some magazines and newspapers articles written by consultants specialized in Supply Chain and logistics, mainly available on the DDI website, have been excluded since their main purpose could be marketing their product to companies and this could change the way of presenting the method, i.e. 33 papers.• The publications without the specified keywords within the article were excluded, finding that they dealt with topics not directly related to the DDMRP (e.g.Decoupled lead time), i.e. 4 papers.• The publications without an exclusive focus on the DDMRP methodology applied to the planning of material requirements in the supply chain (e.g.articles dealing with the broader Demand Driven Enterprise Model) were excluded, i.e. 7 papers.• Since the scope of this review is to analyse the DDMRP related concepts that emerged in the literature in recent years, we defined as review temporal boundaries the period 2016-2021; in fact, starting from 2016 there is an increase in the number of scientific publications (Figure 1), following a few years the first introduction of the DDMRP methodology in 2011 [9].Other 9 papers were thus excluded.Finally, a total of 39 relevant publications were selected for further analyses: 12 scientific articles, 6 conference papers, and 21 theses.
Papers classification
To answer the research questions, the selected publications were classified analysing their content according to three main categories revealed by the review: DDMRP basic principles, comparison with other methodologies, and case studies (Table 1 1.Classification of the selected studies according to the identified categories. Table 1 shows that the research papers (including both the DDMRP basic principles and comparison with other methodologies categories) are more than the case studies, confirming that although some companies are now implementing DDMRP, still very few of them share their data and collaborate to scientifically study the advantages or critical issues related to the method within their specific environment [14].The content of the papers is then analysed and discussed in relation to the three identified categories.
Group 1: DDMRP basic principles
The first group of publications includes 13 items, of which 7 articles were published in scientific journals or conferences and 6 Master degree theses.These publications mainly aim at examining the principles and basic concepts of the DDMRP methodology, showing future developments necessary for the optimization of the methodology.
An increasing interest in the DDMRP field by the Academy is observed, resulting in simulations, studies and methodology analyses.The Polytechnic University of Valencia uses DDMRP for students' group work according to the problem-based learning method [17] and many theses in different countries analyse how the DDMRP works to improve the theoretical knowledge on DDMRP methodology and introduce it to local companies.
Some studies address the main advantages and limitations of the method.According to Cuadra [16], the DDMRP allows to increase sales by a high percentage, to achieve a service rate of 99%, to limit the stock and to reduce the delivery time.Meinzel [21] uses a simulation to address the five steps of implementation of the method, examining the obtained benefits that result in + 13% of average service level, -22% in average lead time and -31% average cost of inventory.De Biasi [25] shows that the DDMRP method allows to improve the information flows within the supply chain and the performance of the warehouses.
Many scholars investigate the reasons leading companies to choose the DDMRP and implement it in the manufacturing environment.According to Perälä [23], the key data needed for the DDMRP implementation within the company must be organized following the next points: to identify the product or item that can benefit from DDMRP application; to place the decoupling points in the BOM of the product; to calculate the zones and buffer levels.Marin [18] considers the novelties introduced by DDMRP the reasons why it finds opposition from some companies' staff members, who do not accept to change their habits.On the other hand, various authors consider essential the development of DDMRP-based software to be integrated into an ERP, for incentivizing organizations to choose the DDMPR method and allowing easier management of the tool by local companies [16], [18], [20].
According to the DDMRP methodology [10], the choice of some variables (namely the positioning of the buffers, the lead time and variability factors, as well as the determination of the safety stock) is based on the experience of the staff who must select the values of the parameters within a certain range.This procedure introduces a subjectivity in the method implementation that should be overcome by analyzing a larger sample of companies using the DDMRP to identify the most suitable sectors of application and provide a set of best practices and mathematical tools supporting the decisionmaking process [20], [18].Lee et al. [19] propose a new safety stock formula for the calculation of the parameter, enhancing existing guidelines for the DDMRP implementation.Velasco Acosta et al. [27] developed a DDMRP model simulated through a discrete event software to evaluate the applicability of the method in a complex manufacturing environment, considering a number of BOM levels up to 4, finding that the customer satisfaction level can be improved by 41% and the average on-hand stock can be reduced by 18%.
Few papers consider the relation of the DDMRP to Supply Chain Management (SCM), though the level of service and the reduction of delivery times can directly affect the customer's relationship with the company.Achergui [26] use the DDMPR buffer positioning approach applied to a hybrid MTO/MTS manufacturing system, demonstrating a viable reduction of the inventory costs for small supply chain networks.Santos [24] evaluates the impact that DDMRP has on the SCM processes, through the AHP (Analytic Hierarchy Process) multi-criteria decision support technique; the author finds that the three most impacted processes are production flow management, demand management and order fulfilment.Pekarcíková et al. [22] analyse the use of multiple product-related BOMs to indicate different levels in the supply chain considering the demand behaviour, with the aim of extending the DDMRP knowledge base in the context of Industry 4.0.
A list of the required future research developments apt to foster the adoption of the DDMRP method, according to the reviewed publications, is sketched in the figure 2. In the circles, the number of papers referring to the required insight.
Group 2: Comparison with other methodologies
The second group contains 8 publications, 6 papers and 2 theses, where DDMRP is compared with other methodologies, namely MRP and MRPII (Manufacturing Resources Planning), that it aims to overcome, the Economic Order Quantity (EOQ) tool and Kanban method.
Miclo et al. [31], [32], [33] use the discrete events simulation and the Kanban game1 data as input data to compare the DDMRP with MRPII, using as main indicators the On-Time Delivery (OTD) and Working Capital (WC).The results obtained show that DDMRP achieves better output indicators than MRPII with less WC and WIP, but with a less satisfactory OTD.Reference orders are much smaller in the case of the DDMRP, but more frequent than in the MRPII; however, the increase in the number of orders does not generate a correspondent increase in the number of setups for the machines, which was set by the execution priorities of the DDMRP.Moreover, the DDMRP method seems to be more stable when facing the variability of real demand, although during the simulation some products went out of stock.As expected, the DDMRP efficiently anticipates the peaks in demand orienting the management to the pull logic for the normal demand and the push logic for the peaks.Still using a discrete events simulation, Shofa et al. [29] compare the DDMRP with the MRP focusing on the inventory level under long lead time and uncertain demand scenarios, finding that using the DDMRP method the inventory level is more stable and the stock level is reduced by 11%.Simulating the behaviour of four production planning and control systems (DDMRP, MRP, Kanban and Optimised Production Technology) applied to multi-stage assembly systems under different levels of bottleneck severity and delivery date tightness, Thurer et al. [34] found that DDMRP maintains a high level of performance also for severe bottleneck, with the lower inventory levels.Miclo et al. [12] simultaneously compare DDMRP with the MRPII and the Kanban systems at different levels of demand variability; they found that Kanban has a slightly lower WIP than DDMRP on average, while MRPII provides the worst results.According to the authors, the innovation of DDMRP lies in the fact that this methodology makes use of the best practices of the two previous approaches.The dynamic buffers have the function of absorbing the variability and allow the effective scheduling of the production, and a constant level of protection from the effect of nervousness.Finally, unlike the Kanban system, DDMRP uses buffers selectively, applying them only where they are needed.The comparison between DDMRP and EOQ, performed through a discrete event simulation in an ideal environment by Tounsi [28], shows how the DDMRP method uses less capital in stocks and WIP, with a higher percentage of orders fulfilled than the EOQ.Table 2 summarises the main advantages of the different methods as highlighted by the reviewed authors.
Group 3: Case studies
The third group collects 17 case studies retrieved from the literature of which 13 theses and 5 research articles, which describe 15 simulations on real data and only 2 cases reporting the method implementation in companies; the case studies explore the benefits and difficulties faced by the manufacturing industry.Table 3 lists the case studies and the company context and motivations for adopting or simulating the DDMRP.
Case study: company name / sector
Company context / needs / issues to overcome REF.
COASA / oils and lubricants for the aviation industry
Obsolescence of the products (90%); Excessive stock; Unnecessary products purchase due to unplanned events [35] GSA / kitchen tools and small appliances Need to improve the service level and reduce the costs to recover market segments [36] Consumer-Packaged Goods (Food sector) Perishable goods and high inventory turns (1.6 to 2.5 weeks); simulating planning under uncertainty at finite capacity [37] White goods Need to improve the service level and reduce the costs to recover market segments [38] Wa-Armonia / cosmetics Need to improve the service level and reduce the inventory costs [39] Automotive Lighting / Outdoor lighting for automotive Variable and unpredictable demand, due to the change in consumers' behaviour; growing structural complexity of the supply chain; architectural complexity of the products. [40] ISEO locks / locking systems Need for higher flexibility and reduced delivery time, confirming the service level [41] Exquise1928 / soft drinks and syrups Interest in improving the company's PCS [42] Dayco Ivrea / components for the automotive sector Management of raw material stock not effective, causing failures in production planning; suppliers lead time and demand variability.[43] Science4you / scientific and educational games/tools Market seasonality; high variability of the demand.Failures in raw materials stock management; long lead times. [44]
Food
Excessive stock; obsolescence of the products. [45]
Automotive
Need to improve inventory level management. [30]
Food -sauces
High fluctuations in the product demand; unstable inventory level.
[46] AeroCo / Aircraft components Interest in analysing the applicability of the DDMRP.[47] Companies CS1 (padlocks), CS2 (components for household appliances), CS3 (special parts for the automotive industry) CS1: considerable purchase lead times; only one experienced responsible for purchase orders.Need to adjust the inventory level to the market requirements, whilst maintaining the service level.CS2: complex supply chain; high levels of raw material at a significant cost; final products stored in the company warehouse before delivery to customers; demand and supply variability and lack of visibility; stock outs and rush orders.CS3: three production plants; complex supply chain, as well as purchasing and production planning.[48] Company CS1 (CS1 as above) [49] [50] Table 3. Case studies included in the review.
As can be seen in the table 3, the application of the DDMRP methodology to the industrial environment is mainly studied to demonstrate its efficacy in responding to demand and lead time variability, improving inventory management, with a resulting economic advantage, and service level.Some criticalities are also revealed.
The simulation performed by Castro J.E.N. [35], starting from an ABC classification of Coasa's products (oils and lubricants for the aviation industry), finds that excess stocks and expired products have been eliminated, reducing the stock by 25% and estimating an economic return of about $ 10,000 per year.A reduction in available stocks, with a simultaneous increase in the ADU and the reduction in stock coverage, is observed also by Kortabarria [48] in the companies where the DDMRP method was implemented.Salazar & Arenas [38] highlight a decrease in total inventories up to 60% with an increase in sales up to 20%, reducing the reactivity time to the market demand up to 80%, in the white goods company they analysed.Brahimi & Saadi [42] compare the planning system of a company bottling and selling soft drinks with and without DDMRP; they note that the DDMRP model improves the stock coverage and makes the inventory level more stable, while it lowers the inventory turnover rate.Leguizamo & Totaitive [32] in simulating a single product, talc, find that the conversion to the DDMRP methodology guarantees a reduction in costs.Dimas et al. [46] show how the DDMRP lower the inventory levels of a company producing food sauce allowing a savings of 53.5%.
According to Ptak and Smith [10], the reduction of stocks corresponds to an unchanged or even better level of service.Salazar & Arenas [38] find a level of service above 99%, while Provenzano [40], in his simulation on a group of finished products (Automotive Lighting), finds that using the DDMRP the customer demand is satisfied without corrective actions.Kortabarria et al. [49], [50] also stress that the reduction in stocks is not followed by a worsening of the level of service.
A reduction of the delivery times is observed by Abith Zachariah [41], together with a higher level of service and satisfaction for the customers in a company producing locking systems.Shofa et al. [30], through a simulation using the data of an automotive company, claim that DDMRP allows a reduction in delivery times and lead time (-94%), improving the service level.Ducrot & Ahmed [37] simulate a European perishable food production company and compare conventional APS software and DDMRP planning, observing the service level and the inventory turns; according to their calculations, DDMRP performs better than the heuristics-based planning in three scenarios out of four and it provides similar results as an advanced mathematical solver.A service level approaching 98% (10% increase over the current service level) is obtained for one production line of the GSA company by Builes Henao through a discrete event simulation [36].Perez Castro [45] argues that thanks to the implementation of DDMRP in a food company, the ROA improves by 2 to 3% and the products remain less time in stock.
Applying the DDMRP methodology to a company selling components for the automotive sector Hincapiè [43] shows that it makes planning management easier since managers can observe the status of stocks on a daily basis, prioritize critical orders and keep the stock in the desired range.According to Kortabarria [48], the implementation of DDMRP entails a fundamental change in the material planning process in each of the three companies (CS1, CS2, and CS3) he analysed after the migration from MRP to DDMRP.In all these companies, day-to-day planning was previously considered not practicable, as the information gathering process was time-consuming and not easy.After the implementation of the DDMRP method, the flow of information along the entire supply chain has significantly improved, and the three companies acquired access to the inventory daily, thus being able to plan in a simple and agile way the needs of materials and to manage the status of supplies, obtaining greater flexibility, saturation and synchronization of the production lines as well as the absence of urgent orders.Both the production and procurement planning should be adapted to the DDMRP implementation: for example, within the ISEO group, the ADU must be evaluated on a weekly instead of monthly basis as carried out previously; the sizing parameters must be continuously updated to dynamically adjust and optimize stock levels; when a new supplier is involved the related information must be promptly updated due to the dynamic nature of the size of the buffers [41].
The alerts dictated by the method, and the priority they represent for the decision-making process, allow to prevent critical situations of shortages or excess stocks, pointed out by Tavares [44] in her simulation of a toys company; however, the author observes that the DDMRP methodology it is not intuitive to apply and requires preparatory steps as well as many input data.Moreover, the definition of the associated decision-making criteria depends on the practitioners' experience and the level of accuracy in defining the DDMPR parameters can impact the method applicability.So, the implementation of the DDMRP requires staff to be trained on the principles and operation of the method, an aspect that is not trivial since generally in the companies the orders and production planning is entrusted to staff members with extensive professional experience in this regard [40].Abith Zachariah analyses the costs for the implementation of the new methodology within the ISEO group, quantifying the costs for the theoretical and practical training of the employees, as well as the costs for upgrading the used management software to support the planning and operational execution of the new method, finding that the overall expenditure is around 40-50 k€ [41].
Other criticalities of the DDMRP are emphasized in the reviewed case studies: the efficiency of DDMRP seems to be affected by the presence of long lead times in the intermediate levels of the supply chain [40], so it cannot be applied to all buffers [41]; moreover, the method is sensitive to the value of the decoupled delivery time and the orders' frequency and variability, which should not be empirically assigned [36].Moreover, it is observed that the DMRP cannot be applied to all products or activities [22]: the purchase of raw materials become critical when delivery times are too long and cannot be related to the effective demand for long time ranges.Fig. 3. DDMRP advantages as retrieved from the reviewed case studies.
The DDMRP supply logic does not apply if the quantity and not the price is negotiated, since if the price is low the company will buy more material than the actual requirement because the cost of keeping it in stock is lower than the higher prices the company would pay in the subsequent purchase period [41].Figures 3 and 4 summarize the advantages and disadvantages of the DDMRP method as retrieved from the reviewed case studies.The bars represent the number of papers citing the considered advantage/disadvantage.Fig. 4. DDMRP disadvantages as retrieved from the reviewed case studies.
Conclusion
The presented review of the available academic studies on the DDMRP methodology highlights the current academic research trends.The main efforts of the scholars are directed to the study of the basic principles of the method, often comparing it to the more established methods.Simulations are widely used to validate the original method and confirm some of the claimed advantages of the DDMRP, while since most of the organizations present complex scenarios the greatest challenge of the simulations is the setting of the parameters for the implementation of the DDMRP, still empirically based and poorly documented.Among these, the enormous subjectivity that dominates the positioning of the buffers is identified as particularly critical and this is still one of the gaps affecting the research papers on the application of this methodology and the implementation in the organizations as well, where a qualified and properly trained specialist should be dedicated to the activities of regularly monitor the demand and consequently adapt the production buffers.
A growing interest in the implementation of the DDMRP in the real industrial environment is proved by the number of case studies, mainly presented in Master or PhD theses after the candidates' internship in local companies; however, most of the retrieved case studies are simulations based on real data and only a few empirical ones.The analyses show that the method allows improving the organisations' production control system overcoming some of the identified weak points, while the costs of its implementation should be taken into account.However, the application of the method to the industrial context is limited to the analysis of the problems of a single company, often considering the possible transition from an MRP system to DDMRP.Moreover, the exiguity of studies reporting the real implementation of the method in the industrial context limits the possibility of compiling a catalogue of best practices.
From this study emerged that two main future research lines can improve the knowledge on the DDMRP applicability and effectiveness into the industrial environment: the development of tools and objective methodologies for the parameters set and the positioning of the buffers and the systematic analysis of the applicability of the method in relation to the sector and the complexity of the organization and its supply chain.
Fig. 2 .
Fig. 2. Future developments of the studies on the DDMRP method, as expected by the papers included in the review.
Table 2 .
Comparison of the different methodologies for managing inventory planning. | 2021-12-16T16:55:30.521Z | 2021-01-01T00:00:00.000 | {
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253044523 | pes2o/s2orc | v3-fos-license | Initial soil organic carbon stocks govern changes in soil carbon: Reality or artifact?
Abstract Changes in soil organic carbon (SOC) storage have the potential to affect global climate; hence identifying environments with a high capacity to gain or lose SOC is of broad interest. Many cross‐site studies have found that SOC‐poor soils tend to gain or retain carbon more readily than SOC‐rich soils. While this pattern may partly reflect reality, here we argue that it can also be created by a pair of statistical artifacts. First, soils that appear SOC‐poor purely due to random variation will tend to yield more moderate SOC estimates upon resampling and hence will appear to accrue or retain more SOC than SOC‐rich soils. This phenomenon is an example of regression to the mean. Second, normalized metrics of SOC change—such as relative rates and response ratios—will by definition show larger changes in SOC at lower initial SOC levels, even when the absolute change in SOC does not depend on initial SOC. These two artifacts create an exaggerated impression that initial SOC stocks are a major control on SOC dynamics. To address this problem, we recommend applying statistical corrections to eliminate the effect of regression to the mean, and avoiding normalized metrics when testing relationships between SOC change and initial SOC. Careful consideration of these issues in future cross‐site studies will support clearer scientific inference that can better inform environmental management.
informed, identifying which underlying soil properties set the potential for SOC sequestration or loss in different environments.
A number of cross-site studies-multi-site experiments and
meta-analyses-have identified a major factor that appears to govern accrual and loss of SOC: standing SOC stocks. For instance, improved agricultural management seems to enhance SOC most strongly in SOC-poor soils, while having a weakly positive-or even negative-effect on SOC in SOC-rich soils (Arndt et al., 2022;Berhane et al., 2020;Deng et al., 2016;Hübner et al., 2021;Iwasaki et al., 2017;Lessmann et al., 2022;Li et al., 2018;Minasny et al., 2017). In a different context, a prominent meta-analysis of soil warming experiments found that SOC-rich soils exhibited stronger losses than SOC-poor soils (Crowther et al., 2016, but see van Gestel et al., 2018, and several regional surveys have indicated that SOC-poor soils tend to retain SOC more readily than SOC-rich soils (Capriel, 2013;Hanegraaf et al., 2009;Riley & Bakkegard, 2006). In aggregate, these studies all point to a general pattern: that changes in SOC are often negatively related to initial SOC stocks, with SOCpoor soils gaining or retaining SOC most readily, and SOC-rich soils exhibiting weaker gains or stronger losses. This pattern may emerge because the capacity of soils to store SOC saturates due to biophysical factors, particularly the amount of silt and clay-sized minerals that protect SOC from microbial decomposers. Consequently, after accounting for the quantity and type of minerals, SOC-poor soils may on-average be farther from saturation and more likely to accrue additional SOC than SOC-rich soils (Cotrufo et al., 2019;Georgiou et al., 2022;Stewart et al., 2007).
The tendency of SOC-poor soils to gain or retain SOC more readily than SOC-rich soils appears to be widespread and has some basis in the carbon saturation concept. However, we suspect that this pattern is often exaggerated by a pair of statistical artifacts. These are: (1) a phenomenon termed regression to the mean; and (2) artifacts that result from normalizing changes in carbon by baseline carbon levels. Here, we illustrate these artifacts using simulated data and suggest more robust approaches to test the relationship between initial SOC stocks and changes in SOC going forward.
| REG RE SS I ON TO THE ME AN
Regression to the mean occurs when random variation affects repeated observations. When initial observations are collected, random variation will tend to produce some extreme low or high values.
When follow-up observations are collected on these extreme cases, the second observations will-more likely than not-produce less extreme values, simply because extreme values are by definition unlikely. This tendency, where extreme values tend to be followed by moderate values that "regress" to the population mean, was classically described by Francis Galton in relation to the inheritance of height in human populations (Galton, 1886). Regression to the mean is a general phenomenon that can occur when paired samples are collected sequentially or simultaneously, regardless of the distribution of a random process. For instance, it can be illustrated by simultaneously rolling two dice of different colors and subtracting the value of one die from the other across repeated trials (Figure 1a). We may safely assume that the dice rolls are independent-and yet, regression to the mean will create a negative relationship between the individual dice rolls and the difference between the rolls (Figure 1b).
Regression to the mean emerges in SOC surveys because all SOC stock estimates are affected by randomness to some degree. This is in part because the measurements required for calculating the SOC stock-carbon content, bulk density, and rock fraction-all carry significant levels of uncertainty (Goidts et al., 2009). In addition to measurement error, soil sampling is inherently random because samples F I G U R E 1 Regression to the mean illustrated with 20-sided dice. A red and a blue 20-sided dies were rolled 75 times (panel a). In panel (b), the difference (blue-red) was plotted against the red die roll. When the red die roll is high, it is most likely that the blue die roll will be lower, and so the difference (blue-red) will tend to be negative. When the red die roll is low, it is most likely that the blue die roll will be higher, and so the difference (blue-red) will tend to be positive. Consequently, random chance will generate a negative relationship between blue-red and red die rolls. In this example, the red die is analogous to an initial SOC estimate, and the blue die is analogous to a final SOC estimate. SOC, soil organic carbon.
Red
Blue Difference are collected at the centimeter scale (e.g. by coring), while SOC stocks can vary substantially (10% or more) at the scale of meters (Goidts et al., 2009;Maillard et al., 2017). In combination, measurement and sampling error will inevitably cause the mean SOC stock estimate at a given site to vary randomly from one sampling campaign to the next. Increasing the number of replicate soil samples taken at a site will reduce this variation, but can never eliminate it completely.
Random variation between sampling events can explain apparent negative relationships between the initial SOC stock (SOC initial ) and change in SOC (SOC final − SOC initial ; ∆SOC). Estimates of SOC initial that are extremely high due to chance will likely coincide with more moderate follow-up estimates, and these paired measurements will hence tend to generate low or negative values of ∆SOC. Conversely, extremely low SOC initial estimates will likely coincide with more moderate follow-up estimates, and will hence yield high values of ∆SOC. This pattern can develop even when samples are not taken sequentially-for instance, regression to the mean will occur in cases where SOC estimates from control plots are substituted for SOC initial , or in cases where SOC initial is approximated using paired "across the fence" or chronosequence sampling designs. The process of extreme baseline values regressing to the mean during repeated sampling will on average produce the appearance that baseline SOC stocks-however, they are defined-are a control on SOC change, regardless of whether any real relationship exists.
The effect of regression to the mean on the interpretation of SOC dynamics has gone largely unnoticed to date. In a notable exception, regression to the mean was discussed in 2006 in the context of repeated soil surveys across the United Kingdom (Lark et al., 2006).
These surveys initially indicated that SOC-rich soils were losing carbon, while SOC-poor soils were not (Bellamy et al., 2005), but this pattern was later partly attributed to regression to the mean (Lark et al., 2006;Potts et al., 2009). Several studies have followed the suggestion of Lark et al. (2006) by correcting for regression to the mean or at least estimating its effect size (Callesen et al., 2015;Hong et al., 2020;Saby et al., 2008;Senthilkumar et al., 2009 in paired control plots. These studies include analyses focused on agricultural practices and land-use change (Arndt et al., 2022;Berhane et al., 2020;Deng et al., 2016;Fujisaki et al., 2018;Haddaway et al., 2017;Hübner et al., 2021;Iwasaki et al., 2017;Sun et al., 2010) but also purely observational studies (Capriel, 2013;Hanegraaf et al., 2009), and meta-analyses of warming experiments (Crowther et al., 2016;van Gestel et al., 2018). The majority of these studies found that ∆SOC and SOC initial are negatively related. This convergence is striking: If the patterns reported in these studies were caused by actual biological processes, this would imply that SOCrich soils lose carbon under a wide range of conditions-including high C input scenarios (Arndt et al., 2022;Berhane et al., 2020)while SOC-poor soils remain unchanged or gain carbon under an equally large range of conditions. However, the extent to which this pattern can be attributed to regression to the mean remains unclear.
The dice example that we presented earlier ( Figure 1) is an extreme case; in practice, the effect of regression to the mean might be moderated by several of factors, such as the range of initial SOC values included in the analysis, or the variance associated with site-level SOC estimates.
To explore the effect of regression to the mean in a more realistic set of scenarios, we used simulated data to replicate the error structure of a typical cross-site study ( Figure 2a). To achieve this, we first created a distribution of "true" SOC initial values, which represented the site level mean SOC stocks across a set of hypothetical sites (n = 200). We generated these data by randomly drawing a set of 200 values from a lognormal distribution with a mean value of 30 tC ha −1 , which is roughly representative of the distribution of SOC initial values featured in several cross-site studies (Arndt et al., 2022;Berhane et al., 2020;Sun et al., 2010). We varied the width of the distribution F I G U R E 2 Simulated SOC data illustrating regression to the mean. (a) Shows a simulated data set in which the true values of SOC initial and SOC final were defined to be equal, and random error was added to each variable before relating SOC initial to ∆SOC (coefficient of variation = 0.1). (b) Shows bias attributable to regression to the mean as a function of the coefficient of variation. Red lines show mean bias when ordinary least squares (OLS) is used to fit the data. The solid lines show results for a "broad" simulated data set, representing a regional-to global-scale analysis with high population variance. The dashed lines show bias for a "narrow" simulated data set, representing a local analysis with low population variance. The yellow lines show bias after applying the correction from Blomqvist (1977).
because regression to the mean has a stronger effect when the population variance (i.e., the variance of SOC initial across sites, or s 2 z ) is small relative to the variance associated with individual estimates (i.e., the variance of SOC initial within sites, s 2 u ) (Blomqvist, 1977). We explored two cases with differing types of variance s 2 z : (1) a "broad" cross-site study representing a regional-scale analysis with a larger s 2 z (standard deviation [SD] of SOC initial = 10 tC ha −1 ), and (2) a "narrow" cross-site study representing a local survey with a smaller s 2 z (SD of SOC initial = 5 tC ha −1 ) (Figure 2b, red lines). SOC distributions were bounded between 10 and 100 tC ha −1 to avoid sampling extremely high or negative values. After generating SOC initial values, we defined the "true" value of SOC final to equal SOC initial , assuming zero management effect. We then added error to SOC initial and SOC final to represent measurement uncertainty plus natural variability in SOC across the sampled area. We generated errors from a normal distribution by varying the coefficient of variation (CV) of the sampling distribution of the mean SOC value at each site between the values of 0 and 0.2. Importantly, this range of values represented the CV of the sampling distribution, that is, the ratio of the standard error to the mean-hence, in practice, larger sample sizes would generate lower CV values. After generating errors, we calculated ∆SOC and modelled its dependence on SOC initial using ordinary least squares regression. We calculated bias by taking the mean difference be- The simulations demonstrate that regression to the mean has the potential to generate negative relationships between ∆SOC and SOC initial under conditions typical of cross-site studies. To apply this approach to a real-world example, we downloaded the data from a global soil warming synthesis study (van Gestel et al., 2018). We found that this data set was somewhat broader than the "broad" example explored above (mean SOC stock = 36 tC ha −1 , SD = 27 tC ha −1 ). We simulated the effect of regression to the mean given these parameters and assumed a within-site CV for the sampling distribution of 0.10. Given the assumed error level, the simulation yielded a slope of −0.04 tC tC −1 . This slope was similar to the slope of the ∆SOC versus SOC initial regression line that we calculated for the entire data set (−0.05 tC tC −1 ), and a significant fraction (24%) of the slope obtained when we fit a regression to the subset of the data from an earlier synthesis (Crowther et al., 2016; −0.17 tC tC −1 ). This result suggests that regression to the mean partly explains the apparent relationship between ∆SOC and SOC initial in this data set (if one exists, see van Gestel et al., 2018). Clearly, future meta-analyses should correct for regression to the mean when testing whether initial carbon stocks mediate changes in SOC.
| CORREC TING FOR REG RE SS I ON TO THE ME AN
Disentangling the effect of regression to the mean from the true underlying relationship between ∆SOC and SOC initial is possible given the right statistical approach. In fact, several studies have found persistent negative relationships between ∆SOC and SOC initial after applying a statistical correction (Hong et al., 2020;Lark et al., 2006;Senthilkumar et al., 2009). One correction approach relies on calculating the regression line between the change value (final − initial) and the initial value, and then correcting the slope derived from this regression (̂ ′ ) using variance estimates to generate an unbiased estimate (̂ ) (Blomqvist, 1977): where is the ratio of s 2 u (the within-site variance of the initial values) to s 2 z (the across-site population variance of the initial values). This equation shows that if s 2 z ≫ s 2 u , (i.e., approaches 0) as in in a large cross-site study, then ̂ approaches ̂ ′ . This corrected slope value can be calculated manually by estimating s 2 z and an average value of s 2 u across the data set, dividing these values to obtain , and obtaining ̂ ′ from a regression of ∆SOC against SOC initial [e.g., using a standard regression calculator, such as the lm() function in R]. In our simulated example, this approach substantially reduced bias in the relationship between ∆SOC and SOC initial (Figure 2b; yellow lines). The usefulness of this correction is limited in the case of soil survey data because it requires knowledge of the uncertainty at individual sampling sites (Lark et al., 2006).
However, in the case of cross-site studies, sampling is often replicated at each site, and so reported SDs and sample sizes can be used to calculate site-level standard errors, and hence the average value of s 2 u across the data set.
(1) =̂ � + 1 − For hypothesis testing, it may also be valuable to estimate the variance associated with ̂ (var ̂ ). This value can be used to construct confidence intervals and determine whether ̂ differs significantly from zero. We have provided R code for performing these calculations and obtaining confidence intervals (see Supplementary Information; Appendix S1). If SOC initial is normally distributed, this variance can be calculated from ̂ , the variance of ̂ ′ (obtained from ordinary least squares regression), the coefficient of variation of s 2 z (CV(s 2 z )) and the coefficient of variation of s 2 u (CV[s 2 u ]) (Blomqvist, 1977): To parametrize this equation, CV(s 2 z ) must be obtained from the SD of SOC initial (s z ) and the overall sample size (total number of sites, n): In this equation, the term 4 represents the fourth central moment of SOC initial . The value of CV(s 2 u ) can be obtained by estimating the standard error of the error variance across sites and dividing it by s 2 u . It is important to note that in practice, SOC initial may not be normally distributed, and s 2 u may be correlated with SOC initial (as was the case in our simulated data sets). Consequently, both ̂ and var ̂ will remain somewhat biased, albeit to a relatively small degree (Figure 2b, yellow lines). While these corrections reduce the artifact caused by regression to the mean to almost zero, clearly there is a need to develop statistical approaches that are tailored for error structures typical of SOC data sets. In the meanwhile, an imperfect correction (Equations 1-3) is preferable to no correction.
Study design can also minimize the effect of regression to the mean when relating ∆SOC to SOC initial . For instance, in the case of cross-site studies that involve collecting new samples (e.g., locally replicated experiments), ensuring the close proximity of final and initial soil samples will reduce variation between sampling events, limiting the effect of regression to the mean. Similarly, increasing the number of samples collected within experimental strata will result in smaller within-site standard errors, further limiting bias. Granted, these remedies will often not be available in cross-site studies that rely on data that have already been collected (i.e., meta-analyses). In these cases, increasing the breadth of initial SOC values across sites (i.e., increasing the population variance, s 2 z ) will dilute the effect of regression to the mean (Equation 1; Figure 2b). Importantly, study design will only incrementally reduce the effect of regression to the mean, and so statistical corrections are essential.
| NORMALIZ ATION ARTIFAC TS
The problem of regression to the mean affects studies that relate differences in SOC stocks to initial SOC, but many studies express changes in SOC in terms of ratios rather than differences. Here, we briefly discuss three types of analysis relying on ratios that are prone to statistical artifacts: (1) analyses that normalize changes in SOC by the initial SOC level (e.g., SOC final /SOC initial , or change in SOC in % or ‰ year −1 ; Li et al., 2018;Minasny et al., 2017); (2) analyses that normalize changes in SOC by the time since a treatment was imposed and then regress this average rate against time (Cai et al., 2022;Han et al., 2016;Liu et al., 2014;Minasny et al., 2017;West & Six, 2007;Xu et al., 2019); and (3) analyses that normalize changes in SOC by SOC levels in an experimental control (as opposed to the initial SOC level), and then relate this value to initial SOC level. (Gross & Glaser, 2021;Han et al., 2016;Liu et al., 2014). We expect that all three of these cases are susceptible to artifacts when relating changes in SOC to initial SOC levels.
Analyses that normalize changes in SOC by the initial SOC level are prone to strong statistical artifacts. These artifacts occur because relative changes in SOC-whether positive or negative-will by definition tend to appear larger in SOC-poor soils, simply due to the fact that the denominator is smaller in these soils. For instance, consider a case in which a positive relative change SOC change in % (100 × ∆SOC/SOC initial ) is regressed against SOC initial . Quite predictably, if the change in SOC is on-average positive, this calculation will generate a decreasing concave curve (a hyperbola) relating the relative rate of change and SOC initial , even when ∆SOC is entirely independent of SOC initial (Figure 3a,b). This pattern is tautological: a fixed increase in carbon will by definition be large relative to a small value of SOC initial , or vice versa, will be small relative to a large value of SOC initial . Consequently, while it may often be true that relative changes in SOC appear larger at low SOC levels, this fact is in no way diagnostic of the actual relationship between the mass balance of SOC and initial SOC levels.
Similarly, analyses that normalize changes in SOC by the time and then regress this average rate against time can produce normalization artifacts. This type of analysis is common in studies of SOC dynamics in agricultural experiments (Cai et al., 2022;Han et al., 2016;Liu et al., 2014;Minasny et al., 2017;West & Six, 2007;Xu et al., 2019), which often aim to characterize the time it takes for changes in SOC to level off after changes in management. Time-averaged sequestration or loss rates are calculated by dividing ∆SOC (Figure 3c, y axis) by the amount of time that has elapsed since new land management practices were adopted. Because time is used as a denominator in this calculation, the average rate will by definition tend to be related to time by a hyperbolic curve that approaches zero, even when ∆SOC is unrelated to time (Figure 3c,d). Consequently, an apparent decline in the average SOC sequestration or loss rate over time is not necessarily indicative of gradual SOC equilibration or saturation; rather, it may emerge even when ∆SOC and time do not have a clear functional relationship.
The third-and most subtle-case that we consider includes analyses that divide SOC values in treatment plots (SOC treat ) or treatment effects (SOC treat − SOC control ) by values in control plots (SOC control ), and then relate this ratio or its logarithm to SOC initial (Han et al., 2016;Li et al., 2018;Liu et al., 2014). Response ratios of this type are invaluable for synthesizing data that are reported on different measurement scales, and it would seem that they are less prone to artifacts, given that SOC initial is not used in calculating the ratio. However, closer consideration indicates that response ratios will often be related to SOC initial when the absolute treatment effect (SOC treat -SOC control ) is unrelated to SOC initial . This possibility emerges because changes in SOC are typically small relative to SOC stocks; consequently, the denominator in the response ratio will tend to be highly correlated with SOC initial . For instance, we can imagine a simplified example in which SOC treat and SOC control always differ by a fixed value, and diverge symmetrically from SOC initial across a range of sites (Figure 4a). In this simplified case, SOC treat − SOC control will be unrelated to SOC initial (Figure 4b), whereas SOC treat /SOC control will tend to be higher at SOC-poor sites and lower at SOC-rich sites ( Figure 4c). Similar (but potentially inverted) patterns will emerge if the pattern of gains and losses across the treatment or control values are changed, provided that SOC treat − SOC control is non-zero and constant across sites. While this example is highly simplified in that it assumes a fixed management effect without error, it shows that a F I G U R E 3 Normalization artifacts.
(a) Shows constant values of ∆SOC as a function of SOC initial . When these constant values are normalized by SOC initial (e.g., as a percentage value), the relationship between the normalized value and SOC initial will by definition be negative (b). Similarly, even if ∆SOC is unrelated to time (c), the average rate of change of SOC will decline as a function of time (d). SOC, soil organic carbon. At each site, SOC in treatment plots (T) increases by 2.5 tC ha −1 and SOC in control plots (C) decreases by 2.5 tC ha −1 . (b) Shows that the difference between SOC in treatment plots and SOC in control plots is constant (5 tC ha −1 ) and does not vary as a function of initial SOC. (c) Shows that the ratio SOC treat /SOC control nonetheless declines as a function of initial SOC. Similar (but potentially inverted) patterns will emerge if the pattern of gains and losses across the treatment or control values is changed, provide that (T)-(C) is non-zero and similar in magnitude across sites.
(c)
relationship between SOC treat /SOC control and SOC initial does not necessarily indicate dependence of SOC sequestration or loss on initial SOC levels.
| CORREC TING FOR NORMALIZ ATION ARTIFAC TS
The best way to avoid normalization artifacts is to avoid normalizing response variables in cases when the independent variable of interest is the same as (or strongly correlated with) the normalizing factor. Absolute metrics like SOC treat − SOC control or ∆SOC are thus likely to be more informative than response ratios in specific cases when SOC control or SOC initial is being used as an independent variable. If ∆SOC is used as a response variable in this context, applying a correction for regression to the mean (Equations 1-3) would be appropriate. In cases where the effect of time on SOC accrual is of interest, the best solution is to treat ∆SOC as a response variable and time as a predictor without first dividing ∆SOC by time (Luo et al., 2010;Poeplau & Don, 2015).
If visualizing the instantaneous rate of change in SOC over time is of interest, the modeled relationship between ∆SOC and time can then be differentiated to visualize SOC dynamics without generating normalization artifacts.
| CON CLUS IONS
We used simulated data to illustrate that changes in SOC will tend to be negatively correlated with initial SOC due to random chance alone. Furthermore, simple calculations indicate that normalized metrics of SOC change will tend to show larger responses at low initial SOC levels, even when the soil carbon balance is insensitive to initial SOC. These statistical artifacts exaggerate the appearance that initial SOC levels are a major control on SOC change, regardless of whether there is a strong underlying relationship. This is far from a purely academic issue. Carbon sequestration in agricultural soils is the object of major policy initiatives, and the basis for contentious new carbon crediting schemes (Oldfield et al., 2022).
Cross-site studies are an important source of information informing these policy debates; hence statistical artifacts in these studies might skew regional estimates of SOC sequestration potential, adversely affecting environmental management and actual climate change mitigation. The scientific community can play a constructive role in environmental management debates not only by synthesizing data but also by adopting existing countermeasures against statistical bias like those outlined here, and developing new statistical approaches that can be applied to cross site studies going forward.
ACK N OWLED G M ENTS
This work was supported by Lawrence Livermore National
Laboratory's Laboratory Directed Research and Development
(LLNL-LDRD) program grant #19-ERD-010 to E.E.N. and the LLNL "Carbon Initiative," with additional support provided from a U.S.
This research was conducted under the auspices of DOE Contract
DE-AC52-07NA27344.
CO N FLI C T O F I NTE R E S T
The authors declare no competing interests. | 2022-10-22T06:16:33.823Z | 2022-10-21T00:00:00.000 | {
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244889811 | pes2o/s2orc | v3-fos-license | A PROTECTED LANDMARK MONUMENT: REINFORCEMENT, REHABILITATION, AND RESTORATION OF THE CATHEDRAL BASILICA OF MANIZALES
. The Cathedral Basilica of Manizales is one of the most representative buildings of the so-called "republican architecture", boosted in a remarkable and singular way after the fires of the 1920s in the city of Manizales, Colombia. Its "eclectic neo-gothic" design was made in Paris, after the fire that destroyed the city's previous cathedral in 1926. This masterpiece of Colombian architecture turned ninety years old in 2018 after the first stone was laid in 1928. Its construction was carried out in "reinforced cement"; few decades after the appearance of reinforced concrete. During its ninety years, the cathedral suffered earthquakes of high intensity, in 1938, 1962 and 1979, which have significantly compromised its structure. Earthquake-resistant rehabilitation studies to preserve the temple, declared a National Monument in 1984, were promoted at the end of the 1990s. They were the diagnosis of the structural conditions of seismic vulnerability and how it could be provided a greater capacity of response in terms of stiffness, resistance and dissipation of energy, as well as the diagnosis of the state and pathology of the materials. This included geotechnical studies of seismic amplification, dynamic behavior using environmental vibrations, 3D virtual modeling, and structural analysis, even with finite elements. For the reinforcement, the intervention of the base of the central spire was proposed, the control of the stability of the four corner spires, the construction of new structural walls joined in strategic
INTRODUCTION
The Cathedral Basilica of Manizales has endured earthquakes of significant intensity, in 1938,1961,1962 and 1979 that they have seriously compromised their structure.The earthquake of 1962 produced partial destruction and the collapse of the northwest tower.In 1979, although the cathedral had been repaired, it suffered serious damage to its main structural walls.The temple originally thought as invulnerable was not and demanded to make new structural reinforcements and interventions that were projected twenty years later (1999) and that have been carried out during the two following decades of the new millennium.The departmental government, with the support of the national government, promoted the accomplishing of these earthquake-resistant rehabilitation studies to preserve the monument.Taking into account similar works carried out in other Latin American countries, such as the intervention of the Cathedral of Mexico City or the experience with churches from Antigua, Guatemala, the objective of this work is to illustrate in synthesis the studies and interventions carried out in the Cathedral Basilica of Manizales, Colombia, between 1979 and 2019.
AN EMBLEM OF LOCAL SEISMIC CULTURE
The Cathedral Basilica of Our Lady of the Rosary of Manizales, a temple declared a National Monument in 1984, today of national public interest, is the symbol of a culture: that of the coffee landscape.Its history represents the evolution and innovation of a technique, of science and a culture, immersed in a context characterized by disasters and risk in the face of intense events.Although the roundwood and the bamboo, guadua, had been used by the Quimbaya people and since the Neolithic period in the pre-Columbian period, the possibility of molding earth walls, using tapia and adobe, the result of the colonization process following the European canon from Spain, entered to be part of the constructive options.The flexible gave way to the rigid and fragile in the search for status with the increase in the thickness of the walls.Thus, between 1854 and 1869 a temple of trodden tapia was built that replaced the first straw chapel.After the foundation of Manizales in the middle of the 20th century, this church built with the splendor of this rigid material, with two lateral towers and a central bell tower, meant the advent of a new but fragile constructive paradigm in the face of earthquakes.In 1875 the strong earthquake that destroyed Cucuta, another city in the Northeast, also had effects in the coffee region of Colombia.The church cracked importantly, as did other tapia and adobe houses that had been built with the same technique.Subsequently, these damages were increased because of the earthquakes that occurred in 1878, 1884 and 1885.The temple of tapia and adobe was repaired several times, but these last earthquakes severely affected its façade.As a result of the observation and awareness of the inhabitants of the region of the possible consequences due to the earthquakes on the tapia and adobe buildings, the community not only made the decision to demolish the temple but to explore new combinations and constructive techniques more appropriate for the city which they called the "tremors architectural style".
The demolition of the temple of trodden tapia was not an isolated solution to the problem of seismic vulnerability.Noting the rediscovery of the earthquake-resistant empirical techniques of the "tremors architectural style" of the houses from the coffee farms and from the urban center, Mariano Santamaría, Bogota architect, proposed a new design for the church that would take advantage of the "bahareque" from the region.Wood and guadua would adopt the forms of a temple that would adapt to the natural peculiarities of this new landscape and its construction was carried out between 1888 and 1897.In the 1920s the novel but precarious electrical network was the cause of they will present short circuits and fires.The fire of 1925 meant the destruction of the city center; tens of blocks were destroyed.The flexible bahareque fell into disrepute because of its vulnerability to fire.In 1926, the wooden Cathedral and two more blocks, which had not been previously affected, suffered the action of a new fire, thus completing a disaster scenario no longer caused by the force of nature.This situation made it necessary to rethink the risk and its different causes and the possibility of exploring new alternatives based on the introduction into the world of the recent Portland cement at that time.In the midst of the controversy about the level of protection against earthquakes and fire, finally, the cemented bahareque replaced the old bahareque and definitively banished the tapia and adobe of the city.The cultural landscape of the rural bahareque evolved into a unique and modern urban bahareque, the result of adaptation and a peculiar awareness of risk in Manizales.Under the name of reinforced cement at that time, concrete technology also became an attractive innovation and an apparent and legitimate alternative for the construction of the new Manizales Cathedral, which should be in principle immune to fires and earthquakes.Angelo Papio & Jean Carlo Bonarda, Italian engineers resident of Manizales at that time, who would be the builders of the new temple, described the construction system proposed by the French architect Julien Auguste Polti, who won the international competition in Paris for the new Cathedral of Manizales, as follows: "The type of construction we use is the monolithic of reinforced cement throughout its structure and the one that offers absolute guarantees of resistance and fire.This system is adopted in countries where earthquakes are frequent, not only by the structure but also the walls come into play in the resistance against the stresses and movements due to the earthquake.This type of construction suits Manizales's floor perfectly.Moreover, it is the most suitable."The new Cathedral of Manizales, in "reinforced cement", became the emblem of local seismic culture, which developed with the curious evolution of the "tremors architectural style"; predominant technique with which the city would develop its "Republican" Architecture.The new Cathedral, by itself, meant a peculiar innovation in that unique process, being the first massive building of reinforced concrete that was built in the country and in the region of the coffee cultural landscape; declared as World Heritage by UNESCO in 2011.
DIAGNOSIS AND DESIGN OF INTERVENTIONS
The new Cathedral of Manizales, of "neo-Gothic eclectic style" in reinforced concrete, was completed in 1939.It suffered its first earthquake in 1938 when its central spire had not yet been built.Subsequently, during the following decades until the end of the 20th century, its structure suffered the action of several earthquakes that significantly affected it.Although reinforcements, repairs were made and the tower that had collapsed in 1962 was rebuilt, it was early concluded that its conservation depended on the degree of earthquake-resistant protection that could be provided, as a result of a study and a preventive and careful intervention that preserved the monument to the future.The earthquakes of 1962 and 1979 had caused serious damage, his condition was critical, and his permanence depended on the correct interventions being carried out in the shortest possible time.Even a moderate earthquake, such as those that occur with some frequency in the region, was known to compromise the partial or total stability of the monument.For this reason, at the request of the Government of Caldas in the mid-1990s and with the support of the Subdirectorate of Monuments of the National Road Institute, today the Directorate of Heritage of the Ministry of Culture, between 1997 and 1999 were carried out studies for the earthquake-resistant rehabilitation of the Cathedral, in order to protect this National Monument.These studies were conducted by a large group of experts [1], according to the state of the art, with the aim of make a detailed diagnosis of the conditions of structural seismic vulnerability and the way in which the monument could be given greater capacity in terms of rigidity, resistance and dissipation of energy to face the seismic demand.
For this purpose, it was based on the realization of a detailed survey and virtual construction of the model of the Cathedral, in order to verify the original plans of Polti, several of which had been restored by the Coffee Cultural Fund (see Figure 1).The plans offered invaluable information for the geometric survey stage, although the information included there did not correspond exactly with the real dimensions of the building.The virtual model was the basis for a three-dimensional discretizing of the temple and analyzing its structural behavior using finite element-based computer programs (e.g.ANSYS, SAP, PDCOMP) (see Figure 2).
The ANSYS program was used to define the structure geometry.In the finite element model, two types of elements were used: Type Beam 4-(elastic beam) and Type Shell 63-(elastic shell).For the construction of the model, it was necessary to use 6437 nodes, 8738 shell-type elements, and 108 beam type elements.For the dynamic analysis, an Eigenvectors analysis was used for the first 10 modes of vibration of the structure.The period of vibration for the fundamental mode was 0.90 sec.and for the second and third mode 0.54 sec.and 0.36 sec respectively.Using the SAP2000 NL Push, a pushover analysis was carried out, which allowed verifying the capacity of the structure.After analyzing the stratigraphic profile, it was established that the soil that controls the local seismic response is silt of high plasticity.Therefore, cyclic triaxial tests were performed, one for each of the samples recovered at different depths.The cyclic properties of the other strata were estimated based on correlations taking into account their index properties.The cyclic triaxial tests were performed for confinement pressures of σ3 = 0.5 kg / cm², σ3 = 1.0 kg / cm² and σ3 = 1.5 kg/cm².The observed behavior of the soil corresponds to that of relatively rigid clays that begin to degrade from deformations of the order of 0.1%.For values of the order of 0.5% they have already lost up to 50% of their stiffness.The values of Go varied between 60 and 130 kg/cm² depending on the confinement pressure which, in this case, varied between 0.5 and 1.5 kg/cm².The damping coefficient with respect to the critic remained at small values until deformations of the order of 0.1% and only from this value were considerable values achieved until reaching values between 12% and 23% for deformations close to 1%.
Taking into account the dynamic properties of the soils, feasible accelerograms and the seismic response spectra for different periods of vibration at the site, the dynamic response of the structure was modeled and evaluated, considering the characteristics and condition of the materials in terms of its resistance and pathology, for which it was necessary to take a large number of samples.This allowed evaluating the structural seismic vulnerability and to design the seismic-resistant reinforcement in such a way that the benefit that these reinforcements would have on the structure could be verified and without generating excessive invasive interventions and preserving the authenticity of the temple.
Initially, with the use of a model of elastic behavior and the definition of a pattern of cracking and fissures simulated by previous earthquakes, obtained from the analysis with environmental vibrations, the inelastic behavior of the structure was assessed.This type of study allowed determining the main deficiencies and weaknesses of the temple to face earthquakes.Efforts could be determined throughout the structure and therefore at critical sites or that could be insufficient to address the most severe seismic actions, a soil-structure interaction analysis was carried out to estimate how the period of vibration of the building and how its structural behavior could relax.The results of these studies indicated that the walls of the Cathedral had cracked in previous earthquakes because they acted in practice in an uncoupled manner.The cracks had arisen precisely because there was no capacity in the walls of the structure to move monolithically in a single set.Most of the cracks and fissures had occurred because of their insufficiency to withstand shear force.On the other hand, since the foundation was a system of joists, some of which are not very rigid due to their height reduction, this would allow, in addition to the seismic action, that the walls would rotate due to lack of embedment.This kind of situation, very unfavorable, explained the decoupling of the walls and their cracks and would have to be corrected.For this reason, it was concluded that it was essential to strengthen the capacity of the existing walls with competent structural elements that would improve the strength and the energy dissipation capacity of the structure and that a new foundation would be carried out that would guarantee its proper embedding in the base.
It was also detected that the central spire induced remarkable efforts at its base when it vibrated at the action of an earthquake.Since near its base where the folded plate that forms the tower was supported in a group of pillars whose reinforcement was insufficient to absorb tension stresses, it was considered that this area was critical and would have to be reinforced.Finally, it could be ratified that the towers or corner spires that had been reinforced after the 1962 earthquake with an internal steel structure were not properly anchored, offering a high possibility of instability in case of a severe earthquake.For this reason, it was considered pertinent to carry out an intervention that could guarantee greater stability and foundation anchorage.
EARTHQUAKE RESISTANT STRUCTURAL REINFORCEMENT
In summary, the diagnostic and design studies had proposed the realization of several new and attached structural walls at strategic points to improve the seismic response and the overall behavior of the structure, the intervention of the central spire base and the control of the stability of the four corner spires.At the beginning of the 2000s, the Foundation "Amor por la Catedral Basílica de Manizales" was established, resources were obtained from citizens, the municipality, and the national territorial finance agency, FINDETER.With unique technical coordination, between June 2002 and August 2004, the proposed earthquake-resistant reinforcement works were carried out by direct administration of said Foundation and through several contractors [2].
For a better behavior before the lateral loads it had been established that it was necessary to reinforce the structure by means of eight new orthogonal structural walls located in the periphery; in the places considered of greater efficiency for structural effects and where they will not cause major changes in the original architecture of the building.These walls could replace existing walls, however, due to the difficulties that this meant for the construction, it was established that said walls should be built attached by means of anchors to the existing walls (see Figure 3).This situation, although minorly modifying the façade, was preferable since the vertical loads were still transferred to the foundation by the existing walls.On the other hand, great difficulties and risks were avoided in the construction process, otherwise it would have been necessary to temporarily hold large vertical loads as well as the thrusts of the arches that converge there.These new walls had to have edge elements capable of supporting the moments that would be generated in case of an earthquake and that must reach the foundation based on a new high foundation beam, located below the existing foundation, which in turn should be built supported by caissons that will guarantee the embedding or non-rotation of the new walls; issue that was very difficult to carry out but that was necessary (see Figure 4).These new structural walls had beams at different levels and at the top to ensure that they could be connected by a network of new beams that formed a ring at the level of the diaphragm between levels 24 and 27 meters and with the purpose to provide an ideal system to absorb tension stresses when the structure would be moved by an earthquake.These beams were built using steel plates attached and bolted to the high ribs of the vaults that reach the heads of the walls and that connect to the dome under the central spire, achieving the connection and transmission of forces and the effect of diaphragm with the new structural walls in the form of L and that finally they were built internally and externally attached to the walls of the structure (see Figure 5).
The central spire was intervened at its base by means of six reinforcing walls attached to the folded plate that forms the tower.In this way, the capacity of the structure at the base of the spire to withstand tension stresses when it becomes subject to lateral forces caused by earthquakes was improved.These walls were built internally so that from the outside it is not possible to see them and because of a recent earthquake they have already presented cracks, which illustrates that they have already been working (see Figure 6).In addition, this internal work was used in the central spire to make a new internal metallic helical staircase that allows people to climb up to the tip of the central spire where is placed the so-called "Polish Corridor".
Finally, in order to improve the stability of the corner spires, a connection was made using eight special rods of 7.5 cm in diameter that connect the base of the internal steel reinforcement structure of each tower at the 11.20 meter level with a new beam located in the foundation to which they must be anchored (see Figure 7).In this way these rods act as tensioners that prevent the turn of the towers in case of a strong earthquake.
TREATMENT AND PROTECTION OF CONCRETE
The treatment of the concrete's state of the Cathedral has been, together with other general maintenance activities, the last interventions that have been made to the monument.The treatment and protection of concrete was one of the interventions that, with special emphasis, should be carried out accordingly with the recommendations of the comprehensive diagnostic and structural reinforcement project in 2000 [1].At that time when the evaluation of the state of the materials and the pathology of the concrete was made, it was concluded that there was already a significant advance in the carbonation of the concrete and that it would surely be aggravated if it was allowed to pass a long time without intervening.This was ratified in a more detailed study that was carried out in 2013 [3] and that resulted in the urgent need to intervene "the skin" of the Cathedral and also carry out another series of relevant interventions related to the repair of decorative elements and cornices, the construction of a new and adequate system of downspouts, the reconstruction of the sewer system and the intervention of the sculptural works and images of the facades and towers [4].This maintenance, which must actually be a periodic activity, was carried out in 2018, following the technical and protection recommendations of the Heritage Directorate of the Ministry of Culture.Two phases of this maintenance were planned.In its first phase, the most urgent activities were included.On the one hand, the removal of mortars from terraces, canals, and walkways, which were replaced with sloping cement to improve adhesion, waterproofing and mechanical resistance to traction and bending.This process was complemented by the application of a product that minimized cracking and where necessary additional reinforcements were made with welded mesh.The slopes were oriented towards the grilles that were projected according to the new hydraulic installations that were projected and made.Also, the washing and disinfection of the façade were carried out by applying a corrosion inhibitor and a water repellent was applied.A type of biocide with properly diluted benzalkonium chloride was applied and for the minimum recommended time, which was also facilitated with the use of plastic bristle brushes to improve the action of this product in the different layers of the dirt layers, particles pollutants, and biofilm that had formed over the years.Then the cleaning was carried out with a pressure washer with a pressure less than 750 psi, with a fan jet and more than 30 cm away between the nozzle and the concrete to avoid damage to the concrete.Once the surface dried, for which at least three sunny days were required, the corrosion inhibitor or passivator was applied to the reinforced concrete at the sites of greatest relevance of the bases and towers.This process had setbacks due to changes in color in the concrete that had to be corrected with abundant rinses and due to the need for the surface to dry, which was not facilitated due to persistent rains.Additionally, due to the lack of water pressure in the highest parts of the temple, a system that kept the pressure constant had to be developed.The final stage was the application of the water repellent in order to repel water, prevent corrosion and achieve a coating that prevents the new appearance of moss.To the extent that this product was applied, gaps and fissures were intervened with another product suitable for this type of repairs, on façades and towers from top to bottom to the 11.20 m level, pending completion of this work in a new phase that allows continuing until this treatment to the base of the temple.Figure 8 illustrates the new clean and clear appearance of the concrete surface of the Cathedral.The dark appearance that it had due to the dirt and carbonation that the concrete had suffered for many years was eliminated.
On the other hand, this was also the opportunity to repair the cornices and decorative elements of the façade, as well as the covering of the downspouts using a mixture of mortars or concrete using proportions of dyes.Multiple trial and error tests were performed in order to simulate the original color of the concrete of the Cathedral.Sixty-five samples and cores were taken to verify the carbonation state of the concrete and dust extraction tests to verify from a chemical point of view in the laboratory the condition of the concrete paste.Cleaning the interior vaults of bird droppings, garbage, fragments and even wood that were from the construction was carried out, and synthetic meshes were also placed to prevent birds from entering these sites as they did before.To improve water management that was very precarious, an appropriate system of siphons, grilles, and downspouts was constructed, and the sewer of the surrounding streets was replaced due to its very poor condition when its respective revision was made.Finally, as a result of the strong deterioration, twenty-two sculptures of the five spires of the temple were changed, replacing them with new ones or repairing them, which allowed preserving the original icons of the Cathedral.These images were made using weather-resistant epoxy resin and the existing cross in the central spire was replaced by the one called the Levitating Christ, which required not only careful technical work on materials, construction processes, platforms and scaffolding in height, but the skill and artistic ability of a renowned sculptor of the city.The concrete sculptures that are located on the roofs were also maintained, as well as the sculptures of the guardian angels located at the base of the central spire.The concrete of these images was washed, disinfected and hydrofuge was applied like in the structure.
CONCLUSIONS
A summary description of the interventions of the structure, the conditions of the concrete, the decorative elements and the sculptures, which have been made to the Cathedral of Manizales, have been presented.These interventions were the result of the Integral Project of Diagnosis and Design of the Structural Reinforcement, which was disclosed in 2000 [5].The reports indicated the works of structural reinforcement and protection of the monument that should be carried out to protect this masterpiece of Colombian architecture, after the study that was carried out on the state of the materials, modeling, and evaluation of the response seismic, vulnerability diagnosis and structural reinforcement design.The earthquake-resistant intervention was carried out between 2002 and 2004 and the treatment of concrete and other necessary interventions were carried out between 2016 and 2017 [6].This illustrates the effort that is necessary in these cases to obtain the necessary economic and professional resources to carry out the protection works of a heritage monument.Although the second phase of maintenance is still needed, which must actually be permanent in such a structure, it can be concluded that the decision of a number of people committed to these efforts, with their expertise and management, have been the ingredient fundamental to ensure that this monument has been protected and that it is preserved for posterity, with the aim of continuing to be an emblematic masterpiece of the local seismic culture of the city of Manizales.
Figure 1 :
Figure 1: Example of original plans of the Cathedral restored and used for structural modeling
Figure 2 :
Figure 2: The virtual model was the basis for a three-dimensional fine-element discretizing of the structure
Figure 3 :
Figure 3: Attached walls were built to provide greater stiffness, resistance and energy dissipation
Figure 4 :
Figure 4: Foundation with caissons to prevent rotation at the base of the structural walls
Figure 5 :
Figure 5: Steel beams make up a diaphragm and connect the heads of the structural walls
Figure 6 :
Figure 6: Internally attached reinforcing walls were constructed at the base of the central spire
Figure 7 :
Figure 7: Eight rods or tensioners provide stability to corner spires in case of an earthquake
Figure 8 :
Figure 8: The current appearance of the Cathedral after washing and concrete treatment and protection | 2021-12-05T16:06:30.776Z | 2021-01-01T00:00:00.000 | {
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254532544 | pes2o/s2orc | v3-fos-license | An integrative exploration of loquat leaf total sesquiterpene glycosides in treating insulin-resistant mice by serum and urine untargeted metabolomics analysis
Loquat leaf is approved to be beneficial in the treatment of diabetes. Total sesquiterpene glycosides (TSG), a major chemical component cluster, has potential ability to improve insulin-resistant diabetes syndrome. Its therapeutic mechanism using metabolomics in vivo is worth to be investigated. This study aimed to reveal the underlying therapeutic mechanism of TSG on insulin-resistant mice by untargeted metabolomics, and to explore the lipid metabolism differences in vivo. High-fat diet was used to induce insulin-resistant mice model. Biochemical indicators were applied to evaluate the model validity and related treatment effect. Ultra-performance liquid chromatography quadrupole-time-of-flight mass spectrometry was utilized to accomplish serum and urine untargeted metabolomics. Oral administration of TSG had a therapeutic effect on high-fat diet induced insulin-resistant mice. Four hundred forty-two metabolites in serum and 1732 metabolites in urine were annotated. Principal component analysis screened 324 differential metabolic signatures in serum sample and 1408 in urine sample. The pathway mainly involved purine metabolism and biosynthesis of unsaturated fatty acids. Lipidomic analysis of urine and serum confirmed that most lipid metabolites were fatty acyls, sterol lipids and polyketides.
Introduction
Loquat leaf, the leaves of Eriobotrya japonica (Thunb.) Lindl., has been approved to possess multiple bioactivities such as anti-inflammatory, anti-diabetes, anti-cancer and antioxidant [1]. The observed chemical components of loquat leaf were flavonoids [2,3], organic acids [4], triterpene acids [5], sesquiterpenes and other micromolecule compounds. Sesquiterpene glycosides, an essential class of effective ingredients in loquat leaf, can be mainly divided into chain, single cyclic and ionone sesquiterpene glycosides (Figure 1 presents their chemical frame structures) according to the structure of aglycone [6,7,8,9,10,11]. Some of the chain sesquiterpenoid glycosides were approved to be pharmacological active substances (substituent type of 4 compounds were named in footnote). Compared with compound 1 and 2, compound 3 had a significant hypoglycemic effect in C57BL/KsJ-db/db/Ola genetic diabetes mice model. Chen et al. [12] had verified that compound 2 could reduce the blood glucose level of type 1 diabetic mice induced by alloxan. In addition, compound 4 was approved to be a histothrombin inhibitor, with potential antithrombotic activity. Currently, studies have tested the efficacy of total sesquiterpenoid glycosides (TSG), which includes such active constituents. It is now well established that TSG was effective in improving lipid accumulation against Nonalcoholic fatty liver disease (NAFLD) in vitro and in vivo [13]. In HepG2 cells of NAFLD induced by oleic acid, TSG was found to reduce lipid deposition. It could also decrease total triglyceride (TG), cholesterol (TC) and intracellular free fatty acid (FFA) contents probably by downregulating CYP2E1 expression and JNK/c-Jun phosphorylation. Furthermore, TSG ameliorated high-fat diet induced excessive fat accumulation in the NAFLD mice [14,15]. Generally, TSG is effective and has potential scope in the improvement of insulin-resistant diabetes syndrome. However, the therapeutic mechanism and metabolism situation of TSG therapy remain unrevealed.
Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia induced by genetic and environments factors. As a common metabolic disease, DM is mainly caused by insulin resistance (type 2 diabetes mellitus, T2DM) and insulin secretion dysfunction (type 1 diabetes mellitus, T1DM). Except for gestational and special types of diabetes, most DM patients are affected by T2DM, mainly caused by defects with relatively insufficient insulin production or blocked receptors [16]. DM is also usually associated with different degrees of sugar, protein and lipid metabolic disorders [17,18].
Lipometabolic disturbance (LD), one of the most common pathophysiological changes in the development of T2DM, is an important risk factor leading to complications such as atherosclerosis, cerebrovascular and coronary heart disease. This kind of lipid abnormality in blood, tissues and organs aggravates insulin resistance and directly acts on islets to cause apoptosis of β cells, resulting in dysfunction and dysregulation [19]. Existing literature has explored a higher fat accumulation and decomposition rate of visceral adipose tissue than subcutaneous adipose tissue [19]. It may cause insufficient oxygen supply in the tissue and increase secretion of adipocytokines and macrophage infiltration [20,21]. Clinical studies have shown that fat accumulation in the visceral is an important cause of metabolic diseases such as insulin resistance and T2DM [22]. Therefore, metabolism including lipid metabolism of T2DM and its therapy need to be evaluated urgently.
Metabolomics is an integral and dynamic technological method to study typical chronic metabolic diseases by discovering abnormal metabolic changes caused by diseases at the molecular level. Therefore, it is often applied in studying diabetes. Typically, the complex metabolism involved in diabetes leads to difficulties in the revelation of pathophysiological process and mechanism. Unignorably, the interaction between traditional Chinese medicine (TCM), essentially their chemical components, and human bodies often benefits from its multi-effect and multitarget mode of mechanism. Therefore, metabolomics presents an obvious advantage in studying diabetes treated with complex TCM components. Analyses on the composition and variation of endogenous metabolites in biological samples can explain the physiological and pathological conditions of subjects and reflect the metabolic rules in the overall metabolism affected by TCM [23,24]. Many metabolites and differential biomarkers related to the pathogenesis of metabolic diseases and TCM treatment have been found through metabolomic studies. TCM therapy may alleviate metabolic disorders by regulating the metabolism of glucose [25], lipid and amino acid [26], reducing lipid peroxidation products [27], improving the body insulin sensitivity [28], and relieving inflammation [29], eventually to be able to ameliorate diabetes [30,31].
The study aimed to reveal the therapeutic mechanism of TSG on insulin-resistant mice by untargeted metabolomics, sketching the changes of metabolites in blood and urine after TSG therapy, digging in the metabolic perturbation pathways, and identifying key compounds in regulation.
Materials and chemicals
Loquat leaves were collected from Xishan Island (Suzhou, China) and authenticated as the leaf of Eriobotrya japonica (Thunb.) Lindl. by Professor Bing-ru Ren (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China). The voucher specimens (No.328636) were deposited in the Herbarium of Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences.
HPLC-grade methanol, formic acid and ammonium acetate were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA).
TC, TG, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), activity assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ELISA Kit for Insulin were obtained from Multisciences Biotech (Hangzhou, China). All the other chemicals and reagents were of analytical grade.
Animals and insulin resistance (IR) model
Male SPF C57BL/6J mice (certificate No. SCXK2013-0016), weighing 18-22 g, were bought from Sino-British SIPPR/BK Lab Animal Co., Ltd (Shanghai, China). The mice were raised in a laboratory environment room under light-dark cycle (12:12 h) with free access to food and water for acclimatization. At the beginning, the mice got a nine-week treatment as follows [32]: regular diet (certificate No. 201401008, Xietong Organism Inc., Nanjing, China) for the control group, high-fat diet (containing 74% feedstuff as control group, 10% sucrose, 10% lard, 5% egg yolk powder, 1% cholesterol, 0.2% pig bile salt) for the others. Then, high-fat diet fed the animals were randomly divided into three groups, each group contained eight mice: IR model group, high dose therapy group (TSH, 200 mg/kg of TSG) and low dose therapy group (TSL, 100 mg/kg of TSG). All the mice were gavaged once daily with equal volume saline or TSG for another 9 weeks. All the animal experiments in this study obeyed the Guide for the Care and Use of Laboratory Animals approved by the Animal Ethics Committee of China Pharmaceutical University (SYXK2016-0011, Jan 27, 2016-Jan 26, 2021).
After treatment, as mentioned in Section 2.4, the mice experienced the oral glucose tolerance test (OGTT) and the insulin tolerance test (ITT) successively during the following week. Then the mice were placed in a metabolic cage. Urine samples were collected during 1 h in the morning.
The mice blood was collected from eyes with sodium pentobarbital anesthesia (50 mg/kg I.P.). After centrifuged, the serum and the urine samples were gently drained and stored at -80 C.
OGTT and ITT
After an overnight fasting, mice were given intragastric administration of 2 g/kg glucose solution (OGTT) or intraperitoneal injection of 0.8 U/kg insulin. Blood sampling was held at 0, 30, 60, 120, 180 min from the vein of the inner canthus, fasting but free to drink in this period The blood glucose was measured by the glucose dehydrogenase method. OGTT and ITT curves were drawn, and areas under the curve (AUC) were calculated, respectively.
Biochemical parameters analysis
Serum glucose, TC, TG, HDL-C and LDL-C levels were quantified by microplate reader (Molecular Device, CA, USA). Serum insulin was determined by ELISA commercial kit. All of the operations were carried out following commercial kits instructions.
Serum and urine sample collection
The whole blood was drawn from the orbit and stored at room temperature for 1 h to solidify and layer. After centrifugation at 3000 rpm for 5 min, the supernatant was transferred and centrifuged again at 12,000 rpm at 4 C for 10 min. Finally, the supernatant serum was removed to store at -80 C until use. The urine was collected in the morning during 1 h and quench immediately with liquid nitrogen for 15 min. After centrifuged at 12,000 rpm, the supernatant was removed to Eppendorf tube and filtrated by 0.22 μm filter. The filtrate was stored in a refrigerator at À80 C for future use. The serum and urine of control group, model group and TSH group were used furtherly in metabolism analysis.
Sample preparation
Prechilled methanol (400 μL) was added to every 100 μL liquid serum or urine sample to precipitate proteins. Then the liquid samples were dried and resuspended with 400 μL prechilled methanol -water (V:V ¼ 4:1, with 0.1% formic acid in water) by well vertexing. The samples were incubated on ice for 5 min and then centrifuged at 15000 rpm, 4 C for 10 min. 300 μL of the supernatant was diluted by 100 μL LC-MS grade water to make the sample containing 60% methanol. The samples were subsequently filtrated by an Eppendorf tube with a 0.22 μm filter. Finally, the filtrate was injected into the LC-MS/MS system analysis.
Blank sample was 60% methanol aqueous solution containing 0.1% formic acid instead of experimental sample, and the pretreatment process was the same as experimental sample. Quality control (QC) sample was obtained by mixing equal aliquots (30 μL) from each experimental sample. Before analysis, 6 QC samples were run to balance the system. QC samples were injected at fixed intervals (every 10 samples) during the analysis process.
UPLC-Q-TOF-MS analysis
Vanquish Horizon UPLC system were used for LC-MS/MS analyses coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Samples were injected and separated by Hyperil Gold column (C18, 100 Â 2.1 mm, 1.9μm). Mobile phase for positive ion monitoring mode were eluent A (0.1% FA in Water) and eluent B (Methanol), for negative mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (Methanol). A 16-min linear gradient was used: 0-1.5 min, 2% B; 12.0 min, 100% B; 14.0 min, 100% B. The flow rate was 0.2 mL/min . Mass spectrometric detection was operated in positive and negative polarity mode separately. The spray voltage was set as 3.2 kV, the capillary temperature was set as 320 C, the sheath gas flow rate and aux gas flow rate were set as 35 arb and 10 arb. Data of mass spectra were obtained within the mass range of 200-1500 m/z.
Data analysis
All the results in animal experiments were presented as means AE SD (standard deviation). Statistical significance was determined using oneway ANOVA followed by Student's t-tests between groups. P value 0.05 was considered statistically significant. For metabolomics analyses, raw data files were generated by UPLC-MS/MS. Then Compound Discoverer 3.0 (CD 3.0, Thermo Fisher) were used to process peak alignment, peak picking, and quantitation Retention time tolerance was set as 0.2 min; signal intensity tolerance was set as 30%; actual mass tolerance was set as 5ppm; minimum intensity, 100000 and SNR (signal to noise ratio), 3. Subsequently, peak intensities were normalized by relative peak abundance towards QC samples. PCA (Principal component analysis) and PLS-DA (partial least squares discriminant analysis) were performed with MetaboAnalyst. The normalized data were used to predict the Molecular formula were predicted according to fragment ions, additive ions and molecular ion peaks. Then peaks were matched with mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.ch emspider.com/) database. Accurate qualitative and relative quantitative results were obtained.
Effects of TSG on high-fat diet induced IR mice model
As shown in Figure 2A after 9 weeks of high-fat diet, compared with the control group, mice in the model group represented significantly increased body weight ( ## P < 0.01). After a 9-week administration with TSG, at the end of the 18th week, the body weight of both TSL and TSH groups persuaded a deep fall compared with the model group (***P < 0.001). In addition, TC, TG and LDL-C levels ( Figure 2B-D) were found decreased (*P < 0.05, ***P < 0.001), while HDL-C levels ( Figure 2E) increased (**P < 0.01, ***P < 0.001) in serum compared with the model group.
As shown in Figure 2F and G, compared with control group, the blood glucose level and the area under the curve (AUC) of model group were significantly increased ( ### P < 0.001). On the contrary, compared with model group, the levels of blood glucose and AUC of TSL and TSH treatment groups came down apparently (***P < 0.001). It indicated an improved glucose tolerance in TSL and TSH treatment groups. Similarly, as shown in Figure 2H and I, compared with control group, the blood insulin level and the AUC of model group were significantly increased ( ### P < 0.001). Compared with model group, the levels of blood insulin and AUC of TSL and TSH treatment groups came down correspondingly (***P < 0.001), indicating a recovery of insulin tolerance in TSL and TSH treatment groups. The findings supported that insulin-resistant mice model was established successfully by nine-week treatment of high-fat diet. It also implied that TSG had a therapeutic effect on high-fat diet induced insulin-resistant mice.
Data quality control
The system stability was validated by QC sample injection occasionally during the whole sample analysis. The Pearson correlation coefficient between QC samples was calculated based on the peak area value.
Metabolite function and annotation
According to the relative standard deviation (RSD) of QC samples (RSD < 30%), in the serum, 793 metabolites were identified under the positive ion mode, 872 under the negative ion mode. These numbers were 3241 and 2616 correspondingly in the urine.
Based on KEGG database, by comparing secondary mass spectrometry information, totally 442 metabolites (206 under positive mode, 236 under negative mode) in serum and 1732 metabolites (952 under positive mode, 780 under negative mode) in urine were annotated. The number of metabolites in different function were figured out and shown in Supplementary Material 2 as reference material. It was found that metabolites found in serum and urine were mostly relevant to the function of amino acid metabolism, lipid metabolism, and digestive system (with annotated metabolites >40).
Differential metabolites
In order to reveal the underlying metabolic perturbation, multivariate statistical analysis was performed. PCA showed an obvious separation between the TSG groups and control group, with PC1 at 11.6%, PC2 at 38. Figure 3A and B). The abscissa represented the expression changes of metabolites in different groups (log2FC, log base two of Fold Change). The ordinate represented the level of significance difference (Àlog10 P-value). As shown in Venn diagram, there were 153 urine metabolites and 64 serum metabolites that had been simultaneously regulated in all the three groups ( Figure 3C and D).
Metabolic pathway annotation of differential metabolites
Hypergeometric test was applied to KEGG (https://www.genome.jp/ kegg) pathway enrichment analysis. Compared with all the identified metabolite backgrounds, pathways of biochemical metabolism and signal transduction which differential metabolites took part in was established. The enrichment KEGG bubble diagram presented the number of differential metabolites in the top 20 pathways and p-value compared TS with Model group in serum (Supplementary Material 4A) and urine (Supplementary Material 4B).
p value under 0.05, in serum. Biosynthesis of unsaturated fatty acids, Metabolism of xenobiotics by cytochrome P450, Drug metabolismcytochrome P450, Ferroptosis were significant interfered in urine (p < 0.05). While Purine metabolism (in serum) and Biosynthesis of unsaturated fatty acids (in urine) were the pathways with the highest concentration of metabolites ( Figure 4A and B). Similarly, both of the pathways were also found interfered in Control group compared with Model group (with p ¼ 0.02, p ¼ 0.4 correspondingly).
Compared Control with Model group, Steroid hormone biosynthesis (in serum) and Ferroptosis (in urine) were the pathways with the highest concentration of metabolites and the most significant p value (with p ¼ 0.0002, p ¼ 0.001 correspondingly). Both of the pathways were also found interfered in TS group compared with Model group (with p ¼ 0.1, p ¼ 0.2 correspondingly). After pathway enrichment analysis of the altered metabolites, the overlap differential metabolisms of TSG group and control group were selected into Tables 1 and 2.
Discussion
The bioactivities of loquat leaf extract TSG were approved, including weight control, reducing TC, TG and LDL-C levels, raising HDL-C level, as well as improving glucose and insulin tolerance. According to the subsequent metabolomics studies of mice serum and urine, most of metabolites were found relevant to the function of amino acid metabolism, posteriorly lipid metabolism. Many studies showed that amino acids mediated the risk of future diabetes and were biomarkers for developing insulin resistance [20,33]. An increase in alanine levels reflects the occurrence of insulin resistance. Some approaches can impact the synthesis of β-alanine in its metabolic pathway. This work found that β-alanine metabolism pathway was affected by up-regulation of spermidine, spermine and 4-amino-butanoate. Furthermore, previous research had investigated the role of polyamines in energy balance and glucose metabolism, indicating that spermidine supplementation improved glucose utilization in obese mice and resulted in significant weight loss [34]. The 4-amino-butanoate prevented and reversed the development of type 1 diabetes by promoting the growth and survival of β cells in mouse models, and exhibited an immunosuppressive effect, thereby protecting β cells from autoimmune damage [35]. Hence the effect of TSG on obesity and type 2 diabetes through the spermine, spermidine and 4-amino-butanoate regulation is worth to be explored in the future study.
As the second most abundant metabolites detected in serum and urine, lipidomic metabolites were annotated according to LIPIDMAPS. Most of them were fatty acyls, 58 of which were found both in serum and urine samples. Among those fatty acyl lipids, fatty acids and conjugates accounted for the majority. Fatty acids may exist as free fatty acids in mammal's body or combine with other molecules to form lipids, such as cholesterol esters, phospholipids and triglycerides [36,37,38,39]. Recent research suggested that fatty acids might have an essential role in insulin resistance and T2DM. In an insulin resistant state, there was an increase in the activity of hormone sensitive lipase releasing free fatty acids into circulation [40]. On the basis, Yang et al. identified 35 potential lipid biomarkers, including 2 fatty acids, 6 triglycerides, 3 cholesterol esters, 1 sphingomyelin, and 23 glycerophospholipids, closely related to the diagnosis of type 2 diabetes [41,42].
In the subsequent pathway analysis, purine metabolism was found to be significantly interfered according to annotating differential metabolites in serum. As demonstrated in Figure 4A, xanthine, hypoxanthine, and adenine were upregulated after dosing TSG. It's worth mentioning that a significantly higher expression of adenine also existed in control groups comparing with model groups. Adenine is an activator of AMPK (AMP-activated protein kinase) [43], and activation of AMPK has been widely used in the treatment of type 2 diabetes. There is substantial evidence suggesting that AMPK is dysregulated in animals and humans with T2D, and AMPK activation (physiological or pharmacological) can improve insulin sensitivity and metabolic health [44]. In purine metabolism pathway, adenine can be synthesized by adenosine or deoxyadenosine under the action of purine-nucleoside phosphorylase, however, differential expression of the two upstream compounds was not observed in our study. We assume that it is more likely the enzyme catalyzed this reaction contributes more. Thus, it is suggested adenine plays an important role in ameliorating insulin resistance by TSG, and its synthetase purine-nucleoside phosphorylase worth to be evaluated in the future research.
Annotation of the metabolites in urine showed that, biosynthesis of unsaturated fatty acids was another pathway mainly inferenced after TSG treatment ( Figure 4B). Ten compounds were assigned in the pathway with 5 of which significantly downregulated, namely eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), arachidonic acid (AA), adrenic acid (AdA), and erucic acid. EPA and DPA were approved to improve diabetes or complications, attenuating hyperglycemia and insulin resistance dramatically in db/db mice [45], attenuating the progression of albuminuria on subjects with type 2 diabetes and coronary artery disease [46]. AA and its extension product AdA are the abundant polyunsaturated fatty acids in mammalian tissues, which are usually esterified into membrane phospholipids. It was well-established that insulin secretion from pancreatic β-cells was stimulated by AA [47,48,49]. Further, the abnormal metabolism of AA may occur in diabetes mellitus patients with vascular disease. To the results of our research, excretion of the 5 unsaturated fatty acids were apparently reduced after dosing TSG, while the situation, 48, in serum stayed unchanged. It was inferred that TSG could improve the utilization of the 5 important unsaturated fatty acids in the process of its pharmacodynamics.
In conclusion, the diagnosis and treatment of type 2 diabetes may be closely related to the metabolic disorders of glycerophospholipids, triglycerides, cholesterol esters and sphingomyelin. Our research similarly showed that, among the lipid metabolite, fatty acyls, sterol lipids, phenol lipids, glycerophospholipids, and polyketides were impacted to varying degrees after TSG treatment. Furthermore, out of 78 annotated fatty acyls, 4 fatty acids and conjugates, 3 fatty esters, 3 octadecanoid, 1 hydrocarbon, 1 fatty amide, and 1 eicosanoid were significantly regulated. Meanwhile, 3 steroids, 2 sterols, 2 steroid conjugates, 1 of bile acids and derivatives were significantly regulated in 42 annotated sterol lipids. Therefore, fatty acyls and sterol lipids metabolism and their significant differential metabolites deserve further investigation to reveal the mechanism of TSG treatment.
Conclusion
This study established that TSG from loquat leaf alleviated insulinresistant diabetes syndrome. Untargeted metabolomics analysis of mice serum and urine found that the metabolites were apparently regulated in insulin-resistant mice, and the alteration was reversed after oral administration of TSG. The therapeutic mechanism mostly involved the regulation of purine metabolism and biosynthesis of unsaturated fatty acids. Furthermore, lipid metabolites in serum and urine were screened and analyzed, these metabolites mainly belonged to the classes of fatty acyl, sterol lipid and polyketide. The present study also provided a utilization of untargeted metabolomics followed by statistical analysis in the research of multicomponent and complex mechanism of traditional medicine.
Author contribution statement
Ya-nan Gai: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper. Funding statement | 2022-12-11T16:05:41.432Z | 2022-12-01T00:00:00.000 | {
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213024453 | pes2o/s2orc | v3-fos-license | Paint Removal with Pulsed Laser: Theory Simulation and Mechanism Analysis
Featured Application: Authors are encouraged to provide a concise description of the specific application or a potential application of the work. This section is not mandatory. Abstract: This paper studies paint removal using laser technology. A finite element model was created using COMSOL Multiphysics software, and the temperature field generated during the cleaning process was analyzed and verified. Laser paint removal behavior was investigated using a fiber laser, and its mechanism studied by combining Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy. In-depth analysis of this relatively new technology could provide the theoretical basis for industrial application. The results of this study show that, when compared to the original paint layer, the infrared absorption spectrum of the cleaned surface had two additional two peaks—1333.36 cm − 1 and 678.82 cm − 1 . In addition, there was a decrease in C element content on the treated surface and an increase in O content. In addition, new organic and complex compounds were formed on the cleaned surface as a result of bond cleavage and rearrangement. Furthermore, paint particles of varying sizes and shapes were produced by the impact of plasma shock. Under high-energy laser irradiation, the paint layer underwent combustion, resulting in spherical nanoparticles of uniform shape. electron microscopy. Compared with the untreated surface, the infrared spectrum had two additional absorption peaks of 1383.36 cm − 1 and 678.82 cm − 1 on the surface of the paint layer cleaned with the pulsed laser. The profile of the C 1s, O1s, and N1s peaks in the cleaned surface indicate that chemical bonds such as C-C, C-H, C-O, C-N are broken and rearranged, resulting in the emergence of new organics and complex compounds. Traces of the impact of plasma was found on the cleaned surface. The removal process produced paint particles of varying sizes and shapes, caused primarily by plasma impact damage. The X-ray photoelectron spectroscopy conducted showed that C element content decreased and that of the O element increased after the cleaning. Smaller amounts of Al and Ti were detected on the cleaning surface, as compared to the untreated area. This means that the paint released nano additives containing metal Al and Ti during the cleaning process. Deconvolution of the Al2p, Ti2p spectra resulted in the formation of new components, such as SiO 2 (Al 2 O 3 ) 0.22 , MgAl 2.7 O / SiO 2 , Al 6 Si 2 O 13 , TiO 0.3 S 1.5 , and Ti 2 S 3 . These results suggest the oxidation of paint and nano additives in air during the laser cleaning process. The formation of spherical nanoparticles is a result of photothermal ablation of the paint, caused by combustion of chemical bonds.
Introduction
The continuous development of laser technology has enabled its widespread application in the communication, medical, and manufacturing fields. Laser paint removal, which is an efficient paint layer treatment method, is a new application. Compared to the traditional physical and chemical paint removal methods, laser paint removal has several advantages: (1) non-contact cleaning with small impact on the substrate; (2) less environmental pollution, and easy disposal of the resultant waste; (3) high cleaning efficiency with low costs; and (4) good controllability. This technology can be used to remove paint off the surface of buildings, bridges, aircraft, vehicles, and various mechanical equipment. Although it has been widely studied, its physical mechanism is complicated, proving to be a challenge for researchers.
Laser paint removal involves a series of complex processes, such as melting, evaporation, and combustion. Watkins [1] summarized and classified the laser cleaning mechanism into six kinds of action mechanisms: ablation, rapid heating and cooling caused by vibration waves, selective vaporization, light pressure, gasification pressure, and plasma burst. Research by Tsunemi [2] and Zhou [3] portrayed the laser process as an ablation effect. Bloisi [4] and Kim [5] highlighted the application of a thermoelastic vibration model in laser cleaning. Yang [6] investigated the influence of thermal stress during the laser paint removal process and found that paint absorbed laser energy, formed a large temperature gradient, and was finally removed by thermal stress. A study by Autric et al. [7] showed that ablation and thermo-mechanical actions were the main mechanisms used for the absorption medium and transparent medium, respectively. Luo et al. [8] studied laser paint removal behavior using a high-power continuous CO 2 laser. They found that the paint removal mechanism was not an independent action, but one that included thermal stress, laser soft ablation, and plasma effects. Several factors-such as the physical and chemical properties of the attachment, physical parameters of the substrate materials and the laser beam, and the cleaning environment-determine which mechanism is dominant.
All of these findings provide important references to study laser paint removal. We found that the use of functional groups and elemental valence changes to study this technology was relatively low.
The experiment performed in this paper aimed at investigating the laser mechanism of paint removal. A finite element model of mobile pulse laser cleaning was established by using COMSOL Multiphysics software. The calculation of temperature fields during laser cleaning was used to aid the analysis. A cleaning experiment was also carried out. Furthermore, to determine the decomposition process, the chemical and physical characteristics of the surface cleaned by the laser were analyzed by XPS. By analyzing changes in the functional groups and chemical valence of C, O, N, Al, and Ti on the surface of the sample before and after cleaning, as well as SEM images of the cleaning surface and the collected particles, the laser paint removal mechanism was comprehensively studied; a high repetition frequency fiber laser was used for the study. Figure 1 shows a finite element model for laser cleaning. To replicate the actual materials, the model was made of an LY12 aluminum alloy substrate measuring 1 mm × 1 mm × 2 mm, an oxide layer with thickness of 7 µm, and a 50 µm polyacrylate resin base paint layer. The moving pulse laser scans the sample in a single pass in the positive direction along the X-axis. The paint layer directly absorbs the laser energy during the cleaning process, and heat is thermally transferred to the oxide layer and the surface of the substrate. By referring to the pre-experiment and experimental equipment conditions, a simulation calculation was carried out using a set of parameters with a fixed wavelength of 1064 nm, pulse width 1 µs, energy density 17.26 J/cm 2 , spot diameter 78 µm, laser repetition frequency 20 kHz, and scanning speed 600 mm/s. Laser cleaning experiments were carried out using a fiber laser with the same parameters. The beam was in Gaussian mode, and spot diameter after pulse laser focusing was about 78 µm.
Materials and Methods
The base material used was LY12 aluminum alloy with size of 15 mm × 15 mm × 2 mm. A yellow polyacrylate resin base (polyisocyanate resin curing agent with a nanoparticle additive such as TiO 2 , Al 2 O 3 ) paint layer on an aluminum alloy surface with thickness of 50 µm was used for the experiment. The surface of the aluminum alloy was anodized to form an oxide layer with thickness of about 7 µm. Table 1 shows the composition of the paint layer, the oxide layer, and the aluminum alloy. The molecular structure of polyacrylate resin and polyisocyanate resin is shown in Figure 2. A silicon wafer was used as the substrate to collect particles, measuring 2 mm × 2 mm, approximately 17 mm from the sample, and parallel to the surface of the sample, facing the ablation port. The silicon wafer was ultrasonically cleaned in an ethanol solution for 5 min to remove surface contaminants. The surface of the paint layer on the 2 mm thick aluminum alloy plate was placed on the laser focal plane. With oscillation of the X and Y axis galvanometers in the scanning galvanometer system, an orderly laser pattern was applied to the paint layer. The surface of the paint layer was scanned once to remove the paint layer. The laser beam emitted by the fiber laser was in the form of a series of discrete circular spots, distributed at a given frequency.
Fourier transform infrared spectroscopy (FT-IR, Nicolet 5700) and X-ray photoelectron spectroscopy (XPS, ESCALAB 250xi) were used to evaluate the functional groups and the chemical valence of C, O, N, Al, and Ti. Scanning electron microscopy (SEM) was used to observe and analyze particle morphology and the morphology of the cleaning surface and the original surface. Figure 3 shows the dynamic change in temperature field distribution of the model during the laser cleaning process. The pulsed laser heat source moved at a speed of 600 mm/s in the X direction, and temperature field distribution appeared as a comet with a tail. The temperature in the center of the pulsed spot was the highest, the edge gradually decreased, and the temperature field conformed to a Gaussian distribution. The uncooled temperature field that was formed by the previous pulse remained on the scanning path. The reason for this is that the temperature field formed by each pulse was connected at a certain overlap rate. Figure 4 shows the temperature change curve at different positions in the x-axis direction at t = 0.0016 s. It can be seen that temperature change of the material is mainly concentrated near the pulse spot irradiation area during the laser cleaning process. The temperature of the paint below surface 15 µm did not change, which indicated that the depth affected by the laser was about 15 µm. When x < 0.0035, the temperature of the paint at different depths increased, which also indicated that there was overlap in the temperature field formed by each pulse, causing an energy accumulation effect. With irradiation of the pulse laser (with high repetition frequency), the paint layer absorbed a large amount of laser energy in a short time, and the surface temperature of the paint layer rose rapidly. Figure 5 shows the temperature change in the direction of depth. At z = 0 µm, the range of temperature change was the largest, which reached maximum value at t = 0.000851 s. The temperature of some parts of the paint layer exceeded the gasification threshold and were eliminated by vaporization and combustion. The pulse laser, however, was t = 0.00083 s. This difference shows that there was an energy accumulation effect on the surface of the paint layer during the cleaning process. Temperature changes at z = 0 µm were found to be zigzag because of the pulsed laser. In addition, an intermittent time pattern was seen during the laser processing. Compared to the z = 0 µm position, it is evident that it took time for the temperature to reach maximum value. This is due to the hysteresis of the maximum temperature caused by thermal conduction in the material. Figure 6 shows the FT-IR spectra of the sample, before and after laser cleaning. The main peaks in the infrared spectrum of the original paint sample are listed in Table 2. After cleaning with the pulsed laser, the FT-IR spectra of the cleaning surface was found to have two additional absorption peaks at 1383.36 cm −1 and 678.82 cm −1 , which are the absorption peaks of C-H single bond vibration in -C(-)-and N-H single bond vibration, respectively. The cleavage of chemical bonds such as C-C and C-H was present during the laser cleaning process. The sharp absorption band at 3677.59 cm −1 was the stretching vibration absorption peak of the free O-H bond. This peak shows that as the surface roughness of the laser cleaning and the surface area increases, the adsorption amount and strength of the absorption peak of the -OH rises. The peak at 1018.23 cm −1 is the absorption peak of the vibration of the C-C single bond skeleton, which increased in strength and had a blue shift. As a result, the C-C single bond skeleton on the cleaning surface increased, and the structure of the polymer was found to be stable. The absorption peak of C-C at 700.03 cm −1 in the long carbon chain saturated hydrocarbon was found to have a blue shift and a decrease in strength, indicating that the content of C-C in carbon chain saturated hydrocarbons was reduced. Intensity of the other absorption peaks on the cleaning surface also decreased, which show that the content of -OH, -C=O, -CH 3 , -CH 2 -, and C-H in the polymer decreased separately. The absorption peak of C-H at 2856.06 cm -1 in -CH 2 -had a red shift, indicating a reduction in the number of electron-withdrawing matrices, such as -C=O, -C-O, -COOH, in the molecular chain. Thus, the electron cloud in the molecular chain shifted and enabled a red shift in the characteristic absorption peak. The above analysis shows that breakage and rearrangement of chemical bonds occur in laser cleaning processes, resulting in new polymers or monomers.
XPS Analysis
The changes in the surface composition of the paint after laser cleaning were determined by XPS. Figure 7 presents the spectra of (a) the cleaned surface and (b) the untreated surface. Peaks of 1s core level of C, O, N and 2p core level of Ti, Al are clearly visible, and the peaks of C, O, and N were found to have changed significantly before and after the cleaning. The results show that the peaks of C and N in the cleaned surface had decreased, while the peak of O had increased. The reason for this difference is most likely due to the following analysis: The surface of the residual paint layer was no longer smooth, and the surface area had obviously increased. In addition, nanoparticle additives such as TiO 2 and Al 2 O 3 were found exposed on the surface. The polymer molecular chain was thermally decomposed and burned by absorbing laser energy, illustrating that the paint had released C and N atoms as simple gas or volatile species of CO and NO 2 during the cleaning process. The peaks of C, N were found to be lower on the cleaning surface and the intensity of the peak of O was enhanced. This is probably due to chemical reactions taking place while the pulsed laser was removing the paint.
The electronic state and existing forms of C, O, N, Ti, and Al can be further studied by examining the fine spectra of each element on the surface of the paint layer before and after laser cleaning. The profiles of the C1s, O1s, N1s, Ti2p and Al2p spectra on the surface of the paint layer before and after laser cleaning are shown in Figures 8-11, respectively.
C1s XPS Analysis
The curv-fitted XPS spectra of C1s obtained from the cleaned and untreated surfaces reveals four peaks (Figure 8). The components of C1s spectra for various binding energy values are listed in Table 3. [9].
Compared with the spectra for the untreated surface, the C1s binding energy of the cleaned surface was found to have a shift, and an increase in binding energy. This was due to breakage of C-H chemical bonds in the polymer molecular chain during the laser cleaning process. The density of the electron clouds around the C atom also reduced, increasing the binding ability of the nucleus to the extranuclear electrons, subsequently resulting in an increase in binding energy. Table 3. Results of the peak separation of XPS C1s spectra, together with the binding energy and relative content of the C1s spectra.
Sample
Binding Energy/eV Components Area%
O1s XPS Analysis
The curve-fitted O1s XPS spectra, obtained from the untreated and cleaned samples, are shown in Figure 9. The components of the O1s spectra for various binding energy values are listed in Table 4. It can be seen from Figure 9b and Table 4 that the O element on the surface of the untreated sample is mainly in the form of metal oxide and oxygen-containing functional group such as O = C, -OH, -COOC-. The relative content of the oxygen-containing functional group is only 17.83%. Compared with the spectra for the untreated surface, the peak of the C1s spectrum obtained from the cleaned sample shows four peaks at 531.05 eV, 531.49 eV, 531.94 eV, and 532.74 eV, respectively. The presence of O element on the surface of the cleaned sample was more complicated. The new metal oxide was found to have complex forms such as TiO 1.65 , Al 2 Si 2 O 7 , SiO 2 (Al 2 O 3 ) 0.22 , indicating that the nano-added particles in the polymer had complicated physicochemical reactions during the cleaning process. At the same time, some nanoparticles were lost. Two new oxygen-containing functional groups appeared at 531.94 eV, 532.74 eV for the O1s spectra, which were related to the (-CH 2 C(CH 3 )(C(O)O(CH 2 ) 3 CH 3 )-) n , (-CH 2 CH(C(O)OH)-) n groups, respectively. The emergence of oxygen-containing functional groups can be because free radicals were generated from the chemical bond and stabilized by oxidation. This demonstrates that after the pulsed laser cleaning, the chemical bonds of C-C, C-O, and C-H in the chemical molecular chains broke and were rearranged. The number of organic molecular chains increased, and the relative content was found to be up to 50%. Figure 10 shows the curve-fitted XPS spectra of N1s obtained from the untreated surface and the cleaned surface. The corresponding binding energy and relative content are shown in Table 5. Four peaks were observed for the N1s spectrum from the cleaned sample. However, the components on the two surfaces were found to be different. The N element in the untreated sample existed in the chemical bonds of C-N and C=N, and the ratio of the peak areas of the two bonds was 4:1.
Al2p, Ti2p XPS Analysis
The curve-fitted XPS spectra in the Al2p, Ti2p regions of the cleaned surface are shown in Figure 11; four peaks are visible. The corresponding binding energy and relative content are shown in Table 6, which also show Al, Ti on the surface of the cleaned layer. Al, Ti were found to have complicated forms-oxides, mixtures with other compounds, and complex compounds with other elements. Al 2 O 3 , SiO 2 (Al 2 O 3 ) 0.22 , and TiO 2 are consistent with the result of the O1s analysis, indicating that the high energy of the laser causes complex physicochemical changes to Al, Ti during the laser cleaning process.
The Surface Morphology Analysis
Morphology of the surface and fracture cross-section produced by the pulsed laser are shown in Figure 12a-d, respectively. It can be seen from Figure 12b,c that there were a large number of cracks and sheets on the cleaning surface, and the fracture cross-section of the residual paint layer had a "cliff-like" shape. There were some regular craters in the bottom of the cleaned areas of the fracture section, as shown in Figure 12c, which are usually observed in laser ablation of metals and organic materials [10]. The craters were a result of mechanical shock. A distinct layered structure was formed inside the paint layer, as shown in Figure 12d. The sheets were folded in the opposite direction of the pulsed laser action, which was a typical plane showing dynamic tensile damage trace of the material. Mechanical action along the direction of the folded sheets was also seen. Stress concentration and whitening of the fracture edge of the sheet layer was also visible.
Morphology Analysis of Collected Particles during the Cleaning Process
A micrograph of particles collected during the laser cleaning process is shown in Figure 13. Figure 13c is a magnified image of the boxed area in Figure 13b. The collected particles were of different sizes (micrometer, submicron, and nanometer). The nanoparticles were irregular in shape and had uneven edges, as shown in Figure 13a. The circular area, as shown in Figure 13b, shows obvious mechanical tear marks and a lamellar structure. The elliptical region from Figure 13b shows a transparent and pleated paint sheet with thickness of about several nanometers. This special structure was due to the cracking that occurred inside the paint layer during the laser cleaning process. As the delamination progressed, the paint layer was finally stripped off the paint base. Figure 13c shows a network structure at the edge of the particle, formed by the incomplete condensation of vaporous material. Spherical nanoparticles (average size of 31.36 nm) of uniform shape are shown in Figure 13d. Particle distribution is shown in Figure 12f: the relatively large particles were about 146 nm, the particles in the network structure were 2 nm in size or smaller, and particles in the 2-16.4 nm range were found to be the most abundant. The percentage of C element in the uncleaned paint layer was found to be 68.17 at%, and fraction of C element in spherical particles was 85.01 at%, indicating that spherical particles were generated due to bond cleavage of the polymer. The paint layer absorbed a large amount of laser energy, resulting in a breakage of chemical bonds such as C-C, C-H, and C-O from the molecular chain. A large amount of C atoms was continuously generated and assembled in the high-energy environment. Combustion also took place during the process and a large number of C atoms formed uniform nanoparticles because they came into contact with cold air and were assimilated into droplets during the ascending process. This process indicated a chemical bond breaking combustion mechanism during laser paint removal, which is consistent with the analysis in Figure 5.
Discussion
Paint removal using laser technology is a complex process. Laser beams are used to work on the paint layer with photons as the energy carrier. The photons transfer energy to the surface of the paint layer, causing a series of physical and chemical changes. The paint layer absorbs most of the laser energy, activating and converting the molecules or atomic groups in the paint to thermal energy. Instantaneous expansion and vaporization, among other processes, occur on the surface of the paint. In this experiment, the photon energy of the fiber laser with wavelength of 1064 nm was found to be 1.16 eV. The laser energy absorbed by the paint surface is transmitted and accumulated in the paint. The area closest to the laser beam's focus shows the fastest response and a rapid rise in temperature. Figure 14 shows the temperature change curves at different depths in the x-axis direction at t = 0.000851 s. The temperature of the paint layer was found to decrease rapidly with increase in depth, as shown in Figure 14. Compared with the temperature of the paint surface, the temperature below surface 5 µm was lower than 600 • C. When the temperature of the paint rises to a certain level, the chemical bonds thermally crack [11][12][13]. The cleavage of chemical bonds, such as C-C, C-H, C-O, and C-N, generate free radicals, which then became intermediates for subsequent chemical reactions. The reaction between activated free radicals and free radicals with oxygen in the air formed new organic compounds, such as (-CH 2 CH(C(O)OCH 3 )-) n , (-CH 2 CH(C(O)OH)-) n , (-CH 2 CHC(CH 3 )CH 2 -) n , (-CH 2 C(CH 3 )(C(O)OCH 2 CH 3 )-) n , (-CH 2 C(CH 3 )(C(O)OC(CH 3 ) 3 -) n , and (-CH 2 CH 2 NH-) n ; oxides such as C 96.8 O 3.2 , and C 93.1 O 6.9 ; and other substances containing N, such as [Mg(C 5 H 4 NCOO) 2 ] and C 48 H 26 N 8 on the laser cleaning surface. The little molecular gas substance generated during the cleaning process became volatile [14][15][16][17]. A large number of C atoms from the gaseous substances formed uniform nanoparticles when they encountered cold air, as shown in Figure 13c,d. The paint removal mechanism is primarily a chemical bond breaking combustion method.
As laser energy is continuously absorbed, as shown in Figure 3, the breaking speed of chemical bonds in the polymer is accelerated. At the same time, gaseous products, such as CO 2 , CO, and H 2 , above the surface of the paint layer, gradually increase. The large amount of steam that is generated strongly absorb the laser energy, and a laser plasma is formed by physical processes such as nuclear collisions and target electron excitation in high-energy gases. This process is accompanied by outward expansion, which produces shock waves that directly act on and generate impact damage on the paint layer [13], as shown in Figure 12a,b. The large amount of bond cleavage causes a gradual increase in electron density and energy in the laser plasma. The high-input energy thus results in a rise in temperature of the electron in the plasma, which is high enough to exceed the Vander Waals force and the chemical bond energy in the polymer. This further promotes the cracking of the inside of the polymer and the breaking of the chemical bond, resulting in destruction of the polymer structure [18,19].
Conclusions
The mechanism of pulsed laser paint removal is studied in this paper from a microscopic angle by means of Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy. Compared with the untreated surface, the infrared spectrum had two additional absorption peaks of 1383.36 cm −1 and 678.82 cm −1 on the surface of the paint layer cleaned with the pulsed laser. The profile of the C 1s, O1s, and N1s peaks in the cleaned surface indicate that chemical bonds such as C-C, C-H, C-O, C-N are broken and rearranged, resulting in the emergence of new organics and complex compounds. Traces of the impact of plasma was found on the cleaned surface. The removal process produced paint particles of varying sizes and shapes, caused primarily by plasma impact damage. The X-ray photoelectron spectroscopy conducted showed that C element content decreased and that of the O element increased after the cleaning. Smaller amounts of Al and Ti were detected on the cleaning surface, as compared to the untreated area. This means that the paint released nano additives containing metal Al and Ti during the cleaning process. Deconvolution of the Al2p, Ti2p spectra resulted in the formation of new components, such as SiO 2 (Al 2 O 3 ) 0.22 , MgAl 2.7 O 5.3 /SiO 2 , Al 6 Si 2 O 13 , TiO 0.3 S 1.5 , and Ti 2 S 3 . These results suggest the oxidation of paint and nano additives in air during the laser cleaning process. The formation of spherical nanoparticles is a result of photothermal ablation of the paint, caused by combustion of chemical bonds. | 2019-12-19T09:15:51.718Z | 2019-12-13T00:00:00.000 | {
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245205363 | pes2o/s2orc | v3-fos-license | Experimental analysis of cutting force during machining difficult to cut materials under dry, mineral oil, and TiO2 nano-lubricant
Difficult-to-machine materials, e.g., Titanium alloys, are highly applicable in diverse industries that yield strength and wear resistance. However, they prove difficult to machine due to high vibration, leading to high cutting forces during the machining process. This vibration occurs from chip discontinuity and thereby leads to high friction between the cutting tool and workpiece. In order to minimize these challenges, lubricants are employed in machining operations to reduce frictional and other unnecessary cutting forces and improve surface finish. This research focuses on studying the nano-lubricant effects in reducing cutting forces in the machining of TI-6AL-4V-ELI alloy. Also, carry out a comparative study of dry, mineral oil, and TiO2 nano-lubricant during face-milling machining for optimal performance. Additionally, the study develops a predictive mathematical model for cutting force using a Taguchi L9 orthogonal array. A two-step approach was employed to develop the nano-lubricant before the machining process. The dynamometer is used to collect the cutting force data at the end of each sample. The Results show that the lubrication conditions play a significant role in the reduction of cutting forces. The mineral oil-based-TiO2 nano-lubricant reduces the cutting force by 19 % compared with the mineral oil during the machining of TI-6AL-4V-ELI alloy. Furthermore, the optimal parameters to reduce cutting forces during face milling of TI-6AL-4V-ELI alloy are cutting speed at 3000 rpm, 200 mm/min feed rate, 0.3 mm depth of cut to obtain the minimum cutting force 30 (N). This study concludes that the application of TiO2 nanoparticles in mineral oil significantly improves the thermal and mechanical properties, which leads to a reduction of cutting force.
Introduction
Complex to cut metals are metals produced for the sole purpose of withstanding extreme conditions and applications [1]. Due to this essential characteristic needed from these metals, the manufacturers increase the hardness, corrosion resistance and reduce thermally conductive to make the metals more preferable than other metals, which has led to the difficulty in machining them during the manufacturing process. Hence they are classified as difficult-to-cut metals [2]. Due to their impressive chemical and mechanical properties, difficult-to-machine materials such as Titanium alloys are highly applicable in the automotive and aerospace industries due to their high strength-to-weight ratio, durability, creep, corrosion resistivity, and wear resistance. According to Hegab et al. [3], the grades of Titanium alloy and their applications are: 1) Grades 1: This is titanium's most ductile and, therefore, the softest category. It possesses high corrosion 2) Grade 5: Ti6Al4V. The most commonly used grade of titanium. Stronger than Grade 1-4 Titanium. It possesses the same grade properties as grades 1-4, except 60 % less thermal conductivity when compared.
10) Grade 38: 2.5 % vanadium, 4 % Aluminium, 1.5 % iron. It is used for armor plating, and it possesses the same properties as grade 5, good cold workability similar to grade 9.
Several problems have been raised during machining Titanium alloy [4]. Furthermore, this study will apply nano-lubricant during the machining process to reduce these problems ranging from short tool life, high heat generated at cutting zone, frictional, and cutting forces. This nano-lubricant will further improve surface finish lubricants and reduce cutting force. Over the years' improvements and continuous research relating to machining operations have given machinists the ability to improve lubricants' quality and performance using nanoparticles [5][6]. Applying nanoparticles to base oil improves the lubricant oil characteristics, improving wear resistance and reducing friction. According to Gaurav et al. [7] and Khalil et al. [8], nanoparticles improve load-carrying capacity and reduce cutting forces. Experiment on a Nickel-Titanium alloy to see the effect of nano-lubricants on the alloy is why studying cutting forces and tool wear. The results show that the nano-lubricant successfully reduced cutting forces by 6-10 % and tool wear by 4.2-34.5 %. The nanoparticle used in this experiment was Al 2 O 3, with soluble cutting oil (Sol-Cut) as the base oil. Hegab et al. [9] carried out a study with promising results on the best way to preserve the process for parameters when cutting 718 Inconel using nano-lubricants minimum quantity lubrication technique. The nanoparticles used are MWCNTs and Al 2 O 3 . Also, Sarhan et al. [10] work on the reduction of power in the milling process using the SiO 2 nano-lubrication system show that: 1) SiO 2 nanoparticles dispersed in mineral oil improve machining process performance by reducing COF and cutting forces.
2) Smaller specific energy and the power required in machining processes are obtained using nano-lubrication systems compared to ordinary lubrication systems.
3) With the nano-lubrication system's application, the increase of cutting forces is reduced compared to ordinary lubrication systems.
Even without the use of base oils, nanoparticles can still be applied dry. A review of Ghaednia and Jackson's [11] work on the dry application of CuO shows that these particles can reduce friction and wear even with lubricants' absence. A review carried out by Zareh-Desari, and Davoodi [12] shows that SiO 2 and CuO nanoparticles dispersed in vegetable oil can reduce friction by 21-31 %. Hence power consumption and cutting forces are substantially reduced. The performance of nano-lubricants was concluded to be superior as compared to conventional machining fluid. The addition of nano-graphene to vegetables produced better surface quality, central tool wear, and flank tool wear juxtaposing to just vegetable oil. This study is in line with the opinion made by [13][14]. Rahman et al. [15] research was carried out on nanofluids to improve base fluids' lubricating effect in turning biomedical grade titanium. The author's results show that the lowest R a (surface roughness) value recorded, i.e. (0.248 µm) for the addition of 0.5 vol%-Al 2 O 3 -CAN nanofluid, 73.1 % less than that recorded for dry cutting.
Furthermore, the lowest temperature was recorded at 875 o C when 0.5 %-MoS 2 was used as the nanoparticle and the most massive Chip thickness (R.C.) at 0.9238, implying a substantial decrease in cutting forces COF.
According to Anand et al. [16], the application of TiO 2 and Graphine nanoparticles in rice bran oil improves the tribological properties of the base fluid. The study also shows that the tool wear rate was reduced due to the application of the nanoparticles. Li et al. [17] performed a quantitative analysis of the lubricating and cooling effects of graphene oxide nano-fluids in the machining of titanium alloy (Ti6Al4V). The results showed that both the frictional force and friction coefficient on the flank face were significantly moderated with graphene oxide nano-sheets. The smaller normal pressure made the nano-sheets easier to make into the tool/workpiece interface, minimizing the tool/workpiece abrasion.
Minimizing cutting forces is one of the essential machining of Titanium alloy issues because less cutting forces would mean cheaper machining costs. High cutting forces are a challenge in machining titanium because of its hardness [18][19]. There is a relationship between cutting force and tool wear rate, as high cutting force will lead to a high cutting tool wear rate [20]. Today, lubricants are used in the reduction of these forces. Research and development in this area have improved these lubricants' properties with nanoparticles' introduction [21]. Although this is undoubtedly a fantastic engineering feat, nano lubrication still leaves many unchartered-water to how relevant it is in manufacturing processes. Not many tests and research has been carried out on nano-lubricants, causing a deficit in the engineering bulk of knowledge. This research is keen on finding out to what extent these improved lubricants reduce the cutting forces in the machining of titanium alloys. The case study nanoparticles are TiO 2 nanoparticles, hence adding to the bulk of knowledge acquired subject matter. Industries are searching for a sustainable way of reducing costs by cutting down unnecessary wastes while preserving the quality and quantity of manufactured goods. High cutting forces prevent the excellent quality finish of machined components and cause premature wear and damage to tools hence running companies at a loss during the manufacturing process.
Furthermore, there is a need to study eco-friendly lubricants and nano-lubricant effects on the cutting force during the machining of the difficult-to-cut materials for the sustainable manufacturing process. Therefore, this research will carry out a comparative analysis on the three machining conditions such as dry machining, mineral oil, and mineral oil-based TiO 2 nanolubricant effect on cutting force reduction during machining of Ti-6AL-4V-ELI alloy, which has not been studied. In aeronautics, aerospace, and medicine, where titanium is a popular metal, a high-quality finish is essential; hence this study is relevant to meeting the need. In order to achieve this, the following objectives are: 1) Analyses the effects of synthesized TiO 2 nano-lubricant on the Ti-6AL-4V-ELI alloy during face-milling machining and also carry out the comparative analysis of the dry and mineral oil with the machining parameters.
2) Develop a predictive mathematical model for cutting force, using Taguchi design.
The study employs an L9 Taguchi design with four factors at three levels of experiments, and the factors are cutting speed, feed rate, depth-of-cut, and lubrication conditions on the response (cutting force).
Materials and methods
The XM1060 CNC machine with the power supply of 400V-50HZ-3PH, spindle speed max of 10000 rpm, rated power of 21 KW, and weight 6980 Kg was used to carry out the face milling operation. This machine is located in the Mechanical Engineering Department of Covenant University, Ota, Nigeria. The specification of the cutting tool's chemical composition is given in Table 1. The material workpiece considered for this experiment is a rectangular Ti-6AL-4V-ELI alloy (Grade 23) Titanium block of 2000 mm×50 mm×5 mm, procured from a local scrap yard in Lagos. The chemical composition, mechanical and physical properties are given in Tables 2 to 4.
The selected nanoparticle employed for this experiment are: Titanium dioxide (TiO 2 ): These nanoparticles possess diameters less than a hundred nanometres, i.e., 15 nm. One everyday use for these nanoparticles is in the production of sunscreen. The significance of the TiO 2 nanoparticle implemented in this study is its high rate of corrosion resistance and the ability to increase the cooling rate of the base oil. The base oil for this experiment is white mineral oil. White mineral oil is accredited for being odorless, colorless, transparent, and possessing a non-fluorescent hydrocarbon blend. It finds its use in the food industry and in dissolving nanoparticles.
Experimental consideration
Determining the Nanoparticle Volume: Nanoparticle volume was obtained by the initial measurements of its dimension using a Transmission electron microscope (TEM).
If the nanoparticles happen to be spherical, the volume is calculated with Eq. (1) [22]: where is the sphere radius. The volume of rod-shaped nanoparticles can be found using Eq. (2): where for length while for rod radius. The volume of nanoparticles that are plate-shaped can be found using Eq (3): where ℎ for thickness while for nanoplate radius. The volume of cube-shaped nanoparticles can be found using Eq. (4): where is known as cube diameter, software such as MiniTab is used to examine the TEM images in getting the dimensions. Measurement is done through various Transmission electron microscope networks. The average is gotten and put in place appropriately using the above equations. Usually, they can all be gotten using only the transmission electron microscope. The nano-lubricant volume concentration can be calculated using Eq. (5) [23]: where -mass of nanoparticles, -density of nanoparticles, -mass of lubricant, density of lubricant. The determine the appropriate quantity of nanoparticles to be mixed with the base oil, Eq. (6) can be used: where -mass of nanoparticles, and -mass of base oil.
Method used for the preparation of nano-lubricant and the machining operation
Ultra-sonication is the chosen method used in this experiment to dispersion the nanoparticles into the base oil. The stages involved in the formulation of the nano-lubricants are as follows: 1) The authors weigh the TiO 2 nanoparticle using a digital electronic scale balance. The desired measurement range is 110 mg, and a maximum error of 0.1 mg was permissible.
2) The TiO 2 nanoparticles are added to the mineral oil.
3) A homogenized mixture is obtained by oscillating the solution in a Branson ultrasonic bath for 3 hours. Surfactants were not added because the nano lubricants are to be used in the experiments immediately, and adding surfactants may decrease the nano lubricant's thermal conductivity and performance. 4) There was good stability of the TiO 2 nanoparticle in the white mineral oil.
The experiment was carried out with the XM1060 CNC machine, and the experimental setup is shown in Fig. 1. Steps taken for the experimental investigations are as follows: 1) The preparation of the vertical CNC milling machine system ready for performing the machining operation, 2) Fixing the milling cutter of 80 mm diameter with carbide insert on the machine's spindle taper.
3) The workpiece is mounted, clamped on vice on top of the table of the machine. 4) CNC programs are produced to determine the cutting tool's travel path. The , , and axes are taken into consideration when programming the machine. Spindle speed, feed rate, axial depth of cut, and radial depth of cut are predetermined and imputed to the machine, then performing end milling operation. 5) A Taguchi orthogonal array is generated in Minitab 16 to give the parameter combination for machining 6) While machining, the cutting forces of the workpiece were measured with the dynamometer. 7) Use the Taguchi method to develop a predictive model for the three (3) machining conditions' performance in machining operations.
8) The cutting parameters chosen for this experiment are derived from the manufacturer's machining recommendations in Table 5.
Procedure employed to develop the mathematical model
For this experiment, the lubrication conditions (Dry, Mineral oil-lubricant, and Mineral oil-based TiO 2 nano-lubricant), Spindle speed, depth of cut, and feed rate are considered the turning parameters. The selection of the machining parameters' value ranges is selected based on the tool manufacturer's recommendations. The Taguchi method (L 9 orthogonal array) is used to organize the experimental setup for cutting parameters (Spindle speed, feed rate, Depth of Cut, and Lubrication Condition) with three levels (3 4 ). Minitab 19 software is used for multiple regression. The response of this study is cutting forces.
Taguchi method is a statistical method with the primary objective of improving quality, and there are two major tools: the orthogonal array and the signal-to-noise ratio. This method significantly reduces the number of experiments, and Taguchi's method accelerates the experimental work. This process is essential for robust experimental design. The optimal setting is the parameter combination, which has the highest S/N ratio. The Taguchi method is known to discover control factors, i.e., the operating parameters [24]. The S/N ratio represented as can be calculated using Eq. (7): = −10 log Output characteristic Mean square deviation .
There are three S/N ratio characteristics in this optimizing process, i.e., the lower, the better; the higher, the better, and the nominal-the better. Nevertheless, in this experiment, the cutting force is best minimized, so the lower, the better ratio is selected. Therefore, in modifying the observed data, the lower-the-better type S/N ratio is represented in Eq. (8) [25]: where being the S/N ratio for the lower-the-better characteristic, represents the measured quality characteristic for the th repetition and, , the number of repetitions in a trial. The subsequent steps for predicting and checking the originality of quality character development occur when established design parameter levels assign the highest design parameter. The approximate number ratio can be calculated using Eq. (9): Taguchi Array L9(3 4 ), Factors 4, and 9 Runs are applied in this study.
Results and discussion
In this section, the Analysis of Signal-to-Noise-Ratio and Analysis of Variance (ANOVA) is applied for the statistical analysis of the experimental results. The Taguchi-based orthogonal experiment was carried out, and the results were recorded and tabulated in Table 6. In Table 7, Cutting Speed, Feed rate, Depth of cut, Lubrication conditions are ranked in order of significance to reducing cutting forces. The Lubrication condition has a delta score of 5.89, followed by the cutting speed of 2.32, feed rate of 2.02, and depth of cut having a delta score of 1.08. Hence, there is an exponential change in the cutting forces when the lubrication conditions are changed. The results show a reduction in cutting forces when cutting speed increases from 2500 to 3000 rpm, the feed rate reduces from 300 to 150, and the depth of cut. Still, when the cutting speed is constant, the feed rate varies from 150 to 200 mm/min, and the depth of cut is changed from 0.9 to 0.3 mm under the nano-lubricants. There is a far more significant reduction in cutting force, based on Figs. 2 and 3, with optimum cutting parameters. As cutting Speed of 3000 rpm, feed rate of 200 mm/min, depth of cut of 0.3 mm, and machining with the Nano-lubricant. Table 8 also shows the response mean analysis. series of trials. Table 9 shows the ANOVA results with the cutting force. % represents the percentage of contribution of each factor on the total variation indicates the degree of influence on the result of the cutting force [26]. According to The Regression model is a mathematical equation used to estimate relationships between a dependent variable and one or more independent variables [27][28][29]. In the regression model for the cutting force Eq. (10), the dependent variable is the cutting force. The independent variables are Cutting Speed, Feed rate, Depth of cut, and machining conditions. Table 11 shows the coefficients relationship of the machining parameters and the cutting force. The variance inflation factor (VIF) proves that the relationship is normal with a multicollinearity set of 1. The P-value from Table 9 shows that the cutting speed is significant with a value of 0.048, and the most significant parameter is the machining condition with 0.001: Cutting Force (N) = 110.7 -0.01267 Cutting Speed (rpm) + 0.0681 Feed Rate (mm/min) -18.17 Machining Condition + 5.00 Depth of Cut (mm). Fig. 2 shows the residual plot for Cutting force; this is a four in one graph derived with the help of the Minitab 16 software. The normal probability plot predicts the cutting force with the cutting parameters. The histogram shows the frequency of occurrence, and the versus order shows the residual against the observation runs. Based on the analysis, it can be said that the Taguchi design of the experiment is linear.
The effects of cutting speed, feed rate, and depth-of-cut and their interaction on the cutting force under the three cutting conditions
The line plot represents the individual variable's influence on the cutting force, resulting in three (3) cutting conditions. The Line plot of the effects of cutting speed, feed rate, and depth of cut on the cutting force is represented in Figs. 3-5. The result analysis in Fig. 3 shows the effect of cutting speed on the cutting force under the three different lubricating conditions. Furthermore, the graph showed that the minimum cutting force was obtained at the highest cutting speed for all three cutting conditions. This result has proven that cutting speed has little or no effect in increasing cutting force during the cutting process via milling machining. Moreover, the nano-lubricant machining condition has a minimum cutting force of 30 (N). Fig. 4 demonstrates the effect of depth of cut on the cutting force under the three different lubricating conditions. The plot shows that the lowest cutting force when the feed rate is 200 (mm/min). In Fig. 5, it is shown that there was a sudden increase of the cutting force at the TiO 2 nano-lubrication condition. This can result from vibration coursed by chips falling back to the cutting region [30]. However, it still has the lowest cutting force obtained when the depth of cut is at 0.3mm, and the nano lubricant is applied. However, from the literature, depth of cut is not a friendly parameter to cutting force. Therefore, applying the depth of cut in cutting Ti-6AL-4V-ELI alloy needs a critical study. It is preferable to apply nano-lubricant with excellent chemical and mechanical properties.
The interaction analysis is another way of studying the relationship between two factors concerning the response value. For example, Fig. 6 illustrates all the cutting parameter's interactions on the cutting force. The cutting force is increased when the machining condition has not interacted with the feed rate. The Interaction Plot is designed to show the relationship between From Fig. 6, when the cutting speed is at 3000 rpm and the Feed rate is 200 mm/min, the cutting force reduces to its lowest. This proves that high cutting speed with low feed rate interactions leads to minimum cutting force during machining of Ti-6AL-4V-ELI alloy. Because the application of the nanoparticles in the mineral oil increases the mechanical property of the base oil, which significantly improves the machining condition during operation to reduce vibration and friction during the face-milling machining, this result is in line with [25,[31][32][33]. The vibration occurs due to the unwanted movement of the chips being removed from the workpiece in the machining process and can affect the workpiece during the machining operation. Therefore, the TiO 2 nano-lubricant in this study assists in removing the chips from the machining region, thereby reducing the cutting force. This reduction process of the cutting force also will assist in prolonging the cutting tool life during operation [34][35][36].
Conclusions
In this research, the effect of the dry, mineral oil, and TiO 2 nano-lubricants on the cutting forces in the machining of Grade 23 Titanium alloy (Ti-6Al-4V) was investigated. The experiment was carried out using the Taguchi experimental design and dynamometer to measure the cutting force during the machining process, and the experimental results were analyzed using Minitab 16. The research considered four parameters such as cutting speed, feed rate, depth-of-cut, and lubrication cutting conditions on the response (cutting force). This study has the following conclusions: 1) Mineral oil-base-TiO 2 nano-lubricant was responsible for the reduction of the cutting force by 19 % compared to mineral oil and the dry machining operations 2) The cutting speed increase with a relatively low feed rate leads to cutting force reduction 3) The optimal parameters with the minimum cutting force when milling Ti-6AL-4V-ELI alloy are cutting speed at 3000 rpm, 200 mm/min feed rate, 0.3 mm depth of cut, under the machining with Mineral oil-base-TiO 2 nano-lubricant with a minimum cutting force of 30 N.
4) The most significant parameters are the machining condition with a 0.001 P-value, with a high contribution ratio of 78.63 %. However, the least significant parameter is the depth of cut with a P-value of 0.542 and a contribution ratio of 0.54 %. | 2021-12-16T17:09:05.876Z | 2021-12-13T00:00:00.000 | {
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259262096 | pes2o/s2orc | v3-fos-license | Experiments with Detecting and Mitigating AI Deception
How to detect and mitigate deceptive AI systems is an open problem for the field of safe and trustworthy AI. We analyse two algorithms for mitigating deception: The first is based on the path-specific objectives framework where paths in the game that incentivise deception are removed. The second is based on shielding, i.e., monitoring for unsafe policies and replacing them with a safe reference policy. We construct two simple games and evaluate our algorithms empirically. We find that both methods ensure that our agent is not deceptive, however, shielding tends to achieve higher reward.
Introduction
Deception is a challenge for building safe and trustworthy AI [18]. Recent advances in reinforcement learning (RL) and language models (LMs) mean that we are increasingly living in a world containing highly capable, goal-directed agents [15]. Deception may be learned as an effective strategy for achieving goals in many environments, especially in multi-agent settings comprised of humans and AI agents [18].
Technical work on deception in AI systems, and how to avoid it, is limited. Deception has been defined within structural causal games (SCGs), which is a framework that applies causal graphs to game theory [18]. Given a causal graph, it is possible to ensure certain safety properties by removing paths in that causal graph [1]. Giving learning agents these kinds of path-specific objectives (PSOs) is also applicable when deception is the property that is considered unsafe. These methods ensure that the agent will not deceive a human providing feedback, however, it will also ensure that the agent will not persuade, teach, or coordinate with the human (i.e., it will not try to influence the human in any way). Shielding is another class of methods for ensuring safety in learning agents [14,6,17,12,7,8,2]. A shield is a kind of monitor. In addition to monitoring, the shield replaces an unsafe action with a safe action if the verification returns that the policy does not satisfy the safety specification.
In this paper, we make three contributions: (1) We introduce a shielding algorithm to ensure that an agent does not deceive other agents. (2) We introduce two simple games to evaluate deception in agents.
(3) We evaluate our algorithm in these environments against an algorithm based on PSO. This paper is organised as follows: In Section 2, we recapitulate the definition of deception in SCGs. In Section 3, we introduce our algorithm and compare it to the PSO algorithm . We then conclude in Section 4.
Defining and detecting deception
Structural Causal Games (SCGs) offer a representation of causality in games [10]. An SCG is a directed acyclic graph containing variables and causal edges between them. There are three types of variables: chance variables (X ), decision variables (D) and utility variables (U ). Along with the graph, an SCG defines the conditional probability distribution (CPD) over each (non-decision) variable, given its parents. Agent's policies choose the CPD over decision variables and agents choose their policies to maximise the expected sum of utility and we use the Nash equilibrium concept. At the beginning of the game a setting is sampled from the prior over the game; given a setting and a policy profile, the value of any variable is uniquely determined. Kenton et al. [11] define agents in SCGs as systems that would adapt their policy if their actions influenced the world in a different way. This is the relevant notion of agency, as we define belief and intent based on how the agent would adapt its behaviour to such changes.
We now introduce a simple signalling game, where an agent that can be weak or strong tries to avoid being attacked by another agent, that wants to attack them only if they're weak. They can defend or retreat, and their decision is observed by the other agent.
Example 1 (War game fig. 1): A signaller S has type X ∈ {strong, weak}. At the start of the game, S observes X , but the target agent T does not. The agents have decisions D S ∈ {retreat, de f end} and D T ∈ {¬attack, attack}. A weak S prefers to retreat whereas a strong S prefers to defend. T prefers to attack only if S is weak. Regardless of type, S does not want to be attacked (and cares more about being attacked than about their own action). X follows a Bernoulli(0.9) distribution so that S is strong with probability 0.9. U T = 1 if T attacks a weak S or does not attack a strong S, and 0 otherwise. S gains utility 2 for not getting attacked, and utility 1 for performing the action preferred by their type (e.g., utility 1 for retreating if they are weak). To deceive is to intentionally cause to have a false belief that is not believed to be true [13]. Past work defines belief, intention, and deception in SCGs [18]; these definitions only refer to agent behaviour.
Belief Agents have beliefs over propositions φ , i.e., Boolean formula of variable assignments (e.g., φ : X = x ∧ ¬Y = y). An agent believes a proposition φ if 1) they act as though they observed φ is true; 2) they would have acted differently if they observed φ was false. An agent has a true/false belief if they believe φ and φ = true/ f alse.
Example 1 (continued): Since S's probability of being weak is low, its optimal policy is to always defend, in order to signal a strong type. T 's best policy in this case is to attack if and only if S retreats. These two policies form a Nash equilibrium. When X = weak, T believes the proposition φ : X = Strong, as 1) if they had observed that X = Strong, they would not have attacked, and 2) if they had observed that X = Weak, they would attack. Therefore, they respond to φ , and they act as if φ = true, so the two conditions for belief are met. When X = weak, φ = f alse, so T has a false belief about φ .
Intention Previous work defines notions of intention, suitable for algorithms, in causal models [9,3,18]. Essentially, an agent intentionally causes the outcomes which provide sufficient reason for it to choose its policy over an alternate policy. What intent to cause means, intuitively, is that if the outcomes the agent wanted were guaranteed to happen anyway, they would not mind choosing an alternative policy [18].
Example 1 (continued): Under the Nash policy, S intends to cause T to not attack, w.r.t. the alternative (honest) policy (de f end when X = strong and retreat when X = weak), because if T 's policy was fixed to ¬attack, then S would choose the honest policy. Hence, S intends to cause D T = ¬attack.
Deception An agent S deceives an agent T about a proposition φ if 1) S intentionally causes T 's decision D T ; 2) T has a false belief about φ ; 3) S does not believe φ .
Example 1 (continued): Under the Nash policies, S deceives T about their type when S is weak. As seen above, T believes that X = Strong, S intends for D T to be ¬attack, and S does not believe that X = weak, so all the conditions for deception are met.
Mitigating Deception
We perform experiments in two examples, optimising agents to play these two games with no mitigation, with PSO, and with shielding. We compare these methods on their optimality and deceptiveness.
Algorithm 1: PATH-SPECIFIC OBJECTIVES Input: An SCG M = (G , θ ), graphical criterion C , policies π −i , natural distributions N . Output: PSO-optimal policy π i 1: Reduce G to G ′ using C . 2: Impute policies π −i and natural distributions from N to those variables with fewer parents in G ′ to obtain θ ′ . 3: Train an agent in M ′ = (G ′ , θ ′ ) to obtain policy π i .
Path Specific Objective (PSO) [1] prevents S learning a deceptive policy by removing S's ability to influence T 's decision during training. In SGSs, this corresponds to removing the path in the graph between D S and D T . The PSO algorithm is shown in algorithm 1.
Example 1 (continued): In the war game, we remove the edge from D S to D T . S is now only interested by the utility it gets directly from its decision, meaning it will learn the honest policy. Therefore, S can be trained with PSO, and it will play the game without being deceptive. However, this removes S's ability to learn to influence T in any way, including positively. In the following example, the only strategy for achieving utility is to influence the other agent, and so the PSO agent does not learn anything. 2): Variables X ∈ {0, 1, 2} is uniformly sampled, and Y ∈ {0, 1} follows a Ber(0.1) distribution. S observes both X and Y , while T does not. D T ∈ {0, 1, 2} and T 's objective is to correctly bet on the value of X . D S ∈ {0, 1, 2}, and S gets some utility when T correctly bets on X , but when Y = 1, S can get more utility if D T = (X + 1) mod 3. T observes D S . This example differs from the first one as S's only way to get utility is to influence T 's decision. S can adopt two sensible policies, which are to always report X (the honest policy), or to, when Y = 1, report on (X + 1) mod 3 instead (a deceptive policy, S intentionally causes T to believe that X has the wrong value). Since Y is rarely 1, as before, T 's optimal strategy remains to follow S's decision even if it is sometimes deceptive. If we use PSO with this game, S will have no way to influence its utility, and T will have no information on the value of X . Therefore, any policies for S and T are optimal and the agents will not learn anything. We introduce the shielding algorithm to solve this problem by preventing deceptive policies from being learned, in a more fine-grained way than PSO.
Algorithm 2: SHIELDING Input: SCG M = (G , θ ), reference policy π ref , T 's policy π T , proposition φ Output: optimal non-deceptive policy π 1: π ← π ref 2: for s in settings do 3: take action a sampled from π(·|s) 4: calculate optimal policy π ⋆ 5: D←DECEPTIONCHECK(M , π π π=(π ⋆ , π T ), π ref =π, S, T , φ ) 6: if D=false then π ← π ⋆ 7: end for Shielding uses a safety constraint, and checks that an agent's action or policy satisfies the constraint before letting it perform that action. In our case, we shield the whole policy rather than individual decisions, and we use the shield during training to prevent the agent learning deceptive policies. The shield used is the deception verification presented in Algorithm 3. We built a simple environment to apply the above definitions and to investigate ways to train non-deceptive agents in SCGs. S (the potentially deceptive agent) is being trained with a minimal version of RL, where S plays several games with random settings and policy. We assume that T (the target of deception) has fixed Nash-policy, because we work with games where the occasions for S to benefit from deception are rare, and the best policy for T remains to believe S's signal, despite it being sometimes false. We implemented algorithm 3, which, given a policy, and a reference policy, indicates whether the policy is deceptive w.r.t the reference policy, by testing every possible setting. Hence algorithm 3 is complete and sound. We initialize a known-safe policy as the reference. As soon as a better performing safe policy is found, we use this as the new reference. if deceptive then return true 8: end for 9: if not deceptive then return false Results are summarised in table 1. For Ex.1, both PSO and shielding learn the optimal non-deceptive policy, whereas when no mitigation is used the optimal (deceptive) policy is learned. For Ex.2, shielding learns the optimal non-deceptive policy, but the PSO-agent cannot learn anything, as in this example the only way for S to gain utility is to influence T .
Conclusion
Summary We introduce a novel shielding algorithm for mitigating deceptive learning agents. We show, in two toy environments, that our algorithm has advantages over previous methods.
Limitations and future work The examples are simplistic, and the optimal policies are very easy to find analytically without doing any training. This work acts as a proof of concept for the idea of automatically detecting and preventing deception while training. Many simplifying assumptions are made, e.g. the fact that the games only have one time-step, or the assumption that one of the policies is fixed. In addition, the verification is exhaustive on the setting space. This works with the small domains of these examples but might become intractable for larger and more realistic problems, which could require Monte-Carlo sampling of the setting, or a latent representation of it. Furthermore, shielding requires an initial safe reference policy, and its convergence to good safe policies is unknown. | 2023-06-28T06:42:56.580Z | 2023-06-26T00:00:00.000 | {
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21208707 | pes2o/s2orc | v3-fos-license | An Update on Implants for Minimally Invasive Glaucoma Surgery (MIGS)
For several years, the gold standard for surgical treatment of glaucoma has been trabeculectomy. Although very successful at reducing intraocular pressure (IOP), there are several potential complications of trabeculectomy, including sight-threatening ones. This has stimulated much research aimed at the development of new and effective procedures to lower IOP with an enhanced safety profile. Minimally invasive glaucoma surgery (MIGS) procedures prioritise patient safety but also demonstrate efficacy in reducing IOP. We performed an online search of peer-reviewed literature using PubMed, entering keywords relevant to this clinical discipline. In summary, there is a lack of long-term safety and efficacy data, a lack of comparative data and a lack of data on standalone (i.e. without simultaneous cataract surgery) procedures. Most implants are not yet FDA approved. Although not exhaustive, since it does not discuss MIGS procedures that are not implants, this article summarises the range of different MIGS implants that are available to the ophthalmic surgeon.
INTRODUCTION
Glaucoma remains the leading cause of irreversible blindness worldwide. It is estimated that 64.3 million people have glaucoma [1]. The only modifiable risk factor is raised intraocular pressure (IOP), with therapy aimed at reducing this by various means [2]. This includes topical hypotensive agents, laser trabeculoplasty and glaucoma drainage surgery. Adherence to drops and their multiple side effects limits their use and efficacy, whereas the effects of laser trabeculoplasty wear off over time, requiring multiple repeat procedures [3,4]. Drainage surgeries such as trabeculectomy and aqueous shunts demonstrate excellent efficacy but have less than ideal risk profiles [5]. Despite their efficacy, tube and trabeculectomy patients have similar rates of vision-threatening complications such as endophthalmitis or choroidal haemorrhage; ocular surface scarring and ocular surface disease can lead to poor patient quality of life [6].
Minimally invasive glaucoma surgery (MIGS) offers a safer, less invasive means of reducing IOP than traditional surgery, with a goal of reducing dependency on topical agents. MIGS can usually be combined with cataract surgery, and most clinical studies have analysed results of combined surgery. With MIGS, there is a trade-off between enhanced safety and less efficacy compared to traditional surgery. MIGS procedures are currently targeted at patients with mild-to-moderate glaucoma.
Generally speaking, MIGS procedures are ab interno, microincisional and conjunctiva-sparing. They share a common approach of minimal tissue trauma and minimal disruption of the normal anatomy and physiology [7]. For the patient, MIGS provides IOP control in the midto low teens with rapid visual rehabilitation and less dependence on topical treatment. This article summarises the current literature on the range of MIGS implants available. This article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by the author.
There are three main groups of implants. These include implants that increase trabecular outflow by bypassing the juxtacanalicular trabecular meshwork (TM), increasing uveoscleral outflow via suprachoroidal pathways, or by creating a subconjunctival drainage pathway. Table 1 summarises the different implants and their mechanisms of action; the ones highlighted in bold are discussed in this review.
TRABECULAR MESHWARK BYPASS STENTS
iStent (Glaukos Corp., Laguna Hills, CA, USA)this is a heparin-coated, nonferromagnetic titanium device with a snorkel shape that is used for implantation into Schlemm's canal Asterisks refer to implants from the same manufacturer (SC). It can be implanted alone (single or multiple), or in combination with cataract surgery (Fig. 1). The iStent Study Group [8] randomised controlled trial (RCT) compared cataract surgery alone to combined surgery with one iStent (n = 239; 116 in a combined/study group and 123 in a cataract surgery only group). The primary outcome measure was defined as IOP (measured in mmHg) B21 mmHg on no topical hypotensive medications at 12 months. The secondary outcome measure was subjects with an IOP reduction from baseline of greater C20% without the use of a topical hypotensive medication at 12 months.
In the study group (combined surgery group), 72% had IOP \21 mmHg at 12 months and 61% at 24 months, which was statistically significant. In the cataract surgery group, the success rate was 50% at 12 and 24 months. Mean IOP reduction was 8.4 ± 3.6 mmHg at 12 and 24 months in the study group. There was a mean decrease in medications, 1.4 ± 0.8, in the study group and 1.0 ± 0.8 in the controls (p = 0.005) at 12 months. In the study group, 15% were taking glaucoma medications at 12 months compared to 35% in the cataract group (p = 0.001).
Although there is more emphasis on using MIGS for earlier stages of glaucoma, the use of iStent in more advanced cases of glaucoma, including post-drainage surgery eyes, has also been studied [9]. At 36 months, the mean IOP in cases with no previous surgery was 15.4 ± 2.2 mmHg, with 13% of eyes on topical treatment. The mean IOP in the group with previous surgery was 14.2 ± 2.3 mmHg, with 44% of eyes on medications.
There is evidence of enhanced IOP reduction with multiple implants. In a prospective study (n = 119) of iStent-only cases, at 18 months follow-up, the IOP was 15.9 ± 0.9 mmHg with one stent, 14.1 ± 1.0 mmHg with two stents and 12.2 ± 1.1 mmHg with three stents [10]. Implantation of each additional stent realised a significant further reduction in IOP (p = 0.001).
This has led to the development of a second-generation iStent, the iStent Inject. This is preloaded with two stents. In one study, implantation of this device resulted in IOP B18 mmHg in 66% of cases at 12 months, with an encouraging safety profile [11].
Hydrus (Ivantis Inc., Irvine, CA, USA)-this is a crescent-shaped trabecular bypass device made of nitinol (an alloy of nickel and titanium), a shape-memory alloy. This means that, when deformed, it returns to its original shape after being heated. Being 8 mm long, it straddles 3 clock hours of SC, with the aim being to access more collector channels and dilate the SC. It acts as a scaffold so that it does not block the collector channel ostia.
A RCT of 100 cases randomised to cataract surgery alone or combined cataract surgery with Hydrus has been completed [12]. There was a washout of glaucoma drops and the results were presented without the influence of glaucoma medications. At 24 months, a significantly greater proportion of the combined surgery cases reached the endpoint of a 20% reduction in diurnal IOP (80% versus 46%, p = 0.0008). The IOP was also significantly lower in the combined surgery group (16.9 ± 3.3 versus 19.2 ± 4.7 mmHg, p = 0.0093), and there was a significant reduction in cases without ocular hypotensive medications in the combined surgery group (73% versus 38%, p = 0.0008).
The safety in the Hydrus group was similar to that in the control. Six of 50 (12%) Hydrus patients had focal peripheral anterior synechiae, but this did not affect device efficacy adversely. The Hydrus stent would have the same indications and relative contraindications as the iStent in theory, but more data are needed to support this.
SUPRACHOROIDAL IMPLANTS
The CyPass (Alcon, Fort Worth, TX, USA), a suprachoroidal/supraciliary implant, is discussed here. The iStent supra (ab interno) and the SOLX Gold microshunt The CyPass is a polyamide implant, 6.35 mm in length and 510 lm in external diameter, that forms a connection between the anterior chamber and the supraciliary space. The collar of the device rests in the anterior chamber angle (Fig. 2), and microholes placed along its length allow for circumferential egress of aqueous into the supraciliary space. Implant position can be confirmed by gonioscopy and/or anterior segment OCT [13].
CyPass with Cataract Extraction and Intraocular Lens Implant (CE/IOL) Hoeh et al. conducted a multicenter prospective study of 57 uncontrolled (C21 mmHg) POAG patients and 41 controlled (\21 mmHg) POAG patients undergoing CyPass implantation and CE/IOL. The safety profile was encouraging. The mean medicated IOP in both groups combined was 21.1 ± 5.91 mmHg (the baseline IOP for each group was not stated). IOP at 6 months was 15.6 ± 0.53 mmHg with 0.9 ± 0.15 medications in the uncontrolled group; i.e. a 37% reduction in IOP (p\0.001) and a 50% reduction in medications (p\0.001). The resulting IOP in controlled patients was 15.6 ± 0.68 mmHg on 0.6 ± 0.07 medications; i.e. a 71.4% reduction in medications (p\0.001) [14].
In the CYCLE study, 136 cases were followed up prospectively for 2 years [15]. At baseline, the uncontrolled group of 51 eyes had IOP C21 mmHg and the controlled group of 85 eyes had IOP\21 mmHg. CyPass implantation and CE/IOL were performed in each case.
The COMPASS Trial was a two-year RCT of 505 subjects randomized to CE/IOL ? CyPass or CE/ IOL alone [16]. The treatment group demonstrated IOP lowering of 7.4 mmHg compared to 5.4 mmHg in the control group (p\0.001), with 85% of treated patients being drop-free at 24 months. There were no vision-threatening AEs in the CyPass group and visual acuity was at least 20/40 in 98% of all cases studied.
CyPass Implantation Alone
In contrast to the COMPASS Trial, the DUETTE study followed patients (n = 65) for 1 year following standalone CyPass implantation [17]. This was a multicentre, single-arm interventional study of 65 eyes with OAG and IOP uncontrolled at [21 mmHg on topical therapy. Baseline IOP was reduced from 24.5 ± 2.8 mmHg with 2.2 ± 1.1 medications to 16.4 ± 5.5 mmHg with 1.4 ± 1.3 medications at 12 months, i.e. a 34.7% reduction in IOP. IOP spikes to[30 mmHg lasted beyond 1 month in 11% of cases; 12.2% had cataract progression at 12 months; four eyes had hyphaema that resolved by month 1. 17% of patients went on to require a trabeculectomy; 6% exited the study by choice, and 4.6% were lost to follow-up.
Viscopass is a term used to describe CyPass implantation combined with the injection of 60 lL of ophthalmic viscosurgical device (OVD) at the end of the lumen to increase the size of the aqueous drainage area created by the CyPass Micro-Stent. A clinical trial comparing this with CyPass alone is underway.
SUBCONJUNCTIVAL FILTRATION
The implants in this group use subconjunctival filtration to establish a nonphysiological route for aqueous outflow akin to traditional trabeculectomy and aqueous shunt surgery. Therefore, they require subconjunctival mitomycin C (MMC) injection pre-insertion to optimise bleb function and survival.
XEN Gel Stent
Subconjunctival filtration represents a familiar route for aqueous outflow, and is the basis of trabeculectomy and aqueous shunt procedures. The XEN gel stent (Allergan, Dublin, Ireland) is an ab interno gelatin stent that is implanted via a clear corneal incision, avoiding conjunctival dissection. It is 6 mm in length and composed of porcine gelatin crosslinked with glutaraldehyde. Three models have been evaluated with inner diameters of 45, 63 and 140 lm [18], with 45 lm being recommended by the manufacturer (Fig. 3). Due to Poiseuille's law of laminar flow, the length and inner diameter of the tube determine the rate of flow, and thus the flow resistance-which prevents hypotony. Preclinical tests established that the implant does not occlude inside the lumen and the implant material does not cause a tissue reaction in the eye [18].
There are few published studies on the XEN 45 implant. Two pilot studies [19,20] have been published. One study investigated the insertion of one XEN implant (the 63 or 140 model) ?MMC combined with cataract surgery, and demonstrated a reduction of IOP from 22.4 (±4.2) mmHg to 15.4 (±3.0) mmHg at 12 months; there was a reduction in glaucoma medications from 2.5 ± 1.4 to 0.9 ± 1.0 [20].
In another pilot study using XEN 140 model implantation alone ?MMC (n = 49 eyes), 40% had unqualified success at 12 months (IOP B18 mmHg and C20% reduction in IOP), 89% had qualified success, and most cases involved a previous failed trabeculectomy [21].
In those studies, there were no serious adverse events such as tube erosion or prolonged hypotony, but some patients had injection of ophthalmic viscosurgical device into the anterior chamber, particularly in the XEN 140 group.
Obviously, larger studies are required to evaluate long-term efficacy and late complications. Results of the phase 4 APEX trial using XEN 45 will be available later this year.
InnFocus
The InnFocus microshunt (Santen Pharmaceutical Company Ltd, Osaka, Japan) is an ab externo drainage device. It involves more steps akin to trabeculectomy compared to other MIGS. The material is SIBS (polystyrene-blockisobutylene-block-styrene) which has been developed specifically for medical implants. It is a biocompatible and biostable thermoplastic elastomer (Fig. 4).
In one study (n = 23 eyes) of microshunt ?MMC, 80% had IOP B14 mmHg at 3 years, but some cases were standalone and others were combined procedures. The mean IOP for the entire group was 10.7 ± 1.5 mmHg at 3 years; the qualified success rate was 95%, with a reduction in medications from 2.6 ± 0.9 to 0.8 ± 1.2 [22].
Transient hypotony and transient choroidal effusion occurred in 13% and 8.7%, respectively, but with spontaneous resolution. There were no serious long-term adverse events, leaks, erosions, migrations or infections.
The results of the RCT of InnFocus microshunt versus trabeculectomy are awaited.
DISCUSSION
MIGS implants targeting different aqueous outflow pathways offer an improved safety profile for glaucoma surgery while preserving modest efficacy (Table 2). An important advantage for patients in addition to the safety element is that comorbid cataract can be treated simultaneously with MIGS implants. The procedures targeting the subconjunctival space appear to be more efficacious in terms of IOP Fig. 4 InnFocus microshunt. Image courtesy of InnFocus reduction. However, there is a lack of comparative studies between the different implants. Although RCTs have been conducted for some of the implants discussed, well-designed randomised clinical trials with an extended follow-up are needed to evaluate the long-term efficacy and late complications of these implants.
MIGS technology has potential advantages that could improve the management of glaucoma. These include reducing the medication burden, which enhances patient quality of life [3], bypassing or delaying the need for more invasive surgery and preserving the conjunctiva if a more-invasive intervention were to be required later on. There are limited data on the cost-effectiveness of MIGS, but one study showed the cost-effectiveness of iStent compared to branded medications [23]. More studies like this are required for a wider range of MIGSs to demonstrate their cost-effectiveness compared to medical treatment.
However, there are several limitations to the current state of MIGS. There is a lack of high-quality data, a lack of study standardization, a lack of cost-effectiveness data, a lack of long-term data and incomplete knowledge of ideal patient selection. Furthermore, many studies have been performed for cases combined with cataract surgery, meaning that they lack robust evidence for the effect of MIGS alone [24]. It is also unclear which established procedures should be compared to the MIGS devices.
Standardisation and improvements in the quality of future MIGS studies will help clinicians to negotiate this ever-expanding area more knowledgably and help them to optimise the selection of the appropriate device for the right patient. With the correct approach to investigating and evaluating new technologies, there is much potential for future generations of MIGS to improve the quality of care for glaucoma patients. authorship for this manuscript, take responsibility for the integrity of the work as a whole, and have given final approval for the version to be published. Table 2 Summary of efficacy and safety data Phaco/iStent [8] Phaco/ Hydrus [13] Phaco/ CyPass [17] Phaco/XEN45 [22] InnFocus [3] Pre Disclosures. Ejaz Ansari has nothing to disclose.
Compliance with Ethics Guidelines. This article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by the author.
Open Access. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits any noncommercial 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. | 2018-04-03T02:13:20.194Z | 2017-07-20T00:00:00.000 | {
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240570128 | pes2o/s2orc | v3-fos-license | Disaster Risk Awareness: The Turkish Migrants Living in Northern Italy
In this study, we analysed the socio-demographic characteristics and disaster risk awareness of the Turkish migrants living in northern Italy. We initiated the study with an extensive face-to-face questionnaire with 544 individual respondents. With the help of the questionnaire, we gathered information on the socio-demographic structure of the Turkish community living in the area and the immigrants’ disaster experience, their level of disaster preparedness and disaster risk awareness, and their potential behaviour during an emergency. Additionally, we conducted focus group meetings in Milan, Lecco, Como and Varese with 49 migrants living in the region. In the focus group meetings, we discussed the migrants’ awareness of disasters and potential behaviour patterns during emergencies. We collected the informative booklets and past event reports prepared by civil protection centres and municipalities and used them in focus group meetings to collect participants’ opinions. The results show that the migrant communities’ disaster risk awareness is low, but their capacity to adapt to suddenly changing conditions is higher than presumed.
Introduction
Scholars have long studied the variety of reasons that people migrate from one place to another. Human mobility has a long history and understanding migration patterns has been central to migration studies for decades. The most significant drivers are often structural (e.g., economic development in the countries of origin) [1]. However, there is no simple explanation such as the pull and push factor, and the migration decision depends on complex interactions of many factors [2]. UNHCR [3] states that migrants and receiver countries benefit from migration, and it enriches their current situations. UNHCR [3] also indicates the situations that leave migrants in vulnerable conditions, and "situational vulnerability" is one of them. Migrants' situational vulnerability is higher than that of the inhabitants for many reasons, the primary one being a lack of knowledge of the local risks and language.
Despite the importance of the issue, many studies on migration and disaster risk management are very limited; they mainly focus on non-European countries and revolve around the specific impact of the hazard, e.g., the effect of storms on migrant communities [4,5]. There are a wide range of studies on post-disaster migration [6]. However, there are few on migrants' perception and awareness of risk and preparedness level [7]. The concepts of risk perception and preparedness have often been associated in many disaster studies not necessarily focused on migrants and framed along the lines of "prediction". Namely, risk perception is a predictor of preparedness behaviour [8].
Migrants are considered the "(in)visible victims of disasters" whose unique needs are often overlooked in disaster planning [9]. Unequal access to disaster preparedness resources is coupled with a poor understanding of migrants' risk perceptions, which Around twenty-five years ago, Susan Cutter asked 'are societies more vulnerable to environmental hazards?' and pioneered the first comprehensive social vulnerability index, which generally includes qualitative indicators rather than quantitative [14]. Social vulnerability is mostly described by individual characteristics of people, such as age, race, health, income, type of dwelling unit and employment [15] (p. 243). The other factors that increase the social vulnerability of a community are a lack of access to resources such as information, knowledge and technology, limited access to political power, absence of social capital, beliefs and customs, deficiency of physical environment, individuals with disabilities, and type and density of infrastructure [16][17][18][19] (cited in [20], p. 245).
Findings from qualitative research, in fact, show that people may be simultaneously vulnerable and resilient [20]. This knowledge led to an important conceptual shift, as it challenges ideas of migrants as passive victims to emphasise their potential role as resourceful agents [20] (p. 6). Studies have also highlighted the key role of social capital in resilience, especially in environments with cultural and language barriers [21].
Additionally, with regard to preparedness, it is a well-known fact that people living in hazard-prone areas are often unprepared, and they fail to take precautionary measures to reduce the impact of a disaster [22,23] (cited in [24]). Communication is one of the many reasons for this. How risk communication is conceived by actors involved in disaster risk management can make a difference [25]. As highlighted by the Intergovernmental Panel on Climate Change [26], taking into account the specific needs of different societal groups is key. Communication processes should be as inclusive as possible, meaning that local risk perceptions and the local framing of risks and needs cannot be ignored since, for instance, language skills influence the levels of disaster preparedness [27]. As Fielding pointed out, different people and different locations require additional warnings [26]. Targeting group-specific information based on the heterogeneity of citizens is crucial [28].
Other relevant terms are place attachment [29], sense of community [30] and sense of place [30]. The study by Misshra et al. [29] asked the question "Does place attachment and the consequent emotional connections and ties that people have with environments affect their preparedness for natural disasters, such as floods?" The authors addressed the research question by considering the three attachment types: "economic, genealogical, and religious". The results show that there is a strong correlation between place attachment and flood preparedness. Place attachment can be considered one of the differences between migrants and local inhabitants. Regarding the sense of community, it requires further studies to investigate the impact of "sense of community" [30] and "sense of place" [31] on the preparedness and awareness of migrants.
We started this study with the problem being that the migrants are invisible victims whose unique needs are not included in disaster planning. Another challenge is that they are often labelled as vulnerable, and their capacities are overlooked in disaster risk studies. In this study, we collect information on the socio-demographic characteristics of migrants and their access to resources such as information, knowledge and technology that helps us to understand the social vulnerability of migrants. The results of the study will help decision makers to adjust disaster risk planning by considering the unique needs of the migrants based on their socio-cultural and economic conditions. It is worth considering that it would be misleading to frame the risk awareness and preparedness of migrants only through the concept of "vulnerability" without considering their capacities as well.
Materials and Methods
In the study, we applied various research methods at three different urban scales: regional, community, and household. The study started with a face-to-face questionnaire conducted during the National Parliamentary Elections in May 2015 in Milan. We administered the questionnaire to 544 individuals. The respondents were selected randomly at the entrance of the Consulate General of the Republic of Turkey in Milan by researchers. The questionnaire gathered information on the Turkish community's socio-demographic characteristics, their disaster experience, disaster preparedness, disaster awareness, and their potential behaviour during an emergency. To collect further information and gather in-depth knowledge on the awareness of disaster, we decided to conduct focus group meetings with various socio-cultural groups, including students, expats, religious minorities, and the members of a religious-political movement. Additionally, we conducted literature research regarding past natural hazards in northern Italy to inquire about visual and written resources during focus group meetings. The risk maps and reports that were prepared by the civil protection authorities and municipalities were examined on a regional scale. We collected the informative booklets and past event reports prepared by civil protection authorities and municipalities to analyse details and share them with participants during focus group meetings to learn more about the participants' experiences and opinions.
Due to the exploratory nature of this study, we started with some generalisations about the socio-demographic characteristics of the Turkish communities living in northern Italy. We presumed that most Turkish communities living in Italy are composed of workers in the food and construction sectors and students. The former has rapidly increased in recent years. The Italian education system became an option for students who do not speak Italian with the launch of English graduate programs. A small portion of the first incoming students decided to work or continue their doctoral or post-doctoral training. The rate, which was significantly small in the first few years, continues to increase every year.
The Survey Area
In Italy, the majority of the Turkish population lives in northern Italy; therefore, the study covers nine administrative regions in the service area of the Consulate General of the Republic of Turkey in Milan, located in the north of Italy. These regions are (1) Lombardia, (2) Valle d'Aosta, (3) Liguria, (4) Piemonte, (5) Veneto, (6) Trentino Alto Adige, (7) Emilia Romagna, (8) Marche, (9) Friuli Venezia Giulia ( Figure 1). According to the information received from the Turkish General Consulate at the beginning of the project, in May 2015, approximately 29,000 citizens had been at the consulate for various consular procedures; however, it is not possible to obtain a concrete number of citizens residing in the functional area of the General Consulate. However, this number was estimated to be approximately 30,000 people by the employees of the Consulate General. The net number of Turkish citizens recorded in the voter roll through address declaration to the Consulate General of the Republic of Turkey in Milan was 10,373 (18 years old or older) as of May 2015, when we started the study. The regions that they were living in and the respective resident numbers of the 19 ject, in May 2015, approximately 29,000 citizens had been at the consulate for various consular procedures; however, it is not possible to obtain a concrete number of citizens residing in the functional area of the General Consulate. However, this number was estimated to be approximately 30,000 people by the employees of the Consulate General. The net number of Turkish citizens recorded in the voter roll through address declaration to the Consulate General of the Republic of Turkey in Milan was 10,373 (18 years old or older) as of May 2015, when we started the study. The regions that they were living in and the respective resident numbers of the 19
Comprehensive Questionnaire: The Size of the Sample
There was no information on the socio-demographic status of the overall Turkish community. According to information obtained in May 2015, 10,373 residents out of 19,936 are registered voters in the Consulate General of the Republic of Turkey in Milan. Based on this number, the sample size was calculated as 544 individuals ( Table 1). The number of families living in the region is unknown, so we used the number of registered voters to decide on the size of the sample. While preparing the questionnaire, the questions were designed to understand the socio-demographic characteristics of migrants, their disaster experience, their awareness of disaster risk and their preparedness level. The questions in the last section of the questionnaire were prepared to gather more information about the Turkish community's socio-demographic characteristics, such as the gender, age, educational status, and language skills of the sample who participated in the comprehensive questionnaire study. A set of
Comprehensive Questionnaire: The Size of the Sample
There was no information on the socio-demographic status of the overall Turkish community. According to information obtained in May 2015, 10,373 residents out of 19,936 are registered voters in the Consulate General of the Republic of Turkey in Milan. Based on this number, the sample size was calculated as 544 individuals ( Table 1). The number of families living in the region is unknown, so we used the number of registered voters to decide on the size of the sample. While preparing the questionnaire, the questions were designed to understand the socio-demographic characteristics of migrants, their disaster experience, their awareness of disaster risk and their preparedness level. The questions in the last section of the questionnaire were prepared to gather more information about the Turkish community's socio-demographic characteristics, such as the gender, age, educational status, and language skills of the sample who participated in the comprehensive questionnaire study. A set of questions were designed to understand the citizens' experience of disasters, disaster preparedness and mitigation actions. The third set of questions were designed to learn more about the participants' preferences for communication media and how often they use them. We wanted to select the most used communication tool to raise awareness about the risks of disasters in their regions of residence by sharing information leaflets and video messages.
The classification used in the survey is as follows: For the classifications with "*", the unit of analysis is individual. For "socio-demographic analysis", the unit of analysis is family. The last page of the questionnaire was composed of questions about the socio-demographic characteristics of the family members living in the same house, such as the number of people living in the house, their ages and education levels, and the languages that the family members speak at home to communicate. During the questionnaire, researchers were present at the site to help the individuals to fill in the questionnaires. In addition to questionnaire forms, we prepared visuals to support the respondents in understanding the questions. Five hundred and forty-four individuals filled out the questionnaires, and 525 families were represented in the study. When the individual disaster experience was different, we let more than one family member fill out the questionnaire. We stapled the questionnaires together when multiple family members filled out the questionnaire, and only one family member filled out the last page. When we calculated the number of family members, we found that we had the socio-demographic data of 1785 individuals.
Focus Group Meetings
We conducted focus group meetings in four different locations (Milan, Lecco, Como and Varese), considering the location and diversity of the Turkish migrants. In total, 49 migrants attended the focus group meetings (for details, please see Appendix A). We decided the contents and locations of the focus group meetings considering the results of the questionnaires. The purpose of the focus group meetings was not to compare the results with the questionnaire, but to gain in-depth knowledge on the awareness, needs, feelings, beliefs, behaviour patterns in a possible emergency, and priorities of various groups. At the beginning of each meeting, the primary investigator (PI) welcomed the participants, introduced herself and the project and the setting of the focus group meetings, mentioned the rights of the participants, and participants signed the consent forms that included information on the study and the participants' rights. Then, the participants were asked to introduce themselves. During focus group meetings, participants were asked ten questions. The meeting began by asking the participants to define "what is a disaster according to them". After these ten questions, participants were asked if they wanted to share anything else or whether they had questions for the researcher or not.
The focus group questions are as follows: • What is a disaster? Please specify. • Do you think that an environmental disaster, such as a flood or earthquake, will happen to you? During the focus group meetings, various hazard maps, disaster photos, and newspaper columns regarding the past events were shared with the participants. The severity and probability of the reoccurrence of these events were discussed to a large extent. In this way, we aimed to attract the participants' attention and carry out a more collaborative and interactive discussion. Furthermore, they had been encouraged to enhance their resilience in disaster risk management. Two MSc students assisted the PI during the focus group meetings. The focus group meetings were as follows; for more details, please consult Appendix A.
Ethical Considerations and Data Management
Ethical aspects were at the centre of our study. We obtained the necessary permissions from the General Consulate of the Turkish Republic in Milan and the Turkish Republic Supreme Election Council to conduct the questionnaires during the parliamentary elections. We ensured honesty and transparency towards research subjects involved in several stages of the study, such as face-to-face questionnaires and focus group meetings. Participants voluntarily engaged in the study, and they were given the project's informed consent form and detailed information sheets in advance. The consent forms were in Turkish. We had two participants who required translation of the documents to Italian, and we translated all the documents for them to Italian. The consent form explicitly stated that participation is voluntary. Anyone has the right to refuse to participate and to withdraw their participation, samples or data at any time without any consequences. Participants gave their consent by signing a separate form from the questionnaire, as the questionnaires were anonymous. We did not collect more data than were necessary to reach the research goal. All data were handled in a manner that respected the rights specified in the agreements (informed consent and transfer of intellectual property).
Results of the Questionnaire
The results of the questionnaire set out the socio-demographic characteristics of the Turkish migrants living in northern Italy. First, the majority of the participants (60% men and 40% women; sample size 544 individuals) reside in the Lombardy (Milan, Como, Lecco, and Varese), Emilia Romagna (Modena and Bologna) and Liguria (Imperia, Turin) regions ( Figure 2). As for the age group of the participants, the highest number of participants was in the 25-34 age group with 38%, and this was followed by the 35-44 age group, with 29% ( Figure 3). As for the employment status, 53% of the participants were employed full-time, 11% were students, and 17% were housewives, of which 95% came to Italy due to marriage (Figure 4). • Socio-demographic characteristics As for the age group of the participants, the highest number of participants was in the 25-34 age group with 38%, and this was followed by the 35-44 age group, with 29% ( Figure 3). As for the employment status, 53% of the participants were employed full-time, 11% were students, and 17% were housewives, of which 95% came to Italy due to marriage (Figure 4).
•
Socio-demographic characteristics As for the age group of the participants, the highest number of participants was i the 25-34 age group with 38%, and this was followed by the 35-44 age group, with 29% ( Figure 3). As for the employment status, 53% of the participants were employed full-time 11% were students, and 17% were housewives, of which 95% came to Italy due to marriag (Figure 4). Regarding the educational status, the majority were high school graduates, with 30% followed by elementary school graduates, with 27%. Some participants had never been t elementary school or had left elementary school. Among them, we encountered two illit erate women (sample size: 544 individuals).
The participants were asked questions to comprehend their level of linguistic skills It was observed that all of the participants can communicate in Italian to various extents Overall, 17% expressed their capability to handle daily tasks with the level of Italian tha they speak, whereas 45% of the participants were confirmed to have a good understand ing of the Italian language. Two participants were observed as being as non-Turkis speakers during the questionnaire. More than 40% of the participants can speak one mor European language in addition to Turkish and Italian. The majority indicated English a Regarding the educational status, the majority were high school graduates, with 30%, followed by elementary school graduates, with 27%. Some participants had never been to elementary school or had left elementary school. Among them, we encountered two illiterate women (sample size: 544 individuals).
The participants were asked questions to comprehend their level of linguistic skills. It was observed that all of the participants can communicate in Italian to various extents. Overall, 17% expressed their capability to handle daily tasks with the level of Italian that they speak, whereas 45% of the participants were confirmed to have a good understanding of the Italian language. Two participants were observed as being as non-Turkish speakers during the questionnaire. More than 40% of the participants can speak one more European language in addition to Turkish and Italian. The majority indicated English as the most widely spoken language among them. French, German and Spanish followed English in this classification (sample size: 544 individuals).
Furthermore, 79% of the participants declared that they speak another language, such as Kurdish or dialect, apart from Turkish, Italian and another European language (sample size: 544 individuals). The participants stated that they speak Turkish, Italian and Kurdish sequentially in their homes. They strongly support the idea of multilingualism by bringing up multi-lingual children who can speak Turkish, Italian, Kurdish and at least one other European language ( Figure 5). • Disaster experience (sample size: 544 individuals) Regarding disaster experience, 31% of the participants confirmed that they had experienced earthquakes and 4% had experienced floods in Italy to ranging extents. In all, 3% of the participants stated that they experienced both disasters ( Figure 6). In particular, the participants from Modena and Milan had incurred monetary and property losses due to earthquake and flood disasters, respectively. One family mentioned that they did not ask for funding from the Italian government as they were not aware of such a mechanism. One person from Modena declared that many families living in Modena returned to Turkey after the occurrence of the Modena Earthquake in 2012. Anxiety about natural hazards was identified in 63% of the participants. While 11% of the participants declared "excessive anxiety", 52% of them expressed "anxiety" in characterising their level of concern against disasters.
•
Disaster preparedness (sample size: 544 individuals) The majority of the participants were opposed to being self-prepared for disasters, propounding the lack of self-preparedness in Italian society. Even if the participants were quite conscious of the drawbacks of unpreparedness, surprisingly, the overwhelming majority were reluctant to take preventive actions. The participants who had been exposed to disasters in Turkey were perceived as being more susceptible and more predisposed towards the behaviour of "preparedness".
Only 23% of the participants informed their children about how to act during a natural hazard. Overall, 71% declared their ignorance about how to use "Fire Extinguisher" equipment. Meanwhile, 92% of the participants stated being self-conscious to switch on/off the gas, electricity, and water valves. In all, 87% expressed that they keep their important documents such as passports, insurance and deed papers in somewhat safe places. Overall, 83% of the participants admitted not having an "Emergency Kit" in their home, while 17% do have a "First Aid Kit". More than 50% of those maintaining a First Aid Kit confessed their ignorance in keeping the necessary medical supplies up to date.
•
Potential behaviour of the respondents during disasters (sample size: 544 individuals) Overall, 83% of the respondents declared not having planned where to reunite in case of an emergency. As a response to the "Where would you prefer going if you were supposed to leave Italy in case of a disaster?" question, while 64% of the participants indicated "Turkey", the remaining 36% answered "other cities of Italy or Europe" based on the relocation of their extended families.
•
Disaster awareness (sample size: 544 individuals) We presented seven disaster scenarios, including earthquake, flood, drought, snowstorm, pandemic, climate change and fire, to the participants. They were asked to classify them from the most probable (1) to the least (7). Participants declared "flood" as the most likely disaster to occur and "drought" as the least likely one in categorising the disasters for the area of interest.
•
The most used communication media tool With the help of this study, we wanted to raise the awareness of Turkish citizens about the risks of natural hazards in their vicinity and enhance their resilience in disaster risk management. For this reason, we asked participants a couple of questions to better understand the most common means of communication to convey "awareness-raising" messages. The responses of the participants showed that not everyone has a smartphone and continuous internet connection. The best means of communication was found to be "SMS" to deliver messages. The participants were asked which social media networks they use the most. More than 400 participants declared having a Facebook account and using it actively in their everyday lives. Therefore, a Facebook account was activated to inform the participants about the recent developments on the topic. We kept the Facebook account active for three years.
During the questionnaire, special topics on which the participants lacked sufficient information in disaster management were revealed, and informative leaflets were prepared to provide accurate information regarding these topics. The leaflets were distributed to the public in the General Consulate of Turkey in Milan. In addition to that, the researchers are currently in collaboration with "Search and Rescue Association" (AKUT) in Istanbul, Turkey, to provide the most accurate responses to the questions such as, "What is a family disaster plan? What are the essential components of an Emergency Kit? How to act during a flood?". The responses obtained from AKUT Team experts are published periodically as a series of videos via the project's Facebook page.
The Results of the Focus Group Meetings
The questionnaire revealed the spatial dispersion of the participants in northern Italy. Most of the population had been identified as settling down in Lombardy (Milan, Lecco, Como and Varese), Emilia-Romagna (Modena and Bologna) and Liguria regions (Imperia). Therefore, we decided to conduct focus group meetings in the Lombardy Region.
During the focus group meetings in Milano, Como, Lecco and Varese, all participants actively participated in the group discussion. We started each focus group meeting with the question of what a disaster is. The generally agreed on definitions are "loss of property", "loss of life", "material loss or damage", and the need for evacuation. During the focus group with women, they defined the disaster as "migration itself is a disaster" and "being prone to Islamophobia" ( Table 2). Table 2. Summary of focus group meetings.
Questions Answers and Reactions
What is a disaster? Please specify. Loss of property, loss of life, material loss or damage, the need for an evacuation, being a migrant, Islamophobia Do you think that an environmental disaster will happen to you?
Most of them said no. There was a difference between the ones who had already experienced an earthquake or flood event and the ones who had never experienced one During an emergency/disaster, how do you reach the information that you need?
Asking my neighbour/friend and family member or calling 112. Participants were not aware of any of the information websites that we shared with them (Information resources covering the regions that they are living were shown to the participants in terms of visual materials and was asked) Do you know these sources of information?
All of them said no How would you be aware of these resources? How do they reach you? Social media (Facebook) and SMS What can you do to protect yourself and your family from a disaster?
The participants in the Varese focus group meeting were very well prepared If you experience a disaster/catastrophe, will you go back to Turkey?
The participants discussed this question, and their final answer was a "yes".
Most of the participants had experienced an earthquake or a flood event in Turkey or Italy. Most of the participants stated that a disaster could happen at any moment; some had a fatalistic approach. The participants in Varese experienced the 1999 Izmit earthquake in Turkey, and one of them was in the earthquake's epicentre. They were still feeling the impact of the event. This group's awareness level was the highest, and they conducted several emergency drills at their home with their children.
It was clear that the priorities during an emergency and reactions to the situation change according to gender, age, and family presence. The first reaction of women was bringing the family together; the first reaction of men was to understand what's happening and the extent of the disaster. On the other hand, all students said that the first thing they would do is reach out for their passports and cash.
Most of the women are dependent on their husbands and do not speak Italian. This linguistic incapability creates a barrier for adaptation, isolates them from local society and increases their vulnerability. They seek word of mouth information and communicate with their neighbours or friends who speak the same language. The focus of mothers is their children. They told us that, first, they would seek their children, and after finding them, they would call their husbands for help.
On the other hand, migrants are tightly connected. Their social network is the main resource, especially those isolated due to the language barrier. However, it is still not possible to conclude that the strong sense of community provides resources that make them resilient in the long run, as in some cases, being isolated might be a barrier to reaching out for essential information and resources.
Discussion
The "City, Migration and Disaster" study was set out to explore the environmental disaster risk awareness of the Turkish community living in the region, as well as their socio-demographic structure. Indirectly, in practice, the study raised awareness of the Turkish migrants and referred them to sources to increase their knowledge and awareness of disaster risk.
Similar to findings from other research, our study confirms a lack of preparedness and a more general lack of interest in preparedness actions. This is aligned with the "invisible" framing arguments [9,10] and can be related to a lack of involvement in disaster decision making processes. This causes a low level of awareness despite the participants living in Italy, which is a country prone to natural disasters, for 10-20 years. Notwithstanding low interest in preparedness, the level of risk awareness with regard to natural hazards seems quite high, since floods and earthquakes were deemed as the most probable risks.
Additionally, our study confirms the role of past experiences in disasters, since the participants who were exposed to disasters in Turkey are perceived being as more susceptible and more predisposed towards the behaviour of "preparedness". However, as shown by Becker et al. [32], the experience-preparedness relationship is a complex one, and may differ in relation to hazards and the socio-economic status of the person. Nonetheless, the importance of past experiences cannot be underestimated, as it is a determinant of future actions and of reslience as well [33][34][35]. The importance of having a multi-faceted approach was also confirmed, as gender and cultural differences seemed to emerge: for instance, concerning gender differences and priorities during the response phase. Moreover, as stated in the introduction, such an approach would be a pre-condition for ensuring proper inclusion in disaster risk reduction (DRR). This seems to be corroborated (even if indirectly) by answers to questions about disasters in general. For "what is a disaster", most of the respondents did not mention a specific hazard but rather referred to, e.g., "migration", "Islamophobia". DDR policies should take into account socio-cultural differences in perceiving disasters.
In line with the findings of this study, we do not seek to label migrants as 'vulnerable', as they have unique capacities that could increase their resilience. The results confirm the studies of Fussell et al. [12] and Guadagno et al. [21], proving that social networks can be a resource in a disaster for migrants. If social capital is important, social competence is also crucial to enhance resilience. In the focus groups, social compentence emerged in relation to the priorities and needs during an emergency, since male participants would rightly call 112. The discussion in the focus group meetings was in line with the Uekusa and Mattheweman [20] study that stated that struggling with the existing inequalities in their daily lives makes migrants resilient. Overall, the results of our study show that there is a high potential for resilience that seems to emerge through some key resilience dimensions that vary from prior experiences with disasters to social capital and competence.
It is also possible to relate the findings of this study with coping mechanisms for trauma. During the focus group meetings, we observed that participants tend to make decisions based on their previous experiences and having a family or not. Additionally, not being attached to the place provides them with the freedom to move in the case of a disaster, but being a part of a close-knit community is one of the main mechanisms to cope with disasters such as floods and earthquakes. More studies can be conducted to relate the findings further with the "Nudge Theory" to improve the resilience of migrants [36][37][38]. The findings from our study suggest that understanding cultural barriers is key for disaster preparedness. Without proper linguistic skills, it is impossible to ensure disaster preparation across all phases of the disaster cycle (mitigation, preparedness, response and recovery).
Conclusions
In this study, we investigated the socio-demographic characteristics, risk awareness, participation in development, prevention and mitigation strategies, education programs, capacity to invest in mitigation, access to flood information and training/experience of the population, perception and awareness of risk condition, awareness of education programs, individual preparation, and understanding of the ways to access flood information among the Turkish migrants living in the area.
In this study, our target group was legal migrants older than 18 years old. However, some marginal groups might be more vulnerable than our samples, such as illegal migrants and close-knit communities that we could not reach out to to conduct focus group meetings. We completed 544 questionnaires with respondents living in nine regions in northern Italy. We limited the geographical focus to the Lombardy region during the focus group meetings because of the high number of Turkish migrants living in the area. The survey was conducted in 2015; the results presented here might be considered "old", but the results can inform future studies on the Turkish community in other European countries. Researchers may benefit from the methodological approach and the findings. For instance, policymakers may be interested in understanding socio-cultural dimensions that should not be overlooked in DRR processes and policies. Moreover, the results are aligned with the findings of previous studies in other countries.
The project succeeded in drawing great attention from both the affiliated institutions and the public, who voluntarily and actively participated in each project stage. It was carried out mainly in the Lombardy region due to limited time and resources. Nevertheless, the project has the opportunity to be extended to cities such as Modena and Imperia, where a remarkable Turkish population that needs to be informed about earthquake and flood risks in their area of settlement is present. Moreover, the project offers insights for further research on the Turkish communities in other European countries. The recent flood events in July 2021 in Belgium, the Netherlands and Austria proved the importance of conducting such studies in hazard-prone areas with a large number of migrants. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. The consent forms were in Turkish. We had two participants who required translation of the documents to Italian, and we translated all the documents for them to Italian. The consent form explicitly stated that participation is voluntary. Anyone has the right to refuse to participate and to withdraw their participation, samples or data at any time without any consequences. Participants gave their consent by signing a separate form from the questionnaire, as the questionnaires were anonymous.
Data Availability Statement:
We did not collect more data than were necessary to reach the research goal. All data were handled in a manner that respected the rights specified in the agreements (informed consent and transfer of intellectual property). Collected data is anonymized and not open source. The data report was prepared in Turkish and submitted to the funding body at the end of the project.
Acknowledgments:
The authors extend their greatest appreciation to the Republic of Turkey Ministry of Culture and Tourism Presidency for Turks Abroad and Related Communities, the General Consulate of the Turkish Republic in Milan and the Turkish Republic Supreme Election Council for the permission and support during the survey, the migrant community who participated in questionnaires and focus group meetings, and AKUT Search and Rescue Association for their informative videos on various hazards and emergencies. The authors would like to extend special thanks to the four MSc Students who supported the research during the fieldwork. Berrak Balcı and Onur Sagır supported the study by conducting the face-to-face questionnaires. We would like to extend our gratitude and thanks to Burcu Koçoglu and Zehra Irem Turksezer for their dedicated support. They conducted the face-to-face questionnaires, took notes during the focus group meetings, and transcribed the recordings after the meetings. In addition, we thank them for their effort to translate the PI's final report from Turkish to English. We included some parts of the translated document in the manuscript. The authors thank Scira Menoni for her support during the "City, Disaster and Migration" Postdoc project. We thank the two reviewers for their valuable comments on the first version of this article.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the study's design, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. The content is the authors' sole responsibility, and the views expressed here are of the authors and not the funding institution. | 2021-10-20T16:39:10.811Z | 2021-09-10T00:00:00.000 | {
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252443227 | pes2o/s2orc | v3-fos-license | Green Public Procurement for Accelerating the Transition towards Sustainable Freight Transport
: Requests for emission reduction in the freight transport sector will be more intense in the coming years. One possible strategy to reduce emissions from freight transport is through utilising zero emission vehicles, which requires substantial investments both by transporters and by authorities. This paper examines how green public procurement (GPP) can be used to push the market in an environmentally sustainable direction. For this purpose, interviews with both public authorities and freight service providers are conducted. The results show that GPP is considered a useful tool for public authorities to both boost the uptake of zero emission vehicles and to share the investment costs with freight service providers. However, our study shows that there are differences between small and large municipalities. Moreover, to succeed with GPP, public authorities must prioritise such tasks in their daily routines through political decisions and strategies. Additionally, barriers related to financial possibilities are crucial to handle, as public support schemes are important to reduce costs for all involved stakeholders. Altogether, our paper shows that with the right tools and willingness among both public and private stakeholders, GPP can contribute to the use of more environmentally friendly solutions in the freight transport sector.
Introduction
Transforming an entire mobility system in a more environmentally sustainable direction requires attention to the many different domains that transport systems comprise. Transitioning personal mobility has already been well explored by scholars in regard to electric cars [1][2][3], while the hard-to-abate shipping sector has been increasingly recognised as a paramount research objective [4][5][6]. One transport domain still insufficiently recognised in research on sustainability transitions is road-based freight transport. Despite accelerated technological innovation within freight transport [7][8][9], it has yet to be explored as a transition site. This paper aims to contribute to enhance knowledge in this area by turning to the Norwegian freight transport sector.
The freight transport sector is characterised by a wide range of companies and operators, both international and domestic, targeting wider or more niche parts of the logistics market, and applying a variety of business models and contracts. On a national level in Norway, road transport in 2020 represented 17% of greenhouse gas (GHG) emissions. Heavy vehicles and vans accounted for 49% of these emissions. Other negative side effects of freight transport are traffic noise, congestion, air pollutions and traffic accidents, which are closely related to vehicle milage travelled [10]. Like any other transport sector, freight transport poses a series of climate and environmental challenges, particularly in cities, that have increasingly compelled local authorities to incorporate freight into climate and planning strategies [11]. However, freight service providers are mainly aware of their own emission, and several large actors have incorporated their own goals for low or zero emissions operations within their self-defined time horizon.
The European unions' (EU) White Paper [12] and the following EU's working document entitled "A call to action on Urban logistics" [13] highlights the need to be aware of logistics and freight transport, thus being a part of public planning processes [14].
Green public procurement (GPP) is an instrument used by public actors to purchase goods and services with a lower environmental impact in their lifetime than goods and services otherwise bought. Green public procurement in the EU is not mandatory, but with several policies, the EU seeks to stimulate for the use of green procurement. In 2016, they launched the Handbook of GPP, which may be applied as a guide for buying green [15]. In Norway, however, green public procurement is one of several strategic instruments for reducing emissions and achieving Norway's environmental goals [16].
In the European context, public procurements have increased in the last 20 years, and now constitute 14% of the EU's gross domestic product (GDP) [17]. In Norway, public authorities spend approximately 600 billion NOK annually on public procurements, and the products and services procured by public authorities represent 16% of the national total emissions [16].
As procurers of large volumes of goods and services, public authorities are able to move the priorities of commercial freight service providers in a greener and more sustainable direction. Although ambitions have been defined at both an international and national level, procurement processes on regional and local levels play an important role in fulfilling the ambitions for sustainability transitions. Along Oslo's Climate strategy [18], the municipality has developed a procurement strategy, as they want to use public procurement as a powerful tool to accelerate the green shift, climate goals and circular economic thinking in accordance with ambitions of being an emission free city [19]. This requires procurement processes that strengthen the competitiveness of climate-and environmentally friendly solutions with a small environmental footprint, high expected quality, long service life and good recycling opportunities [20].
Norwegian green public procurement is anchored in national legislation (e.g., [21]) that obliges public administrations to establish procurement practices that enhance sustainability; for instance, by requiring specific technological solutions or setting specific emission limits for the products or services that are procured. Although green public procurement is increasingly emphasised as a climate measure, The Office of the Auditor General in Norway has concluded that current public procurement practices are not contributing enough to diminish GHG emissions and to provide the use of climate friendly solutions. They also point to a lack of holistic approaches for procurement, which are needed to take sufficient environmental actions [17].
The paper delves into the governance of sustainability transitions in freight transport by studying practices with green public procurement. Specifically, this paper explores preliminary experiences with green public procurement in freight transport and discusses how such practices should be developed to accelerate sustainability transitions in freight transport. The study focuses on procurements where freight transport is a result of the procurement, but not the main purpose. To do so, we study practices and perspectives among Norwegian freight service procurers as well as freight service providers through qualitative interviews and nation-wide surveys.
In the following section, we introduce green public procurement as a governance instrument for progressing sustainable technologies in freight and provide an overview of existing knowledge about green public procurement in sustainability transitions. We then account for the methods and data used to explore the research questions in Section 3. We present the results in Section 4, following a discussion based on the results in Section 5. Finally, we conclude in Section 6.
Transition through Green Public Procurement
The governance of sustainability transitions is an increasingly important research topic, as the pace of transition depends on the degrees to which transitions are managed or incentivised [22]. Within the field of sustainability transitions, Transition Management has been particularly prominent in devising governance strategies for steering and accelerating transitions in different sectors [23,24]. Transition Management is a "prescriptive framework" that allows for creating "space for short-term innovation and develop long-term sustainability visions linked to desired societal transitions" [25]. Specifically, Transition Management points to particular governance activities (i.e., strategic, tactical, operational, reflexive) that allow for dealing with the complexity of sustainability problems and the societies that they are enmeshed in. Within the framework of Transition Management, green public procurement (GPP) could be an example of tactical governance activities, which intervene into established structures such as rules and regulations, routines, and institutions.
Within the scholarly field of sustainability transitions, green public procurement could be considered an expression of Strategic Niche Management. This field of research tends to focus on two ways in which emerging technologies and innovations can diffuse and become competitive in existing markets or become dominant in new niche markets. Scholars of Strategic Niche Management are thus concerned with how emergent innovations could be nurtured and matured in protective spaces that shield them from market competition until they are sufficiently competitive [26]. Thus, green public procurement could also be a fruitful strategy for accelerating transitions when following ideas of Strategic Niche Management. Green public procurement could nurture niche innovations by creating markets for specific technologies or for any technologies that reduce emissions. Green public procurement is therefore an example on how sustainable transitions could be steered on low-level governance.
The potential for stimulating sustainable innovations and technological changes by public procurement is high in the transport and logistics sector. The sector is undergoing rapid changes due to digitalization in the supply chain and forcing changes in the individual behavioural pattern such as increased online shopping, home delivery services, and new consumer patterns. It is therefore a golden opportunity to link green public procurement to the transport sector and identify its possibilities and challenges as a tool towards achieving CO 2 -free city logistics by 2030 [13,18,27].
Ref. [28] has investigated the success factors in implementing GPP and identified four criteria to succeed with the implementation of GPP: (1) consistent and operational policy goals; (2) nation-wide campaigns for GPP; (3) ethics, professionalism, capacity, and knowledge among employers; and (4) systems for checks and balances among actors in the entire purchasing process. Ref. [29] has examined GPP in Swedish municipalities and observed that most previous measures to curb emissions from transport in urban areas focussed on passenger transport, and that urban freight has almost been forgotten. Ref. [29] also mentions that there are exceptions, and that knowledge from the few studied municipalities might be transferred to other municipalities to accelerate the transition towards more sustainable freight transport. Ref. [30] identifies significant differences in GPP uptake between countries and national levels: the countries working most with GPP are characterised by a large governmental sector, and municipalities and local administrations are more prone to work with GPP than authorities on EU and national levels.
Ref. [31] has developed a method for GPP, which was tested on public procurers of transport in Sweden. This method is a participatory method, seeking to enhance the procurer's understanding and knowledge about practical needs. Their results emphasise that the participatory method and improved insight for the procurer's contribute to broad environmental assessments, enhancing green procurements and to a more equitable assessment of alternative technologies. With Rotterdam and The Hague as examples, [32] studied public procurement as a driver for more sustainable freight transport. They state that it is hard to determine the exact number of trips that are generated from procurements, and that procurements are first applied as an administrative activity, and not as an activity to meet policy.
Green Vehicles in Freight
When choosing a vehicle, transporters consider criteria such as the size of the vehicle, its capacity, the range, fuel usage, etc. such that they match with their requirements. With the new technological advances, use of battery electric vehicles is supported as a measure to decarbonise freight transport, especially in urban areas in which transport is characterised by short distances, many stops and low driving speed [33].
Studies show that despite the high investment cost of battery electric freight vehicles, they are cost competitive with conventional diesel vehicles [34]. However, in some cases, transporters need to implement measures such as reducing the scope of service, mixing the fleet with conventional vehicles, using opportunity charging, etc., to increase the profitability of EVs as a compensation to high investment costs [35,36]. Moreover, external factors such as the availability of charging stations on public spaces [37] or exemption from paying road-toll or parking fees [38] are also recognised as influential factors in promoting the use of BEVs in urban freight transport.
Norway has been successful in introducing policies for boosting the uptake of EVs in passenger transport. However, the incentives such as financial support schemes administered by the public agency Enova are less comprehensive for the BEVs in freight. Exemption from value added tax and discounts for registration taxes are already offered for fossil fuel vehicles [39] and support for purchasing the vehicles of establishing charging infrastructure covers a subtle part of the investment costs [40].
Considering the limited options to offer as financial support for freight transporters in Norway, the role of Green Public Procurement is highlighted as a tool to accelerate the transition towards more sustainable freight transport. However, the barriers for effectively using the tool to promote zero emission vehicles into freight system is not well studied in the literature.
This paper aims to fill this research gap by investigating the potentials of green public procurement from both procurers and transporters points of view. It highlights the important factors to succeed with the GPP as a tool for transitioning towards more environmentally sustainable freight transport and how it is perceived both by public officials and transporters by providing empirical data.
Materials and Methods
This study is based on a combination of qualitative (interviews) and quantitative (survey) methods. Qualitative methods were used to shed light on the topic and gain deeper insight into whether and how environmental criteria are used in public procurements within the Norwegian municipalities today. Interviews were chosen because they allow us to interact with the informant and explore the reasoning behind their answers. Quantitative methods, here represented by surveys, allow us to examine if specific findings from the interviews are also viable in a broader context. Table 1 shows the number of interviews and respondents in the respective survey used in the further analyses. Two of the municipalities are regional centres and two are smaller municipalities. The interviewees from public procurers were staff responsible for procurement processes in the respective municipality. Two of the three interviewees from freight service providers operate in the business-to-business market, while the third also delivers to private households. The informants were persons with responsibilities for vehicle fleets, daily businesses, and submitting tenders for public procurements. Interview guides were developed based on national documents [41,42] and the Public Procurement Act [43]. The interview guide for municipalities revolves around the following topics: (1) strategy and collaboration, (2) experience with public procurements, and (3) public support schemes and national goals. The interviews with freight service providers highlighted the following themes: (1) technology, (2) logistic solutions and (3) policy to explore drivers and barriers impacting the freight operators in the transition of urban freight. The interviews were conducted as digital meetings
Interviews
The results from the interviews were further used as an input to design survey questions, as well as a basis for analysis in this study.
Surveys
Two separate surveys were designed and distributed among potential participants: (1) survey for public procurers distributed among municipalities' staff, and (2) survey for freight transport providers distributed among transporters. The design and distribution of the two surveys are briefly explained in this section.
The survey for municipalities is structured according to the same three main topics as in the interviews, while the questions are reformulated to fit into the format of a survey. Findings from the interviews were used to create response categories. The survey was distributed via e-mail to all 356 Norwegian municipalities with a request to forward it to administrative personnel responsible for or experienced with procurements. A reminder was sent out to non-response municipalities after two weeks. In total, we received 71 individual responses from 61 different municipalities, implying that the request had been forwarded to more than one person in some municipalities, and both had responded. However, as the roles and the answers from the same-municipality respondents are not identical, all responses are included in the analysis. There was no record of more than two answers from identical municipalities. Although only 46 participants responded to all the questions in the survey, all 71 responses were included in the analysis since those who were not able to complete the survey also provided useful information about why they did not include requirements for low or zero emission solutions.
Using the classification of municipalities by population size from Statistic Norway [44], and comparing to national statistics for 2022, we found that the smallest municipalities were somewhat underrepresented, and the largest somewhat overrepresented in the survey sample, while the remaining categories were proportionally represented ( Table 2). The informants represent a range of roles related to public procurement ( Table 3).
The survey among freight service providers was distributed to the members of the Norwegian association for lorry owners (NLF), targeting freight service providers at different levels. In the survey, we asked whether they submit tenders for public procurements, together with other questions about public procurements, how they relate to it, and their opinion on its effects on sustainable transitions. The survey was initially distributed to over 3000 members of the NLF, of which 220 responded. Roughly one third (39%) of the respondents provide services for the public sector, and one out of four (27%) are personally involved in tenders for the public sector ( Table 4). The results presented in this paper are based on responses from companies which do provide services to the public sector (N = 87).
Results
The results are presented in three sub sections. The first section presents existing requirements on public procurements and how they are currently used. The second section gives an overview of the municipalities' perspectives and their requirements on public procurements. The last section describes freight service providers' perspectives and their obligations for public procurements.
Existing Low and Zero Emission Requirements in Freight Transport
The in-depth interviews and comments in the survey show that there are differences, both in the types of requirements and how requirements are used in practice in the municipalities' procurement process. The most common requirement, related to transport in Norwegian public procurements for goods and service, is for the type of propulsion technology in the vehicles. One reason for this might be its simplicity to require and control, compared to requiring a specific emission level, which is the second most common requirement. Some municipalities also put restrictions on specific types of deliveries: for example, by demanding smaller vehicles, consolidation, or deliveries outside of rush hour. However, all such requirements are considered more demanding, both for the public procurer to control and the freight service provider to document.
There are also variations in which part of the transport leg such requirements apply. Although many goods are produced outside of Norway and therefore include transport activities abroad, the transport-related requirements are mainly only applied for the last mile in Norway. This might also be explained by the tendency to ask for requirements which are easier to monitor, as the transport activities outside of Norway are not under the control of a Norwegian distributor and there might be limited alternatives with low or zero emission options in the market abroad.
The third difference is whether the application of requirements depends on the value of the tender. The results are presented in Table 5. The response categories (specifically values NOK 100,000 and NOK 500,000) are based on answers in the interviews. Although these limits are also viable for other municipalities, the majority using requirements always include them ( Table 5). The motivation for applying value thresholds for including requirements is related both to the possibilities for monitoring what is delivered, and how large the emissions from the transport in that procurement is. The Public Procurement Acts says requirements should be in proportion to both costs and emissions.
The Perspective of Municipalities
The results are cross tabulated with the size of municipalities to see if there are any differences that may depend on the number of inhabitants in each municipality.
Observing the statistics in Table 6 reveals a relationship between adoption of strategies and number of inhabitants in the municipality. Municipalities with a large population are more probable to have an implemented strategy for promoting green public procurement (GPP). Political decisions seem to be more important than the Public Procurement Act from 2017. An important insight from the interviews is that in smaller municipalities, both political will and personal interest in the administrations play more important roles in success with GPP for freight transport. Municipalities were also asked what kind of commodities and services they included as requirements related to sustainable transport. The responses show that requirements apply for all kind of goods, but are more common for office supplies, furniture, consumables, and food.
As shown in Table 7, dialogue with freight service providers is the most common way to gain knowledge about low emission solutions or delivery alternatives. Responses summarised in Table 7 are based on a multiple-response question, therefore the total sum of responses is higher than the number of respondents. Percentages show the share of the respondents choosing each of the alternative responses. The technological maturity for low and zero emission vehicles varies between large and small vehicles [46]. The technology for small vehicles is mature enough to compete with conventional vehicles regarding range and payload. For heavy vehicles, however, the new technologies could currently only be competitive within urban areas. Considering the significantly higher purchase price compared to vehicles with internal combustion engines, it is reasonable to expect that requirements for low or zero emission vehicles would negatively affect the tender price. Results presented in Table 8, however, show that the municipalities do not clearly acknowledge this. A large share of the respondents from municipalities state that they do not know the effect on prices, and the share stating that the price remains unchanged is almost the same the share stating that the price increases due to environmental requirements in tenders. Different public support schemes to cover extra costs have been launched such as Klimasats by the Norwegian Environment Agency or support to infrastructure by the local municipality. This is because there is a difference in price for fossil fuelled vehicles and low or zero emission vehicles. The new Public Procurements Act also tries to speed up the transition in sustainable solutions when it asks public authorities to always examine possibilities to include requirements for low or zero emission solutions. For such support schemes to be useful, they must be communicated to potential users, e.g., freight service providers. According to the responding procurers, the knowledge level among smaller actors must be enhanced (Table 9). It is worth mentioning that Tables 8 and 9 are based on answers from respondents who include requirements for low or zero emission transport in their procurements. Therefore, the number of analysed respondents is lower than Tables 5-7.
The Perspective of Freight Service Providers (FSP)
When it comes to what affects the freight industry's acquisition of vehicles running on alternative fuels, environmental requirements in public procurement are assessed to be significant, although not the most important factor (Table 10). As shown in Table 11, findings from interviews with freight service providers indicate that an orientation towards public clients seems to both affect their experiences with procurements processes and their internal goals for low-and zero emission vehicles. Further findings from the interviews are embedded in discussions where it is appropriate.
Respondents directly involved in providing bids for GPP tend to consider the environmental requirements to represent a challenge for the industry with negative effects on price and quality of services offered, although they also tend to agree that such requirements have a positive effect on sustainable transition (Table 12). Table 11. Findings from interviews with freight service providers.
FSP Experience with Public Procurement Goal
A Experience a growing interest for FSP opinion/knowledge regarding requirements. Poor evaluation and monitoring of requirements.
Zero emission city logistics in 2026 B PP is time-consuming; therefore, they concentrate on the private market. Euro VI fleet C Experience a growing interest for FSP opinion/knowledge regarding requirements. Poor evaluation and monitoring of requirements.
Fossil free vehicles and buildings in 2025 Findings from the in-depth interviews with freight service providers indicate that the largest drawback with low or zero emission vehicles requirements is how it affects the operational routines, and, consequently, the price and quality of deliveries. In addition, freight service providers are experiencing a growing interest for market dialogues with public procurers and are mainly positive to share their knowledge about possibilities and barriers in the market.
The interviews also include statements from the industry about the importance of follow-up routines after a tender has been awarded. Since offered solutions are part of the competitive basis in the procurement, it is important to make sure that the winner delivers according to the tender. If not, other bidders are able to deliver a more cost-effective solution. From the experience of the interviewees, today's follow-up practice has the potential to be improved.
Discussion
As findings from the surveys indicate, a large proportion of Norwegian municipalities are already working with green public procurement (GPP). Today's practices of GPP involving freight services have been presented above and the results indicate potential for further refining of the practices. Part two of our research question is «how should GPP practices be developed further to accelerate sustainability transitions in freight transport? This section starts with a brief overview of the most important issues recognised through the analyses as a basis for the following discussion, which also relates to other studies within the same field.
According to the results, factors such as political decisions, strategies, requirements, capacity/budget, competence, and methods for monitoring/follow-up are the most prominent issues in public procurements of goods and services for accelerating the transition towards a more sustainable freight transport. These issues are important for both public procurers and freight service providers, but in different ways.
Political decisions and strategies are identified as the most prominent reasons for municipalities to work with GPP in this sample. However, there are differences between the larger and smaller municipalities, where the smaller tend to be lagging behind regarding the implementation of GPP. One reason for not implementing the GPP in smaller municipalities could be related to the lack of competence and personal interest. Statements from interviews point to personal interest as often being essential when prioritizing different tasks if public procurements is one of several responsibilities of the respondent. In contrast, the larger municipalities can work with GPP in a more professional way, since they often have designated procurement departments, in line with studies where professionalism and knowledge are mentioned as success criteria in GPP [28].
It is worth noting that a large administration is not mandatory to succeed with GPP. One possibility for empowering the smaller municipalities and enhancing their involvement in GPP could be to increase their access to resources or more efficient use of existing resources, e.g., through inter-municipal collaboration in procurement processes. Some of the municipalities already use networks on a local and/or regional level as a source for acquiring information about low emission solutions and delivery alternatives. Enhanced collaboration through such networks could help smaller municipalities to increase their competency for implementation of GPP. As understood in [16,29], enhancing knowledge through transfer it between actors is important, especially in smaller municipalities. Easy access to or already acquired competence about GPP probably facilitates its inclusion in daily routines. Capacity can also be related to access of vehicles running on alternative fuels. This is mentioned by both freight service providers and small municipalities. Access to appropriate vehicles and corresponding fuelling/charging stations is crucial for succeeding with transitioning of the freight transport.
In addition, our results highlight the importance of dialogue between the freight industry and municipalities for enhancing procurers' knowledge about new solutions and alternatives. At the same time, the procurers indicate that there is room for improvement when it comes to the freight service providers' knowledge of legislation and support schemes. Utilising the dialogue between procurers and the freight industry to provide suppliers with information about legislation and support schemes could be worth exploring.
An active strategy is not merely important for municipalities. Setting strategies and aiming to achieve them requires generating requirements that both encourage and force freight service providers to develop solutions for meeting the requirements. Although the results here have shown that economic incentives are the most important parameter for freight service providers, findings from the in-depth interviews show that the freight service providers working for the public sector have the most ambitious goals for delivering low or zero emission solutions. This may not only be due to the orientation in the market affecting internal strategies, but also due to internal budgets and fleet sizes which reflect the capacity of the companies in terms of both financial and administrational capacity. To become an early adopter freight service provider, they need financial resources, just as municipalities. Requirements for low or zero emission vehicles may therefore favour larger freight service providers already in possession of adequate vehicles. Investments on new technologies or dealing with the risks involved for ordering new vehicles are easier to face for larger freight providers.
Thus, GPP could be combined with public support schemes to secure that both small and large municipalities and freight service providers can engage in the green shift. Public support schemes covering some of the extra expenses often associated with the introduction of new technologies are examples of strategic niche management, which has proved to be a useful tool in transition management based on both our study and previous findings [23,26]. However, many small municipalities state that the funding from current public support schemes is scarce and that it often includes cumbersome application processes. So, to accelerate the transition, and reduce the bias for larger actors, easier application processes, and more financial support to cover for both risks and extra work would be beneficial.
Otherwise, less competitions may contribute to a negative effect on price and quality when fewer bids are delivered, something freight service providers already have reported.
Finally, proper methodologies for monitoring the compliance of requirements are needed to ensure fair competition. It is important from a competitive point of view that the winning freight service provider deliver what is promised. The development of standardised monitoring methodologies may facilitate the engagement of smaller enterprises and municipalities as it reduces the need for follow-up resources. Moreover, increased use of value-based inclusion of environmental requirements, as many municipalities already apply, can also be a measure to accelerate more sustainable transport solutions associated with the larger purchases of goods and services.
The results presented in this paper confide to a Norwegian context, which is characterised by a large public sector, and therefore might not be transferable to other contexts without modification. However, this study sheds light on how municipalities and freight providers perceive GPP, and the success factors related to it. The results reveal that despite highlighting the topic by National legislation, local willingness is essential to effectively include GPP in public procurement. Similarly, there exist tools and networks in a wider perspective, such as the EU, though mixed national contexts. This study presents a useful approach to enhance knowledge about the topic, which can be further used in other contexts.
Conclusions
This study aimed to gain knowledge on GPP, its practices in Norwegian contexts and how it is perceived from both public procurers and freight transport providers. The results show that Norwegian municipalities in all sizes have already started to implement requirements for low and zero emission transport in their procurement of goods and services. However, there are still some barriers to overcome, especially in the smaller municipalities. Building and utilising networks to enhance and disseminate knowledge and resources among municipalities and between municipalities and the freight service operators should be further explored. Future research can probably contribute to enhance knowledge on how to establish attractive networks for smaller actors, providing them with relevant and practical knowledge about GPP. Moreover, carrying out research on GPP in countries with a smaller public sector will also be of huge relevance, since Scandinavian countries are characterized by a larger public sector than many others.
Finally, several barriers are directly or indirectly related to financial resources. Therefore, extended public support schemes adapted to the needs of the smaller actors, both private and public, should be considered to avoid only larger freight service providers being able to compete on procurements, including requirements for low or zero emission solutions. Other relevant measures include simplifying application routines to reduce the work related to the process of applying for financial support for extra expenses related to low or zero emission transport. | 2022-09-23T15:06:33.686Z | 2022-09-16T00:00:00.000 | {
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54478203 | pes2o/s2orc | v3-fos-license | Efficacy of video game-based interventions for active aging. A systematic literature review and meta-analysis
Background Due to the appeal and recent technological advances of video games, the games have gained interest as an intervention tool for active aging. The aim of this systematic literature review and meta-analysis was to determine the efficacy of video games for active aging and to examine the influence of potential moderator variables. Methods A systematic search was done using the following databases: Medline, PsycINFO, EMBASE, CINAHL and the Cochrane Central Register of Controlled Trials. In addition, previous reviews and meta-analyses were used to identify randomized controlled trials (RCT) of video game-based interventions for active aging published through February 28, 2018. An evaluation of the methodological quality of the articles and a meta-analysis and moderator analysis was conducted. Results A total of 22 articles depicting 21 RCT with 1125 participants were included. The results indicated that video game-based interventions produced positive effects on objectively measured physical health, negative affect and social health, with small effect sizes (d = 0.41, d = 0.26 and d = 0.40, respectively). The magnitude of this effect was moderated by the presence of subclinical conditions of participants, the type of game (exergames), the presence of physical activity, the type of prevention (indicated), non-blinded assignation, and older age of participants. The methodological quality of the studies was acceptable, the weakest area being external validity. Conclusion These finding indicate that video game-based interventions may assist adults in leading active aging processes and preventing secondary aging. Although more research is needed, video game-based interventions are a promising and accessible tool for active aging promotion.
Introduction
To the best of our knowledge, no previous systematic literature review or meta-analysis has analyzed the efficacy of video game-based interventions for active aging through a life course preventive perspective, despite the World Health Organization recommendations [3]. Moreover, none of them based their findings on randomized controlled trials (RCT), nor included all kinds of video games or analyzed diverse health areas. The main aim of this meta-analysis was to determine the efficacy of video game-based interventions for active aging from middle adulthood (i.e., � 45 years-old) in RCT. The secondary aim was to identify the specific moderating variables for the efficacy of the interventions.
Methods
This systematic literature review and meta-analysis was developed in adherence with the guidelines by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [18] (see PRISMA checklist at S1 File). The protocol for this review was registered in the International Prospective Register of Systematic Reviews (CRD42018086870) and fulfilled the AMSTAR quality criteria for Systematic Reviews [19].
Search strategy
Studies published through February 28, 2018 were retrieved through systematic literature searches in the databases of Medline, PsycINFO, EMBASE, CINAHL and the Cochrane Central Register of Controlled Trials. The search terms were: (game � OR gami � ) AND (serious OR interactive OR computer OR video OR multimedia OR internet OR Wii OR online) AND (health � OR behav � OR wellbeing OR prevent � OR social OR exer � OR acti � OR edu � OR optimal OR positive OR successful OR engagement OR habit � OR affect � OR mood OR emotion � OR self-efficacy OR self-esteem OR nutrition OR diet OR food OR cognitive OR physical OR mental) AND (adult � OR old � OR eld � OR geront � OR aged OR aging) AND (RCT OR randomi � controlled trial). Further studies were included through hand search, tracking cited references in other studies and relevant previous literature reviews.
Selection procedure
Studies identified in electronic searches, after exclusion of duplicates, were screened for relevance based on titles, abstracts and keywords. Full texts of articles considered relevant were obtained and fulfillment of inclusion and exclusion criteria was evaluated independently by two reviewers. Any disagreement was discussed in a consensus meeting. If consensus was not achieved, a third independent reviewer adopted a decision.
Studies were included if: (a) they were a RCT; (b) they assessed the efficacy of interventions for active aging; (c) the intervention received by the experimental group (EG) was delivered through a video game format; (d) the participants were healthy adults older than 44; (e) they used at least one standardized outcome measure; (f) they reported at least pre-treatment and post-treatment quantitative results that permitted computation of the effect size; and (g) were written in English or Spanish language.
Studies were excluded if they: (a) were pilot, feasibility, preliminary or proof of concept studies; (b) included mixed participants (e.g. young and older adults) without differentiating the results of each group; (c) reported multimodal interventions, not being able to discriminate which outcomes were associated with the video game intervention.
Data extraction
Data of the selected studies were extracted independently by two reviewers using a standardized data extraction protocol and coded based on a coding manual (S2 File) as suggested by the Cochrane Handbook for Systematic Reviews of Interventions [20].
Data coding
Descriptive information extracted from selected studies (when available) was comprised of the following: type of technology/device; name and type of video game (serious, casual, exergame); participants' demographic characteristics (sample size, age, gender, education, civil status, socioeconomic status, urban or rural context and attrition); characteristics of the interventions received by the experimental and control groups (format, duration, number of sessions, presence of professional, individual tailoring, dosage, time of outcome assessment, follow up); outcome measures and findings.
For the purpose of this review, a video game was considered "serious" when it included gaming features aimed to promote behavior change and/or improve health [9]; "casual" when it was used in a leisure context with the sole aim of entertaining and without a specific aim of improving health; and "exergame" if it required physical activity when played [21].
To select primary outcomes, we based on health and social services, behavioral, personal and social determinant factors of active aging in an individual level according to the theoretical model of the World Health Organization [3]. In addition, we focused on health area of action of active aging [22]. Health was defined as a state of complete physical, mental and social wellbeing and not merely the absence of disease [23], and conceptualized as a three domain concept including physical, mental and social health [24]. Therefore, the primary outcomes were a change from baseline to post-treatment of physical, mental and social health domains.
Physical health was divided into objective (e.g., motor functioning, cardiovascular functioning) and self-reported health measures. Mental health was divided into cognitive health and emotional health, as recommended by Duncan and Barret [25]. Cognitive health included the cognitive domains described by Strauss, Sherman and Spreen [26]: executive functioning (working memory, inhibitory control, task switching/flexibility and reasoning/problem solving), visuospatial skills, immediate memory, delayed memory, language, attention and processing speed. Emotional health included positive and negative affect, according to the affective structure established by Watson, Clark and Tellegen [27]. Social health included the capacity to fulfil one's potential and obligations, the ability to manage life with some degree of security and independence despite a medical condition, and the ability to participate in social activities [24]. When the authors did not report a global measure of a particular domain, a composite change score was calculated as a combined average of the mean change (and variance) across all outcomes reported for that specific domain, as suggested in previous metaanalyses [28,29].
To analyze the influence of the characteristics of the studies on the effect sizes, potential moderating variables of participants, interventions, methods, context and extrinsic were coded [30]. Note that the potential moderating variable "type of prevention" distinguished between universal, selective, and indicated prevention, according to the US Institute of Medicine [31]. Universal prevention targets the general population; selective prevention targets segments of the population with an increased risk of developing a disorder because they have been exposed to risk factors; and indicated prevention targets people who have some symptoms of the disorder but do not yet meet the full diagnostic criteria. The moderator analysis was performed for a composite construct, combining the three health domains together (physical health, mental health and social health). See S2 File for a comprehensive list of the moderating variables and the outcome measures used to calculate the composite scores.
To assess the methodological quality of the included studies, we used Downs and Black's checklist [32], which assesses reporting, external and internal validity, bias, confounding variables and power, comprising a total of 27 items and a maximum score of 32. Risk of bias of the selected studies was assessed using the instrument of the Cochrane Collaboration, which evaluates selection (random sequence generation and allocation concealment), performance, detection, attrition, reporting and other bias [20]. Studies were considered of low risk if none of the items were considered as high risk and not more than one item was coded as unclear or not reported. If one or more items were considered as high risk, the risk of bias of the study was considered high.
Inter-rater reliability of the data coding was evaluated using Cohen's Kappa concordance index. It was excellent for the moderating variables coding (Kappa = 0.89), moderate for Downs and Black checklist (Kappa = 0.51) and substantial for Cochrane risk of bias assessment (Kappa = 0.78).
Data analysis
A meta-analysis was conducted with a fixed effect model if there was not heterogeneity (I 2 � 50%) or random effect model if there was heterogeneity (I 2 > 50%) [33] through the Cochrane Review Manager software RevMan 5.3. Publication bias was assessed with the Begg's test. Effect sizes for each meta-analysis were estimated as Standardized Mean Change Index (d MR ) for within-group pre-posttreatment comparisons, and as Standardized Mean Index difference (d) for between-group comparisons at posttreatment [34]. Effect sizes of 0.2 were considered small, 0.5 medium and 0.8 large [35], and outliers were excluded. For those studies that involved more than one experimental group (EG), the same control group (CG) was used for the calculation of separate effect sizes (comparisons between experimental and control groups). For studies reporting more than one CG, data for the control with the most neutral activity (i.e., usual care if available) were selected for computing the effect size. Effect sizes of follow-up assessments were not included in the meta-analysis because they were scarce (only four studies conducted follow ups) and follow up times were not comparable (ranging from 4 to 48 weeks).
A meta-analysis of variance (meta-ANOVA) statistic for categorical variables and a metaregression analysis for quantitative variables (using IBM SPSS software, Version 21.0) was used to examine the contribution of moderating variables to the variance.
Study selection
A total of 661 articles were identified after removing duplicates. Of these, full texts of 44 articles were assessed for inclusion (Fig 1). We requested additional data from authors of 12 studies, of which 8 provided the additional data [36][37][38][39][40][41][42][43]. Twenty-two articles were excluded: two because they were not RCT [42,44]; one because the intervention was not delivered through a video game format [45]; four because the participants were not healthy older adults [46-49]; four because they did not use standardized outcome measures [50-53]; three because they did not report the minimum data necessary to calculate the effect sizes and it could not be obtained from the authors [54-56]; four because they were pilot, feasibility or proof of concept studies [57-60]; and four because they reported on multimodal interventions and were not able to discriminate which outcomes were associated to video games only [41,[61][62][63]. Two articles reporting outcomes from the same RCT were combined into one study [36,64]. Finally, 22 articles depicting 21 RCT (22 EG and 23 CG) were included. Their characteristics and findings are presented in Table 1. ). In addition, the participants had 13.21 mean years (SD = 1.15) of education, and urban provenance [68]. None of the studies provided information about the civil status or the socioeconomic characteristics. Although all the studies included healthy older adults, three of them included participants with sub-clinical conditions such as subthreshold depression [69] or reduced mobility [38,66]. The aims of the interventions were diverse and mainly focused on mental and physical health. Almost half (47.6%) of the studies were focused on improving mental health or preventing cognitive deterioration, 19.2% on improving physical functions and 33.3% were multidomain, targeting physical and mental health. None of the studies focused on social health; however, two studies assessed social-health related outcomes [38,64]. The majority (85.7%) of the studies assessed interventions considered as universal prevention programs, and 14.3% were indicated prevention programs [38,66,69]. Only five (23.8%) of the interventions were designed based on a theoretical model; specifically, the cognitive psychology theoretical models [67,[70][71][72][73].
The 57.1% of the interventions were delivered in individual format, 19.0% in a group format [39,69,74,79], 9.5% in dyads with a partner [40,78], and 9.5% were individual but simultaneously played in the same room as other participants [65,70].
A professional was present in 81.0% of the interventions, but in 28.6% of cases only during the participants' training [37,39,43,67,71,75]; this information was not available in 14.3% of the studies [64,70,79], and in one the intervention was completely self-administered [68]. The type of professional who delivered the intervention was a researcher in 33.3% of studies, and a health professional in 28.6%; 19.0% of studies did not specify the type of professional [37,67,71,73]. Only one study [37] stated that the professionals had been trained, and 47.6% of the studies trained the participants before starting the intervention [37,39,43,64,65,67,[70][71][72]75], through training sessions ranging from 15 to 60 minutes long (M = 43.38; SD = 16.86).
Most studies (81.0%) evaluated the participants only at the end of the intervention. Followup assessments were infrequent and mostly brief. Only 19.0% of the studies conducted a follow up assessment: 9.5% of them at four weeks [37,72], 4.7% at 12 weeks [64], and 4.7% at 48 weeks [70]. Longer term benefits were evident in one study [72], partially present in another study [64], and not maintained in one study [37]. Additionally, in one study, the follow up assessment was only conducted with non-standardized measures [70]. Attrition was assessed in all studies, and ranged from 0% in five (23.8%) [65,68,70,73,79] to 25% in one [64], with a mean of 7.6% of dropouts.
Meta-analysis of the efficacy of video game-based interventions for active aging
Begg's rank correlation test confirmed the absence of publication bias (Kendall's tau b = 0.01; p = .454, 1-tailed). A total of 14 studies assessed physical health; 13 of these were included in the meta-analysis. Of the 14 studies, 10 studies assessed objective measures between experimental and control groups and 4 studies assessed self-reported measures. Only one study that assessed physical health was excluded from the meta-analysis because it was identified as an outlier [73]. This study found that adapted physical activities training (alone or combined with Wii Fit) was more effective than Wii Fit alone at improving the balance of independent senior participants. Additionally, 18 studies assessing mental health were included in the meta-analysis (16 comparisons for cognitive mental health and 12 for emotional mental health), and 2 studies assessing social health were included in the meta-analysis. No study was excluded from the meta-analysis for mental or social health.
Finally, in social health data were pooled from two comparisons [38,64] (with 65 participants in EG and 55 in CG). Participants of the EG experienced higher beneficial effects from video game-based interventions than those from the CG (SMD 0.40, 95% CI = 0.04 to 0.77, p = .03; I 2 = 0%, p = .49) (Fig 5).
Discussion
In this systematic review and meta-analysis, we analyzed the efficacy of video game-based interventions for active aging in adults older than 44. Based on 21 RCTs, it was found that video game-based interventions produced small positive effects on objectively measured physical health, negative affect and social health. These findings are similar to a previous systematic Efficacy of video game-based interventions for active aging review for older adults [81], which found significant mental health outcomes in the majority of the reviewed studies, followed by some physical and social health benefits. However, the results of the current meta-analysis also contradict findings from a previous meta-analysis which reported that video game training enhanced several aspects of cognition in older adults including reaction time, attention, memory, and global cognition [17], although in the current metaanalysis there was a trend in this direction. There are some possible explanations for this trend. Firstly, the conclusions of our meta-analysis may be more rigorous and conservative, because it included only RCTs, while the meta-analysis by [17] included RCTs and other studies. Secondly, while the most frequently assessed cognitive outcomes in the current meta-analysis were global cognition and executive functioning followed by memory and lastly attention and speed of processing, attention and speed of processing have demonstrated to be the cognitive functions that improve the most after video game training [17,68].
Regarding physical health, the benefits of video games are encouraging. They seem to improve some physical health variables in the older adults, for whom aging-related progressive degeneration in muscle strength and balance control system can lead to motor impairment, disability and falls [82,83]. Although the effect sizes found were small, this may be due to the healthy status of the participants. In addition, while objectively measured physical health had significant benefits, self-reported physical health did not. One explanation for this finding is that self-reported measures refer to subjective health issues that are subject to high personal variability. These subjective health issues can also be confounded with changes that occur during normal aging (perceived exertion, perceived health, pain intensity). Furthermore, most of the studies in the current review assessed the same objectively measured variables that were trained during the video game-based intervention, which may have resulted in more positive outcomes. Physical health was assessed as muscle strength [77], balance [38,39,74,76,77], falls [66,75], functional fitness [78] postural control and gait [72].
The positive effects of video games on negative affect and social health are of particular importance as depression and social isolation in older adults are risk factors that double the risk of subsequent dementia [84] and mortality [85]. Previous research has demonstrated that playing video games could lead to greater social interaction, less loneliness, a sense of accomplishment, and positive mood [40]. Our results confirm that video games can play a protective role in this area. However, it is unknown if the quality of mood enhancement and social participation derived from playing videogames is equivalent to non-virtual social participation. Social health is an emerging domain, and would greatly benefit from future research. Some of the outcome measures used in the reviewed studies, like The Short Form Health Survey [86], or the World Health Organization Quality of Life Scale Brief Version [87] include subdomains assessing social health (e.g. Social Role Functioning, Social relationships), though information about social health cannot be assessed if the authors only report the total score. This was the case in two of the studies analyzed for inclusion in the current study [40,63].
The magnitude of the effects of video game-based interventions were moderated by the health status of participants, the type of game, the presence of physical activity, the type of prevention program, blinded assignment and participants' age. Specifically, participants with subclinical conditions benefited more from the interventions than healthy ones, which is consistent with the larger effect size obtained by studies on indicated prevention programs [88] and could also be caused by their greater statistical power.
Exergames resulted in better outcomes than other types of video game interventions. This finding may be due to exergames accounting for the beneficial effects in cognition and physical condition, while serious and casual video games lost explanatory power when this variable was controlled. This can be explained by the fact that exercise training induced functional brain plasticity and prefrontal adaptations that were correlated with improved performance in executive functions and processing speed. This is likely a result of reducing the need for prefrontal resources of executive functions and attention in dual tasks [74]. Similarly, cognitive decline is associated with impaired gait in older adults [89]. Another hypothesis is that cerebral metabolic activity that occurs with physical activity training requires increased availability of oxygen [90]. In addition, exergames train different motor and cognitive abilities such as multidirectional displacements, weight transfer, attention, planning, decision making and concentration [91]. This is consistent with previous research that demonstrated that combining not only different cognitive abilities but also combining cognitive and physical training improved cognitive performance in older age to a greater extent, suggesting the implementation of combined cognitive-physical interventions [41]. Previous reviews on exergames in adults and older adults concluded that exergaming provided a novel method for increasing or substituting physical activity, and resulted in improved physical function, depression and cognitive function [16,92,93]. The significant effects still existed when excluding waitlist-only controlled studies, and when comparing to physical activity interventions [29]. However, our results partially contradict a previous study that found that serious games have small positive effects for healthy lifestyle promotion in all ages [21]. Our results might be due to the fact that our study focused on adults older than 45, and that serious games seem to be less effective than exergames in healthy life style promotion.
Furthermore, the fact that non-blinded participants had better outcomes could be explained as a placebo effect. However, due to the few blinded studies in this review (n = 6) this should be further explored in future studies. Lastly, the fact that older participants benefited more from the intervention may be related to age-related decline in physical, cognitive, emotional and social functioning that video game interventions can prevent. In addition, these benefits may also be due to the fact that older adults may start the training program with lower physical, cognitive and emotional functioning scores related to aging decline [94], which result in larger effect sizes after the intervention. This finding is consistent with a previous meta-analysis on video games aimed at older adults [17].
However, no moderating effects were found in the other studies' participant characteristics (e.g., gender, education, marital status, socioeconomic class, or region), intervention variables (e.g., number of sessions, play duration, dosage of interventions, format, interface) or methodological variables (e.g., randomization method, type of control group, drop outs). These results indicate that video game-based interventions are broadly applicable across a wide range of participants and are equally effective on different dosages and formats. One reason for this may be that video game-based interventions are usually friendly and intuitive so people of any educational, social class or region can play them. In contrast to face to face interventions, video game based-interventions maintain fun, motivation, commitment with the task and allow for different activity levels, preventing fatigue [8]. No heterogeneity of the results was found, except for objective measured physical health, due to two studies [72,74] which had results inconsistent with those of the other studies included in the meta-analysis. These differences may be due to the small sample size (n = 33 and n = 46, respectively) and the younger age of participants in these two studies (M ages 75.3 and 69.3, respectively). The small sample size and younger ages could prevent the generalization of the results and result in a ground effect that could impede the appreciation of improvements. The results of those studies should be considered with caution. Consequently, in the current meta-analysis a random effect model was applied to correct for these effects [33].
This review has a number of strengths, including a registered protocol, rigorous evaluation of the quality and risk of bias of the studies and rigorous methods of quantitative synthesis. As far as we know, it is the first literature review and meta-analysis focused on video game-based interventions for active aging. Some limitations of the reviewed studies must be considered: most of the reviewed studies had small sample sizes, a lack of theoretical model-based interventions, none of the interventions were based on a manualized treatment and only 42.9% were based on a standardized protocol. Furthermore, there was a wide use of non-standardized measures, especially of criterion outcome measures (e.g., playing score, reaction time) and computerized non-validated adaptations of tests (e.g., Stroop test, Wisconsin Card Sorting Test), although it must be noted that only the results emerging from standardized instruments were included in this study. Followups were scarce and generally brief and in 57.1% of studies the risk of bias was unclear. Maybe for these reasons, the effect sizes in significant health domains were small.
In addition, conclusions drawn from this meta-analysis must be considered in the context of some limitations. Firstly, we included RCT which did not use intent-to-treat analyses, introducing the possibility of survival bias. In order to control them, we carried out a moderator analysis including attrition as moderator. Secondly, social health domain was only measured in two studies; therefore, social health results should be interpreted with caution.
Conclusions
Despite these limitations, the findings suggest that video game-based interventions are a promising and effective intervention for active aging promotion. Future studies to increase methodological rigor are needed. Additionally, more studies are suggested to assess adults older than 44 but younger than 60, with longitudinal studies analyzing the preventive efficacy of interventions in the aging process. RCT of serious video-game-based interventions targeting health domains other than cognition are recommended. | 2018-12-13T14:06:43.538Z | 2018-12-11T00:00:00.000 | {
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17014245 | pes2o/s2orc | v3-fos-license | Wnt signaling activation and mammary gland hyperplasia in MMTV-LRP6 transgenic mice: implication for breast cancer tumorigenesis
Although Wnt signaling activation is frequently observed in human breast cancer, mutations in the genes encoding intracellular components of the Wnt signaling pathway are rare. We found that expression of Wnt signaling co-receptor LRP6 is up-regulated in a subset of human breast cancer tissues and cell lines. To examine whether overexpression of LRP6 in mammary epithelial cells is sufficient to activate Wnt signaling and promote cell proliferation, we generated transgenic mice overexpressing LRP6 in mammary epithelial cells driven by the mouse mammary tumor virus (MMTV) promoter. We found that mammary glands from MMTV-LRP6 mice exhibit significant Wnt activation evidenced by the translocation of β-catenin from membrane to cytoplasmic/nuclear fractions. Expression of several Wnt-target genes including Axin2, Cyclin D1 and c-Myc was also increased in MMTV-LRP6 mice. More importantly, mammary glands from virgin MMTV-LRP6 mice exhibit significant hyperplasia, a precursor to breast cancer, when compared to wild-type littermate controls. Several matrix metalloproteinases are up-regulated in MMTV-LRP6 mice that could contribute to the hyperplasia phenotype. Our results suggest that Wnt signaling activation at the cell surface receptor level can contribute to breast cancer tumorigenesis.
Introduction
The defining feature of the canonical Wnt pathway is the stabilization of cytosolic β-catenin, which enters the nucleus and activates Wnt target genes by binding to transcription factors of the T-cell factor/lymphoid enhancing factor (TCF/LEF) family (Giles et al., 2003;Moon et al., 2004). In the absence of Wnt ligands, β-catenin is phosphorylated by a multi-protein complex that marks it for ubiquitination and degradation by the proteasome. This β-catenin degradation complex contains the adenomatous polyposis coli (APC) tumor suppressor, scaffold protein Axin, glycogen synthase kinase 3β (GSK3β), and casein kinase 1 (Ck1). The action of this complex is inhibited upon binding of Wnt to its receptors. Experiments performed in Drosophila (Wehrli et al., 2000), Xenopus (Tamai et al., 2000) and mice (Pinson et al., 2000) demonstrated that the low-density lipoprotein receptor-related protein 5 (LRP5)/LRP6 (termed Arrow in Drosophila) acts as a co-receptor for Wnts, which interact with both the seven transmembrane receptor of the Frizzled (Fz) family and LRP5/6 to activate the canonical Wnt signaling pathway.
The role of Wnt/β-catenin signaling in cell proliferation indicates that dysregulation of this pathway may result in cancer. Indeed, several components of the Wnt/β-catenin signaling pathway have been identified as oncogenes or tumor suppressors (showing gain-of-function or loss-of-function mutations, respectively) in human cancers (Giles et al., 2003;Moon et al., 2004). Mutations in these genes are most evident in colorectal cancer. About 85% of all colorectal cancers contain mutations in the tumor suppressor gene APC. Mutations in the oncogene encoding β-catenin (CTNNB1) are present in approximately 10% of the colorectal cancers. The consequence of either APC inactivation or β-catenin mutation is similar: failure of proper β-catenin degradation leads to its cytosolic accumulation, nuclear translocation, and constitutive activation of β-catenin-responsive genes (Giles et al., 2003;Moon et al., 2004). Although genetic mutations of APC or CTNNB1 are rarely observed in breast cancer, compelling evidence has indicated abnormal regulation of Wnt/β-catenin signaling in breast cancer tumorigenesis (Turashvili et al., 2006;Lindvall et al., 2007). Wnt1, the founding member of the Wnt genefamily, was initially identified as a mammary oncogene insertionallyactivated by mouse mammary tumor virus (Nusse and Varmus, 1982;Peters et al., 1983;Nusse et al., 1984). Overexpressionof Wnt1, Wnt10b or an activated form of βcatenin in vivo results in mammary tumorigenesis (Tsukamoto, et al., 1988;Lane and Leder, 1997), while mice deficient in LRP5 are resistant to Wnt1-induced mammary tumors (Lindvall et al., 2006). Mammary tumors were also observed in heterozygous APC Min mice (Moser et al., 1993). In human breast cancer, secreted Frizzled-related protein1 (sFRP1), a member of the secreted Wnt antagonist family, is down-regulated in malignant tissues (Ugolini et al., 2001;Klopocki et al., 2004). More importantly, β-catenin levels are significantly upregulated and correlate with poor prognosis, acting as a strong and independent prognostic factor in human breast cancer patients (Lin et al., 2000).
LRP6 is expressed in human cancer cell lines and human malignant tissues (Li et al., 2004), and is elevated in testicular germ cell tumors (Rodriguez et al., 2003). Bafico et al. reported that there is an autocrine mechanism for constitutive Wnt pathway activation in human cancer cells, and that the autocrine Wnt signaling can be inhibited by siRNA directed against LRP6 (Bafico et al., 2004). This is the first demonstration that Wnt signaling may be activated in cancerous cells via cell surface Wnt receptors and not due to mutations in one of the downstream signaling components. Our previous studies demonstrated that stable expression of LRP6 in human fibrosarcoma HT1080 cells alters subcellular β-catenin distribution such that the cytosolic β-catenin level is significantly increased. This is accompanied by a significant increase in Wnt/β-catenin signaling and cell proliferation in vitro, and tumor growth in vivo (Li et al., 2004). To investigate the role of LRP6 in mammary tumorigenesis, we generated MMTV-LRP6 transgenic mice and found that overexpression of LRP6 in the mouse mammary gland is sufficient to induce mammary hyperplasia.
Generation of MMTV-LRP6 Transgenic Mice
To assess the potential role of LRP6 in mammary tumorigenesis, we generated a mouse model in which human LRP6 is overexpressed in the mammary gland. The transgenic construct consists of a mouse mammary tumor virus (MMTV) promoter placed upstream of human LRP6 cDNA followed by an SV40 polyadenylation (polyA) site. MMTV-LRP6 mice were generated (named Founder 1-5) and three founders (Founder 1, 4 and 5) carried germline transmission of the LRP6 transgene were identified by RT-PCR ( Figure 1a). Realtime quantitative PCR analysis showed that Founder 4 has the highest level of LRP6 transgene expression, whereas Founder 5 has the lowest level ( Figure 1b).
To confirm LRP6 expression at the protein level, Western blot analysis of mammary gland lysates prepared from 10-week old MMTV-LRP6 virgin mice was performed. An antibody to detect both endogenous mouse LRP6 as well as the transgenic human LRP6 was used. We found that mammary glands from the transgenic MMTV-LRP6 Founder 4 and 1 displayed 2.6 fold and 1.7 fold greater LRP6 expression levels, respectively, compared to mammary glands from WT littermate controls ( Figure 1c). Furthermore, immunohistochemical staining confirmed that LRP6 expression in transgenic MMTV-LRP6 virgin glands was higher than WT virgin glands (Figures 2a, b).
Mammary Gland Hyperplasia in MMTV-LRP6 Transgenic Mice
Whole mount preparations are a well-established method to identify early premalignant lesions of the mammary epithelium (Cardiff et al., 2000). Thus, we performed whole-mount staining of virgin glands to examine the ductal structure of mammary glands in MMTV-LRP6 transgenic mice and WT littermate controls. As shown in Figure 3a, examination of a mammary gland taken from a wild-type littermate control female mouse revealed a branching ductal structure typical of a virgin female. In contrast, inspection of the MMTV-LRP6 mammary gland revealed an unusual number of secondary and tertiary branches and small, spiculated side buds. Quantification of terminal end buds (TEBs) from mammary glands revealed that TEBs from MMTV-LRP6 virgin mice (Founder 4) at 14 weeks (n=4) and 21 weeks (n=4) of age are 3.3 and 6 folds higher, respectively, than those from WT littermate control glands (Figure 3b). Both the founder 1 and 4 lines displayed similar mammary epithelial abnormalities with the founder 4 line having more significant hyperplasia. Furthermore, histological sections of virgin glands showed more individual ducts lined with cuboidal epithelium in transgenic mammary glands than WT littermate control glands (Figures 2c, d). These findings indicate that overexpression of LRP6 in the mouse mammary gland is sufficient to induce mammary gland hyperplasia.
Wnt Signaling Activation in MMTV-LRP6 Transgenic Mice
The ability of LRP6 overexpression to induce mammary gland hyperplasia is likely the result of activation of the Wnt/β-catenin signaling pathway. Transactivation of gene expression by the β-catenin/TCF/LEF complex represents the nuclear target of the Wnt/β-Catenin signaling pathway. To measure TCF/LEF-dependent transcriptional activation we performed reporter gene expression analysis with the Wnt/β-catenin signaling reporter TOPFlash in primary human mammary epithelial cells (HMECs) (Figure 4a). A vector with mutated copies of the TCF/LEF (FOPFlash) was used for measuring non-specific transactivation ( Figure 4b). We found that transient transfection of LRP6 into HMECs results in a significant increase of TOPFlash luciferase activity, an effect blocked by cotransfection with Dkk1 ( Figure 4a). In addition, Wnt3A treatment greatly enhanced the effect of LRP6 on TOPFlash luciferase activity in HMECs (Figure 4a).
Uncomplexed cytosolic β-catenin (free β-catenin) is the active form of β-catenin that is translocated to the cell nucleus, which activates transcription factors of the TCF/LEF family, leading to the transcription of Wnt target genes (Bafico et al., 1998(Bafico et al., & 2004. We then examined the extent of Wnt/β-catenin signaling in mammary glands from MMTV-LRP6 mice or WT littermate controls by determining the levels of free β-catenin present in the cytoplasma of mammary tissues. While there was no significant difference of total cellular β-catenin in mammary glands between MMTV-LRP6 transgenic mice and WT littermate controls, virgin glands from MMTV-LRP6 transgenic mice (founder 4) exhibited higher levels of cytoplasmic free β-catenin than WT littermate controls Figure 5a). Quantification of Western blot signals revealed that expression levels of cytoplasmic free β-catenin in mammary glands from MMTV-LRP6 transgenic mice are 1.9 folds higher than those from WT littermate controls. Furthermore, we prepared mammary gland nuclear extracts and found that the levels of both total β-catenin and unphosphorylated, active β-catenin in mammary nuclear extracts from MMTV-LRP6 transgenic mice were higher than those from WT littermate controls (Figures 5b,c). All together, these data suggest that increased LRP6 expression in mammary glands promotes Wnt/β-catenin signaling by altering β-catenin subcellular distribution.
Axin2 (Yan et al., 2001;Jho et al., 2002;Leung et al., 2002;Lustig et al., 2002) is a direct and specific transcriptional target of the Wnt/β-catenin signaling pathway. It is well recognized that the expression level of Axin2 is the signatureof the activation of Wnt/βcatenin signaling. To further confirm that Wnt/β-catenin signaling is up-regulated in MMTV-LRP6 transgenic mammary glands, we examined the expression of Axin2 by q-PCR. As expected, Axin2 in mammary glands from MMTV-LRP6 transgenic mice were 3.3 folds higher than those from WT littermate controls (Figure 6a). c-Myc and Cyclin D1 are key transcriptional targets of the Wnt/β-catenin pathway (He et al., 1998;Shtutman et al., 1999;Tetsu and McCormick, 1999). To identify a potential mechanism by which LRP6 accelerates the development of dysplastic mammary lesions, we examined the expression of c-Myc and Cyclin D1 in mammary tissues. As expected, virgin glands from MMTV-LRP6 transgenic mice exhibited higher levels of c-Myc and Cyclin D1 expression than WT littermate controls ( Figure 6b). Quantification of the Western blot signals revealed that expression levels of c-Myc and Cyclin D1 in mammary glands from Zhang et al.
C-Myc and Cyclin D1 are two important cell cycle regulators. Having established that the expression levels of c-Myc and Cyclin D1 in mammary glands from MMTV-LRP6 transgenic mice are up-regulated, we then examined the proliferation status of mammary epithelial cells. Ki67 is a nuclear protein that is tightly linked to the cell cycle, and a marker of cell proliferation. Indeed, immunohistochemical staining revealed that Ki67 expression in transgenic MMTV-LRP6 virgin glands was higher than WT virgin glands (Figures 6d, e). In contrast, TUNEL staining revealed that there was no significant difference in apoptosis status between transgenic MMTV-LRP6 virgin glands and WT virgin glands (Supplement Figure 1), suggesting that the regulation of apoptosis is not involved in mammary gland hyperplasia of MMTV-LRP6 mice.
Discussion
While genetic mutations of certain intracellular components of the Wnt/β-catenin pathway, such as APC and CTNNB1, are significant contributing factors for colorectal cancers, they are typically not the predominate mechanism associated with other cancer types such as breast cancer. Instead, it appears that dysregulation of cell surface Wnt/β-catenin signaling components leads to aberrant activation of this pathway in breast cancer (Turashvili et al., 2006;Lindvall et al., 2007). We found that expression of the Wnt signaling co-receptor LRP6 is up-regulated in a subset of human breast cancer tissues and cell lines (unpublished data). In the present study, we demonstrated that transgenic mice over-expressing LRP6 in mammary epithelial cells driven by the MMTV promoter is sufficient to induce mammary gland hyperplasia, a precursor to breast cancer. During the revision process of the manuscript, Lindvall et al. reported that canonical Wnt signaling through LRP6 is required for normal mouse mammary gland development, and that LRP6 expression is increased in basal-like human breast cancer, a triple-negative phenotype associated with high grade, poor prognosis, and younger patient age (Lindvall et al., 2009). Altogether, these findings indicate that mammary tumorigenesis can be initiated at the cell surface receptor level in mammary epithelium, and that LRP6 is a potential target for breast cancer therapy.
Cyclin D1 and c-Myc are two important cell cycle regulators. Clinically, the Cyclin D1 gene is amplified in up to 20% of human breast cancers and Cyclin D1 protein is overexpressed in >50% of human mammary carcinomas (Bartkova et al., 1994;Gillett et al., 1994;McIntosh et al., 1995). For c-Myc, a comprehensive meta-analysis suggests that at least 15% of breast cancers present with significant amplification of c-Myc, and that c-Myc amplification is significantly associated with a poor prognosis in breast cancer (Deming et al., 2000). In animal studies, transgenic mice over-expressing Cyclin D1 in mammary epithelium (MMTV-Cyclin D1) develop mammary hyperplasia and mammary carcinomas (Wang et al., 1994). Similarly, constitutive c-Myc expression under the control of MMTV or the whey acidic protein promoters is oncogenic in transgenic mice (reviewed in Amundadottir et al., 1996;Nass and Dickson, 1997). Amplification of c-Myc or Cyclin D1 has been identified as a downstream step at the end of Wnt/β-catenin pathway activation (He et al., 1998;Shtutman et al., 1999;Tetsu and McCormick, 1999). Therefore, it is expected that LRP6 overexpression would cause the activation of Wnt/β-catenin signaling, up-regulation of Cyclin D1 and c-Myc expression levels, and increases the expression of the cell proliferation marker Ki67, all of which we observed experimentally in MMTV-LRP6 mice. As such, these events could account for mammary hyperplasia in MMTV-LRP6 mice.
MMPs are multifunctional enzymes capable of targeting the extracellular matrix, growth factors, cytokines and cell surface-associated adhesion and signaling receptors. Clinically, MMPs have been associated with advanced-stage cancer and contribute to tumor progression, invasion, and metastasis. In ductal breast carcinomas, it has been demonstrated that MMP-2, -3, -9, -11, -13 and -14 are synthesized either by stromal fibroblasts, infiltrating macrophages or vascular pericytes (Wolf et al., 1993;Okada et al., 1995;Heppner et al., 1996;Nielsen et al., 1997Nielsen et al., & 2001Chenard et al., 1999). In animal models, it has been found that expression of an autoactivating form of MMP-3 under the control of the whey acidic protein promoter induces premalignant and malignant lesions in the mammary glands. Moreover overexpression of a natural inhibitor of MMPs, tissue inhibitor of MMP (TIMP)-1, inhibits tumor formation in MMP-3 transgenic mice (Sternlicht et al., 1999). More interestingly, Blavier et al. recently reported that the expression of several MMPs including MMP-2, -3, -9,-13, and -14 was increased in hyperplastic glands and mammary tumors of MMTV-Wnt1 transgenic mice. Furthermore, when MMTV-Wnt1 mice were crossed with transgenic mice overexpressing a natural MMP inhibitor, TIMP2, in the mammary gland, the double transgenic mice displayed an increase in tumor latency and a reduction in tumor formation (Blavier et al., 2006). MMP-2, -3, -7, -9, -13 and -14 are all known target genes of the Wnt/β-catenin pathway (Crawford et al., 1999;Takahashi et al., 2002;Tamamura et al., 2005;Wu et al., 2007). In the present study, we demonstrated that mammary glands from MMTV-LRP6 transgenic mice exhibit significantly higher levels of MMPs than WT littermate controls. Therefore, upregulation of MMPs could also account for mammary hyperplasia in MMTV-LRP6 mice.
Whole mount staining of mammary gland is a well-established and recommended method to identify early premalignant lesions of the mammary epithelium (Cardiff et al., 2000). In our mammary gland model, we found that the MMTV-LRP6 female mice exhibit mammary gland hyperplasia. However, none of the MMTV-LRP6 mice developed adenocarcinoma of breast for more than an 18-month span. Therefore, it is possible that the extent of the Wnt/βcatenin activation upon LRP6 overexpression is sufficient to cause mammary gland hyperplasia but not sufficient to lead to breast cancer in virgin mice. It will be interesting to examine whether multiparous MMTV-LRP6 mice develop breast cancer. Crossing MMTV-LRP6 mice with MMTV-Wnt mice will allow us to test whether LRP6 and Wnt1 synergistically promote breast cancer tumorigenesis. Together, these studies should help to define whether LRP6 and Wnt ligands are novel targets for breast cancer therapy.
Generation of MMTV-LRP6 mice
To generate the MMTV-hLRP6 construct, human LRP6 cDNA fragment was removed from the pCS-myc-hLRP6 vector (kindly provided by Dr. Christof Niehrs, German Cancer Research Center) by digestion with Cla and XbaI, followed by n-fill with Klenow enzyme. The LRP6 fragment was then blunt-end ligated into the EcoRI site (n-filled) of the MMTV-SV40-Bssk vector (kindly provided by Dr. Philip Leder, Harvard Medical School). Purified MMTV-LRP6 DNA fragment was microinjected into the pronuclei of fertilized mouse eggs, and transferred to pseudopregnant mothers by the Transgenic and ES Cell Core of Washington University School of Medicine. Mice were genotyped for the presence of the transgenes by PCR with primers 5′-GGCTATACCAGTGACTTGAACTATGATT-3′, and 5′-GGTTCCTTCACAAGATCCTCTAGAGTC-3′ to identify the MMTV-LRP6 transgene.
RNA isolation and quantitative PCR
Total RNA from inguinal mammary gland (number 4) of 14-week old mice was extracted by SV total RNA isolation kit (Promega, Z3100). Total RNA was dissolved in nuclease-free water and stored at −80°C. Reverse transcription was performed using a SuperScript II RNase H-reverse transcriptase (Invitrogen), and the reaction mixture was subjected to either reverse transcription-PCR (RT-PCR) or quantitative real-time PCR (q-PCR) to detect levels of human LRP6, MMPs, Axin2 and actin, which was used as an internal control. For a 50 μl of q-PCR reaction, 25 μl of SYBR Supermix from BioRad, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, 2 μl of mixture of reverse transcription reaction, and 21 μl of water were included. After 40 cycles, the relative levels of gene expression were quantified with Bio-Rad iCycler iQ software. The primers used to amplify target genes by RT-PCR and q-PCR were as following: hLRP6-F (5′ TAGCATTGAAAGAGTTCATAAACGA 3′), hLRP6-R (5′ CACTCGATGAACATTTGTAGCCTTT 3′); Axin2-F (5′
Western blot analysis
Thoracic and inguinal mammary glands from both wild type and transgenic mice were lysed with phosphate-buffered saline containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride and the protease inhibitor cocktail from Roche. Protein concentrations were determined with Protein Assay kit from Bio-Rad. Equal amount of proteins from each sample was used for SDS-PAGE. The immunoreactive bands were visualized by enhanced chemiluminescence and exposure to film. For densitometric analyses, immunoreactive bands were scanned using a Kodak Digital Science DC120 Zoom camera and quantified using Kodak Digital Science image analysis software. Human LRP6 antibody was from R&D. c-Myc and Cyclin D1 antibodies were from Santa Cruz. β-catenin antibody was from BD Transduction Laboratories, active β-catenin antibody was from Millipore. β-Actin antibody was from Sigma.
Luciferase reporter assay
Wnt3A conditioned medium (Wnt3A CM) and L cell control CM were prepared as previously described (Lu et al., 2008). Normal human mammary epithelial cells (HMECs) were purchased from Lonza. HMECs were plated into 24-well plates. For each well, 0.05 μg of the TOPFlash TCF luciferase construct (Upstate Biotechnology) or the negative control FOPFlash TCF luciferase construct was co-transfected with LRP6-, Dkk1-expressing vector, or empty pcDNA3 vector. A β-galactosidase-expressing vector (Promega, Madison, WI) was included as an internal control for transfection efficiency. After 24 h incubation, the cells were treated with 5% of Wnt3A CM or L cell control CM. After further 24 h incubation, cells were lysed and both luciferase and β-galactosidase activities were determined with enzyme assay kits (Promega). Luciferase activity was normalized to the activity of the β-galactosidase.
GST-E-cadherin binding assay for cytoplasmic free β-catenin
The GST-E-cadherin binding assay was carried out as previously described (Lu et al., 2008). Briefly, recombinant GST-E-Cadherin protein was expressed, purified and conjugated with agarose beads. The GST E-cadherin beads were incubated with 100 μg of total cell lysates prepared from mammary gland tissues of MMTV-LRP6 mice and WT littermate controls for 4 h at 4°C. This process allows uncomplexed cytoplasmic freeβ-catenin in total cell lysates to bind to GST E-Cadherin beads. The cytoplasmic free β-catenin was then eluted from the GST E-Cadherin beads, subjected to SDS-PAGE, and detected using a monoclonal antibody to β-catenin.
Preparation of crude membrane fractions
Membrane fractionation can enrich membrane proteins to be detected. Thoracic and inguinal mammary glands from both MMTV-LRP6 transgenic mice and WT littermate controls were harvested and immediately put into prechilled 20 mM Tris pH 8.0 containing 150 mM NaCl, 1 mM CaCl 2 and EDTA-free Complete proteinase inhibitors. After homogenization with a dounce homogenizer and low-speed centrifugation at 1500g for 5 mins, a small portion of homogenate was kept at 4°C as whole tissue lysate, while the majority of homogenate was further centrifuged at 100,000g for 30 min at 4°C to separate the membrane fraction Zhang et al.
(insoluble pellet) from cytoplasmic fraction (supernatant). The insoluble pellet was resuspended in 50 mM Tris pH 8.0 containing 80 mM NaCl, 2 mM CaCl 2 and EDTA-free complete proteinase inhibitors. Triton X-100 was then added to whole tissue lysate, membrane and cytoplasmic fractions at 1% final concentration. The membrane fraction was passed several times through a 28-gauge needle and cleared further by centrifugation at 100,000g for 15 min. Total protein concentrations were determined with Protein Assay kit (Bio-Rad). Equal amounts of total protein were separated by SDS-PAGE under reducing condition, and analyzed by Western Blotting. To validate the membrane protein preparation, Western blot analysis was performed with antibodies against cell surface receptor LRP1 (Bu et al., 1995) and nuclear marker Lamin B1 (Abcam) (Supplement Figure 2a).
Nuclear extraction
NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo scientific) was used for preparation of nuclear and cytoplasmic fractions from mammary gland tissues. To validate the purity of the nuclear and cytoplasmic fractions, Western blot analysis was performed with antibodies against nuclear marker Lamin B1 (Abcam) and cytoplasmic marker HSP90 (Cell Signaling Technology) (Supplement Figure 2b).
Whole mount staining of mammary gland
Inguinal mammary glands (number 4) were dissected at the indicated times of development and spread on glass slides. After fixation in Carnoy's fixative for 4 h at room temperature, the tissues were hydrated and stained in carmine alum as described on http:// mammary.nih.gov. Samples were then dehydrated, cleared in Histoclear, and photographed. Counting of terminal end buds (TEBs) was performed by defining counting areas first, drawing 1mmX1mm squares on whole mount pictures, and taking the average number of TEBs from 5 randomly picked areas.
Immunohistochemistry
Inguinal mammary glands (number 4) were dissected from both MMTV-LRP6 transgenic mice and WT littermate controls, fixed in 4% phosphate-buffered paraformadehyde overnight, transferred to 70% ethanol, embedded in paraffin, and sectioned at 5μm. After deparaffinization, rehydration and antigen retrieval by heating in antigen unmasking solution (Dako, Carpinteria, CA), tissue slices were blocked with serum using Vectastain ABC kit (Vector Laboratories, Burlingame, CA), incubated with primary anti-hLRP6 antibody (R&D, Indianapolis, IN) or Ki67 antibody (Abcam, Cambridge, CA) at 4°C overnight. After washing with PBS, the slices were incubated with biotinylated secondary antibody for 30 min at room temperature, and detected by Histostain Plus DAB kit (Zymed Laboratories, CA).
H & E staining
Inguinal mammary glands (number 4) were excised, fixed with paraformadehyde, and embedded in paraffin. Sections were cut at 5 μm, dewaxed, rehydrated, and stained with hematoxylin and eosin.
TUNEL staining
Paraffin sections at 5 μm of mammary glands from MMVT-LRP6 and WT littermate controls were depariffinized. TUNEL staining was performed with TUNEL Apoptosis Detection kit (Upstate) following manufacturer's instructions, and nuclei were counterstained with DAPI 5 μg/ml for 5 min.
Statistical Analysis
All quantified data represent an average of at least three samples. Error bars represent standard deviation. Statistical significance was determined by Student's test, and p<0.05 was considered as significant.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material. Oncogene. Author manuscript; available in PMC 2010 July 28.
significant difference compared to HMECs transfected with LRP6 and Dkk1, or with empty vector only; **P<0.01 indicates a significant difference compared to HMECs transfected with LRP6 or Dkk1 only, or to HMECs transfected with LRP6 and Dkk1 and treated with Wnt3A CM.
Figure 5.
Wnt/β-catenin signaling activation in the mammary glands of MMTV-LRP6 mice. (a) Virgin glands from MMTV-LRP6 mice or WT littermate controls (14 weeks old, n=3) were dissected and lysates were prepared. Cytoplasmic free β-catenin was pulled down from 200 μg of cell lysate using pGST-E-Cadherin, and then examined by Western blotting with a specific β-catenin antibody. The levels of total cellular β-catenin and actin were also analyzed by Western blotting. Right panel, quantification of the Western blotting signals of cytoplasmic free β-catenin. (b-c) Nuclear extracts from mammary glands of MMTV-LRP6 mice or WT littermate controls (14 weeks old, n=3) were prepared by fractionation, and examined by Western Blotting with either regular β-catenin antibody (b) or activeβ-catenin (unphosphorated β-catenin) antibody (c). Lamin B1 was blotted as a loading control. Lower panel, quantification of the Western blot signals of β-catenin from mammary gland nuclear extracts. The levels of β-catenin were normalized to the Lamin B1 levels. Error bars represent SD. *P<0.05 indicates a significant difference compared to mammary glands from WT littermate controls.
Zhang et al.
Page 20
Oncogene. Author manuscript; available in PMC 2010 July 28. MMPs are up-regulated in MMTV-LRP6 mice. RNA from mammary glands of 14-month old MMTV-LRP6 virgin mice and WT littermate controls were prepared, and the expression levels of 6 MMPs were measured by q-PCR. Fold changes compared to WT littermate controls were plotted. All the values are the average of triple determinations with the SD indicated by error bars. *P<0.05 indicates a significant difference compared to mammary glands from WT littermate controls.
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257562162 | pes2o/s2orc | v3-fos-license | Exploring the role of entitlement, Social Dominance Orientation, Right-Wing authoritarianism, and the moderating role of being single on misogynistic attitudes
Abstract This article aimed to explore individual level factors as predictors of misogynistic attitudes. Given that misogyny and activity on online forums related to so called incel-dom is growing and has been identified as a terrorist threat, it becomes important to better understand the underpinnings of misogynistic attitudes, also in a normal population. Based on previous research and theory, entitlement, Social Dominance Orientation and Right-wing authoritarianism was explored as well as the moderating role of being single among American men (N = 302). Results from an online survey showed that all three predictors as well as being single (compared to being in a relationship) significantly predicted misogynistic attitudes. The effect of SDO was moderated by relationship status such that singles who were high SDO expressed most misogyny. The results contribute to a better understanding of who may come to adhere to a more radical view of women.
to be related to misogyny, but also takes current relationship status into account.Given that involuntary celibacy by default imply the lack of a romantic partner, it is important to take relationship status into account when trying to understand the psychological underpinnings of misogyny.
The article aims to explore the role of status legitimizing ideologies and entitlement on misogynistic attitudes.Further, I aim to explore how relationship status may be an important moderator in this relation.Both Right-wing authoritarianism (RWA) and Social Dominance Orientation (SDO) are status legitimating ideologies, which have been connected to sexism.Particularly, SDO has been related to hostile sexism (Christopher & Mull, 2006;Lee, 2013;Sibley et al., 2007;Sibley & Becker, 2012), which comes close to misogyny.In trying to understand why men adopt misogynistic ideas and attitudes, the manosphere plays an important role providing support and camaraderie, but also a narrative for why the men active on these online forums feel rejected by women.Because the notion of one being unable to find a romantic partner is at the core of the incel-ideology (Ging, 2019), one important potential moderator is being single.While there are indications that statuslegitimizing ideologies should correlate with misogyny, the literature has not yet explored if this relation may be particularly pronounced among single men.
Individual factors explaining misogyny
Before discussing individual factors that may explain misogyny, a note on misogyny and sexism is needed since much research has used sexism as an outcome variable.
Sexism is defined as a prejudice or discrimination based on sex, not necessarily against women, but that is the context in which sexism is almost exclusively used.While sexism is a prejudice (Glick & Fiske, 1996) it contrasts to many other prejudices such as racism because relations between women and men are more complicated as they also entail some form of attraction between the groups often missing in other (hostile) intergroup contexts.This gave rise to the idea behind ambivalent sexism (Glick & Fiske, 1996), where sexism is seen as two complimentary dimensions of benevolent and hostile sexism.Benevolent sexism refers to sexism that is positive in tone, presenting women as fragile and in need of male protection (Glick et al., 2004).While it is presented as something positive, benevolent sexism still functions to position women in stereotypical roles, as well as positioning men as dominating (Glick & Fiske, 1996) and should therefore be considered normatively problematic.
Hostile sexism is a prejudice in a traditional meaning of antipathy (Allport, 1958).Hostile sexism reflects hostility against women who challenge male power (Glick et al., 2004).The dimension contains three areas; paternalism, gender differentiation and heterosexism.Paternalism refers to the dominant control of women, gender differentiation refers to the superiority of men over women and heterosexism refers to that women are sexual objects who may manipulate men through sex.Hostile sexism is therefore closely linked to misogyny.
Misogyny is defined as hatred of, aversion to, or prejudice against women (Srivastava et al., 2017).As such, it shares some aspects with sexism, but has a stronger focus on the hatred part.The definitional boundary of misogyny remains relatively loose (Rottweiler & Gill, 2020).Misogyny can take different shapes such as male privilege, patriarchy, sexual harassment of women, violence against women and objectification (Srivastava et al., 2017).Recently, Rottweiler and Gill (2020) tried to create a scale for measuring misogyny and in doing so they also included research on hostile sexism.In the final scale, there are obvious connections to hostile sexism, such as items about women trying to manipulate men by using sex.Because hostile sexism as defined and measured is very similar to misogyny, the literature review will comprise literature on hostile sexism as well as misogyny.
Entitlement
Entitlement is a central theme on the manosphere (Ging, 2019;Hoffman et al., 2020), and is closely connected to ideas about hegemonic masculinity.Although not specifically related to misogyny, the concept "aggrieved entitlement" captures the ideas surrounding the gendered aspect of masculine entitlement (Kalish & Kimmel, 2010).Aggrieved entitlement is a gendered sense of entitlement thwarted by societal, political, or economical forces, such as women's liberation and feminist progress.Aggrieved entitlement is gendered because it is anchored in the cultural norms and ideas about manhood, masculinity, and what men are entitled to given this identity.These include not just ideas about what a "real man" is (powerful, athletic, non-feminine, heterosexual) but more importantly how masculinity is performed and maintained (Vandello & Bosson, 2013).Specifically, it prescribes violent vengeance to maintain and reinforce masculine status when it is perceived to be threatened (M€ oller-Leimk€ uhler 2018; Kalish & Kimmel, 2010).The manosphere flaunts many different aspects of male entitlement and how it is thwarted in modern society.The Incel culture, for example, is characterized by ideas of men's entitlement to women and sex, and of how some men are robbed of this mainly based on women's pickiness (Bates, 2020;European Commission, 2021;Guy, 2021).
Ideological predictors
Both Social dominance orientation (SDO) and Right-wing authoritarianism (RWA) are status legitimizing ideologies, meaning that they entail world views where inequalities are seen as legitimate (Major & Kaiser, 2017).They have been shown to predict prejudice including sexism (Duckitt & Sibley, 2010;Van Assche et al., 2019).Most previous studies explore the effects of these ideological convictions on sexism in samples composed of both women and men (e. g.Van Assche et al., 2019) and find that both ideologies seem to be equally strong predictors of hostile sexism.However, in the present study, the main goal is to explore how this relationship pans out among heterosexual men specifically.
Social Dominance Orientation is an expression of the motivational goal for group-based dominance and superiority (Duckitt, 2001;Pratto et al., 1994).People high in SDO see the world as a competitive jungle, which entails a struggle for resources (Duckitt et al., 2002)a sort of zero-sum game.In terms of gender relations this means that if women gain status and privileges, men loose out, and thus men should be motivated to combat feminist progress.Because men who score high on SDO should hence be particularly sensitive to competitiveness in gender relations, they should also perceive that women are trying to challenge the male dominance leading to hostile sexism.This is also what research has found (Sibley et al., 2007).
Right-wing authoritarianism is a concept that captures dimensions of authoritarian tendencies (Altemeyer, 1981;Duckitt, 2001).RWA is related to a preference for traditional values, submission to authorities and endorsement of punitive practices to ensure that these values and submission is upheld (Manzi et al., 2017;Perry et al., 2013).People high in RWA tend to uncritically submit to and respect authorities and oppose deviance from the norms, prescribing a traditional way of life.They also have a strong proclivity for social control, such as restrictive laws and harsh punishment (Duckitt et al., 2010).In relation to sexist attitudes, RWA has been shown to consistently predict sexism (Austin & Jackson, 2019;Christopher & Mull, 2006;Lee, 2013).Specifically, RWA seems to be related to benevolent sexism among men (Sibley et al., 2007).
In sum, both SDO and RWA are strong predictors of sexism.Most studies suggest that SDO is more strongly related to hostile sexism (Christopher & Mull, 2006;Lee, 2013;Sibley et al., 2007;Sibley & Becker, 2012, although see Hannover et al., 2018 for an exception).
Relationship status
Misogyny may be dependent upon an individual's relationship status and history.Considering that the central theme for the incel movement is the difficulty these men have in finding a romantic partner, or in having sex (Ging, 2019) it can be assumed that most users on these forums are single.Indeed, the term involuntary celibacy implies that these men do not have a partner.
As women have come to put higher standards on the men they are dating, it has become increasingly difficult for men to find a partner.While being single may in fact be beneficial for women, single and divorced men experience poorer mental health compared to married men (Grundstr€ om et al., 2021;Williams et al., 2010).Poor mental health among men has in turn been associated with misogynistic attitudes (Fleming et al., 2018).Thus, it seems logical to include relationship status as a variable when explaining misogyny.While it is not assumed that single men by default are more misogynistic, the argument here is that individuals being single will moderate the effect of entitlement and SDO on misogynistic attitudes such that their effect will be stronger for single men compared to men who are in a relationship.The following hypotheses were formulated: H1: Entitlement and SDO predict misogynistic attitudes independently, controlling for RWA.H2: Relationship status moderates the effects of entitlement and SDO on misogynistic attitudes, such that the effects will be stronger for single men compared to men in a relationship.
For exploratory purposes, the interaction between relationship status and RWA is also included.
Method Overview
The present study was an online survey performed with American, heterosexual men as participants.Data collection was performed via the platform Prolific, which is an online platform connecting researchers and participants.Invitations to participate was sent to the subject pool that met the criteria of inclusion, which were being an American citizen, being a man and being heterosexual.All selection criteria are self-reported.Eligible participants were invited to a survey on social issues.The survey was available to all potential participants based on the selection criteria until the quota of 300 was filled 1 .Data was collected in February 2022.
Participants were invited to the survey and informed that it was about societal issues.They were further informed that some questions might feel extreme, but that these questions still reflect opinions in our society and hence are important to include.This was to safeguard against some people reacting to the questions about misogyny, where some items can be perceived as offensive.Then the participants were informed about ethics, such as voluntary participation, anonymity and data handling.They were required to provide informed consent by ticking a box stating that they had understood the information and agreed to participate.Following this, the survey started.Participants were first asked some background variables.Then followed the SDO and RWA scales, and finally the misogyny scale.
Measures
The dependent variable misogyny was assessed using the misogyny scale by Rottweiler and Gill (2020).The scale contains 10 items indicating that women seek to control men by sex, that women are deceitful, and items related to the devaluation of women.Some sample items are: "Women seek to gain power by getting control over men", "I think that most women would lie just to get ahead" and "I feel uncomfortable when a woman dominates the conversation".Responses were made on 7-point scales from 1 ¼ Strongly disagree to 7 ¼ Strongly agree.Inter-item correlations ranged from .39-84.Cronbach's alpha was high, 0.95.This measure is relatively newly developed and has shown good convergent and discriminant validity by Rottweiler and Gill (2020).While it can be used for both women and men, Rottweiler and Gill (2020) show that the effects are significantly higher among men compared to women.
The focal independent variables were entitlement and Social dominance orientation (SDO).Entitlement was measured using a measure of psychological entitlement, by Campbell et al. (2004), which is defined as " … a stable and pervasive sense that one deserves more and is entitled to more than others" (p.31).A benefit with this measure is that it a general sense of entitlement not only restricted to the sexual or relationship arena, which fits nicely with the aim of this article to provide general predictors of misogyny.Some sample items are: "I honestly feel I'm just more deserving than others", "Things should go my way" and "I deserve more things in my life".The scale has shown to be both reliable and valid, not associated with social desirability, according to Campbell et al. (2004).
Nine items were used, and answers ranged from 1 ¼ Strongly disagree to 7 ¼ Strongly agree.Inter-item correlations ranged from .39 À .71.Cronbach's alpha was high, 0.92.
Social dominance orientation was measured using a short version with 4 items, by Pratto et al. (2013).The scale has been tested in several cultural contexts and proved to be both reliable and valid.The items read: "In setting priorities, we must consider all groups" (R); "We should not push for group equality"; "Group equality should be our ideal" (R); "Superior groups should dominate inferior groups".and Answers ranged from 1 ¼ Strongly disagree to 7 ¼ Strongly agree.Inter-item correlations ranged from .52 À .83,and Cronbach's alpha was high, 0.87.This short measure has been tested for internal reliability and predictive validity across 15 languages, by Pratto and colleagues (2013).
Right-wing authoritarianism (RWA) was included as a control variable.It was measured using the short version of the RWA scale by Bizumic and Duckitt (2018).It contains 6 items and has shown to be both reliable and valid.Some sample items are: "The facts on crime and the recent public disorders show we have to crack down harder on troublemakers, if we are going to preserve law and order" and "It's great that many young people today are prepared to defy authority" (R).Answers ranged from 1 ¼ Strongly disagree to 7 ¼ Strongly agree.Inter-item correlations ranged from .32 À .62.Cronbach's alpha was high, 0.80.
Relationship status was assessed with the question "What is your current relationship status?" Response options were single, dating, married, other, prefer not to say.
Statistical analyses
All 302 participants were included in the data set.Because all participants had answered most of the questions, no observations were removed.There were however internal missing values since all items were voluntary.When a participant had a missing value on an item included in an index, the whole index was coded as missing for that person.
The relationship status measure was dichotomized into "single" and "in a relationship" (comprised of both dating and married).The options for "other" and "prefer not to say" were coded as missing values (total 9 persons).The final variable had a distribution of 167 (57%) in a relationship and 126 (43%) single.
The education variable was recoded into the following order: 1 ¼ Elementary school, 2 ¼ high school, 3 ¼ trade/technical/vocational education, 4 ¼ college, 5 ¼ graduate school and used as a continuous variable in the analyses.
Statistical analyses were performed using SPSS and Stata.
Results
First, some descriptive results are presented.Table 1 shows means, standard deviations, skewness and kurtosis for the main variables.As can be seen in Table 1, the means were fairly low considering the scale ranged to 7, which is to be expected in a normal population when assessing these variables.Skewness was also within acceptable ranges and should not pose a major problem to the statistical analyses (George & Mallery, 2010).
Table 2 shows the correlations between all variables in the study.The main dependent variable, misogyny was correlated with all other variables except education.Age was negatively associated with misogyny, such that younger men were generally more misogynistic than older.Relationship status was associated with misogyny such that single men displayed more misogynistic attitudes, compared to men who were in a relationship.Entitlement, SDO and RWA were all positively correlated with misogyny.Single men were younger compared to men in a relationship, and they were less educated.While younger men generally displayed higher entitlement, they were lower in both SDO and RWA compared to older men.All the focal predictors of the study, entitlement, SDO and RWA were positively correlated.
Next, t-tests were run to test for potential differences between men in a relationship and men who were currently single on the main outcome variable misogyny, as well as the independent variables entitlement, SDO and RWA.The results are presented in Table 3.As can be seen, only misogynistic attitudes differed significantly between men in a relationship and men who were single.
To test the hypotheses that entitlement and SDO would predict misogynistic attitudes independently and while controlling for RWA (H1), and that relationship status would moderate the effects (H2) a hierarchical regression analysis was run.In model 1, the control variables age and education level were included.In model 2 the focal predictors entitlement and SDO were added as well as the control variable RWA.In Model 3, relationship status was added and in Model 4, the interactions between relationship status and entitlement, SDO and RWA were added.The results are presented in Table 4.
As can be seen in Table 4, when adding more relevant predictors, the weak effect of age found in Model 1 disappear.Model 2 shows that entitlement, SDO and RWA have uniquely separate effects on misogynistic attitudes.Explained variance significantly increased from Model 1 to Model 2 when adding these predictors.These results support H1 stating that entitlement and SDO should have unique separate effects while controlling for RWA.That RWA also had a unique effect was somewhat unexpected as RWA previously has mainly been associated with benevolent sexism (Sibley et al., 2007), but some research show that RWA have an equally strong effect on hostile sexism (Van Assche et al., 2019).
In Model 3, relationship status was added and had a unique effect of its own on misogynistic attitudes while controlling for entitlement, SDO and RWA.Single men showed more misogynistic attitudes compared to those in a relationship.This effect, although relatively Exploring the role of entitlement, social Dominance Orientation, Right-wing authoritarianism weak (explained variance increased only 1% from Model 2 to Model 3) is noteworthy and is further reflected on in the Discussion.Finally, Model 4 included the interactions between relationship status and the focal predictors of entitlement and SDO, and exploratively also with RWA.Only the interaction between relationship status and SDO was significant, and the effect was weak (explained variance increased only 1% from Model 3 to Model 4). 2 The interaction is decomposed in Figure 1.
The significant interaction between SDO and relationship status is shown in Figure 1, where slopes are plotted with 95% Confidence intervals.As can be seen in the figure the confidence intervals do not overlap from about 3 on the SDO scale, even though they are close.A simple slope analysis showed that the slope for single men (coded as 1) was significantly different from 0, B ¼ 0.36, SE ¼ 0.07, t ¼ 4.74, p < 0.001, indicating that among single men, social dominance orientation had a positive relation with misogynistic attitudes which was absent among men in a relationship, B ¼ .13,SE ¼ .07,p ¼ .06.Admittedly, the slope for the men in a relationship was close to significant indicating that also in this group does it seem that SDO increase misogyny, but the effect is stronger among single men.This supports H2 stating that relationship status would moderate the effect of SDO on misogynistic attitudes.However, there was no significant interaction with entitlement, which was expected.RWA did not interact with relationship status.
Discussion
This article aimed to provide a better understanding of how individual level variables can explain misogyny.As most Western societies have taken steps towards gender egalitarianism, a gender backlash has occurred where some men become increasingly misogynistic.A central theme that flourishes on misogynistic online forums is masculine entitlement-men should have the right to women and sex.This is for instance seen in discussions about legalizing rape as a means to control and dominate women (DeCook & Kelly, 2021).Further, ideological orientations such as social dominance orientation (Pratto et al., 1994) and right- wing authoritarianism have both previously been connected to sexism, where SDO has been specifically connected to hostile sexism (Sibley et al., 2007).Hostile sexism comes very close to misogyny in that it entails a view of men as superior, and women are seen as a group that should be dominated.The results from the present study also included relationship status, arguing that being single should exacerbate the effects of entitlement and SDO on misogyny.In a survey with American men, it was found that entitlement, SDO and RWA predicted misogynistic attitudes uniquely.Only the effect of SDO was stronger for single men compared to those in a relationship.Interestingly, being single had a unique, albeit weak, effect on misogyny indicating that single men are in general more misogynistic than men in a relationship.While the present study does not allow for causal conclusions, one could speculate about why these men are single.Given that the last decades' equality work has led to that women are less reliant on men and freer to choose who they want to engage with and if they want it at all, it seems reasonable that misogynistic men are left to their own.This is also what the incel ideology prescribes-that feminist progress has enabled women to reject men, and these men wish to revert to a time when women were dependent upon men.However, given that the present study utilized a normal population sample it is noteworthy that being single had such a unique effect on misogyny.
The present study sheds additional light to the ideological variables predicting misogyny.The results are in line with previous research that has shown a relation between SDO and hostile sexism among men (Sibley et al., 2007).These relations are highly relevant given that SDO has also been connected to being positive to abusing women (Kiral Ucar & € Ozdemir, 2021)-a relation that was mediated by hostile sexism-men that desire dominance over women support sexist practices by accepting legitimizing myths that justify the inequality and hierarchy (Kiral Ucar & € Ozdemir, 2021).In contrast to previous research, RWA predicted misogyny with about equal magnitude as SDO did (Sibley et al., 2007).In a recent study, similar effects were found in a mixed gender-sample (Van Assche et al., 2019) and Hannover et al. (2018) found that among Muslim men, RWA, but not SDO predicted hostile sexism.In line with the results of the present article, Hellmer et al. (2018) showed that both SDO and RWA predicted hostile sexism in a sample of men.According to Glick and Fiske (1996;Fiske et al., 1999), hostile sexism results when men feel that their male dominance is threatened, while benevolent sexism is rather the result of men's dependence on women for intimacy.Thus, it may be that only some facets of RWA are related to benevolent sexism and some related to hostile sexism, which was also found in a mixed gender sample (Austin & Jackson, 2019).Specifically, conservatism, which entails the submission to authorities and obedience was related to hostile sexism, which was argued to be due to that women were seen as defying traditional gender roles.Hence, the results for RWA seem somewhat mixed and future research may explore the different sub-dimensions of RWA to better understand its effects on misogyny.
The fact that both SDO and RWA had unique and relatively strong effects on misogynistic attitudes is theoretically relevant as it highlights that misogynistic attitudes are driven by different motivations.SDO is concerned with the legitimization of group inequalities in society, including women as an inferior group.SDO has also been connected to lower empathic concern (B€ ackstr€ om & Bj€ orklund, 2007;Hellmer et al., 2018) which is in line with a misogynistic and dehumanizing view of women.RWA is rather concerned with submission to authorities, a strong preference for the traditional and value of conformity, and authoritarian aggression against transgressors of the traditional RWA has been connected to racist attitudes and that line of literature often argue that RWA entails a heightened sense of threat against the own group, and that other ethnic groups compose that threat (Peresman et al., 2021).Hence, it seems logical that men high in RWA may also see women as a potential threat to the ingroup of men, which could lead to more misogynistic attitudes.For instance, RWA has been shown to predict hostility against immigrants that do not assimilate with the majority culture (Thomsen et al., 2008).Thus, RWA may have a more complicated relation to misogynistic attitudes, where RWA mainly predicts misogyny when men high in RWA think about women that do not conform to traditional gender roles.
Only SDO was significantly moderated by relationship status.This means that single men who are high in SDO are those that express most misogynistic attitudes.Considering the misogynistic ideas flourishing the incel community, such a relation is not surprising.The main message of the incel ideology is group differentiation and dominance (Ging, 2019).
Further, misogyny has been connected to the alt-right movement and white supremacy, even labelled white male supremacy (Cottee, 2020).For instance, themes that masculinity or racial pureness is under threat, is sometimes collectively labelled aggrieved entitlement, a term that signifies the loss of certain privileges which have been reserved for identities in the intersection of race and gender, namely white men (Kalish & Kimmel, 2010).While these movements lack organization, being mainly composed of blogs, forums, and influencers (Forshcer & Kteily, 2019;Jones et al., 2020;Ranstorp & Ahlin, 2020), they do have a clear ideological goal, where they share the idea that some groups (essentially all minority groups, but particularly women and immigrants) are to be dominated over by others (white men) (Kaati et al. 2019).Their violence is designed to have far-reaching societal effects on the hierarchies and structure of social groups.Thus, incel and alt-right violence conforms to an emergent trend in terrorism with a more salient hate crime dimension that necessitates greater scrutiny and analysis (Hoffman et al., 2020).Given these interconnections it becomes a highly prioritized issue to better our understanding of who may be predisposed to such ideas.
Limitations and future directions Some limitations are worth to note.First, the present study is correlational, so even if it is assumed that ideological positions underlie misogyny, the causal path could also be the other way around-that is, misogyny as driving ideology.However, in an attempt to remedy this problem, Sibley et al. (2007) conducted a longitudinal study where SDO measured at Time 1 predicted hostile sexism measured at Time 2 (5 months later).Hence, it can be assumed that the same direction is applicable in the present study as well.
The conceptuatlization of misogyny is still not clear in the literature (Rottweiler & Gill, 2020).In the present study, a scale of misogyny was used (Rottweiler & Gill, 2020).However, upon inspection of the items, they do not differ greatly from hostile sexism as measured and conceptualized in the ambivalent sexism literature (Glick & Fiske, 1996).Both scales encompass items regarding women as sexual objects, that men are superior and should dominate women, and that women are decietful (Glick & Fiske, 1996;Rottweiler & Gill, 2020).In other research, other items have been used (Fleming et al., 2018).These items were more strongly associated with misogyny as often presented on incel forums, such as women are only good for having sex, that physically hurting woman would not problematic and that women have mistreated them in the past.One problem with measuring misogynistic tendencies in a normal population is that the variabels become very skewed if the items are too radical.An important challenge for future research is to reach a common conceptualization of misogyny that can be measured in a normal population.In relation, while it should be seen as a strength that the present study used a normal population sample of men, it would have been desireable to have some measure of activity on the manosphere or incel-forums included.It would help to further understand the individual factors associated with men seeking out the manosphere.
The effects in the present study were relatively small, except for the main effects of the individual predictors.This is not surprising as it comprised a normal population sample.As variables measuring relatively extreme opinions tend to be skewed in such samples, standard errors increase and explained variance decrease.Therefore, it should be seen as highly important that significant effects were indeed found, and they do point to that these relations carry some real-life validity.
Conclusions
The present study should be seen as a first important step in assessing correlates of misogyny, especially in a normal population.We need a better understanding of who is likely to come to endorse misogynistic attitudes and potentially violence against women.It is worrisome that we find effects on misogynistic attitudes of being single and SDO in a normal population of men, which indicate that these ideas are not limited to incel-forums, but seem both wide-spread and accepted among the male part of the population.
Figure 1 .
Figure 1.Interaction between Social dominance orientation and relationship status on misogynistic attitudes.Confidence bands represent 95% confidence intervals.
Figure A. 2 .
Figure A.2. Interaction between relationship status and psychological entitlement on misogynistic attitudes.Confidence bands represent 95% confidence intervals.
Figure A. 1 .
Figure A.1.Interaction between relationship status and right-wing authoritarianism on misogynistic attitudes.Confidence bands represent 95% confidence intervals.
Table 1 .
Means, standard deviations, skewness and kurtosis for the main variables.
Table 2 .
Correlations between all variables in the study.
Table 4 .
Hierarchical regression analyses predicting misogyny, unstandardized regression coefficients and standard errors are presented.
Table 3 .
Means and standard deviations for men in a relationship and single, and t-tests between the groups on misogyny, entitlement, Social dominance orientation and Right-wing authoritarianism. | 2023-03-17T15:08:35.328Z | 2023-03-15T00:00:00.000 | {
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15189218 | pes2o/s2orc | v3-fos-license | Pre-transplant Evaluation of Donor Urinary Biomarkers can Predict Reduced Graft Function After Deceased Donor Kidney Transplantation
Supplemental Digital Content is available in the text
INTRODUCTION
T he number of end-stage renal disease patients on the waiting lists for deceased donor kidney transplantation (DDKT) has grown extensively while that of available kidneys remains static. As a result, the waiting time for DDKT is increasing. Several strategies have been developed to increase the supply of organs, including the use of suboptimal donors -such as expanded criteria donors (ECDs). However, donors with acute kidney injury (AKI) at the time of organ procurement are often rejected for kidney transplantation because of variable outcomes. The quality of the donor kidney affects short-and long-term allograft outcomes. 1,2 Although various algorithms or scoring systems have been developed to evaluate kidneys from deceased donors, [3][4][5][6][7][8] arbitrary risk categorizations, unclear decision thresholds, excessive variables, and small differences in overall scores limit their usefulness. Serum creatinine or estimated glomerular filtration rate (eGFR) are commonly considered as assessment tools for evaluating deceased donor kidneys; however, during AKI, these levels do not represent the steady-state, and these values do not predict posttransplant outcomes very well. 9,10 Recently, new biomarkers such as urinary neutrophil gelatinase-associated lipocalin (uNGAL), urinary kidney injury molecule-1 (uKIM-1), and urinary L-type fatty acid-binding protein (uL-FABP) reflecting early pathological processes have been developed for the early diagnosis of AKI or ischemic injury. [11][12][13][14][15][16] Several attempts have been made to use recipient biomarker data for the prediction of graft dysfunction. 9,14,[16][17][18] However, surveying recipient biomarkers is not useful in decision making for the acceptance or allocation of deceased donor kidneys before DDKT. In contrast, donor biomarker analysis could serve as a valuable assessment tool for the quality of donor kidneys because these reflect multiple potential events for donor kidney injuries before kidney donation. Early graft dysfunction -defined as delayed or slow graft function (SGF) -has increasingly occurred since kidneys from ECD and those with acute injuries were used. 1 Early graft dysfunction is associated with a higher incidence of acute rejection, worse early posttransplant renal function, and poorer long-term graft survival. [19][20][21][22][23] Therefore, we investigated the usefulness of donor uNGAL, uKIM-1, and uL-FABP levels in predicting early graft dysfunction. Additionally, we developed a prediction model of early graft dysfunction using these donor biomarkers.
Patients
We prospectively enrolled deceased donors after brain death who successfully donated their kidneys between July 2010 and June 2013 at 4 Korean transplantation centers (Seoul National University Hospital, Chonbuk National University Hospital, Samsung Medical Center, and Inje University Busan Paik Hospital). All kidneys from the deceased donors were transplanted; however, 79 kidneys were delivered to hospitals that did not participate in this study. As a result, 94 deceased donors and 109 their recipients were enrolled. The Institutional Review Board approved the study protocol (H-1105-110-363), and informed consent was obtained from the legal guardians of donors. This study was performed in accordance with the Helsinki Declaration of 2000 and the Declaration of Istanbul 2008.
Data Collection
All clinical data were obtained from medical records. Donor renal function was evaluated at admission for organ donation and in the morning on the day of transplantation. eGFR was estimated using the Modification of Diet in Renal Disease (MDRD) equation for GFR 24 in adults and the Schwartz equation 25 in the 10 pediatric donors. ECDs were defined as previously described. 26 Recipient renal function was evaluated on 1 week and 1, 3, 6, and 12 months posttransplantation. Acute rejection episodes were defined as either biopsy-proven rejection or clinically suspected acute rejection improved by empirical steroid pulse therapy. Allograft loss was defined as the need to return to dialysis. Early graft function was classified as delayed graft function (DGF) for recipients requiring dialysis within the 7 days after transplantation, SGF or immediate graft function (IGF) for patients with serum creatinine levels !2.5 mg/dL 2 or 2.5 mg/dL without dialysis 7 days after transplantation, respectively. Several studies reported that not only patients with DGF but also patients with SGF showed worse short-and long-term outcomes than those with IGF, with the increased incidence of acute rejection in the first 6 months and a negative impact on long-term graft and patient survival rates. [19][20][21][22][23] Therefore, we adopted reduced graft function (RGF) which includes DGF and SGF as our primary outcome. 23 Donor AKI was defined on the basis of final serum creatinine as !2.0 mg/dL. 27 The primary outcome was the incidence of RGF, and the secondary outcome was donor AKI and 1-year graft function.
Measurement of uNGAL, uKIM-1, and uL-FABP Levels Pretransplant urine samples were obtained from all deceased donors at admission for organ donation and in the morning on the day of operation (D0). Fresh urine samples from both donors and recipients were immediately centrifuged at 5000 Â g to remove insoluble elements. Aliquots of the supernatant were stored at À80 8C and thawed at 37 8C before analysis. Urine creatinine levels were measured by standard enzymatic methods with an automated analyzer. All donor biomarker values were normalized to urine creatinine levels.
Immunohistochemical Staining for NGAL, KIM-1, and L-FABP Zero time (D0) protocol biopsies were performed before perfusion and collected at 1 center. Specimens were graded according to the Banff' 2007 classification. 28 Renal graft tissue immunostaining was performed with antibodies against NGAL (Sigma Aldrich, St. Louis, MO), KIM-1 (R&D Systems), and L-FABP (R&D Systems). Their expression levels were evaluated based on the proportion of immunopositive cells in the total tubular area as previously described. 14,15,29
Statistical Analysis
Chi-squared tests were used for categorical variables and Student's t-tests were used for continuous variables. Mann-Whitney U-tests were used for continuous variables with a skewed distribution. Correlations between urine biomarkers and continuous variables were assessed by Spearman correlation. The relationship between biomarkers and donor AKI was analyzed by multiple logistic regression analyses. Multiple linear regression analysis with a stepwise variable selection was used to assess the relationship between biomarkers and eGFR at 1 year after transplantation. The diagnostic performances of biomarkers to predict RGF were evaluated by receiveroperating characteristic (ROC) curves analyses. Multiple logistic regression analyses with a backward variable selection were used to assess the association between biomarkers and RGF in the presence of covariates and to produce the best-fit model for predicting RGF. First, we produced a full model including biomarkers and all relevant clinical factors as covariates. Then, we estimated Akaike information criterion in the each model of the backward selection process, and reduced the model until Akaike information criterion no longer improved to minimize the overfitting of the models and select the best-fit model. 30 Validation and calibration of the best-fit model were performed, internally in the full model with 1000 bootstrap resamples drawn using the 0.632 bootstrap resampling technique. 31,32 The bootstrap resampling technique is an effective technique for internal validation and calibration of a prediction model. 31 Validation and calibration using bootstrap resampling would estimate the likely performance of the model on a new sample of patients from a similar clinical setting, and measure the degree of error between the predictive probability and observed probability of the model. The estimate of optimism and the amounts of errors between the predicted probability and the actual probability of RGF in the best-fit model were calculated through the validation and calibration procedure previously described. 33 The RGF prediction score was established using the bestfit model, and the nomogram points for the score were depicted proportionally to the unstandardized b coefficients of the significant predictors in the best-fit model. The points of all significant predictors were summed to a total point. ROC curve analyses were used to assess diagnostic performances of the score and the cut-off point for RGF occurrence was determined using the Youden-J index. The goodness of fit of the scoring method was determined by Hosmer-Lemeshow test and Levenberg-Marquardt nonlinear regression analysis. The diagnostic performance of the score was compared with that of the kidney donor profile index (KDPI) 34 and the DGF risk calculator 7 using the DeLong test. 35 All statistical analyses were performed using R.v.3.1.3 for Windows and designated R packages including rms, pROC, ROCR and OptimalCutpoints for logistic regression analysis, validation, calibration, identifying optimal cut-off values, and comparisons between ROC curves.
Characteristics of the Study Population
The multicenter, prospective, and observational study enrolled 94 deceased donors and 109 corresponding recipients.
The clinical characteristics of the donors are shown in Table 1, recipient characteristics and transplantation details are summarized in Table 2. Ten pediatric donors (14-20 years old) were included, and there was no en bloc transplantation. DGF and SGF occurred in 15.6% and 4.6% of recipients, respectively. The overall 1-year patient and graft survival rate was 99.1%, and 95.4%, respectively. Acute rejection occurred in 21.1% of recipients. Mean cold ischemic time was 177 minutes, which was markedly shorter than that in other western countries.
The renal function of donors at admission for organ donation and D0 was significantly lower in the RGF group than in the IGF group (Table 1). Cold ischemic time was longer, and tacrolimus was used less often in the RGF group than in the IGF group (Table 2). Posttransplant renal functions during the 1st year after transplantation were also significantly worse in the RGF group than in the IGF group (Table 2). However, there were no significant differences between the RGF group and the IGF group in other characteristics of donor, recipient, or transplantation-related characteristics (Tables 1 and 2).
Expression of Biomarkers in D0 Protocol Biopsies
D0 protocol biopsies were collected from 46 recipients. There were no abnormalities in 80.4% of patients. Mild interstitial infiltration and acute tubular necrosis were found in 2.2% and 8.7% of patients, respectively. No significant difference in Remuzzi score was observed between the RGF and the IGF groups (data not shown). 36 NGAL, KIM-1, and L-FABP were detected in 66.7%, 14.3%, and 95.2% of samples, respectively (Supplemental Figure 1, http://links.lww.com/MD/A779). Overall, 90.5% of the samples had a KIM-1 expression score of 0.5, 81.0% had a score of 1, 66.7% had a score of 2, and 14.3% had a score of 3. However, tubular expression of NGAL, KIM-1, or L-FABP was not significantly different between the RGF and IGF groups. Furthermore, no correlation was observed between renal function and NGAL or L-FABP staining intensity or KIM-1 score at any time point after transplantation.
Urine Biomarkers of Donors
Donor uNGAL and uL-FABP levels were significantly higher in the RGF group than in the IGF group at admission for organ donation and D0; however, the statistical strength of associations at admission for organ donation was lower than those at D0 (Table 3). Furthermore, both uNGAL at D0 (r ¼ À0.419, P < 0.001) and uL-FABP at D0 (r ¼ À0.190,
Association of Biomarkers with Donor Acute Kidney Injury
Before transplantation, 18% of donors had AKI on the basis of final serum creatinine level. 27 37 showed that donor uNGAL was also significantly associated with donor AKI (OR 8.99, 95% CI 1.25-7.14, P ¼ 0.014).
To evaluate the usefulness of the donor biomarkers as pretransplant RGF predictors, we generated a prediction model that included donor biomarkers and other relevant clinical factors already known to be predictive for RGF. 4 -7,34 Univariate logistic regression analyses showed that donor serum creatinine, cold ischemic time, and uNGAL and uL-FABP at D0 were significantly associated with RGF. Multivariate logistic regression analysis demonstrated that the reduced best-fit model still included donor serum creatinine, and uNGAL and uL-FABP at D0 as RGF predictors after other variables were adjusted ( Table 4).
Validation of the Model Including Biomarkers for RGF
Using predictors relatively associated with RGF as indicated by the multivariate logistic regression analysis, we performed ROC curve analysis to assess the diagnostic performance of the best-fit model for RGF development. The AUROC of the best-fit model was 0.816 (95% CI 0.716-0.917, P < 0.001), which was significantly better than the diagnostic performance of donor creatinine alone for RGF ( Figure 1E, Table 4). Validation of the best-fit model with 1000 bootstrap resamples showed that the estimate of optimism was 0.064 and the bias-adjusted AUROC of the best-fit model was 0.752 (Supplemental Figure 2, http://links.lww.com/MD/A779). The estimates of both models were generally linear and agreed well with the ideal line. The mean absolute error and 0.9 quantile absolute error of the predicted probability were 0.029 and 0.039, respectively, suggesting only a small degree of bias from overfitting.
Prediction Score for RGF
Using the best-fit model, we generated a nomogram for a scoring method predictive for RGF ( Figure 2, Table 5). uNGAL and uL-FABP were log 10 -transformed before they were included in the model. The prediction score ranged from 0 to 220. The AUROC of the prediction score was 0.808 (95% CI 0.707-0.909, P < 0.001). We compared our model with other well-known models, including the DGF calculator 7 and KDPI. 34 Notably, the AUROCs of the DGF calculator and KDPI for predicting RGF were 0.627 (95% CI 0.481-0.772, P ¼ 0.016) and 0.606 (95% CI 0.471-0.740, P ¼ 0.190), respectively. The diagnostic performance of the RGF prediction score was significantly better than those for both the DGF calculator and KDPI (P ¼ 0.046 and 0.019, respectively; DeLong tests). The optimal cut-off value of the prediction score was !144 points, as determined by the maximum values of the Youden-J indexes. The sensitivity and the specificity of the prediction score at this value were 86.4% and 65.5%, respectively. Since the prevalence of RGF was 20.2%, the positive and negative predictive values of the prediction score were 38.7% and 95.0%, respectively ( Figure 3). The Hosmer-Lemeshow goodness-of-fit test showed a P value of 0.85 for the prediction score. When the The data were presented as the median (the 1st quartile, the 3rd quartile). Cr ¼ urine creatinine, IGF ¼ immediate graft function, KIM-1 ¼ kidney injury molecule-1, L-FABP ¼ L-type fatty acid binding protein, NGAL ¼ neutrophil gelatinase-associated lipocalin, RGF ¼ reduced graft function.
à Comparison between the IGF group and the RGF group using Mann-Whitney U-tests. D0, in the morning of the day of transplantation operation.
patients were equally divided into 5 groups based on their prediction score, the observed probability of RGF corresponding to the average prediction score of each group was also wellmatched with the probability predicted from the average prediction score (Levenberg-Marquardt nonlinear regression coefficient R 2 ¼ 0.967; Figure 3).
In the univariate linear regression analyses, donor age, KDPI, recipient BMI, acute rejection, uNGAL at D0, uKIM-1 at D0, and uL-FABP at D0 were significantly associated with 1year eGFR. Multiple linear regression analyses adjusting for donor age, donor gender, KDPI, cold ischemic time, recipient age, recipient gender, recipient BMI, recipient diabetes history, number of HLA mismatches, induction therapy, use of calcineurin inhibitor, acute rejection, and donor urinary biomarkers at D0 showed that donor uL-FABP, donor age, and acute rejection were significant predictors of 1-year graft function (Table 6).
DISCUSSION
Both uNGAL and uL-FABP of donors were reliable for RGF prediction and correlated with donor renal function. The best-fit model including donor uNGAL, uL-FABP, and serum creatinine at D0 provided better predictive values for RGF than To find the best-fit model for predicting RGF, a backward variable selection procedure was performed during the multivariate logistic regression analysis. The best-fit model included donor creatinine, uNGAL at D0 and uL-FABP at D0 as significant predictors of RGF after the other variables were adjusted. D0, in the morning on the day of transplantation operation. BMI ¼ body mass index, CI ¼ confidence interval, CVA ¼ cerebrovascular accident, KT ¼ kidney transplantation, L-FABP ¼ L-type fatty acid-binding protein, NGAL ¼ neutrophil gelatinase-associated lipocalin, OR ¼ odds ratio. The prediction score included donor uNGAL, uL-FABP, and serum creatinine. The score for each patient was the sum of the points designated to the 3 predictors. The gradations between 2 numbers in the rulers designated to uNGAL and uL-FABP equally divided the differences between the 2 numbers (e.g., the gradation next to 10 2 indicates 2 Â 10 2 ). (B) Goodness of fit of the prediction score for RGF. The dashed line indicates the predicted probability according to the score, and the black dots indicate the observed frequency of the RGF at the mean scores of each quintile. The observed frequency agreed well with the predicted probability according to the score. (Levenberg-Marquardt nonlinear regression coefficient R 2 ¼ 0.967). RGF ¼ reduced graft function, uKIM-1 ¼ urinary kidney injury molecule-1, uL-FABP ¼ L-type fatty acid-binding protein, uNGAL ¼ urinary neutrophil gelatinaseassociated lipocalin.
donor serum creatinine alone. Based on the best-fit model, we generated a nomogram for predicting RGF. The diagnostic performance of the prediction score for RGF was significantly better than those for the DGF calculator and KDPI. Donor urinary NGAL levels at D0 were also associated with donor AKI. Furthermore, uL-FABP at D0 was predictive for 1-year allograft function.
Because of organ shortages, kidneys from deceased donors with high risk for graft dysfunction and graft failure have been increasingly used. 1,2 Patients with RGF, including DGF or SGF, showed poorer short-and long-term outcomes than those with IGF, with a higher incidence of acute rejection episode, diminished posttransplant graft function, and an increased risk of graft failure. [19][20][21][22][23] Therefore, it is important to predict graft dysfunction with an early and reliable biomarker when determining the acceptance or allocation of deceased donor kidneys. Several studies have identified early biomarkers for predicting graft dysfunction. 9,12,14,[16][17][18] Urinary biomarkers directly reflect tubule damage in kidneys, and sampling is easy and noninvasive. The recipient urinary NGAL levels within the 1st week after transplantation are an early marker of DGF and are independently associated with 1-year graft function. 9 The recipient urinary L-FABP level can serve as an early indicator of ischemia and a predictor of early allograft function. 17 However, while previous studies have demonstrated the prognostic value of recipient urinary biomarkers for DGF, 9,17 these cannot be used to assess deceased donor kidneys with AKI before DDKT. Therefore, there is still a need for good donor markers.
A recent study reported that the uNGAL levels in AKI group were higher than those in non-AKI group in stable hospitalized patients awaiting cardiac surgery. 38 Reese et al 37 also showed a significant association between donor urinary NGAL and donor AKI. Thus, our findings on the association between donor uNGAL levels and donor AKI are in agreement with these previous studies. 37,38 Based on the previous studies, donor urinary biomarker reflected the multiple potential pathways associated with acute kidney injuries including trauma, hypotension, and exposure to nephrotoxic agents.
Recently, Hollmen et al 12 investigated the prognostic value of the uNGAL levels in deceased donors for the 1st time. In patients with high donor uNGAL (!18 ng/mL), prolonged DGF (!14 days) occurred more often than in those with low uNGAL (<18 ng/mL), and 1-year graft survival was worse. uNGAL was an independent risk factor for prolonged DGF, but failed to predict DGF itself. Reese et al 37 also analyzed the associations between biomarkers in deceased donors and outcomes including donor AKI, DGF, and 6-month eGFR. Notably, uNGAL, uKIM-1, and uL-FABP in donors were strongly associated with donor AKI, and uNGAL was associated with DGF. Furthermore, both uNGAL and uL-FABP were associated with 6-month eGFR only among recipients without DGF. In this study, DGF incidence was lower (15.6%) than those in the previous studies (31%-39.8%), most likely because both donors and recipients were younger, ECDs were less common, and cold ischemic time was shorter. 12, 37 We used RGF as an outcome in this study due to this low DGF incidence. In contrast, the duration of dialysis before transplantation was longer, donor serum creatinine was higher, and uNGAL levels were higher in this study. Both the D0 biopsy findings and 1year graft survival rates were similar between this study and the study by Hollmen et al. 12 Despite differences in donor characteristics of each study population or country, our findings that donor uNGAL or uL-FABP can be associated with donor AKI, as well as predict RGF and posttransplant graft function were in agreement with those of previous studies. 12,37 As such, donor urinary biomarkers, including uNGAL and uL-FABP, may sufficiently predict posttransplant graft outcomes irrespective of DGF incidence. KIM-1 levels in recipients have been reported to correlate with renal graft function. 16,18 However, the prognostic values of KIM-1 were modest, and the combination of KIM-1 with other biomarkers rather than KIM-1 alone showed a strong ability to predict AKI. 39 This study demonstrated that donor uKIM-1 failed to predict RGF or correlate with 1-year allograft function. KIM-1 is upregulated at 48 hours after ischemia-reperfusion injury and peaks at 2-3 days after injury, persisting thereafter. 40 Both uNGAL and uL-FAPB are elevated within 3 hours of injury, and peak within 6 hours. 41 Although the increased levels of NGAL and L-FABP represent a response to kidney injury, increased KIM-1 might indicate ongoing recovery and regeneration after injury. These differences may have contributed to the poor predictability of uKIM-1 for RGF in contrast to uNGAL and uL-FABP.
Increased NGAL expression in renal allograft tissues was associated with poor graft function after transplantation, 14 whereas KIM-1 did not predict DGF. 29 In this study, tissue biomarkers expression in D0 biopsies did not discriminate between patients with RGF and IGF. Superficial wedge biopsy sampling often results in markedly variable samples, and hamper the evaluation of injuries in the outer medulla, the main area of acute injury. Thus, relatively mild tissue injury and the lower sample sizes of this study might contribute to the negative results for tissue biomarkers.
We further proposed a simple scoring system to predict RGF based on only 3 donor parameters available before transplantation, which were selected based on their contribution to the AUROC and P value. The diagnostic performance of the RGF prediction score was better than that for the DGF calculator and KDPI. Additionally, we proposed additional cutoffs for medical decision-making based on these predictive values. Among kidneys with a prediction score for RGF < 144, 95% will likely have IGF after KT. This information could be useful for clinicians when deciding whether to accept or discard a donor kidney and when deciding to whom the kidney should be allocated.
In this study, donor urine biomarkers were measured at admission and in the morning on the day of operation. Because donors may experience several kidney injuries, including trauma, hypotension, and exposure to nephrotoxic agents, before and during hospitalization, biomarker levels on the day of operation may be better to assess the quality of the donor kidney than those obtained at admission. However, Multivariate linear regression analysis was performed with a stepwise variable selection procedure. uL-FABP at D0, donor age and acute rejection correlated with 1-year graft function after the other variables were adjusted. D0, in the morning on the day of transplantation operation.
ATG ¼ antithymocyte globulin, BMI ¼ body mass index, CI ¼ confidence interval, CNI ¼ calcineurin inhibitor, DM ¼ diabetes mellitus, HLA ¼ human leukocyte antigen, KDPI ¼ kidney donor profile index, KIM-1 ¼ kidney injury molecule-1, KT ¼ kidney transplantation, L-FABP ¼ L-type fatty acid binding protein, NGAL ¼ neutrophil gelatinase associated lipocalin. biomarker levels on the day of operation might be impractical for organ allocation decision. Unfortunately, we did not evaluate the exact longitudinal changes of donor biomarkers between the time of admission and organ procurement; thus, further longitudinal studies are needed to decide the best time-point for the measurement of donor urinary biomarkers.
To our knowledge, this study is the 1st to investigate the association of donor urinary and tissue biomarkers with RGF and 1-year graft function in DDKT, and introduce a practical scoring method including donor urinary biomarkers to predict RGF. This study is also unique in that it was based on an Asian population with short cold ischemic times and a low overall incidence of DGF, unlike Western populations investigated in previous studies. Further, larger-scale, long-term studies including external validation are needed to confirm our results.
In conclusion, both urinary NGAL and L-FABP of donors are useful biomarkers for RGF after DDKT, and a new score based on donor biomarkers can predict RGF well. The prediction score for RGF can help guide allocation of deceased donors, and acceptance of kidneys from deceased donors by clinicians and transplant candidates, and can contribute to maximal organ utilization. | 2018-04-03T05:01:01.519Z | 2016-03-01T00:00:00.000 | {
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243149994 | pes2o/s2orc | v3-fos-license | Bedding Plane Effects on Mechanical Behavior of Surrounding Rock in Mountain Tunneling
)e layered rock showed the characteristics inMountain tunnel, Yunnan. A series of uniaxial compression tests and variable angle shear tests were carried out, and the aim was to investigate the effect of the bedding on its mechanical parameters and failure modes. )e test results show that the uniaxial compressive strength, elastic modulus, and Poisson’s ratio of layered rock present a U-shaped distribution with the increase in bedding orientation from 0° to 90°. All of them have a maximum when the bedding orientation is 0° and a minimum when the bedding orientation is 45°. )e failure modes of layered rock can be summarized into three types: the fracture tensile failure parallel to the weak plane of bedding; the shear slip failure along bedding weak plane; and tension-shear composite failure between bedding weak plane and matrix. Based on the testing data and analysis results, it can be concluded that the layered rock specimen with different bedding orientations is an important reason for the anisotropy of mechanical parameters and failure modes.
Introduction
e layered rock is usually interbedded with soft and hard rock. e main structural plane is bedding plane, and there are weak structural planes such as interlayer dislocation and argillaceous intercalation. e deformation and failure are mainly controlled by geology and rock combination. e heterogeneity of the rock with bedding plane is enhanced, and the minerals on the internal plane are easy to form weak structural plane. When there is bedding plane in the rock mass, it makes the axial strain larger. In the construction of tunnels in layered rock, the anisotropy of surrounding rock is very important to the safety and stability [1,2]. When drilling in layered rock formation, the permeability of fractures between bedding is much higher than that of rock matrix. Fluid is easy to immerse into the formation along the microfractures between bedding, which changes the effective stress state around the well, reducing the strength of layered rock and affecting the stability of wellbore during drilling [3]. erefore, it is of great engineering application value to study that bedding orientation effects on mechanical parameters and failure mode of layered rock.
At present, many scholars have obtained lots of achievements in the study of the effect of bedding orientation on layered rock. Wang et al. [4] conducted Brazilian splitting tests on slates with different bedding orientations and clarified the failure mechanism by analyzing the changes of tensile strength and failure behavior. Yang et al. [5] carried out the direct shear test on layered rock specimens, and the results show that the bedding orientation has a significant effect on the cohesion and internal friction angle of rock. Xia et al. [6] carried out the direct shear test of layered rock, and the results show that with the increase in the bedding orientation, the shear strength index of rock mass first increases, then decreases, and finally increases. Fan et al. [7] used theoretical analysis and numerical simulation to study the macroscopic mechanical behavior of layered rock and discussed transversely the isotropy strength criterion of layered rock. Li et al. [8] without considering the influence of c and φ, when the bedding orientation of layered rock is 45°, the crack initiation and propagation are most likely to occur; when β is known, the corresponding fracture toughness value can be obtained. Liu et al. [9] conducted uniaxial compression tests to show that there are two forms of compression failure of layered rock: compression shear failure occurs from the interior of layered rock and the shear plane is rough; the surface of layered rock is smooth, and sliding failure occurs along the bedding plane. Gao et al. [10] conducted compressive and tensile strength tests of shale and analyzed the transversely isotropy characteristics of shale specimens. e results show that the tensile strength perpendicular to the bedding plane is about 300-360 times that parallel to the bedding plane, and the velocity anisotropy ratio of compression wave is about 1.3-1.4. Liu et al. [11] obtained three failure mechanisms of layered rock: tensile failure along bedding plane, shear failure along bedding plane, and tensile failure of matrix in the direct tensile test. rough numerical simulation, Tan et al. [12,13] analyzed the stability of layered rock with different bedding directions, the range of surrounding rock loose area, and the key parts of instability, which has important theoretical significance for further understanding the instability mechanism of tunnel in layered rock. Xia et al. [14] carried out numerical simulation of rock with different bedding orientations under the uniaxial compression test and obtained that when the bedding bond strength is very different from the bedrock, the effect of bedding can be ignored, while when the two are close, bedding has a great influence. Fu et al. [15] conducted the uniaxial compression test on layered rock and obtained that there are three failure forms of rock mass with the change of bedding orientation. Yuan et al. [16] studied the influence of loading rate on the mechanical properties of layered rock and obtained that the total energy, elastic strain energy, and dissipated energy of rock mass after exceeding the critical value of loading rate have S-shaped curve variation law similar to the strength.
In this paper, the transverse isotropy and anisotropy of layered rocks in Mountain tunnel, Yunnan, were studied.
rough the uniaxial compression test and variable angle shear test, the variation law of peak strength and mechanical parameters of layered rocks with different bedding orientations was explored, and the failure modes were classified and analyzed. When layered rocks were treated as transversely isotropic bodies, five independent material parameters were determined. e relationship among bedding orientation, cohesion, and internal friction angle was established.
Calculation Principle of Transversely
Isotropic Constitutive Model Parameters e physical parameters, mechanical parameters, and hydraulic parameters of rock mass usually show anisotropy, and these parameters will change with the change of direction. Although layered rock shows different mechanical properties at different bedding orientations, it is usually regarded as transversely isotropic in practical engineering applications. As shown in Figure 1, the coordinate system X, Y, and Z is set, where the Z axis is the longitudinal axis direction of the specimen. In the general coordinate system, the stress-strain relationship is as follows [17]: where ε is the strain tensor, S is the flexibility matrix, and σ is the stress tensor. e matrix is expressed as follows: In equation (2), there are five independent elastic parameters, namely, E 1 , E 2 , v 1 , v 2 , and G 12 , which together represent the deformation performance. E 1 and E 2 are the elastic modulus in the transversely isotropic plane and the direction perpendicular to the plane, respectively; Poisson's ratios v 1 and v 2 describe the reflection of the transverse strain in the transversely isotropic plane and the stress parallel or perpendicular to the isotropic plane, respectively; G 12 is the shear modulus perpendicular to the transversely isotropic plane.
In this experiment, v 1 and v 2 are Poisson's ratio when the bedding orientation is 90°and 0°, respectively. In order to determine these five independent material parameters, at least one specimen with any bedding orientation is needed in addition to the specimens with 0°and 90°bedding orientation. erefore, the sample with 30°bedding orientation is selected. e formula of the elastic modulus with β between loading direction and bedding plane is as follows [17]: where E β is the elastic modulus when the angle between loading direction and bedding plane is β.
Specimen Preparation.
e rock core used in the test is taken from the tunnel of Xiangli Expressway Tunnel in Yunnan Province. e structural characteristics of the rock mass are plate-shaped or flaky-shaped. ere is a small amount of clay and silty filler in the rock fracture (as shown in Figure 2). e specimens were taken from the whole large rock mass and cut into the whole cube. In order to study the anisotropic characteristics of rock mass under different bedding orientations, core drilling sampling was carried out from five directions of 0°, 30°, 45°, 60°, and 90°, respectively. e schematic diagram of core drilling sampling is shown in Figure 3.
According to the specific requirements, the specimens used in the test were processed into test blocks of different sizes and shapes by drilling and grinding machines. e number of test blocks in each group should not be less than 3.
e standard specimens were made as follows: Φ25 × 50 mm cylinder specimens were used for the uniaxial compression test (as shown in Figure 4), and cube specimens with 30 mm side length were used for the variable angle shear test (as shown in Figure 5).
Test Equipment and Scheme.
e uniaxial compression test and variable angle shear test were carried out on cylinder specimens and cube specimens, respectively. GAW-2000 electrohydraulic servocontrolled uniaxial test machine and hydraulic screen display universal testing machine (a set of 20°, 30°, and 40°variable angle shear fixture) are used, and these equipments have stable performance and high test accuracy. In the uniaxial compression test, the displacement control mode was adopted, and the axial load was applied to the specimens at the loading speed of 0.015 mm/min, and the stress-strain curve was collected at the same time. In the variable angle shear test, the stress control method was used to apply the load at the loading rate of 100 N/s until the specimens failed, and the failure load P was recorded, as shown in Figure 6.
Influence of Bedding Structure on Rock Mechanical
Parameters.
e results of uniaxial compression tests of layered rock with different bedding orientations are shown in Figure 7.
It can be seen from Figure 7(a) that when the bedding orientation changes from 0°to 90°, the peak strength of layered rock first decreases and then increases. e peak strength decreases between 0°and 45°and increases between 45°and 90°. On the whole, there is a "U-shaped" change trend of high on both sides and low in the middle. e peak strength is the highest at 0°and the lowest at 45°. e results are consistent with the previous layered rock test results [18][19][20]. e maximum peak strength is more than three times of the minimum peak strength. erefore, the peak strength of layered rock is affected by bedding orientation, showing obvious anisotropy characteristics. According to the concept of "anisotropy ratio" proposed by J. Singh et al. [21] in 1989, the anisotropy ratio of peak strength of layered rock is defined as follows: where R c is the anisotropy ratio of peak strength; σ c max is the maximum peak strength; and σ c min is the minimum peak strength. According to Figure 3, when the bedding orientation is 0°, the peak strength is the maximum (94.90 MPa), and when the bedding angle is 45°, the peak strength is the minimum (27.26 MPa); that is, R c is 3.48, so the anisotropy ratio of the peak strength of layered rock is medium anisotropy. According to the test results and equation (3), five independent parameters of layered rock can be obtained, as shown in Table 1.
After the transverse isotropy material parameters of layered rock are determined, more accurate engineering analysis can be carried out for the rock engineering with layered rock as the main lithologic characteristics, such as the change of rock stress state and the deformation of rock mass caused by tunnel excavation in layered rock stratum.
Influence of Bedding Structure on Rock Fracture
Characteristics.
e anisotropy of mechanical parameters of layered rock is closely related to its fracture mode. With the change of bedding orientation, the fracture of specimens shows different failure modes. In the uniaxial compression test, the specimen is subjected to load and goes through compaction stage, elastic stage, expansion stage, and failure stage. When the axial stress reaches the specimen peak strength, the accumulated energy inside the specimen is released suddenly and the failure occurs. Obvious macrocracks appear on the surface and run through the whole (1) β � 0°. Shear slip failure is the main failure form. For the specimens with 0°bedding orientation, the crack direction is almost horizontal, only a few splitting cracks appear at the end of the specimen. e reason is that the loading direction is orthogonal to the bedding orientation, and the two ends of the specimen are subject to friction resistance, which limits its lateral deformation. e tensile failure through the weak plane of the bedding is formed in the middle of the specimen under the action of large lateral tension, which leads to the obvious shear slip phenomenon of the upper and lower parts of the specimen along the direction approximately parallel to the weak plane of the bedding, and causes the local surface fracture of the specimen. Under the action of large lateral tension in the middle of the specimen, the tensile failure through the weak plane of the bedding is formed, which leads to the obvious shear slip phenomenon of the upper and lower parts of the specimen. Under the action of load, obvious shear failure is formed along the bedding weak plane at the upper and lower ends of the specimen, and many flat failure planes are formed at the upper and lower ends of the specimen, and the shear slip phenomenon parallel to the bedding weak plane occurs. is is because the shear stress on the bedding weak plane is greater than the shear strength of the specimen itself.
(5) β � 90°. Tension and shear failure coexist, but tension failure is the main failure form. e crack has a small vertical inclination and through the entire rock specimen. Under the action of load, the specimen gradually appears tensile cracks parallel to the bedding plane and finally forms the splitting tension failure along the bedding plane. e specimen has a tensile fracture plane which is obviously parallel to the bedding weak plane and runs through the matrix and through the ends of the entire rock specimen. Since the entire specimen is divided into two parts, one large and one small, their bearing capacity is reduced, respectively. e smaller part of the thin slab-shaped rock bears greater load than its own compressive strength. It leads to buckling instability and secondary rupture along the bedding weak plane.
From the above analysis, the main controlling factors of layered rock fracture with different bedding orientations can be obtained. It can be seen from the analysis that for any rock specimen failure mechanism with different bedding orientations, the bedding weak plane plays a leading role. erefore, the bedding weak plane has become a key factor affecting the anisotropy of the failure mechanism of layered rock specimens. e elastic modulus is an important performance parameter of engineering materials. From the macroscopic point of view, the elastic modulus is a measure of the ability of an object to resist elastic deformation. e elastic modulus of the material can be affected by all the factors that affect the bonding strength. When there is bedding plane in the rock, the bedding plane belongs to the weak plane of the rock, so compared with the matrix material, the stiffness of the weak plane of bedding is small and the deformation is large. Compared with the rock mass without bedding plane, this kind of rock is prone to failure under uniaxial compression. e variation of elastic modulus and Poisson's ratio with bedding orientation is shown in Figures 7(b) and 7(c). It can be seen from Figure 7(b) that under the uniaxial compression test of layered rock, the elastic modulus first decreases and then increases with the increase in the angle between bedding orientation and horizontal plane. On the whole, it is similar to the change trend of compressive strength and also presents a U-shaped change trend of high on both sides and low in the middle. When the bedding orientation is 0°(parallel to the bedding), the elastic modulus of layered rock is the largest; when the bedding orientation is 45°, the elastic modulus of layered rock is the smallest. e vertical bedding is easy to be tensioned and split along the bedding plane under the action of axial stress; in parallel bedding, because the axial stress is perpendicular to the bedding plane, the stacking reaction occurs on the bedding plane during loading, resulting in the compression of the bedding plane, and the axial strain changes relatively large. e deformation modulus is used to define the anisotropy ratio of layered rock: where R E is the anisotropy ratio of the elastic modulus; E max is the maximum elastic modulus of layered rock between 0°a nd 90°bedding orientation; and E min is the minimum elastic modulus of layered rock between 0°and 90°bedding orientation. e ratio of the maximum to the minimum is 2.8, which is the anisotropy ratio of the deformation modulus of layered rock.
Poisson's ratio of layered rock specimens, as shown in Figure 7(c), is similar to the compressive strength and elastic modulus of layered rock. It shows a U-shaped change trend of high on both sides and low in the middle. On the whole, Poisson's ratio decreases or increases but the amplitude is small at the low bedding orientation (from 0°t o 30°) and the high bedding orientation (from 60°to 90°), which indicates that the bedding anisotropy is weak at this time; at the slightly higher bedding orientation (from 30°to 60°), Poisson's ratio changes greatly with the bedding orientation, which indicates that the bedding anisotropy is strong at this time.
Effect of Bedding Plane on Rock Shear Properties.
e results of the variable angle shear test of layered rock are shown in Table 2.
According to the measured data of normal stress and shear stress in Table 2, the fitting results are shown in Figure 9.
It can be seen from Figure 9 that the overall fitting degree of the Mohr-Coulomb fitting curve is very high. According to Figure 9, the cohesion and internal friction angles with different bedding orientations can be obtained, and the results are shown in Table 3.
According to the above, the cohesion and internal friction angle with different bedding orientations are obtained, and the relationship among bedding orientation, cohesion, and internal friction angle is established, as shown in Figure 10.
It can be seen from Figure 10 that with the increase in the angle between bedding plane and horizontal plane, the cohesion and internal friction angle of layered rock specimens first decrease and then increase, showing a U-shaped variation trend of high at both sides and low in the middle similar to the compressive strength curve. When the bedding orientation is 0°and 90°, the cohesion and internal friction angle are relatively large, and when the bedding orientation is 60°, they are relatively small. e failure modes are shown in Figure 11. When the bedding orientation is 0°, there is the composite failure of tension through weak bedding plane and shear slip along bedding plane; when the bedding orientation is 30°, the shear failure along the weak bedding plane and through the bedding plane occurs; when the bedding orientation is 60°, the shear slip failure along the weak bedding plane occurs, and the ends present the phenomenon of crossing the matrix; when the bedding orientation is 90°, the splitting tensile failure along the weak bedding plane occurs, and the failure surface is relatively regular.
Conclusions
In this paper, uniaxial compression and variable angle shear tests are carried out on rocks with different bedding orientations. By analyzing the evolution trend of mechanical parameters and failure modes of with different bedding orientations, the following conclusions are drawn: (1) e compressive strength of layered rock has obvious anisotropy. With the increase in the angle between bedding orientation and horizontal plane, the uniaxial compressive strength first decreases and then increases and generally presents a U-shaped variation trend of high on both sides and low in the middle. R C � 4.8, indicating that the specimen is moderately transverse isotropic. (2) e elastic modulus and Poisson's ratio of layered rock first decrease and then increase with the increase in the bedding orientation. On the whole, the variation trend of both is similar to the compressive strength of layered rock. (3) In the uniaxial compression test of layered rock, the anisotropy of failure mechanism of layered rock is closely related to the bedding orientations, which is mainly reflected in different failure modes with different bedding orientations. On the whole, the failure modes of Figure 1specimens can be divided into three types: the fracture tensile failure parallel to the weak plane of bedding; the shear slip failure along bedding weak plane; and tension-shear composite failure between bedding weak plane and matrix. (4) e cohesion and internal friction angle of layered rock specimens show a U-shaped change trend of high at both sides and low in the middle similar to the compressive strength curve. For the variable angle shear test, the 0°specimen shows the composite failure of tension through the bedding weak plane and shear slip along the bedding plane. Shear failure along bedding plane and through bedding plane occurs in the 30°specimen. e shear slip failure along the bedding plane occurs in the 60°specimen, and the end of the specimen runs through the matrix. e 90°specimen shows splitting tensile failure along the bedding weak plane.
Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper. | 2021-10-15T15:04:24.629Z | 2021-10-13T00:00:00.000 | {
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248005906 | pes2o/s2orc | v3-fos-license | A local phase space stochastic quantization?
I examine whether Nelson's stochastic formulation of Schr\"{o}dinger equation could be derived from a phase space process through a colored noise smoothing. If this conjecture is true, it would yield a local stochastic hidden variable theory. I discuss how this does not necessarily contradict Bell type theorems as general local stochastic theories can violate local causality assumptions. I also discuss the generalization to quantization of fields and speculate about the gravitational origins of noise.
Introduction
Ever since the advent of quantum theory, the question of whether there exists a local deterministic or stochastic theory underlying quantum mechanics has been of considerable interest. One important direction was provided by Nelson's seminal work on the reformulation of quantum mechanics in terms of stochastic mechanics of particles [1][2][3]. Stochastic mechanics is an exact reformulation of quantum theory like the path integral formulation. However, when applied to multiple particles and fields, it is manifestly non-local. Nelson suggested the important question [3]: Can one give a local model which would reduce to stochastic mechanics in an appropriate limit?. This is the central question, I am concerned with in this note. Although, it is folklore to believe that it is impossible, I argue following Nelson that Bell type theorems do not actually rule out local stochastic theories which in general can violate local causality assumptions. I outline the construction of a simple local theory and formulate conjectures which would enable such a theory to exist first for the single particle case, then for multiple particles and finally for the prototypical example of the scalar field. Of course, existence of such a theory does not mean that the universe obeys it. Assuming that the theory is physical, I will also speculate about potential gravitational origins of noise. In the absence of evidence for the quantization of the gravitational field, this could provide a consistent view of the universe where quantum theory arises out of gravitational interactions for all the fields in the universe. This is similar to that Brownian motion of a single particle arises from the microscopic Newtonian dynamics of a gas.
To see the main difficulty, consider the Madelung formulation of quantum theory: where ρ(x, t) is the probability of finding the particle at (x, t) and S(x, t) is the phase of the wave function. The second equation would be the classical Hamilton-Jacobi equation if the last term (quantum potential) was absent, and the equations would describe an ensemble of classical particles. Thus, a derivation of Schrödinger equation must somehow generate the quantum potential term either implicitly or explicitly. Nelson's stochastic mechanics in terms of particles trajectories (see section 2.1) provides an implicit understanding of this term in terms of averaged stochastic derivatives. If there are multiple particles, the quantum potential term in general depends on all the positions of the particles. A stochastic unraveling such as Nelson's theory then becomes non-local. The central question is equivalent to asking whether one can induce the quantum potential term while retaining locality. Nelson argued that Markovianity of his theory might be the main obstacle against formulating a local underlying theory and a non-Markovian theory is not necessarily non-local in [3]. I will propose a way to circumvent this problem through replacing the white noise assumption in Nelson's theory with colored noise. One important objection against the existence of a such a local theory comes from believes surrounding Bell-type theorems or inequalities. These theorems assumes two kinds of locality: the usual locality in the sense of relativity and local causality. Local causality assumptions do not have to be satisfied by general local stochastic theories. I will discuss these issues in section 4. The outline is as follows. In section 2, I give an overview of Nelson's theory for a single particle then propose a phase space formulation based on colored noise. I formulate conjectures related to the existence of such a theory. In section 3, I generalize the arguments given for the single particle case to the multiple particle case. In section 4, I elaborate on the issues of locality and local causality. In section 5, I generalize the ideas to the case of the scalar field and speculate about the gravitational origin of noise.
2 Single particle 2.1 Nelson's stochastic mechanics for a single particle I give a brief review of Nelson's stochastic formulation of non-relativistic quantum mechanics in one dimension. For more details see Nelson's original paper [1], his two books [2,3] and Guerra's review [4]. Consider the Schrödinger equation: Putting ψ(x, t) = ρ(x, t)e i S(x,t) we get the Madelung equations: where ρ(x, t) is the probability of finding the particle at (x, t) and S(x, t) is the phase of the wave function. We recognize first of the equations as the continuity equation with velocity 1 m ∂S ∂x . The second of the equations apart from the last term (quantum potential) on the right hand side is the Hamilton-Jacobi equation. Thus if = 0, we have the classical ensemble of particles. The Newton's equations of motion are then the equations that characteristic curves obey corresponding to this set of Madelung partial differential equations. Since the quantum potential term depends on the probability ρ(x, t), giving deterministic characteristics seems not possible. However as Nelson proved [1][2][3], it is possible to give a Markovian stochastic process associated to the solution of Madelung equations in position space. We start by assuming that a particle obeys the following stochastic differential equation: where b(x(t), t) is a general function and dW is the Wiener process. We will always interpret the stochastic differential equations in this paper in Itô sense. We call this as Nelson's first postulate. The diffusion equation associated to this is [5,6] ∂ρ(x, t) ∂t where ρ(x, t) is the probability of finding the particle at x at time t. In order to match with the continuity equation we define where we assumed that ρ(x, t) is nowhere zero. For a discussion of what happens at zeros see [3] and references therein. Indeed, the probability of the particle arriving at places where ρ(x, t) = 0 is zero since b(x, t) is singular at zeros and acts repulsively on the stochastic particle trajectory pushing the particle away from the zeros. We want S(x, t) just defined in this way to satisfy the quantum Hamilton-Jacobi equation. We could postulate it as a partial differential equation but Nelson found a way to write this solely in terms of the stochastic particle trajectory. The quantum Hamilton-Jacobi equation can be shown to be equivalent to the following equation: where D + and D − are forward and backward derivatives which will be defined below, the right hand side is the classical acceleration of the particle evaluated on the stochastic trajectory and the left hand side is the time-symmetric stochastic acceleration. This is the stochastic analogue of Newton's second law. Thus we call this as Newton-Nelson law or Nelson's second law. The forward and backward derivatives are defined to be where E[f |x(t)] denotes the expectation of f conditioned on x(t). For any function F (x, t) we can write its forward and backward derivatives explicitly as follows The derivation of the formula for D + is straightforward but the calculation of D − is subtler [2][3][4]. Using these formulas it is straightforward to show that the Newton-Nelson law is equivalent to the x derivative of the second Madelung equation (eq.2.3). It has been shown that for each solution of the Schrödinger equation there is an associated stochastic process satisfying Nelson's postulates and if Nelson's postulates are satisfied that one can construct a wave function which satisfies the Schrödinger equation with its absolute square the probability density of the position of particle. The stochastic formulation can be generalized to particles propagating in higher dimensions, multiple particles, fields and particles with spin [3,4].
Colored noise smoothed quantum process
Let A β be the colored noise defined by the equation where dW is the Wiener process. Heuristically as β → ∞, the left hand side can be neglected yielding where · denotes expactation. Thus, τ = 1/β is the correlation time. If x β is driven by A β as in then as β → ∞, x β converges to the solution of where x β (0) = x(0). This can be made mathematically rigorous [2,7] Although more general colored noises are possible, I will restrict to those described by eq. 2.12 in this note for simplicity. Suppose a quantum process x q (t) is associated with a solution of Schrödinger equation through Nelson's formulation. An equivalent characterization is the pair (b(x, t), ρ(x, 0)) (one can solve for x q (t) using eq. 2.4). Now replace the white noise in eq. 2.4 with colored noise A β to define the new process where ǫ = /m, with the same initial density ρ(x, 0). As β → ∞, this should be a good approximation to x q (t). However the stochastic acceleration of x(t) will violate Nelson's second law: as can be seen by direct calculation (see next section). The violation of Nelson's second law is of the same order of the quantum potential. As x(t) and x q (t) are close to each other for large β in an appropriate norm, their stochastic derivatives differ. Of course, this is common as even two deterministic functions with wildly differing derivatives can be close in norm (e.g. take a reasonably smooth function and consider a function rapidly fluctuating around it). This is why I call x(t) as a colored noise smoothing of x q (t).
If on the other hand, we consider the white noise limit of A β and calculate small correlation time τ = 1/β expansion, we get a process of the form [8] dx β (t) = b(x β (t), t)dt + ǫ(1 + τ g(x β (t), t))dW (t) (2.18) for some g(x, t) related to b(x, t). Now we see that for large β, x β approximately satisfies Nelson's second law and it is also close to x q (t). The singular nature of the white noise limit is evident as D + and D − are sensitive to the presence of Wiener terms as can be seen from the definitions (eqs. 2.10 and 2.11).
Calculation of stochastic acceleration for a colored noise process
Consider the process of the form for given b(x, t): where initially x and A β are uncorrelated. We will make use of the following formulas for forward and backward derivatives conditioned on fixed (x(t), A β (t)) of a function G(x, A β , t) which can be found in section 5 of Guerra's review [4]: (2.20) (2.21) We also need the following result on conditional expectations for a set of random variables (x, A, z): for any function F (z). We see that and Using eq. 2.22, we calculate the stochastic acceleration of x(t) as For large β, A β is close to white noise so we can assume ρ t (A β |x) ≈ ρ t (A β ) and neglect the last term after the transient time as A β ≈ 0. Thus we see that Nelson's second law is violated if b(x, t) corresponds to a quantum process.
A phase space derivation?
Now consider the phase space process where v β (t) is the velocity of the particle and a(x β (t)) is the acceleration on the particle. One can show through a similar calculation as last section that Nelson's second law is satisfied by x β (t) for large but finite β. Also for large β, integrating out v β and A β , x β (t) approximately satisfies where we identify b(x, t) = vρ t (v|x)dv as the drift velocity. Hence, x β (t) also approximately satisfies Nelson's first law. Can we say that as β goes large then x β (t) becomes a quantum process satisfying both Nelson's first and second law exactly? More precisely, given x β (t) satisfying eq. 2.26 for large β, does there exist a quantum process x q (t) near x β (t)? Conversely for a given quantum process, does there exist x(t) near it satisfying eq. 2.26? I don't know the answers to these questions. Here is the subtlety similar to that we encountered in the colored noise smoothing of a quantum process. In the singular limit when we replace A β (t) by white noise we have If we calculate the stochastic acceleration we see that due to the presence of the Wiener term, extra terms are induced. Thus x ∞ (t) violates the second law. We can trace this back to the ordering of limits ∆t → 0 in the definition of D + and D − and β → ∞. In general What have we learned until now? We know that given a quantum process we can construct a colored noise smoothing close to it albeit violating Nelson's second law. If we make a correlation time expansion around the singular white noise limit, we get a process close to the quantum process and approximately satisfying Nelson's second law. The phase space process on the other hand exactly satisfies Nelson's second law for all finite and large β and approximates Nelson's first law. However in the singular limit, it fails to satisfy Nelson's second law. Therefore it is not obvious whether the questions raised above have affirmative answers. Hence I formulate two conjectures: , v(t), A β (t)) satisfy eq. 2.26. Assume that A β is initially uncorrelated to x and v. Then as β becomes large, there exists a quantum process x q (t), satisfying both of Nelson's laws (equivalent to a solution of the Schrödinger equation), which is close to x(t) projection of the phase space process in some appropriate norm.
Conjecture 2. Converse of Conjecture 1. Let x q (t) be a quantum process. Then there exist a phase space process of the form given by eq. 2.26, which is close to x q (t) in some appropriate norm, as β becomes large.
I will argue in the following section that Conjecture 1 implies Conjecture 2.
Conjecture 1 implies Conjecture 2
Given a solution ψ(x, t) to the Schrödinger equation, split it into probability and phase functions as . Then ρ(x, t) and S(x, t) satisfy the Madelung equations: which is equivalent to Nelson's first postulate: Therefore given a solution to the Schrödinger equation, the associated Nelson process is completely specified by ρ(x, t) and b(x, t). We would like to construct an approximate solution to eq.2.26 to match with the Fokker-Planck equation (eq.2.33). Since ρ(x, t) is already fixed by the solution to the Schrödinger equation, the only free function isρ(v|x, t) as these two determine the joint densitŷ The mean ofρ(v|x, t) is fixed by b(x, t) (eq.2.27) which is determined from the solution of the Schrödinger equation via eq.2.32. Furthermore, we have the natural probability normalization constraint ρ(v|x, t)dv = 1. Therefore, the problem becomes the following: Given a solution to the Schrödinger equation represented by ρ(x, t) and b(x, t), construct an approximate solution Assuming conjecture 1 is true, the solutions to eq.2.26 is close to x q (t) satisfying Nelson's both laws. Therefore, eq.2.26 approximately propagates the initial (ρ( according to the Schrödinger equation. Since (ρ(x, t = 0), b(x, t = 0)) completely specifies the initial conditions for the Schrödinger equation (up to an additive constant in the initial phase S(x, t = 0)), the solutions are unique. Therefore (ρ(x, t),b(x, t)) = (ρ(x, t), b(x, t)). Hence given a solution to the Schrödinger equation specified by ρ(x, t) and b(x, t), we see that any initial densityρ(x, v, t = 0) satisfying eq.2.35 and ρ(x, v, t = 0)dv = ρ(x, t = 0) yields an approximate solutionρ(x, v, t) to eq.2.26 such that vρ(v|x, t)dv = b(x, t) and ρ(x, v, t)dv = ρ(x, t). Note that there are infinitely many ways to choose the initial conditional densityρ(v|x, t = 0) compatible with eq.2.35.
3 Multiple particles
Nelson's mechanics for multiple particles
We have worked with a single particle until now. In this section, I give a review of Nelson's stochastic formulation of non-relativistic quantum mechanics for n particles with position variables x = (x 1 , ..., x n ) and masses m 1 , ..., m n , interacting via the potential U ( x). Consider the Schrödinger equation: Putting ψ( x, t) = ρ( x, t)e i S( x,t) , we get the Madelung equations: where ρ( x, t) is the probability of finding the particles at x at time t and S( x, t) is the phase of the wave function. We recognize first of the equations as the continuity equation with velocity ( 1 . The second of the equations apart from the last term (quantum potential) on the right hand side is the Hamilton-Jacobi equation. Thus if = 0, we have the classical ensemble of particles. We start by assuming that a particles obey the following stochastic differential equations: is a general function of positions and time, and (dW 1 , ..., dW n ) are independent Wiener processes. We call this as Nelson's first law or postulate. The diffusion equation associated to this is [5,6] ∂ρ where ρ( x, t) is the probability of finding the particles at x at time t. In order to match with the continuity equation, we define where we assumed that ρ( x, t) is nowhere zero. For a discussion of what happens at zeros, see Section 2. Note that in eq.3.6, we assume that (b 1 , ..., b n ) is a gradient. At first sight, this seems like an independent assumption. However, it can be shown that the gradient assumption is implied by the Newton-Nelson law using a variational argument (see Theorem 6 of [9] and [3]). Newton-Nelson law is introduced below. We want S( x, t) just defined in this way to satisfy the quantum Hamilton-Jacobi equation. The quantum Hamilton-Jacobi equation can be shown to be equivalent to the following equations: where D + and D − are forward and backward derivatives which will be defined below, the right hand side is the classical acceleration of the particle evaluated on the stochastic trajectory and the left hand side is the time-symmetric stochastic acceleration. This is the stochastic analogue of Newton's second law. Thus we call this as Newton-Nelson law or Nelson's second law. The forward and backward derivatives are defined to be where E[f | x(t)] denotes the expectation of f conditioned on x(t). For any function F ( x, t) we can write its forward and backward derivatives explicitly as follows 3.2 A phase space derivation for multiple particles?
The multi-particle case is a straightforward generalization of the single particle case. Consider the multi-particle phase space process ., x n , t) are the position, velocity and the acceleration of particle i. Like the single particle case, one can show that Newton-Nelson law is satisfied for large β i . Also integrating out the noises and velocities, we can approximate x i (t) by ., x n )dv i as the drift velocity for particle i. Here we assumed that close to the white noise limit with large β i , A i 1 and A i 2 become independent for i 1 = i 2 . Subtleties similar to that in the single particle case remain here at the white noise limit in calculating the stochastic acceleration. I formulate conjectures which parallel the single particle case.
)} i satisfy eq. 3.12. Assume that A i is initially uncorrelated to x and v. Then as {β i } i become large, there exists a quantum process x q (t), satisfying both of Nelson's laws (equivalent to a solution of the Schrödinger equation), which is close to x(t) projection of the phase space process in some appropriate norm.
Conjecture 4. Converse of Conjecture 1. Let x(t) be a quantum process. Then there exist a phase space process of the form given by eq. 3.12, which is close to x q (t) in some appropriate norm, as {β i } i become large.
Next, I argue that Conjecture 3 implies Conjecture 4 with by a similar argument given for the single particle case.
Locality and local causality 4.1 Nonlocality of Nelson's theory vs locality of phase space model
The multi-particle Nelson's theory is manifestly non-local as is well known [3]. It is easy to see that the drift velocity b i of each particle depends on the positions of all other particles in general. Even if the particles are no longer interacting, a potential acting on one particle affects the drift velocities of other particles. In this sense, Nelson's formulation is non-local. As argued by Nelson in [3] this could be due to the Markovian nature of noise and in a non-Markovian theory non-locality may not be required as non-locality could be traded off with temporal correlations. The phase space model presented here is non-Markovian due to the colored nature of the noises introduced. Is it also local? Assume that Conjectures 3 and 4 hold. The following simple argument is in favor of locality. Let the particles interact for some finite time until t = 0 and we turn off the interactions (keeping single particle potentials) and separate the particles. Close to the white noise limit each particle obeys the equation As A i 's will be uncorrelated for large β i 's, every particle is subject to independent forces and independent potentials. The model is local in this sense. However the initial correlations can be preserved and this would give rise to quantum correlations. When the velocities are integrated out, we would obtain Nelson's theory which is non-local. The non-locality arises from integrating out degrees of freedom. We know from Bell type inequalities that certain theories cannot simulate quantum theory. Assuming that the measurement settings are independent from the variables of the theory, since the phase space is local, if it is simulate quantum theory, it cannot be locally causal [10]. In the next sections, I elaborate on this: local stochastic theories need not to be locally causal, therefore Belltype inequalities provide no obstacle against the existence of a local stochastic underlying theory for quantum mechanics which is itself not locally causal.
Multi-time measurements in stochastic mechanics
In order to make contact with Bell-type inequalities, we need to review how to make sense of multitime measurements in Nelson's theory. It is well known that for position measurements at a single time, the predictions of quantum mechanics and that of stochastic mechanics agree [1]. It is quite easy to see this is the case. The quantum mechanical expectation value of functions f and g of the positions x 1 and x 2 at time t is given by which is the same as that given by stochastic mechanics. Since the Schrödinger equation and stochastic equations agree on the evolution of ρ(x 1 , x 2 , t), the quantum expectation value is equal to the classical expectation value, therefore the theories are completely equivalent when position measurements are considered at a single time. However, it is also well known that the predictions of stochastic mechanics differ from that of quantum mechanics for measurements of commuting position variables at different times. The quantum mechanical expectation value of functions f and g of positions x 1 at time t 1 and x 2 at time t 2 is given by where ρ(x 1 ,x 2 , t 2 |x 1 , x 2 , t 2 ) is the quantum mechanical probability obtained from solving the Schrödinger equation, for the particles reaching to (x 1 ,x 2 ), at time t 2 given that their positions were (x 1 , x 2 ) at time t 1 . In general, this multi-time expectation value contradicts the expectation value x 1 (t 1 )x 2 (t 2 ) obtained in stochastic mechanics. All these generalize to multiple particles and measurements at more than 2 times, or multi-time measurements. For multi-time measurements, in order to make the quantum mechanical expectation values compatible with the expectation values calculated from stochastic mechanics, Blanchard et al [11] proposed that when the stochastic mechanical expectation values are computed, the probability density must be updated upon measurement. This process is the stochastic mechanical counterpart of the wavefunction collapse. To be concrete, consider the two particle example. Assume that t 2 > t 1 . When x 1 is measured at time t 1 , the following reduction takes place in the probability density: ρ(x 1 , x 2 , t 1 ) is replaced by ρ m (x 1 , x 2 , t 1 |x 1 ) = ρ(x 2 |x 1 , t 1 )δ(x 1 −x 1 ), wherex 1 is the measured value of x 1 . The stochastic equations are propagated with the new initial density ρ m (x 1 , x 2 , t 1 |x 1 ) to ρ m (x 1 , x 2 , t 2 |x 1 ). When the expectation values are computed, one first compute averages fixingx 1 , then averages over the density ρ(x 1 , t 1 ) = ρ(x 1 =x 1 , t 1 ). This gives the quantum mechanical expectation value (eq. 4.3) [11]. However, if one does not take into account the probability density reduction and propagates the initial density ρ(x 1 , x 2 , t 2 ), one would get correlations in stochastic mechanics which would contradict the quantum expectation values. It must be stressed that this property is very peculiar to stochastic mechanics. In a usual diffusion process which is governed by a partial differential equation linear in probability, collapsing the initial probability, propagating the solution to a later time and averaging over the initial probability gives the same results for the statistics when the initial density is directly propagated. However, in stochastic mechanics, the drift velocities b i depend on the probability density, hence the diffusions are non-linear. When the probability is updated upon measurement and propagated, the drift velocities would be different from the the ones obtained by propagating the initial density directly. It is this non-linearity of stochastic mechanics seems to enable the probability collapse to make the quantum and stochastic mechanical expectation values equal for multi-time position measurements. For details, see [11].
Is the above probability collapse scheme non-local? Assume Conjecture 3 and 4 hold. Nelson's stochastic mechanics is manifestly non-local: anything done on the first particle will be felt by the second particle instantly because of the drift b 2 of the second particle depends on the position x 1 of the first particle in a general entangled state. The non-locality of Nelson's mechanics emerges from integrating out the velocity variables of the phase model, which is local. If one takes the point of view that the probabilities only represent the knowledge that one has about the underlying particle trajectories, there is nothing non-local in terms of how the particles evolve. It is the probability distribution that collapses, which is not part of ontology. The real quantities, which are the phase space stochastic trajectories of the particles, are not affected by each other, as is clear from that they are dynamically uncoupled and the random forces driving them are independent.
How to make contact with Bell type theorems? Consider the two particle case. Suppose that Albert has the two particles with phase coordinates (x 1 , v 1 ) and (x 2 , v 2 ) and he prepares them in the state ρ 0 (x 1 , v 1 , x 2 , v 2 ), which corresponds to an entangled quantum states, by some local procedure. Then he sends the first particle to John and the second particle to Edward. Let John and Edward synchronize their clocks. John makes a measurement of position x 1 with outcomex 1 at time t 1 and Edward makes a measurement of position x 2 with outcomex 2 at a later time t 2 . After this procedure is repeated many times, John and Edward report back their measurement outcomes to Albert for him to calculate empirical expectation values of functions ofx 1 andx 2 . Since ρ(x 1 , x 2 , t) evolves according to the Schrödinger equation, and since the quantum Heisenberg position operators X 1 (t 1 ) and X 2 (t 2 ) commute, the quantum mechanical expectation value f (X 1 (t 1 ))g(X 2 (t 2 )) is given by eq.4.3. This must coincide with the empirical expectation values that Albert has computed since the particles obey the Schrödinger equation. How would Albert compute expectation values in the stochastic model? He has two major choices. Either for each experiment, he assumes that John measured x 1 at t 1 and it yieldedx 1 and he updates the probability density to make a refined estimate of Edward's measurement of x 2 at t 2 conditioned onx 1 , then he averages over the probability density ρ(x 1 ), or he calculate the expectations directly without collapsing the probability distribution. We discussed earlier in this section that these would in general give different results. If Albert chooses to update the probability density of two particles upon measurement, then he would obtain the quantum mechanical expectation values. Therefore, we assume that Albert calculate the expectation values in this way to make sure that multi-time measurements in quantum theory and stochastic mechanics coincide without violating locality.
Local causality
With the above measurement prescription, we obtain a correspondence between stochastic mechanics and quantum theory with multi-time measurements. Assuming Conjectures 3 and 4 are true and the measurement settings are independent from the particle variables (non-conspiring), this provides a local stochastic theory for quantum theory. Bell-type theorems [10] imply that local, locally causal and non-conspiring deterministic or stochastic hidden variable theories satisfy inequalities that are violated by quantum mechanics. As quantum mechanics is not locally causal [10], neither the phase space model can be if the conjectures 3 and 4 are true.
Although local deterministic theories are locally causal, these two versions of locality are separate for stochastic theories. There is no reason that a local stochastic theory be locally causal automatically. See chapter 23 of [3]. To elaborate on this, lets remind ourselves what locality and local causality means in the sense of Bell [10]. Locality means that influences cannot propagate with a speed greater than that of light. This is the usual relativistic notion of locality. Local causality is subtler. Let R1 and R2 be spacelike separated regions. Let A(B) be stochastic variables (beables) in R1(R2). Denote the beables of the past of R1 (R2) minus the intersection of pasts of R1 and R2 as N (M ). Denote the beables in the intersections of the pasts of R1 and R2 as Λ. Local causality states that p(A|Λ, N, B) = p(A|Λ, N ): the beables at R2 does not affect the outcomes at space-like seperated R1 given the past beables Λ and N of R1. This is of course true in deterministic local theory as the past of R1 determines what happens at R1 completely. In a stochastic theory where the dynamics is deterministic and initial conditions are unknown, local causality still holds. However, in general local causality is independent from locality for stochastic theories [3]. A sufficiently chaotic theory can cause loss of information from the past of R1 to R1, still retaining useful information in R2 as correlations. This chaotic dynamics can emerge from deterministic dynamics of many degrees of freedom or it could be due to external noise. For a general stochastic theory, information in R2 could be non-redundant given the past of R1 without any causal connection between R1 and R2. Hence if conjectures 3 and 4 are to be true, the local phase space model cannot be locally causal.
Potential physical sources of noise
How physical is to assume that the noise appears as velocity rather than a force in the phase space process? A possible answer to this comes from consideration of canonical momentum rather than kinematic momentum. A similar problem appears when one quantize a charged non-relativistic particle in an electromagnetic field. We know that canonical commutation relations should be imposed on canonical rather than kinematic variables. Consider the Lagrangian of a unit charged particle with unit mass with position and velocity (x, v) in an electromagnetic field (φ, A): The canonical momentum is and the equation of motion isṗ With the canonical variables x, p we have Restricting to one dimension we have Now we see that if the electromagnetic potential A is the colored noise A β , we have induced noise as velocity. On the other hand if we have used kinematic momentum v, we would not have the noise term as velocity but acceleration. Having the noise term as a velocity seems to be important to make direct contact with Nelson's first law. A similar argument can be given for a non-relativistic particle in a weak gravitational field where again the canonical and kinematic momenta differ and the source of noise would be gravitational. In the next sections, I provide a quantization of the free scalar field in a stochastic gravitational field assuming conjectures 3 and 4.
Stochastic quantization of the free scalar field
The phase space model for multiple particles enables a generalization to fields. The simplest case is the free scalar field though interacting fields and fields with spin could be treated in similar ways with some technical complications. First, I review stochastic quantization of the free scalar field following [4]. Consider the Klein-Gordon field φ satisfying where ∆ is the three dimensional Laplace operator. Put the system in a box B with appropriate boundary conditions with a real complete orthonormal basis of eigenfunctions u i of ∆ satisfying Then each q i (t) is a harmonic oscillator with frequency ω i = m 2 + k 2 i : Assume the Hamiltonian of the field is 1 To stochastically quantize the field, we promote each of the harmonic oscillators to quantum oscillators unit mass (not to be confused with the field mass m): where dW i (t)'s are independent Wiener processes and ǫ = √ . Does the noise {dW i (t)} i act locally on φ? To see this, calculate the stochastic derivative of φ using eq.5.8: for each x ∈ B. The noise term is again Gaussian since it is a linear sum of Gaussian processes with mean and covariance ǫ i u i (x)dW i (t) = 0 (5.14) in the infinite volume limit where we used dW i (t) = 0 and dW i (t)dW j (t) = δ ij dt. Hence, we can write with dW (x, t) = 0 and dW (x, t)dW (x ′ , t) = δ(x − x ′ )dt. In this sense, the noise acts locally on φ(x, t). Note that each b i (q 1 (t), ..., q n (t), t) can be written in terms of φ(x, t) using eq.5.9. Nelson's second law has a simple expression in terms of for the field: as can be seen using eq.5.8 and eq.5.12. This can be also obtained by replacing the acceleration term ∂ 2 t φ by its stochastic counterpart 1/2(D + D − + D − D + )φ in eq.5.6. Does this stochastic quantization depend on the time slicing? Yes, it is obviously dependent on coordinates. For a different time slicing, one gets a different stochastic process. All these stochastic processes together should preserve the relativistic covariance of the resulting quantum theory [4]. Now, I give a phase space model for the stochastic field. Instead of eq.5.11, we have as we had for the multi-particle case. Assuming Conjectures 3 and 4 are true, for large β, this provides a phase space model for the stochastic field. Is this model local? To see this, write the above equations in terms of φ(x, t) using eq.5.8 and defining V ( for each x ∈ B. In the large β i limit, assuming A i 's should become independent Wiener processes. Thus, as above, i u i (x)A i (t) converges to a Wiener process with zero mean and covariance δ(x−x ′ )dt in the infinite volume limit. The dynamical equation is obtained in the infinite volume and large β i limit.
Gravitational origins of noise
Can the source of noise be gravitational? I outline a model below. Consider the scalar field φ in a weak stochastic background gravitational field g µν with the action S = 1 2 d 4 x √ −g(g µν ∂ µ φ∂ ν φ − m 2 φ 2 − 2ξRφ). (5.20) where R is the Ricci scalar, g = detg and η µν = diag(1, −1, −1, −1). Putting g µν = η µν + h µν , expanding S to first order in h µν and integrating the non-minimal coupling term ξRφ by parts we have The canonical momentum of the field is In the quantum regime assume that the minimal coupling part can be neglected as compared to the non-minimal coupling part. Of course for the experimentally tested classical regime, the minimal coupling part should dominate the non-minimal part. We have in the quantum regime The acceleration equation can be obtained from the Euler-Lagrange equation yielding where F µν is some linear differential operator depending on φ and ∂ σ φ. In the Newtonian gauge h µν = diag(θ, θ, θ, θ) assuming some isotropy condition on the stress-energy tensor generating it. Hence, we have the system of equations In order to stochastically quantize the field, we need to make the identification (assuming conjectures 3 and 4): ξ∂ 0 θ = ǫA β where A β is a space-time colored noise, which is characterized by some set of parameters β, converging to space-time white noise (time derivative of the zero mean Wiener process with covariance δ(x − x ′ )dt as above in the infinite volume limit) as β becomes large. Assuming that the statistics of θ becomes independent of φ when β is large and θ = 0, eq.5.25 yields Nelson's second law (eq.5.17). This finalizes the sketch of a phase space quantization of the scalar field in a stochastic background gravitational field. Note that, this cannot be achieved with minimal coupling in the framework considered here since the noise terms induced from minimal coupling would depend on the field itself. Can this background gravitational field be sourced by a matter distribution? In the limit of large β, in the infinite volume limit, θ is characterized by a space-time Gaussian field with zero mean and covariance: dθ(x, t)dθ(x ′ , t) = ǫ 2 ξ 2 δ(x − x ′ )dt (5.26) In a Newtonian universe, assume small fluctuations around a constant mean (dark) matter density. A zero mean homogeneous isotropic Gaussian matter distribution can be described by the two-point function of the fluctuating part of the matter density δρ(x): c(x − x ′ , t) = δρ(x, t)δρ(x ′ , t) (5.27) whose Fourier transform P (k) is a power law: The spectrum of the gravitational potential θ can be calculated from the Poisson equation: ∆φ = 4πGδρ (5.29) where G is the Newton's constant. Taking the Fourier transform of both sides we can show that the spectrum P θ (k) of θ is given by P θ (k, t) = (4πG) 2 |k| 4 P (k, t). (5.30) The Fourier transform of the covariance of θ is: P θ (k, t) = ǫ 2 ξ 2 t (5.31) from which we obtain P (k, t) = |k| 4 (4πG) 2 ǫ 2 ξ 2 t.
(5.32) Analogous expressions can be written for the case of expanding universe. Of course, this is a tall order: fluctuations should remain up until where quantum field theory is tested in particle accelerators. Also, Planck's constant is emergent and can depend on time in principle. Note that the matter distribution should temporally fluctuate as white noise. This is consistent assuming that the fluctuations arise from the gravitational interactions of all particles (fields) in the universe as Brownian motion of single particles emerge from the microscopic dynamics in a gas or plasma [12]. | 2022-04-08T01:15:34.304Z | 2022-04-06T00:00:00.000 | {
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52880390 | pes2o/s2orc | v3-fos-license | Long-term follow up of tandem autologous-allogeneic hematopoietic cell transplantation for multiple myeloma
We previously reported initial results in 102 multiple myeloma (MM) patients treated with sequential high-dose melphalan and autologous hematopoietic cell transplantation followed by 200 cGy total body irradiation with or without fludarabine 90 mg/m2 and allogeneic hematopoietic cell transplantation. Here we present long-term clinical outcomes among the 102 initial patients and among 142 additional patients, with a median follow up of 8.3 (range 1.0-18.1) years. Donors included human leukocyte antigen identical siblings (n=179) and HLA-matched unrelated donors (n=65). A total of 209 patients (86%) received tandem autologous-allogeneic upfront, while thirty-five patients (14%) had failed a previous autologous hematopoietic cell transplantation before the planned autologous-allogeneic transplantation. Thirty-one patients received maintenance treatment at a median of 86 days (range, 61-150) after allogeneic transplantation. Five-year rates of overall survival (OS) and progression-free survival (PFS) were 54% and 31%, respectively. Ten-year OS and PFS were 41% and 19%, respectively. Overall non-relapse mortality was 2% at 100 days and 14% at five years. Patients with induction-refractory disease and those with high-risk biological features experienced shorter OS and PFS. A total of 152 patients experienced disease relapse and 117 of those received salvage treatment. Eighty-three of the 117 patients achieved a clinical response, and for those, the median duration of survival after relapse was 7.8 years. Moreover, a subset of patients who became negative for minimal residual disease (MRD) by flow cytometry experienced a significantly lower relapse rate as compared with MRD-positive patients (P=0.03). Our study showed that the graft-versus-myeloma effect after non-myeloablative allografting allowed long-term disease control in standard and high-risk patient subsets. Ultra-high-risk patients did not appear to benefit from tandem autologous/allogeneic hematopoietic cell transplantation because of early disease relapse. Incorporation of newer anti-MM agents into the initial induction treatments before tandem hematopoietic cell transplantation and during maintenance might improve outcomes of ultra-high-risk patients. Clinical trials included in this study are registered at: clinicaltrials.gov identifiers: 00075478, 00005799, 01251575, 00078858, 00105001, 00027820, 00089011, 00003196, 00006251, 00793572, 00054353, 00014235, 00003954.
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/2/380
Ferrata Storti Foundation W e previously reported initial results in 102 multiple myeloma (MM) patients treated with sequential high-dose melphalan and autologous hematopoietic cell transplantation followed by 200 cGy total body irradiation with or without fludarabine 90 mg/m 2 and allogeneic hematopoietic cell transplantation. Here we present longterm clinical outcomes among the 102 initial patients and among 142 additional patients, with a median follow up of 8.3 (range 1.0-18.1) years. Donors included human leukocyte antigen identical siblings (n=179) and HLA-matched unrelated donors (n=65). A total of 209 patients (86%) received tandem autologous-allogeneic upfront, while thirty-five patients (14%) had failed a previous autologous hematopoietic cell transplantation before the planned autologous-allogeneic transplantation. Thirty-one patients received maintenance treatment at a median of 86 days (range, 61-150) after allogeneic transplantation. Fiveyear rates of overall survival (OS) and progression-free survival (PFS) were 54% and 31%, respectively. Ten-year OS and PFS were 41% and 19%, respectively. Overall non-relapse mortality was 2% at 100 days and 14% at five years. Patients with induction-refractory disease and those with high-risk biological features experienced shorter OS and PFS. A total of 152 patients experienced disease relapse and 117 of those received salvage treatment. Eighty-three of the 117 patients achieved a clinical response, and for those, the median duration of survival after relapse was 7.8 years. Moreover, a subset of patients who became negative for minimal residual disease (MRD) by flow cytometry experienced a significantly lower relapse rate as compared with MRD-positive patients (P=0.03). Our study showed that the graft-versus-myeloma effect after non-myeloablative allografting allowed long-term disease control in standard and high-risk patient subsets. Ultra-high-risk patients did not appear to benefit from tandem autologous/allogeneic hematopoietic cell transplantation because of early disease relapse. Incorporation of newer anti-MM agents into the initial induction treatments before tandem hematopoietic cell transplantation and during maintenance might improve outcomes of ultra-high-risk patients. Clinical trials included in this study are registered at: clinicaltrials.gov identifiers: 00075478, 00005799, 01251575, 00078858, 00105001, 00027820, 00089011, 00003196, 00006251, 00793572, 00054353, 00014235, 00003954.
Long-term follow up of tandem autologous-allogeneic hematopoietic cell transplantation for multiple myeloma Introduction
Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment for multiple myeloma (MM) but its role is controversial. The first clinical experience with myeloablative regimens proved to be curative for a small proportion of patients but was accompanied by unacceptably high non-relapse mortality (NRM) rates. 1,2 The introduction of less intensive conditioning regimens for allogeneic HCT, which relied on graft-versus-tumor (GvT) effects for tumor eradication, lowered NRM but at the expense of higher disease relapse rates. 3,4 In the late 1990s, combining cytoreductive high-dose chemotherapy before autologous HCT with subsequent minimal intensity conditioning allogeneic HCT, an approach aimed at inducing GvT effects, proved to be less toxic than myeloablative allogeneic HCT and was well tolerated. 5,6 Seven prospective trials compared clinical outcomes of autologous HCT versus tandem autologous/minimal intensity allogeneic HCT in newly diagnosed MM patients and yielded discordant results regarding depth of response, overall survival (OS), and progression-free survival (PFS). Differences in conditioning regimens, as well as graft-versus-host disease (GvHD) prophylaxis, including ATG use, patient selection, definition of MM risk profiles, and duration of follow up, made meaningful comparisons between trials difficult. [7][8][9][10][11][12][13][14][15] We previously reported initial results in 102 MM patients given tandem high-dose melphalan and autologous HCT followed by 200 cGy total body irradiation (TBI) with or without fludarabine 90mg/m 2 and HLA-matched HCT from related or unrelated donors. 16 Here we update the early observations and add results from 142 additional patients treated with the same approach for a total of 244 patients with a median follow up of 8.3 years (range, 1.0-18.1).
Patients
From August 1998 to January 2016, 244 MM patients completed sequential treatment with high-dose melphalan and autologous HCT followed by 200 cGy TBI ± fludarabine and allogeneic, granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cell (PBMC) infusion. One hundred and sixty-four (67%) patients were transplanted at the Fred Hutchinson Cancer Research Center (Fred Hutch), Seattle, WA, USA, and 80 (33%) patients received their transplant at eight other institutions. All patients included in the analysis were treated under eighteen clinical trials that were co-ordinated by Fred Hutch, approved by each institution's review board, and registered at "clinicaltrials.gov". All patients and donors signed written informed consent in accordance with the Declaration of Helsinki. The nature of the analysis is retrospective, and we present clinical data for those 244 patients who received both autologous and allogeneic HCT. Patients' characteristics are detailed in Table 1. Median age at diagnosis was 51 years (range, . Ninetyseven (42%) patients had high-risk cytogenetics. Fifty-seven (25%) had high-risk disease according to International Staging System (ISS) stage III and 36 (16%) according to Revised ISS (R-ISS) stage. Ninety-one (37%) had received more than one induction therapy line for unresponsive disease. A total of 209 patients (86%) received tandem autologous-allogeneic upfront while 35 patients (14%) had failed a previous autologous HCT before the planned autologous-allogeneic HCT.
Definitions and risk assessment
Beta-2 (β2)-microglobulin and serum albumin values at diagnosis were available for 225 (92%) patients and were used to calculate risk according to the ISS. 17 The R-ISS was introduced in 2015, 18 and we calculated it retrospectively for 217 (89%) patients. Lactate dehydrogenase (LDH) serum levels 19 at diagnosis were available in 216 patients. Conventional cytogenetics and/or fluorescence in situ hybridization (FISH) studies at diagnosis and at any time before allogeneic HCT were available for 232 patients. Highrisk cytogenetics were defined as follows: t(4;14); 20 t(14;16); 21 t(14;20) 22 by FISH; del (17/17p), 23 1q21 amplifications 24 both by FISH and conventional karyotyping; and non-hyperdiploid karyotype 25 by conventional cytogenetics. Plasma cell leukemia included circulating plasma cells ≥ 20% of complete blood count or ≥2000 plasma cells per microliter. 26 Extramedullary disease at diagnosis was defined as extramedullary plasmacytomas. 27 Patients were considered high risk if they had one of the following: ISS stage III, high-risk genetic lesions, extramedullary disease presentation, plasma cell leukemia, LDH levels ≥ 2 upper normal limits or failed previous autologous HCT. Ultra-high-risk was defined as having ≥ 2 adverse factors. 23,28 All patients not meeting previous criteria were considered standard risk.
HLA-typing
Patients and donors were matched for HLA-A, HLA-B and HLA-C by at least intermediate resolution DNA typing and for HLA-DRB1 and DQB1 by high-resolution techniques, as previously described. 29 Donors were HLA-identical siblings in 179 cases and HLA-matched unrelated in 65 cases; 11 unrelated donors were mismatched with their recipients for a single HLA allele (n=7) or antigen (n=4).
Allogeneic hematopoietic cell transplantation
After complete recovery from autologous HCT, patients proceeded to allogeneic HCT at a median of 75 days (range, . No further therapy was given between autologous and allogeneic HCT. The conditioning regimen for allogeneic HCT consisted of 200 cGy TBI at 7 cGy/minute from a linear accelerator (n=163) or two opposing Cobalt-60 sources (n=81). Recipients of unrelated grafts (n=65) received in addition three daily doses of fludarabine for a total of 90 mg/m 2 . PBMC grafts contained a median of 9.0 (range, 1.7-24.0) x 10 6 CD34 + cells/kg actual body weight. Postgrafting immunosuppression included mycophenolate mofetil 9.00 x 10 6 (1.7-24.0) Median CD3 + /kg infused, n (range) 3.28 x 10 8 (0.4-11.7) continued on the next page (MMF) (from a minimum of 28 days for sibling recipients to a maximum of 180 days for unrelated donors) and a calcineurin inhibitor (CNI) of either cyclosporine (n=176) or tacrolimus (n=56) for a minimum of 80 days with a subsequent taper to 180 days, as previously described. 5 Twelve patients received sirolimus in addition to MMF and CNI at the dose of 2 mg orally once daily from day -3 to day +80 (n=4), day +180 (n=6), and day +365 (n=2). 30 Thirty-one patients included in the analysis also received bortezomib (n=21; either at 1.6 mg/m 2 intravenously or 2.6 mg/m 2 subcutaneously every 14 days for up to 9 months) or lenalidomide (n=10; starting dose of 10 mg per day, range: 5-25 mg per day, on days 1-21 of each 28-day cycle, for 12 cycles of planned treatment) as maintenance treatment after allogeneic HCT, per protocol, as specified in the Results section.
Chimerism evaluation
Donor chimerism was assessed at days 28, 56, 84, 180 and 365 after allogeneic HCT on peripheral blood CD3 + T lymphocytes and CD33 + myeloid cells, while unfractionated marrow was analyzed only on day +84. This involved FISH analyses in sex-mismatched pairs and polymerase chain reaction-based studies of polymorphic microsatellite regions in all other patients. 31
Disease response assessment
Disease responses were based on the 2016 Uniform Response Criteria developed by the International Multiple Myeloma Working Group 32 with some minor modifications. Complete response (CR) required negative immunofixation (IFIX) on the serum and urine, disappearance of any soft tissue plasmacytomas and/or osteolytic bone lesions, <5% plasma cells in bone marrow aspirates, and no evidence of clonal disease on flow cytometry analysis; very good partial response (VGPR) was defined as serum and urine M-protein detectable by IFIX but not on electrophoresis (SPEP) or ≥90% reduction in serum M-protein plus urine M-protein level <100 mg/24 hours (h). Partial response (PR) required ≥75% reduction of serum M-protein and reduction in 24 h urinary M-protein by ≥90% or to <200 mg/24 h and no increases in sizes or numbers of soft tissue plasmacytomas and/or lytic bone lesions; stable or progressive disease (PD) before autologous HCT was defined as chemo-refractory disease, while the achievement of at least a PR as chemotherapy-sensitivity disease. Patients were evaluated both before autologous HCT and before allogeneic HCT in order to estimate the baseline levels of disease activity before each transplantation, again on days 28, 56, 84 and 180 after allogeneic HCT, and thereafter on a clinical basis. Disease evaluation includ- ed serum and urine SPEP and IFIX for M-protein detection and quantification, plasma cell quantification, cytogenetics and FISH studies in the marrow, and radiological imaging to assess for osteolytic lesions/plasmacytomas whenever appropriate. Six-color multi-parameter flow cytometry analysis of marrow cells for detection of minimal residual disease (MRD) was carried out for a subset of patients who achieved IFIX-negative CR after tandem autologous-allogeneic HCT and were treated at Fred Hutch (n=28). Samples were analyzed at the University of Washington Hematopathology Laboratory. The sensitivity of the flow cytometry assay for plasma cell neoplasms ranged from 0.01 to 0.001%. MRD negativity (MRD NEG ) status was defined as no evidence of quantifiably detectable disease.
Graft-versus-host disease evaluation
Grading of acute and chronic GvHD was performed according to previously described methods. 33,34 Information regarding the administration of systemic immunosuppressive treatment for GvHD was collected prospectively.
End points and statistical methods
Primary objectives of this study were OS and PFS. Secondary end points included: cumulative incidences of acute GvHD, chronic GvHD, NRM, disease response, and disease relapse. We also examined response to treatment and survival among those patients who experienced disease relapse after allogeneic HCT. OS, PFS, and NRM were defined as the times from allogeneic HCT to death, death or progression, and death without progres-sion, respectively. Probabilities of OS and PFS were estimated using the Kaplan-Meier method; cumulative incidences of relapse, NRM, and GvHD were estimated taking competing risks into account. Cox and Fine & Gray regression models were used to
Disease response before and after autologous hematopoietic cell transplantation
Disease responses are summarized in
A B
86) after allogeneic HCT. Seventeen patients completed the planned treatment (lenalidomide, n=7; bortezomib, n=10). Disease progression was the reason for early treatment discontinuation in 9 of the treated patients. One patient on lenalidomide stopped the treatment due to an acute GvHD flare which was successfully treated. Other causes included: patient choice (n=1), diarrhea not GvHDrelated (n=1), severe headache (n=1), and liver function abnormalities (n=1). By univariate analysis, maintenance therapy was not associated with any clinical outcome. Median OS was 6.3 (95%CI: 3.6-not reached). There was no difference in terms of median PFS between those patients who received maintenance treatment (n=31) after allogeneic HCT (2.56 years; 95%CI: 0.88-4.22) and those (n=175) who achieved a response after allogeneic HCT but did not receive maintenance (2.59 years; 95%CI: 1.86-3.50).
Survival after disease progression -One hundred and fiftytwo patients (62%) experienced relapse or progression. Median survival after the first relapse/progression was 2.9 years (95%CI: 1.9-3.7) for the entire cohort (n=152) (Figure 4). Twenty-eight of the 152 received palliative best supportive care and died after a median of 2.1 months. One patient died during conditioning for a planned subsequent allogeneic HCT. Data on salvage therapy were not available for 5 patients (3%). One hundred and nineteen patients received a total of 228 lines of treatment, with a median of two lines (range, 1-9) of therapy each. Treatments included lenalidomide (n=67), bortezomib (n=54), thalidomide (n=36), alkylators/anthracyclines (n=27), pomalidomide (n=10), carfilzomib (n=9), daratumumab (n=1), and others (n=6). Twenty-seven (23%) of the 119 treated patients were unresponsive to salvage treatments and died after a median of 9.2 months (95%CI: 5.5-12.0), while the 83 patients who achieved at least a PR had a median survival of 7.8 (95%CI: 5.9-10.6) years. Disease responses for those 83 patients included: 29 CR (15 of these patients are still in CR, 4 are alive with PD, and 10 have since died), 13 VGPR (3 are still in VGPR, 2 are alive with PD, and 8 have since died), and 41 PR (9 currently with stable disease, 10 alive with PD, while 22 have since died). Data on disease response after salvage treatments were not available for 9 patients. Eighteen patients received a median of 2 donor lymphocyte infusions (range, 1-4) (preceded by chemotherapy in 9 patients) after a median of 1.4 years post-allografting (range, 0.9-8.3). Four of these achieved a partial response, while the remaining 14 did not show a response. Patients who relapsed during the first 18 months after allogeneic HCT (n=82) had a worsened prognosis (median survival after relapse/progression: 1.1 years; 95%CI: 0.6-1.9) compared to those (n=70) who relapsed beyond 18 months after allogeneic HCT (median survival after relapse/progression: 7.2 years; 95%CI: 4.3-10.6; P<0.0001).
Discussion
Advances in the understanding of MM biology have led to novel treatments that have dramatically prolonged PFS and OS. First-line autologous HCT has remained standard of care for eligible patients. Three randomized trials reported significantly superior median PFS, ranging between 25 and 32 months, as compared to conventional chemotherapy. [35][36][37] PFS was further improved up to 43-50 months after "new drugs" were employed both in the induction and consolidation/maintenance phases. [38][39][40] Whether double autologous transplants are superior to a single autograft remains to be determined. [41][42][43] In our series, an overall median PFS of 1.9 years may appear modest. However, when applying retrospective risk stratification, median PFS was 6.5 years in standard-risk patients, 2.5 year in high-risk patients, and 0.7 years in ultra-high-risk patients. Extramedullary relapse without marrow relapse has been frequent after allografting. [44][45][46] Sanctuary sites may be less accessible to graft-versus-myeloma effects than marrow. Of note, extra-medullary relapse occurred in 25% of current patients who did not have extramedullary involvement at diagnosis. Overall NRM was low (2% at 100 days and 14% at 5 years) and, with a median follow up of 8.3 years, median OS was 6.4 years. By risk stratification, median OS was not reached in standard-risk patients, while high-risk and ultra-high-risk patients had worse outcomes with median OS of 8.4 and 2.3 years, respectively. Of note, only a minority of our patients achieved a complete remission after induction treatments, and a subset of them received tandem autologous-allogeneic HCT beyond first line. Moreover, none of our patients received recently Food and Drug Administrationapproved monoclonal antibodies, such as daratumumab and elotuzumab, which have been associated with remarkable response rates. Although restricted to a small group of patients, we demonstrated that the achievement of MRD NEG predicted long-term CR among patients with IFIX-negative CR after autologous HCT. Whether long-term persistence of MRD NEG indicates disease eradication is unclear. Multiparameter flow cytometry and PCR-based methods are two sensitive techniques currently used to evaluate MRD in MM. Evaluation of MRD through immuno-phenotyping is more broadly available than PCR-based methods. In the present series, patients who achieved MRD NEG by flow cytometry experienced a significantly lower relapse rate as compared with MRD POS patients (P=0.03). These findings confirm previous observations by Giaccone et al. who reported on the clinical impact of immuno-phenotypic remission after allografting in 66 MM patients. 47 Conditioning was 2 Gy TBI-based in 55 of the 66 patients. After a median follow up of 7.1 years, patients who achieved conventional CR and MRD NEG disease status had better clinical outcomes in terms of OS (median not reached) and PFS (median 59 months). Moreover, Ladetto et al. reported a PCR-based molecular analysis of MRD after minimal-intensity TBI-based conditioning in newly diagnosed patients who had not been exposed to new drugs. 48 After a median follow up of 12.1 years, the median OS and PFS were not reached in patients who achieved PCR MRD negativity. Overall, MRD studies support the hypothesis that potentially curative graft-versus-myeloma effects after minimal intensity conditionings allowed longterm disease control and persistent yet non-progressive MRD in a subset of MM patients.
Whether graft-versus-myeloma effects are associated with chronic GvHD is still a subject of debate. Ringdén et al. evaluated the impact of acute and chronic GvHD on relapse and survival in 177 patients transplanted from HLA-identical siblings after non-myeloablative or reduced-intensity conditioning. 49 Acute GvHD was associated with a significantly higher risk of TRM, while limited chronic GvHD significantly reduced the risk of relapse. However, the reduced relapse risk did not translate into better OS. Crawley et al. reported that chronic GvHD was associated with better PFS and OS after reduced-intensity conditioning. 3 In the present study, we report an incidence of chronic GvHD of 55%, which, as in other comparative prospective trials, 7,9 was not associated with better disease control. A trend towards higher chronic GvHD rates after the introduction of minimal/reduced-intensity conditioning was shown in a Center for International Blood and Marrow Transplant Research (CIBMTR) analysis on 1207 MM recipients between 1989 and 2005. Overall, 50% of the patients who survived at least five years in the 2001-2005 cohort developed chronic GvHD, 65% of whom had extensive involvement. 4 Discontinuation of all immuno-suppressive agents is a surrogate for achieving immunotolerance, and is associated with improved quality of life. Importantly, only a minority of current survivors remained on immunosuppressive drugs long-term: 24%, 12%, and 4% at five, ten, and 15 years, respectively.
Indications for allografting in MM have greatly changed over the years due to remarkable advances in the understanding of disease biology and new treatment modalities that increased median survival rates up to 8-10 years in standard-risk patients. However, relapse has remained a major issue, and poor outcomes have been observed in patients with high-risk/ultra-high-risk disease. 50 Interestingly, Sobh et al. described trends and clinical outcomes of allogeneic HCT for MM in Europe between 1990 and 2012. 51 The study included 7333 patients who were divided into 3 groups: 1) allogeneic HCT upfront (n=1924); 2) tandem autologous-allogeneic HCT (n=2004); and 3) allogeneic HCT as a second-line treatment or beyond (n=3405). A steady increase in numbers of allogeneic HCT over the years was observed. The use of upfront allogeneic HCT increased up to the year 2000, followed by a decline thereafter, representing 12% of allogeneic HCT performed in 2012. Tandem autologous-allogeneic HCT peaked around the year 2004 and represented 19% of allogeneic HCTs in 2012. Allogeneic HCT as salvage after at least one autograft has steadily increased over recent years and represented 69% of allogeneic HCTs in 2012. Unfortunately, only a minority of these patients were enrolled in controlled trials and remarkable heterogeneity in using allogeneic HCT was observed among different European countries.
The potential role combining "new drugs" with graft-versus-myeloma effects has not yet fully been explored. In a Phase II study, the feasibility of using bortezomib within a reduced-intensity conditioning regimen and as maintenance therapy post allograft was evaluated. 52 Conditioning consisted of fludarabine, melphalan, and bortezomib, while maintenance treatment consisted of 7 cycles of bortezomib. Sixteen high-risk patients who had relapsed after an autograft were prospectively enrolled. Nine of 16 patients (56%) achieved CR and 5 of 16 (31%) achieved PR after allogeneic HCT. In this heavily pretreated high-risk population, 3-year cumulative incidences of NRM, relapse, and OS were 25%, 54% and 41%, respectively. For the first time, this trial showed safety and efficacy of an intensified conditioning with a "new drug" in poor prognosis patients. Moreover, the concept of maintenance treatment after an allograft was also introduced. Our group recently published the results of a prospective Phase II single-center trial evaluating bortezomib as maintenance treatment after tandem autologous/allogeneic HCT for high-risk MM. At a median follow up of 51 months, a net benefit in terms of OS and PFS was shown among newly diagnosed patients over those with relapsed/persistent disease, suggesting that bortezomib maintenance may add a survival benefit among untreated patients. Treatment-related toxicity was limited, without any GvHD exacerbations. 53 Different observations were reported with immunomodulatory drugs. Somewhat compromised by an unacceptably high dropout rate, the Phase III BMT CTN 0102 trial did not show a benefit of thalidomide maintenance after tandem autologous/allogeneic HCT. 13 Lenalidomide maintenance was evaluated in a study by the HOVON Group 54 where the unexpectedly high toxicity profile, mainly exacerbation of acute GvHD, led to early discontinuation in 87% of the patients. In a Phase II CIBMTR trial on 30 patients, 55 the use of lenalidomide was feasible if given at lower doses. A lower toxicity profile of lenalidomide maintenance was also reported by Kroger et al. in relapsed patients after an autograft and rescued with a myeloablative allograft. 56 Importantly, a synergy between new drugs and graftversus-myeloma effects with a far safer toxicity profile has been described in the relapsed setting, suggesting that allogeneic-HCT and new drugs may be complementary. In our study, the median duration of survival of 7.8 years from the first relapse/progression among patients who achieved a response to salvage treatments supports this concept. These findings have been confirmed by two other recent reports. 57,58 An update of an Italian study focused on the role of "new drugs" in long-term clinical outcomes. 57 Median OS from first relapse was 7.5 years in the autologous/allogeneic group and two years in the tandem autologous group (P=0.01). Htut et al. compared the post-relapse OS after autologous/allogeneic HCT versus tandem autografts in patients reported to CIBMTR between 2000 and 2010. 58 Six-year post-relapse OS was significantly better in the autologous/allogeneic group as compared with the tandem autografts group, 44% versus 35%, respectively (P=005). Taken together, these findings suggest a synergy between new agents and the donorderived immunological milieu.
The current role of allografting in multiple myeloma is controversial though the procedure is still used at many centers. Prospective studies were designed before agents with potent anti-myeloma activity became readily available. However, despite the recent introduction of very effective pharmacological therapies, there remains a subset of high-risk patients accounting for about 10-15% of new diagnoses, whose dismal prognosis is further compounded when early relapse (within 18 months from firstline treatment) is observed. 59,60 The negative impact of adverse cytogenetics on clinical outcomes was not overcome by allogeneic HCT in our series. More aggressive plasma cell clones may have escaped graft-versus-myeloma effects after non-myeloablative 2 Gy TBI. Instead, the impact of certain high-risk cytogenetics was partly neutralized by graft-versus-myeloma effects in a study by Kröger et al. 61 on 73 patients treated with autologous HCT followed by reduced-intensity melphalan 140 mg/m 2 plus fludarabine, where no significant differences in PFS between patients with del(17p13) and/or t(4;14) and those without these abnormalities were observed after a median follow up of six years. A French trial also showed no differences in clinical outcomes between t(4;14) and non-t(4;14) patients. 62 Another study by Rasche et al. 63 showed no differences in survival outcomes in patients carrying del(17p), t(4; 14) or amp(1q21) as compared to those with normal cytogenetics. We speculated that the incorporation of melphalan 140 mg/m 2 , as employed in the studies by Kroger and Rasche, in the conditioning regimen before allogeneic HCT added more cytotoxicity and might have resulted in superior tumor cell-kill and, therefore, better survival among those with high-risk cytogenetics. Moreover, almost 50% of our patients with adverse cytogenetics did not receive "new agents" as part of induction treatment that, in part, are able to overcome certain high-risk genetic features. 64 In summary, our study showed that tandem autologous/minimal intensity allogeneic HCT for MM was safe and characterized by low acute and long-term toxicities. Patients among standard and high-risk categories were able to achieve long-term sustained remissions, while patients with ultra-high-risk disease did not benefit from the tandem HCT. Similarly, patients with progressive disease after autologous HCT failed to respond to allogeneic HCT and succumbed to the disease, suggesting that graft-versus-myeloma effects alone are inadequate to control refractory and high-tumor burden disease states. Allogeneic HCT may be employed as a platform for posttransplant immune-based strategies such as novel immunomodulatory drugs or proteasome inhibitors, CAR T-and N-cell infusions, and bispecific T-cell engagers in selected high-risk populations where prognosis remains poor even in the era of new drugs. [65][66][67] Funding Research reported in this article was supported by the National Cancer Institute of the National Institutes of Health under award numbers CA078902, CA018029 and CA015704. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, which had no involvement in the in study design; the collection, analysis and interpretation of data; the writing of the report; nor in the decision to submit the article for publication. | 2018-10-11T13:15:26.218Z | 2018-09-27T00:00:00.000 | {
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220064848 | pes2o/s2orc | v3-fos-license | Weight change among repeat participants of an Aboriginal community-based weight loss program
Background Community-based weight loss programs may have potential to address overweight and obesity at the population level. However, participation patterns and individual outcomes from these programs are understudied. This study examined repeat participation patterns and participant weight change between contests over seven years of an Aboriginal Australian team-based program in order to identify (1) predictors of repeat participation and (2) associations with weight change between contests. Methods Data for the 12 contests from 2012 to 2018 were merged, with probabilistic record matching. A total of 7510 enrolments were registered for the 12 contests, representing 4438 unique people. Contest lengths varied from 10 to 16 weeks in duration. Non-repeat participants were those who only competed once in the program by the end of 2018, and repeaters were those who competed in at least two contests. Associations between repeat participation and participant baseline (i.e., first participation occasion) characteristics, change in diet and physical activity and percent change in weight during the first participation occasion were examined using crossed random effects (for person and team) regression adjusted for exposure to the program. Weight percentage change between contests was calculated for consecutive participation occasions occurring at least three months apart, converted to percent change per month. Weight change was regressed on number of repeat participation occasions adjusted for age, gender, baseline weight at first participation occasion, and weight percent change in the immediately preceding contest. Results One-third of the 4433 participants participated more than once, with women more likely than men to repeat. A 1% reduction in weight during a competition was associated with an increase in weight of 0.05% per month between competition end and subsequent participation. Regain was smaller the heavier participants were at their first participation. Conclusions While individuals benefit from weight loss through program participation, strengthening strategies for weight loss maintenance within or following the program could improve long-term weight outcomes and reduce weight cycling.
Introduction
Weight loss interventions among Aboriginal Australians have proven effective in reducing weight and noncommunicable disease risk [1][2][3][4][5]. Effective interventions are particularly needed for a population of whom over two-thirds are overweight or obese and whose mortality rate is 1.6 times that of non-Indigenous Australians [6]. Community-based programs offer easy and affordable access and have potential to both strengthen community ties and improve physical health [7][8][9]. Outcomes from community-based weight loss programs can be difficult to establish due to the variety of program designs and reporting inconsistencies [10]; never-the-less, weight reduction and reduced risk of non-communicable disease have been demonstrated in other populations [11][12][13][14]. For Aboriginal Australians there is limited but positive evidence of effectiveness for community-based programs [15].
Losing excess weight has health benefits for cardiovascular and other risk factors [16] but the health effects from any weight regain are less clear, with some studies suggesting loss of health benefits attained through weight loss [17], increased health risks [18], or improved risks relative to those who remain at a stable weight [19] or maintain the lost weight [20]. Whilst there is a large literature documenting weight loss, there are far fewer studies examining maintenance of weight loss, with most finding poor maintenance rates and regain common [21,22]. Additionally, little is known about weight maintenance among Aboriginal Australians.
Attendance in an intervention and in any postintervention program maintenance phase can aid weight loss and maintenance [23,24]. Repeat program participation may also improve weight outcomes [11,25,26], particularly if the repetition is consecutive [27]. However, repeat participation may also indicate repeated loss/regain (weight cycling) if weight loss is not maintained in the interim, and weight cycling may be detrimental to health [28][29][30][31][32]. Thus, investigating characteristics of repeat participants and their weight changes in and between programs has potential public health implications.
Team-based weight-loss competitions are often used in wellness programming as they have wide reach for population impact [33]. There are some successful, repeated, team-based, community weight-loss competitions, but individual outcomes of community competitions are infrequently evaluated in peer-reviewed literature [33]. There are three notable exceptions based in the United States of America. Repeat participants in Shape Up Rhode Island, an annual 16-week state-wide team-based competition, were more likely to be older (> 40 yrs), obese and meet guidelines for attaining moderate physical activity (PA) and vegetable consumption compared to non-repeaters [27]. Completion rates and weight loss correlated with social influence [12,14], and both more minutes/week in PA and more steps/day correlated to weight loss [14]. In the second study, employees in an annual 5-week team-based competition at a Michigan hospital lost weight, with their outcomes differing with participation patterns over the program's 5 years; over two-thirds of the employees gained weight between contests [34]. In the third study, individuals' weight changes within and between an annual 14-week community competition in Texas were documented [11]. Average weight loss among first-time participants who lost weight during their first participation was approximately 5% and lessened with each subsequent participation; weight regain between first and second participations was approximately 3%, with nonconsecutive repeat participants experiencing more regain [11]. Factors associated with weight changes included gender, starting weight, and the number of repeat participations [11].
We recently evaluated a community-based team weight loss competition, the NSW Aboriginal Knockout Health Challenge (KHC) [15], a program open to Aboriginal Australians and Torres Strait Islander peoples aged 16 years or older. KHC contests have been held once each in 2012 and 2013, and twice per year since 2014. Contest lengths have varied from 10 to 16 weeks in duration. The time between the start of the first (or only) contest in 1 year and the end of the previous contest ranged between 178 and 245 days, with shorter periods (17-94 days) between contests held in the same year. (Contests lengths and dates can be found in the additional file, supplemental Table 1). The program has been described in detail elsewhere [35]; briefly, teams of 20 or more persons compete to lose weight through PA and healthy eating. Participants may enrol as a member of a team at the beginning of any contest and teams selfdetermine their activities to support healthy lifestyle behaviours. The program capitalizes on Aboriginal Australians' values (e.g., community) and is structured for prize money to be directed to environmental and socioeconomic factors which disproportionally affect Aboriginal Australians and contribute to their poor health [6]. Our recent evaluation of the KHC found increased participation and significant average weight loss [15] but did not explore within-individual outcomes. Therefore, we analysed participation patterns, particularly repeat participation patterns, and participant weight change between contests over 7 years (2012-2018) of the KHC. The study aims were to identify (1) predictors of repeat attendance and (2) the effects of repeat attendance on weight change. Ethics approval for the secondary analysis was provided by the Aboriginal Health and Medical Research Council (Project 1125/15) and the University of Sydney (2019/425).
Data collection and treatment
Participant outcomes are measured pre-post contest for prize allocation; written consent (provided by the participant, and, for those under 18 years of age, by their parent/legal guardian with the underage participant providing written assent) allows use of the data for prize calculation and research purposes. Participants join teams by submitting data and consent to a team manager; data are collated and sent to a central database. Data comprise name, date of birth and gender as well as objective measures of weight (to nearest 0.1 kg) and height (cm) which were collected by a health professional. From 2013, self-reported current smoking status, fruit and vegetable intake (servings of each on a typical day), and from 2014, PA (frequency in last 7 days of 20 min or more vigorous PA, 30 min or more of walking, and 30 min or more of moderate PA) were also recorded using validated questions [36]. At the conclusion of the contest, participants' weights are recorded by a health professional and self-reported lifestyle risk factors are reported via questionnaire using the same questions as at registration.
Data for the 12 contests were merged, with probabilistic record matching by participant name, sex and date of birth through an independent data linkage agency (The Centre for Health Record Linkagehttp://www.cherel. org.au/; 0.05% false positive rate). The start and end dates of the 12 contests were then merged with the contest data.
Participants
A total of 7510 enrolments were registered for the 12 contests from 2012 to 2018, representing 4438 unique people. Six records (n = 5 people) were excluded because the participant was aged younger than 18 years and did not have parental consent for their data to be used, leaving 4433 unique people with 7504 enrolments.
Primary outcomes
This study had two primary outcomes: to identify (1) predictors of repeat participation and (2) associations with weight change between contests. Non-repeat participants were those who only competed once in the KHC by the end of 2018, and repeaters were those who competed in at least two contests. In order to account for the changing probability of repeating across time (because those whose first participation is later in the KHC series have less opportunity to repeat than those who start earlier [37]) an exposure variable was calculated as the number of contests between first and second occasion of participation or, for non-repeaters, the number of contests from first participation through to the 12th contest. Weight percentage change between contests was calculated for consecutive participation occasions which occurred a minimum of 3 months apart (i.e., between one contest's end and start of the next contest the individual competed in). The end weight of the previous contest was subtracted from the start weight of the next and the difference divided by the end weight of the previous. For example, a participant weighing 100 kg at the start of contest 7 and 95 kg at the end of contest 6 (~7 months prior), would have a change of 5.3%; a positive number therefore demonstrates a weight gain between contests. This was converted to a rate per month by dividing by the number of months between the two contests using the contest start and end dates.
Data analysis
Participation patterns were ranked from most to least common among the 4433 unique participants.
Further exclusions of the 4433 participants occurred at analysis. Two records were excluded from analyses as their weight change exceeded limits (e.g., a weight reduction greater than 30%) used previously in a weight management program of a similar duration as the KHC [23]. All other weights were within limits (leaving 4432 people with 7502 records). For likelihood of repeat participation, data comprised individuals who had formed teams of ≥20 persons at their first participation occasion (leaving 4312 people) and those whose first participation was prior to contest 12 (n = 4104 people). For analysis on weight changes between competitions, analysis was limited to participants who had 2 or more participation occasions (leaving 1532 people with 3071 records) and to those occasions where contest participation had a minimum three-month gap (leaving 1132 people with 1794 records). Further, a complete case approach was used for all analyses (i.e., relevant records with missing data were excluded).
The associations between repeat participation and participant baseline (defined as first participation occasion) characteristics, change in diet and PA, and percent change in weight during the first participation occasion were assessed using bivariate analyses. Baseline characteristics included gender (male, female), age (years), weight (kg), fruit and vegetable intake (per serve), and minutes of vigorous and moderate PA and walking. Fruit and vegetable intake were also categorised dichotomously as meeting (or not meeting) current dietary recommended levels of two serves of fruit and five serves of vegetables [38]. PA was categorised dichotomously as having (or not having) adequate PA (defined as three or more vigorous sessions/week; or five or more walking or moderate sessions/week; or 1-2 vigorous sessions/week and 3-4 walking or moderate sessions/week according to previous procedures) [39]. Associations between repeat participation and change in diet and PA (calculated by subtracting baseline from post-intervention scores for each contest) and percent change in weight during the first participation occasion were further adjusted for gender and age, as these variables have been shown previously to affect contest outcomes [15]. Crossed random effects Poisson models with random effects for person and team were used to account for the clustering of observations and were adjusted for the exposure to the KHC. Crossed random effects were used for all analyses to account for the clustering of observations within person and within team because participants did not uniquely nest within teams but changed teams across contests [40]. For repeat participation analyses only, results are reported as rate ratios (RR) for at least one occasion of repeat participation.
The analysis of the effect of repeat participation on weight percent change between contests was restricted to sequential participation occasions at least 3 months apart (to allow for weight regain following weight loss [22]). Cumulative repeat participation was operationalised as a categorical variable ranging from 2 to 6+ participation occasions to examine non-linear effects; 6-12 cumulative participation occasions were collapsed to form the category 6+ due to small numbers. Covariates were age, gender, baseline weight at first participation occasion, and weight percent change in the immediately preceding contest. Linear crossed random effects models were used with random effects for person and team. Results are presented as percent change in weight per month for a one unit increase in the independent variable.
All analyses were conducted using Stata 15.1 (College Station, TX, USA).
Participation patterns
Over the 12 contests comprising the challenge, 4433 individuals participated, yielding a total of 7504 participation occasions. Participant baseline characteristics and participation patterns are shown in Tables 1 and 2, respectively. (An additional file contains further contest and participation characteristics not shown here.) Single-contest participation was the most common pattern, with 2901 (65.4%) participants. Contest 5 had the highest single-participation participants (n = 384, 8.7%) and contest 8 had the lowest (n = 75, 1.7%). Among those who had participated more than once (n = 1532, 34.3%), participating twice (n = 816, 18.4%) was the most common frequency and no individual competed in all 12 contests. The number of days between the official end of one contest to the start of the next contest ranged between 17 and 245 days.
The participation pattern for most repeaters included some consecutive contest participation (n = 1147, 74.9% of repeaters). The most frequent pattern was competing in contest 11 and contest 12 (n = 85, 10.4% of two-time participants). Consecutive participation was more frequent (n = 489, 59.9% of two-time participants) than skipping one or more contests in between. The number of two-time participants decreased with increasing gap between contests, with just 4 persons (0.5% of twotime participants) competing in only the first and last contests. Among those competing three or four times, most (84.5 and 98.1%, respectively) had at least two consecutive participations and few (25.2 and 28.4%, respectively) had no skipped contest between their first and last participation.
Likelihood of repeating Table 3 shows the bivariate rate ratios (with 95% CI) of repeating at least once for characteristics of participants on their first occasion of participating, adjusting for the number of possible contests they could enter as described in the methods. Among the demographic, weight and behaviour measures only gender and, marginally, amount of vigorous PA showed significant relationships with the likelihood of repeat participation. Female participants had a 34% higher likelihood of repeating, and the likelihood of repeating increased by 2% for every extra session of vigorous PA reported at registration in the first participation. Neither the number of fruit nor vegetable serves nor PA behaviour bore much relation to repeat participation. Further, meeting dietary and PA recommendations at first participation start, versus not meeting these, was not related to repeat participation (see additional file, supplemental Table 4). Table 4 shows the rate ratio for repeating given participants' outcomes in their first contest, adjusted for age and gender and possible repetition occasions. The likelihood of repeat participation increased with increased weight loss and with a higher number of sessions of walking and moderate PA as measured at the end of their first occasion of participation. Specifically, the likelihood of repeating increased by 3% per percentage of registration weight lost and by 7 and 6%, respectively, for each session increase of walking and moderate PA. There were marginally significant results for number of vegetable serves (p = 0.071) and of vigorous PA sessions (p = 0.065), both demonstrating an incremental effect as the number increased. Figure 1 shows participants' raw mean weight at start and end of each contest occasion, for those competing two or more times. It appears that there is a decrease in weight during each contest and a subsequent increase during the time before the subsequent participation. Table 5 shows weight changes between contests, adjusted for age, gender, percentage weight lost during the previous contest, the starting weight on the first occasion of participating, and the number of participation occasions at that point. Due to small numbers, participation occasions 6-11 were combined. Holding all else equal, for every percentage increase in the amount of weight lost, there was a 0.05 percentage gain per month in between contests. However, for every kilogram heavier the participant was on their first occasion of competing, there was a small (0.002) decrement in the percentage weight regain. There appeared to be somewhat of a dose-response effect for repeat participation whereby the amount of weight gain between contests increased as the number of participation occasions increased. For example, predicted percentage gain per month generated using average values on all other covariates showed that the weight gain per month after two participation occasions was 0.30%, after four was 0.57%, and after six was 0.70% per month (data not shown). A supplementary analysis using participation as a continuous variable found that the average increase in weight percent per month was 0.10 (.06, .13, p < .001) per participation occasion.
Discussion
This study analysed participation patterns and participant weight change between contests over 7 years (2012-2018) of a community-based weight loss program for Aboriginal Australians. About one-third of participants repeated the community weight loss program. Gender was a strong predictor of whether someone participated more than once, with behavioural measures contributing minimally to this prediction. Between competitions, repeat participants regained some of the weight they had previously lost, with smaller regain the heavier a participant was when they first joined the KHC.
Participation
The KHC is a long-running program with increasing appeal [15]. While the most common participation pattern in the KHC was a single participation occasion, repeaters accounted for about one-third of the participants. In our study, almost no baseline characteristic predicted repeat participation. The observed increased likelihood of repeat participation among women contrasts with results from studies of online PA and other behaviour change programs which found no gender difference [27,41] or a higher likelihood for repeat participation among men [41]. In those studies and this, males comprised a minority of the study population, consistent with findings that men are less likely to seek assistance for weight loss [42]. Reports of age [27,41] predicting repeat participation contrast with our findings but could be explained by the internet-based program format of those programs. Previous research found attaining sufficient vigorous PA did not determine likelihood of repeat participation [27], whereas here the amount of vigorous PA reported at baseline was marginally (p = .052) related to the likelihood of repeat participation. Again, the different program formats may explain variabilities in health behaviours as participation predictors. Additionally, the KHC serves a unique population (Aboriginal Australians) and its incorporation of their values (e.g., reflecting the strong value in family and community by having participation based on teams) may influence repeat participation likelihood [43]. Investigating participant motives for involvement in the KHC and satisfaction with the program format may elucidate why a gender difference in repeat participation exists and help tailor the program to maximize its reach. Whilst we identified predictors of repeat participation, we did not measure goals and expectations. We did model the likelihood of repeating on changes in lifestyle outcomes during participants' first participation Fig. 1 Weight changes over four participation occasions. Participants' raw mean weight at start and end of each contest occasion, for those competing two or more times, censored at 4 participations; n = 1532 occasion. Greater increases in walking and moderate PA sessions and greater weight loss were associated with increased likelihood of repeating by between 3 and 7%. Previous research has also found higher levels of participation [11,25,26] are associated with improved weight outcomes. Identifying whether actualized behaviour changes were participant goals could provide insight into the relationships between goals, behaviour changes and repeat participation. Further, there are a variety of other motives for program participation, including health concerns and social support [44], and these directly relate to the secondary goals of the KHC. Motives may differ between first-time and repeat participants in physical activity events [45]. Investigating repeat and non-repeat participant motives may be helpful for further program development or to elucidate other strategies needed to combat weight loss.
Weight change between repeat contests
Repeat participants gained weight between contests at a rate of 0.05% body weight per month. Similarly, weight regain, of approximately 3%, was also found during the 40-week length gap between first and second participations in another community-based weight loss competition [11]. That weight is regained between contests, increases with each participation occasion, and correlated with a higher percent weight lost during the program suggests weight cycling may be a problem among repeat participants. Figure 1 also lends itself to such an interpretation. Positively, regain may be less of a problem for heavier participants (here and in the American program [11]), which suggests community-based programs may have a positive health impact among obese individuals. Further, this type of community program may confer additional benefit for weight loss through its team-based format as weight loss can be clustered within teams [12] and team-based participation may increase the amount weight lost [11,14]. On the other hand, team-based participation may also increase the likelihood for regain as the social support available from teammates during participation disperses [11]. Social support has been implicated as having a potential role in weight loss [14] while individual coping skills, such as self-monitoring [46,47] and emotional control [48], may be key to maintenance. Thus, structuring a community program to maximize the benefits of social support during competition while teaching individual skills for maintenance may be important to minimize weight cycling. Investigating coping skills of repeaters and non-repeaters may indicate where skill differences exist and may be more valuable for weight maintenance among this population. Males comprise an equal proportion of the overweight/obese Aboriginal population [49]. They may be less likely to lose weight in the KHC [35] and have lower participation and probability of repeating than females, but our results indicate they are no more likely than females to regain lost weight. Similarly, the observed gender difference in weight regain disappeared with participation cycles in the American program [11] and a recent systematic review found participant demographics were not associated with weight loss maintenance [22]. Therefore, addressing avenues to increase male participation and weight loss success in the KHC could have long-term population health benefits.
That weight increases with time between participation occasions reinforces the need for weight maintenance support. Evidence suggests self-monitoring assists with weight maintenance [46,47], so incorporating this as part of the program may mitigate weight regain among participants. Notably, the 2013 KHC contest included a maintenance phase comprising individual (e.g., monthly weigh-ins) and team-based (e.g., team sports carnival competition) components [35]. While few KHC participants were evaluated in the maintenance phase, the results were promising: the mean weight at contest end and 9 months thereafter were comparable [35]. There is some evidence that web-based programs can assist maintenance as well, particularly in the short-term [50], and the KHC already has a social media presence which could act as a platform for post and/or between competition weight loss maintenance support. Weight loss is best maintained with continued support following program end. To this end, the KHC encourages participants to enrol in a free 6-month telephone coaching service and facilitates enrolment through an opt-in option at contest registration for participant contact details to be shared with the service. Evaluating participant uptake and outcomes through the coaching service would provide a more complete picture of long-term weight loss outcomes of KHC participants.
Strengths and limitations
This is the first study to explore weight maintenance among repeat participants of an Aboriginal communitybased team weight loss competition. Strengths of this study include the inclusion of 12 contests over 7 years, using a minimum gap of 3 months between participation occasions to allow sufficient time for weight regain, the large sample size, and the inclusion of nutrition and PA behaviours.
Limitations include the ability to measure weight maintenance only among those who repeated the program. The KHC has been shown to be effective for short-term weight loss and healthy lifestyle adaptation [15], and it may be that non-repeaters are able to maintain their weight loss and health behaviours. Among repeaters, we cannot state to what extent the subsequent participation might have mitigated an otherwise larger increase in weight.
Conclusion
The KHC has been successful in attracting increasingly large numbers of participants, starting with 324 in its first year and increasing to 4433 individuals having participated over the first 7 years of operation. Many participants return to the competition repeatedly, especially women, who had a 34% higher likelihood of repeat than men. Both program weight loss and increases in number of sessions of walking and moderate PA were associated with small (3-7%) but significant increases in likelihood of repeat participation. However, the association between contest weight reduction and subsequent regain (0.05% regain per month for every 1% body weight reduced) might signal a need for the program to incorporate weight loss maintenance strategies to better conserve the benefits accrued during competition. Tracking the longer-term outcomes of those not returning to the competition would assist in understanding the full impact of the KHC. Further, qualitative research with those who do and do not repeat would yield insights into people's patterns of engagement with the program.
Additional file 1. Weight_change_among_repeat_Additional_file_1.docx. The additional file contains contest and participation data in tabular form: contest dates (including number of days between each contest), most frequent participation patterns, non-repeater participant characteristics, and further rate ratios for repeat participation. | 2020-06-26T14:48:16.886Z | 2020-06-26T00:00:00.000 | {
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255846029 | pes2o/s2orc | v3-fos-license | Intro 50 years anniversary of Applied Physics
After Helmut Lotsch, and after the division of Applied Physics into Appl. Phys. A (APA) and Appl. Phys. B (APB), Michael Stuke took over APA as Editor-in-Chief. He served in this role until end of 2016 and developed APA into a wellrecognized journal in the field: the number of publications increased steadily from around 250 papers per year in 1996 until about an average 750 when he left. Also, the Julius Springer Prize for Applied Physics and the Julius Springer Forum in Applied Physics were created in this period. In December 2016, Michael Stuke stepped down, and Thomas Lippert took over the role as Editor-in-Chief of APA in January 2017, with the goal of keeping the high standards of APA, but also to try to increase the impact factor and to slowly “modernize” the journal. There were a number of measures and changes which took place, during the first years, i.e.
After Helmut Lotsch, and after the division of Applied Physics into Appl. Phys. A (APA) and Appl. Phys. B (APB), Michael Stuke took over APA as Editor-in-Chief. He served in this role until end of 2016 and developed APA into a wellrecognized journal in the field: the number of publications increased steadily from around 250 papers per year in 1996 until about an average 750 when he left. Also, the Julius Springer Prize for Applied Physics and the Julius Springer Forum in Applied Physics were created in this period.
In December 2016, Michael Stuke stepped down, and Thomas Lippert took over the role as Editor-in-Chief of APA in January 2017, with the goal of keeping the high standards of APA, but also to try to increase the impact factor and to slowly "modernize" the journal.
There were a number of measures and changes which took place, during the first years, i.e.
• A number of editors stepped down together when M.
Stuke retired • We replaced them and even increased the number of editors, according to the topics we defined for the journal, also with the goal to have for each topic at least one expert editor • Topics and key words were defined in more detail, and certain topics were not any more present. Our current topics cover: which now have the role of a first quality/topic check, and then assigning the papers to the corresponding editors • plagiarism checking was introduced as standard approach for each submitted paper, and other integrity measures, e.g. to limit the number of self-citations or reviewers insisting on own papers to be cited. • the editor pick was established, with certain papers highlighted for the journal • a new appearance was introduced for APA and APB, now having a cover image for each issue • increased presence of the journal in social media with support from the Springer Physics platforms • Highlight journal metrics in 2021: more than 3600 submissions, with an acceptance rate of 26%; a median 18 days until the first decision; almost 818,000 downloads. • the sum of these measures may have contributed to a steady increase of our Impact Factor, which more than doubled in 5 years, reaching 2.983 in 2021 (as compared to 1.455 in 2016) Of course, we faced also challenges, e.g. the increased number of misconduct and research integrity cases (sometimes needing support from the Springer Research Integrity Team), the increasing problems to find two or more available reviewers, but we are working on this to have all these issues resolved. Our main goal is to keep high standards for the journal (e.g. high quality articles and fast turn-around time) to ensure that the journal will thrive for many more years.
On the occasion of the 50th anniversary of the journal, we have put this special issue together, where we collected papers from: • recipients of the Julius Springer Prize in Applied Physics • authors of our most cited papers • former editors/senior editors • present editors/senior editors • authors invited by our editors.
We hope that we managed to put an interesting special issue together with a mixture of reviews and original articles, that highlight the broad selection of topics of Applied Physics A.
Finally, Thomas would like to thank the people at Springer, which take care of the journal behind the scenes, and of course mainly the editors present and past, without them the journal would not be there where it is.
Thomas Lippert. Editor-in-Chief of Applied Physics A. | 2023-01-16T16:03:09.562Z | 2023-01-13T00:00:00.000 | {
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258937864 | pes2o/s2orc | v3-fos-license | An Atypical presentation of pulmonary embolism in a critically ill patient
A pulmonary embolism (PE) occurs when a venous thrombotic material from the lower extremities embolizes to the pulmonary vasculature. Common presenting symptoms include shortness of breath and pleuritic chest pain with vital signs demonstrating hypoxia, tachycardia, and tachypnea. In this paper, we describe a unique presentation of a critically ill patient who developed a saddle pulmonary embolism despite being on prophylactic anticoagulation.
Introduction
A pulmonary embolism (PE) occurs when venous thrombi dislodge from the lower extremities and causes a downstream obstruction in the branching pulmonary vasculature. It is associated with a significant mortality burden and continues to be the third most common cause of death from cardiovascular disease [1] . The incidence of PE diagnoses has increased with increased use of computer tomographic pulmonary an-
Case report
A 66-year-old female with a past medical history of hypertension, hyperlipidemia, diabetes mellitus type 2, and tobacco dependence presented to the emergency room complaining of shortness of breath, headache, productive cough with clear sputum and a small amount of bright red blood, as well as fever and nausea. On exam, her blood pressure was slightly elevated at 133 systolic and 80 diastolic, she was tachycardic at 130, she was febrile at 101.3 °F (38.4 °C), her respiratory rate was normal at 19 breaths per minute, and her oxygen saturation was normal at 98% on room air. She was ill appearing and had rhonchi and decreased breath sounds in the lower lung fields bilaterally. Her white blood cell count was elevated at 12 K/uL (normal range: 4-11 K/uL), hemoglobin was slightly low at 11.5 (normal range 11.7-16.1 g/dL), and proBNP was increased at 6344 (normal range: less than or equal to 125 pg/mL). Urinalysis demonstrated negative nitrites, moderate leukocyte esterase, 10-20 white blood cells, and rare bacteria. Chest radiograph revealed ground glass opacities in the right lung base ( Fig. 1 ). The patient's presentation was attributed to sepsis secondary to urinary tract infection or pneumonia. Echocardiogram revealed an ejection fraction of 11% (normal range greater than 55%) and right ventricular systolic dysfunction. Her symptoms were then believed to be secondary to acute systolic heart failure rather than pneumonia and her antibiotic was modified to only treat possible urinary tract infection. The patient later developed altered mental status and arterial blood gas was ordered, which revealed respiratory alkalosis, with a pH of 7.467 (normal range: 7.35-7.45), a pCO2 of 31.5 mm Hg (normal range: 34-45 mm Hg), a pO2 of 49 mm Hg (normal range: 80-100 mm Hg), a HCO3-of 22.8 (normal range: 22-26 mm Hg), and an arterial O 2 saturation of 88%. Her symptoms improved on nasal cannula and cardiology planned for a coronary angiogram to evaluate for coronary artery disease,
Fig. 2 -Repeat chest radiograph (day 4 of admission) demonstrating increased density of air opacities in the right lung base.
however this was canceled due to development of new onset atrial fibrillation. She was found to have a decreased thyroid stimulating hormone of less than 0.01 mcU/mL (normal range: 0.27-4.20 mcU/mL) and an increased free T4 of 3.5 ng/dL (normal range: 0.9-1.8 ng/dL). She was started on methimazole per endocrinology and beta blockers were started for rate control per cardiology. Her white blood cell count increased to 16.8 K/uL (normal range: 4-11 K/uL) and repeat chest radiograph demonstrated slightly increased opacity in the peripheral right midlung zone ( Fig. 2 ). Therefore, her antibiotic regimen was further modified to again cover pneumonia. The patient remained persistently hypoxic and tachycardic, and repeat chest radiograph was obtained, which revealed a wedgeshaped peripheral opacity in the right middle lung, concerning for pneumonia or pulmonary embolism ( Fig. 3 ). Computer tomography (CT) of the chest was ordered for further evaluation, which revealed dome shaped opacities in the right middle and right lower lobes, which could represent pneumonia or pulmonary infarction ( Fig. 4 ). D-dimer performed at the time was 3.27 mg/L (normal range: 0-1.2 mg/L). Computer tomographic pulmonary angiogram (CTA) revealed saddle pulmonary embolus with lobar and segmental pulmonary emboli bilaterally, right greater than left ( Fig. 5 A-C).
Discussion
Pulmonary embolisms have diverse presentations, ranging from largely asymptomatic to the presence of more characteristic symptoms. It is crucial to consider pulmonary embolism in the presence of consistent symptoms, clinical findings, and risk of venous thromboembolic disease [4] . When considering the risk for venous thromboembolic disease, it is important to consider Virchow's triad of blood hypercoagulability, venous stasis, and endothelial damage [5] . Common presentations include immobilized patients who are hypoxic, tachycardic, and complain of sudden onset severe pleuritic chest pain and dyspnea. Patients with high risk of death may also present with syncope, hemodynamic instability, cardiac arrest, shock, or hypotension [6] .
Clinical consideration of PEs may be delayed if presentation is atypical, such as in our patient, who uniquely presented with initial complaints of productive cough with leukocytosis and reduced ejection fraction, and developed altered mental status due to respiratory alkalosis, atrial fibrillation, persistent hypoxia, and tachycardia later in the course of her hospitalization. Diagnosis may further be delayed if multiple comorbidities exist, such as in our patient, where her systolic heart failure, underlying pneumonia, and hyperthyroidism seemed to plausibly explain her presentation, and further investigation was prompted by the patient's lack of clinical improvement despite treatment efforts. It is also important to note, that the absence of pleuritic chest pain should not prevent consideration of a PE, as seen by the complete absence of pleuritic chest pain throughout our patient's clinical course. Clinical suspicion of a PE was also low due to our patient being on prophylactic anticoagulation. However, patients may still develop PEs despite being anticoagulated, termed anticoagulation failure, and further hematological work up is indicated in these patients [7] .
Chest radiographs, while nonspecific, may assist in excluding other causes of chest pain and dyspnea. In rare cases, such as in our patient, a wedge-shaped consolidation mimicking pneumonia may be seen. This is colloquially referred to as Hampton's Hump sign [8] . Approximately 25% of patients with PEs have normal EKG findings. However, those with EKG abnormalities typically have sinus tachycardia, P pulmonale, atrial fibrillation, or right axis deviation. S1Q3T3 may also be seen in some patients, which describes a large S wave in lead I, a Q wave in lead III, and an inverted T wave in lead III, together which represent acute right heart strain [9] . D-dimer levels may exclude pulmonary embolism; however, it must be qualified in patients with baseline elevations. Echocardiograms may show evidence of right ventricular dysfunction; however, these findings may be attributed to other cardiovascular diseases or may be incidentally found in otherwise healthy patients. Specific echocardiographic findings, such as the 60/60 sign, McConnel sign or direct visualization of right heart thrombotic material are rarely seen, but when present, are diagnostic for PE [4] . CTA is the gold standard of diagnosis, as it allows direct visualization of the thrombosed vessels, while also ruling out aortic dissections and myocardial infarctions, which may present similarly. Ventilation perfusion scans examine lung areas that remain ventilated without adequate perfusion, but use is limited by high cost and frequency of inconclusive results [10] .
Therapeutic anticoagulation is the primary treatment for patients with an acute PE, because it significantly improves patient outcomes. First line therapy typically involves anticoagulation with low molecular weight heparin followed by novel oral anticoagulants with apixaban or rivaroxaban for at least 3 months [10] . In patients with contraindications to thrombolysis, percutaneous mechanical thrombectomy may be considered [11] .
Patient consent
The authors obtained informed consent from the patient whose case was discussed in this report. | 2023-05-28T15:05:24.279Z | 2023-05-26T00:00:00.000 | {
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219927667 | pes2o/s2orc | v3-fos-license | Sustainable Lighting and Light Pollution: A Critical Issue for the Present Generation, a Challenge to the Future
: Human beings’ poor night vision and primitive fear of the dark are reflected in an imperative need to use artificial light to illuminate their environment. Outdoor illumination undoubtedly contributes to the enhancement of practical opportunities for social and economic developments. Considered as a necessity, a means of security, and an attraction or valorization, city lighting growth has been literally exponential in the last half century. Beyond the financial and energy resources that it absorbs, the artificial lighting of urban spaces overflows its objective by polluting our nights to the point that, in our modern megacities, the stars disappear. Apart from the fact that stars are no longer visible, the scientific community is increasingly interested in the direct and indirect impacts of artificial lighting on biodiversity. In parallel, some studies have shown recently that stray light may have direct or indirect e ff ects on human health and mood. The scope of this Special Issue, dedicated to the memory of Prof. Abraham Haim and Dr. Thomas Posch, is to put together a series of high-level papers treating light pollution in a holistic manner that goes from technological advances to policies, passing through impacts on biotopes and human health. Beyond its evident scientific interest, this Special Issue is also contributing to awareness raising, aimed at decision- and policy-makers.
The human being is not a night animal, even if the night sky has fascinated our species since the dawn of our existence. Our poor night vision and our primitive fear of the dark are reflected in an imperative need to use artificial light to illuminate our environment. Here, the term "artificial light" refers to man-made night-time light. The power to artificially override the natural cycle of light and dark is recent, but decisive, event for human society. For sure, outdoor illumination undoubtedly provides enhanced practical opportunities for social life development and contributes to an extensive range of evening and night-time activities that would otherwise be difficult, if not impossible, without artificial light.
Considered as a necessity, a means of security, and an attraction or valorization, city lighting growth has been literally exponential, especially since World War II. The phenomenal rise of public lighting is manifested today by at least 100 million lighting points illuminating our cities worldwide, whose annual electricity consumption would represent, roughly, more than 200 TWh. Further, lighting constitutes up to 25% of the budget of rural small-size cites, corresponding to roughly 50% of their electricity bill. This uncontrollable growth is no longer sustainable and a drastic reduction in energy consumption must be imposed.
Beyond the financial and energy resources that it absorbs, the artificial lighting of urban spaces overflows its objective by polluting our nights to the point that, in our modern megacities, the stars disappear from the night sky when, just 100 years ago, the milky way could be observed from almost everywhere. However, the loss of the night skies has happened gradually over the course of the last century and this has resulted in a collective ignorance of the degradation, about which none but astronomers complained about for a long time.
Apart from the fact that stars are no longer visible, the scientific community is increasingly interested in the direct and indirect impacts of artificial lighting on biodiversity. We know that, for more than 100 years now, all living organisms have evolved varying degrees of sensitivity to light. Light stimulates hormone production, regulating circadian rhythms in all living species, and it also influences phototropism in plants.
In fact, the biological adaptation of all animal species to natural light conditions (sun, moon, and stars) has evolved over billions of years in all animal species. Paul Beier underlined that "most nocturnal mammals react to increasing moonlight by reducing their use of open areas, restricting foraging activity and movements, reducing total duration of activity, or concentrating foraging and longer movements during the darkest periods of night." [1] Several experimental studies have shown that artificial light at night-time disturbs ecosystems and could play a significant role in the decline of species whose roles in the ecosystem, already weakened by human presence, are not yet fully known. There are some examples, among many others: Sea turtle hatchlings emerge from the nest at night and use visual cues to direct themselves towards the brightest and lowest horizon, eventually leading them to the sea. Night-light pollution could alter the cues perceived, disorienting the fragile hatchlings [2].
Seabirds of the order Procellariformes (albatrosses, petrels . . . ) are known to be highly affected by light pollution near their breeding colonies. The prime and most serious impact is related to the abandonment of colonies [3]. The second impact relates to the disorienting effect that light pollution has on immature individuals that disperse for the first time from their colonies [4].
Kerbiriou et al. showed that the loss or the gain in bat activity when lamp types (i.e., spectrum) are switched strongly depends on the initial and the new lamp intensities. Their results stress the need to consider simultaneously the effects of changes in different light characteristics when street lighting changes [5].
We know that invertebrates make up the majority of biodiversity on earth and are vital to ecosystems. Artificial lighting attracts large numbers of a wide range of invertebrates, but moths are perhaps best known for this behavior [6]. UV, green, and blue light, which have short wavelengths and high frequencies, are best discriminated by most insects and are highly attractive to them [7].
Current research highlights the importance of artificial light with smaller wavelengths in attracting moths, yet the effect of the spectral composition of artificial light on species' richness and on the abundance of moths has not been studied systematically [8]. Further, the role of moths in pollination processes is not fully known.
In parallel, some studies have recently shown that stray light may have direct or indirect effects on human health and mood. As an example, people report negative health impacts from sleep disturbances due to light intrusion into their homes. In addition, human health problems have been associated with exposure to artificial light at night, inhibiting the production of melatonin, which is associated with the incidence of certain breast cancers [9]. In any case, even low levels of illuminance in the blue region of the light spectrum may disrupt melatonin secretion with catastrophic effects to sleep-wake cycles. Further, artificial lighting may also result in night-time glare, including disability glare, which affects driving and pedestrian safety. However, its detection and quantification are generally subjective and relative to existing conditions. Further, it is often said that outdoor lighting can reduce criminality in modern cities; however, there is no systematic study that can provide an evidence to this "urban legend".
Thus, scientific evidence highlighting the negative impacts of artificial lighting in connection with strong actions from the associates of militant people contributed to a spectacular increase in awareness at a political level in many countries. As a consequence, light pollution is now taken into consideration in more and more national environmental protection and territorial development policies. In addition, some locations are particularly sensitive to light pollution and lighting schemes, and these areas should be carefully planned around to avoid negatively affecting fragile ecosystems.
Globally, today we can say with certitude that artificial light in the wrong place or at the wrong time is light pollution. Artificial light has the potential to significantly disrupt ecosystems and natural light and dark patterns. Light pollution, unlike other forms of contamination and waste, remains largely overlooked and unregulated in many countries. It is often exacerbated by excessive, misdirected, or obtrusive uses of light that is difficult to explain today. It is necessary to measure light pollution, quantify the impact on biodiversity, build reliable and robust simulation tools that allow us to predict the impact of new technologies in light pollution, and open questions that need rapid and precise answers. Today, we know that even if a part of light pollution (called usually "sky glow") is unavoidable due the diffusion of light in the atmosphere (linked mainly to the reflection of moisture and very fine particulate matter in suspension), most of the pollution is coming from poor lighting design, often exacerbated by poor installation and maintenance. That part is avoidable, but initial costs for high-quality lighting systems may rise rapidly to the point of become prohibitive for countries with developing economies. On the other hand, in developed economies, the private lighting of external space is a growing cause for concern, because it is rather difficult to regulate with policies. Population awareness raising is most likely the solution here.
The scope of this Special Issue is to put together a series of high-level papers treating light pollution in a holistic manner that goes from technological advances to policies, passing through the impacts on biotopes and human health. Nine excellent papers are included in this Special Issue, placing it in a prime position in today's literature in the domain of light pollution. Beyond evident scientific interest, this Special Issue is also contributing to awareness raising, aimed at decision-and policy-makers. | 2020-06-11T09:10:58.270Z | 2020-06-03T00:00:00.000 | {
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255967058 | pes2o/s2orc | v3-fos-license | Finite Element Analysis to Study Tensile Strength Differences between Free and Attached Ear Lobules
Summary Recurrent ear lobule deformity is a chronic condition with aesthetic implications. The problem is normally addressed by certain improvisations of the traditional lobuloplasty technique. These include introduction of autologous tissue components like cartilage pieces to improve the structural integrity. Certain authors also advocate a different site for repiercing of the ear hole away from the lobuloplasty scar. Our study tries to understand the differences in the tensile strength between free and attached ear lobules, using finite element analysis. Eighteen healthy female volunteers with attached (eight subjects) and free ear lobules (10 subjects) were chosen, and the lobules were scanned using Artec 3D scanner. The model was then converted to free form or attached form (opposite to the form in which it was present originally) by decreasing or increasing the area of contact using geomagic software. Finite element analysis was then performed on both the models, and their yield max and, hence, the maximum load at the yield max at 0.7 strains according to previous studies were estimated and compared. The yield max and the corresponding load were found to be lesser in the free variety than in the attached variety. This experiment helps us to understand that when a structural difference in the ear lobule surgically may bring about a change in the tensile strength of the lobules. However, further clinical trials are required to clinically translate the same.
INTRODUCTION
Ear deformities such as a stretched ear hole or split ear lobule are a common cosmetic problem. Apart from the classical lobuloplasty 1 techniques, the introduction of autologous tissues like dermofat or cartilage to strengthen the structure of the ear lobule has been described in the literature. The traction 2 injuries are usually affected by the load applied to the ear studs. The naturally existing lobules are of two different categories, namely free and attached. 3 In the attached lobule, the ear lobule is attached to the side of the face up to a tangent drawn from the lowest point of the ear lobule, whereas in the free lobule this tangent is not attached to the side of the face. Since the incidence of ear deformities in the attached variety is lower than that of the free lobule, it would be logical to hypothesize that their physical structure is a contributing factor. Hence, the effect of the shape and support on the ear lobule's structural response using finite element analysis (FEA) 4 is studied in this work. FEA is a computational model, which is a mathematical method that can be used to document the deformation due to stress applied on tissue geometry.
MATERIALS AND METHODS
The research methodology conformed to the Declaration of Helsinki. Eighteen healthy female subjects of Asian descent of ages between 20 and 50 years old were selected among attendants accompanying patients visiting our hospital after written informed consent. An institutional ethical clearance was sought, and the subjects' right ear lobules (for uniformity) were scanned using an Artec 3D scanner. This 3D scanner captures the geometry of an object and produces a three-dimensional digital model. Of the 18 ear lobules scanned, 10 belonged to the free category and eight belonged to the attached category. Subsequently, the solid models were generated using Geomagic Freeform software, 5 and were then converted to finite element (FE) models using an FEA software (COMSOL Multiphysics software). 6 An FE model was generated through a series of operations that include generation of FE mesh for the solid model, definition of material properties, and application of loads/boundary conditions. To enable comparison, the FE models of ear lobules that were initially free were converted to attached lobules and vice versa using the Geomagic Freeform software in the following way. This software incorporates the basic geometry of the structure acquired using 3D scanning in question, and its tools can merge the boundaries between the ear lobule and the lateral border of face to mimic an attached lobule and conversely can introduce boundaries by erasing the portion of the lobule geometry that attaches the lobule to the side of the face to mimic a free lobule. (See figure 1, Supplemental Digital Content 1, which displays conversion of free to attached and attached to free using Geomagic free form software. http://links.lww.com/ PRSGO/C342.) The analysis using FE models was done as follows. A free tetrahedral mesh was used for the discretization of the models. Using the previously published literature, a hyperelastic material with Ogden model was adopted with shear modulus G = 0.5 MPa and α = 6, presuming that the ear lobule tissue is identical to skin. 7 Skin shows nonlinear stress-strain behavior between the strain range of 0.3-0.6 and linear stress-strain outside this strain range with a yield strain of 0.7. The boundary surfaces on the FE model were selected to apply boundary conditions (Fig. 1). A vertical downward load on the ear hole was applied to its vertical projected area, assuming the hole was a regular cylinder (Fig. 1). The results obtained from these FE analyses were validated using the vertical displacement of the ear holes obtained from the clinical experiments by loading a 25 g weight on a hook-like instrument (designed by the principal author) mounted on the subject's ear hole. 8 Once validation was agreed upon, loading was started with 20 g and gradually increased to 200 g in load steps of 5 g in FEA. The von Mises equivalent strain was simultaneously computed in each step. When the von Mises equivalent strain at any point exceeded the strain of 0.7, then, that point was considered to have yielded. Thus, a stressstrain graph was developed. (See Video [online], which displays a stress-strain graph.) Figures 1-3 show the comparison of results obtained from FEA and clinical experiments. For comparison, the observed vertical displacements (in mm) of the ear hole are recorded, and their mean is computed. In Figure 1, the comparison of vertical displacement is shown for attached lobules. It is observed that the mean of the FEA is almost similar to the mean of clinical experiments. In a similar comparison for the free lobules presented in Figure 2, it is observed that the deviation of FEA concerning the clinical experiments is around 1.2 mm, which is relatively small compared with the total deformation of the ear lobules.
RESULTS
Therefore, the FEA is further used to estimate the yield max (max load before rupture) of the ear lobules to evaluate their structural integrity. The results of FEA for the estimate of the yield max are shown in Figure 3. It is observed that the yield max for the attached lobules was higher than that of the free lobules.
Because the observation of values from the experimental and FEA study involve different subjects, such that the number of subjects in experimental observations differs from the number of subjects in FEA, any further statistical Takeaways Question: Will a structural modification from a free to an attached ear lobule bring changes to the tensile strength in the lobule and thus decrease the incidence of split ear lobule?
Findings: An experimental modification of a free to an attached lobule using computational analysis showed increase in tensile strength in the subjects.
Meaning: An attached ear lobule is stronger than a free lobule in terms of its tensile strength.
DISCUSSION
FEA is found to be a suitable method to test the surgical hypothesis in many scenarios. However, to the best of the authors' knowledge, published works on experimental or FEA studies for ear lobules are nonexistent.
This study helps understand that when the geometry of a lobule is altered concerning its constraints or area of attachment to the face, its structural response 9 changes and so does the maximum load for yielding or rupture (yield max). 10 These observations can be clinically translated after randomized control trials to find a solution to the recurrent ear deformity problem. So, if a free lobule is surgically modified into an attached lobule (by freshening the medial border and attaching it to a raw area created on the lateral part of the face), then the maximum load-bearing capacity of the lobule should increase.
The limitations of the study are: Generalizability: The subjects included in the study belong to a community in the place of study. However, whether genetic, racial, and communal differences have any repercussion needs to be tested further.
Selection bias: The subjects who were included in the study belonged to the age group of 20-50 years.
Confounder: In all the subjects, the fatty tissue interspersed between the skin layers was not considered in the analysis. However, because the assumptions are the same for both free and attached models, the implication may not be significant.
Application: Although the experimental study shows higher structural strength in terms of tensile strength in the attached lobule than the free variety, the exact results need to be tested in clinical patients, which would require studies in the form of randomized controlled trials.
The FEA showed that the attached variety of ear lobules could bear stress much higher than free ones, as demonstrated by their higher yield max values. This suggests that a surgical modification of free to attached variety can change the tensile strength of the lobule. But to clinically translate the results, randomized control trials on real patients, and standardizing them, is warranted. If proved, then this surgical option can be offered to patients with recurrent split ear lobules. | 2023-01-19T20:43:53.723Z | 2023-01-01T00:00:00.000 | {
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266233493 | pes2o/s2orc | v3-fos-license | Costs of endovascular and open repair of thoracic aortic aneurysms
Abstract Background Repair of thoracic aortic aneurysms with either endovascular repair (TEVAR) or open surgical repair (OSR) represents major surgery, is costly and associated with significant complications. The aim of this study was to establish accurate costs of delivering TEVAR and OSR in a cohort of UK NHS patients suitable for open and endovascular treatment for the whole treatment pathway from admission and to discharge and 12-month follow-up. Methods A prospective study of UK NHS patients from 30 NHS vascular/cardiothoracic units in England aged ≥18, with distal arch/descending thoracic aortic aneurysms (CTAA) was undertaken. A multicentre prospective cost analysis of patients (recruited March 2014–July 2018, follow-up until July 2019) undergoing TEVAR or OSR was performed. Patients deemed suitable for open or endovascular repair were included in this study. A micro-costing approach was adopted. Results Some 115 patients having undergone TEVAR and 35 patients with OSR were identified. The mean (s.d.) cost of a TEVAR procedure was higher £26 536 (£9877) versus OSR £17 239 (£8043). Postoperative costs until discharge were lower for TEVAR £7484 (£7848) versus OSR £28 636 (£23 083). Therefore, total NHS costs from admission to discharge were lower for TEVAR £34 020 (£14 301), versus OSR £45 875 (£43 023). However, mean NHS costs for 12 months following the procedure were slightly higher for the TEVAR £5206 (£11 585) versus OSR £5039 (£11 994). Conclusions Surgical procedure costs were higher for TEVAR due to device costs. Total in-hospital costs were higher for OSR due to longer hospital and critical care stay. Follow-up costs over 12 months were slightly higher for TEVAR due to hospital readmissions.
Introduction
Patients with chronic thoracic aortic aneurysm disease (CTAA) are most often elderly with significant and multiple co-morbidity 1,2 .Nevertheless, patients are offered major surgical repair procedures such as endovascular stent grafting (TEVAR), potentially involving expensive technology, or open surgical repair (OSR), with costly intensive care stays and rehabilitation.Despite being effective for some CTAA patients, TEVAR and OSR are associated with significant complications [3][4][5] .Currently, there are no UK-specific economic studies assessing outcomes beyond the chosen procedure.
There was significant controversy regarding NICE recommendations for infrarenal aneurysm repair techniques in 2019 6 .Draft recommendations, driven largely by a lack of cost effectiveness and since revised, stated that TEVAR should not be used in the elective setting.The effect of similar interventions is therefore in the spotlight.Furthermore, centralization of specialist services has been a major policy focus in the UK in recent years, with evidence that outcomes for vascular surgery can be improved through centralization 7 .Services have undergone a substantial reorganization with amalgamation of smaller units and single-handed surgeons to form larger units.However, the structured organization of complex aneurysm surgery is still in its infancy.The costs of TEVAR and OSR procedures must be accurately determined to understand the tariffs that are required to allow services to be set up and managed and to identify where resources need to be pooled into specialist complex aortic hubs.With these issues in mind, and given the increasing demand for treatment due to an ageing population with rising prevalence of CTAA 2 , and limited NHS resources, further evidence regarding accurate costs of TEVAR and OSR procedures is needed.
Cost data is an important factor in developing clinical guidelines 8,9 , yet there is a sparsity of large multicentre micro-costing studies in Europe regarding TEVAR and OSR procedures that captures the whole treatment pathway from the procedure to discharge and subsequent follow-up.The Effective Treatment of Thoracic Aortic Aneurysms (ETTAA) study provided a unique opportunity to undertake a prospective micro-costing of a large cohort of patients with CTAA repaired https://doi.org/10.1093/bjs/znad378 Original Article with open and endovascular techniques.This study aims to establish accurate costs of delivering endovascular stent TEVAR and OSR in a cohort of UK NHS patients suitable for open and endovascular treatment for whole treatment pathway from admission and to discharge and 12-month follow-up.
Methods
The ETTAA study ETTAA was a large prospective observational cohort study of routine practice which recruited patients across 30 NHS vascular/cardiothoracic units in England.The protocol and funder's report are both published 10,11 with further details of the inclusion criteria but, briefly, consisted of patients ≥18 years of age who attended NHS hospitals in England between March 2014 and July 2018.Patients were included if they had a previously or newly diagnosed aneurysm with a diameter ≥4 cm in the arch or descending thoracic aorta.Exclusion criteria were acute dissection and previous surgical intervention for an aneurysm in the same segment of the aorta.Recruited patients were divided into four groups: watchful waits; conservative management; TEVAR; or OSR.
The micro-costing study
Important differences between the populations undergoing TEVAR and OSR in the ETTAA study raised concerns that any comparisons are biased because of unobserved or inadequately controlled confounding 11 .Therefore, this micro-costing study was based on a subset of the larger ETTAA patient population who, based on recorded study data, had no contraindication to either OSR or TEVAR, ensuring a fair comparison in terms of patient resource use and associated costs.Eligibility to receive either procedure was assessed by clinical experts.Reasons for OSR patients being ineligible for TEVAR included: aneurysm repair extending into the ascending aorta; concomitant cardiac procedures; and unsuitable aortic morphology.Reasons for TEVAR patients being ineligible for OSR included: BMI below 20 or above 35; NYHA IV dyspnoea; or age over 85.Additionally, 10 TEVAR patients had index procedures prior to enrolment in the study; these patients were excluded to maintain equipoise.
A multicentre prospective cost analysis of ETTAA patients undergoing surgical intervention with either OSR or TEVAR for patients with CTAA was performed and reported in accordance to the Consolidated Health Economic Evaluation reporting Standards (CHEERS) statement 12 and STROBE guidelines 13 .The micro-costing approach was adopted from the perspective of the UK NHS in order to improve the precision and accuracy of the cost estimate 14,15 and to undertake the 'direct enumeration and the costing of every resource input consumed in the treatment of a particular patient' 16,17 .The process involves the identification of all resources required for the provision of care; accurate measurement of each resource; and valuation of the resources used.The cost analysis focused on three stages based on chronological sequence of events: index (first) surgical procedure, post-procedure until discharge, and 12-months follow-up.For each patient, and for each stage of the cost analysis, all components of costs stratified by category of resource use were computed by multiplying units of resource use by their unit costs and summed.
Although capital equipment costs are incurred at a single point, it is typical to derive a cost for economic evaluation, whereby the initial cost of a capital asset is converted to annual equivalent sums over their expected lifetime.These annual costs are then discounted to reflect alternative investment or consumption opportunities forgone.In this study, capital equipment was discounted by 3.5 % per year 18 and the sum of these amounts was divided by their expected annual usage to obtain a cost per procedure 19 .
Identification and measurement of resource use
Resource utilization was identified and measured using information derived from expert clinical opinion and data collected in case report forms (CRFs), which were designed with expert guidance from members of the ETTAA collaboration including clinicians, statisticians and health economists.Details of resource use are presented in Tables S1, S2, S3 20 .Resources necessary to undertake the surgical procedures included staff, medical devices, reusable surgical equipment, consumables and overheads.A procedure CRF captured patient-level data on theatre time, type of graft, blood products used and perioperative complications, with other information such as surgical equipment provided by clinical experts based on a 'typical' procedure.
Resources necessary to provide postoperative care until discharge were collected using two CRFs.A post-procedure and discharge CRF captured the number of days in hospital, days in an intensive care unit or a high-dependency unit, postoperative blood product use, the use of any diagnostic investigations and any adverse events (including cardiac and renal failure).If a patient suffered an adverse event requiring a return to theatre, theatre time and reason for return to theatre were captured in a return-to-theatre CRF.These events were micro-costed using the same methods as described previously.
Use of NHS resources during a 12-month post-procedure follow-up including readmissions related to the aneurysm was collected at patient level using a study-specific follow-up CRF at 3, 6 and 12 months post-surgery.This included use of primary and community care and secondary care.If a patient was readmitted to hospital for reasons related to aneurysms, a hospital admission CRF captured length of stay by level of care.If a patient underwent another procedure during follow-up, this was captured using the same CRFs as the index procedure and was recorded as an additional procedure.Following discussion with experts, it was assumed that each patient who underwent TEVAR had a CT scan and a vascular outpatient visit at 1-month post discharge and annually thereafter, with OSR patients assumed to have a CT scan and a cardiology outpatient appointment at 6 months post-discharge and annually thereafter.
Unit costs
Unit costs were obtained in pounds Sterling from a variety of sources including national databases 21 , published studies 22 , stent graft device manufacturers and were inflated to 2018-19 prices using the healthcare and community health services inflation index 22 .Details of unit costs are reported in Tables S4, S5, S6 20 .
Statistical analysis
Baseline characteristics were summarized with cohorts compared using Student's t-test and Pearson's χ 2 test as appropriate.Costs for three stages (index procedure, post-procedure until discharge and follow-up) of the cost analysis were summed over all resource categories to obtain a total annual cost for each patient at 12 months.Costs were summarized and presented as mean (s.d.).Analysis was conducted using Stata v15.1.
Results
A total of 886 patients were recruited to the ETTAA study (Fig. S1).Of these, 601 patients did not have any surgical procedure consisting of watchful waits (n = 489) and conservative management (n = 112).The remaining patients had at least one type of surgical procedure, TEVAR (n = 150) and OSR (n = 135), as reported elsewhere 11 .Overall, 115 TEVAR and 35 OSR patients were judged potentially eligible for both procedures (no contraindications) and were included for primary comparison.These patients were recruited from 30 ETTAA sites, with participant numbers from each site presented in Table S8.Of the TEVAR patients, two required aortic arch endovascular repair, 97 required descending thoracic aortic repair and 16 required complex repair with thoracoabdominal stent with fenestrated or branch grafting.Of the 35 OSR patients, 13 required hybrid grafts (for example, using frozen elephant trunk grafts), 20 patients required standard thoracic repair, and two patients required thoracoabdominal aneurysm repair.A subgroup analysis was also performed highlighting the cost differences between procedures of differing complexity.
Patient characteristics
The patient characteristics of those deemed suitable for open and endovascular repair at baseline are presented in Table 1.
Subgroup analysis
Subgroup analysis was conducted to compare costs of procedures that differed in terms of complexity within TEVAR and OSR (Table 3).Within the TEVAR cohort, those undergoing aortic arch repair or complex repair thoracoabdominal stent with fenestrated or branch grafting had higher costs from admission until discharge £70 231 (£27 406) and £49 768 (£9 120) compared to TEVAR descending thoracic aortic repair £30 675 (£11 920).Both complex procedure types had higher cost estimated across all resource components compared to TEVAR descending thoracic aortic repair with aortic arch repair also being much higher than complex repair thoracoabdominal stent with fenestrated or branch grafting.The largest cost difference for the index procedure was due to the stent graft device cost (aortic arch £31 845 (£4320), complex repair thoracoabdominal stent with fenestrated or branch grafting £29 915 (£4753), descending thoracic aortic repair £19 266 (£8587)).The largest cost differences in the post-procedure until discharge period was due to length of stay, particularly critical care (aortic arch £16 171 (£18 860), complex repair thoracoabdominal stent with fenestrated or branch grafting £6695 (£4809), descending thoracic aortic repair £2929 (£4348)).
Within the OSR group, those with hybrid surgical grafts (n = 13) had a mean (s.d.) procedure cost almost twice as much as those with standard thoracic repair, £24 854 (£8258) and £12 757 (£5678), respectively.The two patients with thoracoabdominal repair had slightly higher procedure costs of £15 394 (£6598) versus standard thoracic repair.Higher procedure costs for hybrid graft patients were driven by the high expense of the surgical graft £13 545 (£3766).Costs until discharge were much lower for the hybrid group £17 199 (£11 259) compared to standard repair £26 191 (£27 351).However, thoracoabdominal repair had very high costs until discharge recorded, £127 421 (£152 054), driven by high critical care and length of stay costs.Overall, hybrid costs were the lowest of the OSR subgroups £42 023 (£14 218), followed by standard thoracic repair £48 151 (£53 541), Due to small numbers, it is only appropriate to compare between descending thoracic aortic repair (TEVAR n = 97) and standard thoracic repair (OSR n = 20).Overall costs are higher in the standard thoracic repair OSR subgroup, with costs driven by critical care and ward days that occur in the post-procedure period.For descending thoracic aortic repair costs were driven by the high stent device costs that occur during the procedure.
Subgroup analysis
Subgroup analysis was conducted to compare costs of follow-up that differed in terms of complexity within TEVAR and OSR (Table 5).Within the TEVAR cohort, those undergoing complex repair thoracoabdominal stent with fenestrated or branch grafting had higher costs from follow-up £5912 (£11 152) compared to TEVAR descending thoracic aortic repair £4 794 (£11 180), with aortic arch endovascular repair having a much lower follow-up cost £684 (£274).Primary care and secondary care costs were similar between the different TEVAR groups.No hospital admissions or re-interventions were recorded for the aortic arch patients.There were five hospital readmissions recorded for those undergoing complex thoracoabdominal TEVAR with a mean (s.d.) cost of £11 112 (£2038) per readmission and 13 hospital readmissions recorded for descending thoracic repair with a mean (s.d.) cost of £4466 (£4267) per admission.Overall, aneurysm-related hospital admissions costs were higher for the complex TEVAR group £5051 (£11 077) versus descending thoracic repair £915 (£2881).Only the descending thoracic repair group had re-interventions recorded with a mean (s.d.) cost of £39 467 (£8527) per reintervention, resulting in a mean (s.d.) cost of £3087 (£10 882) across the cohort.
Within the OSR group, those with hybrid grafts (n = 8) had a much higher 12-month follow-up cost of £12 445 (£19 406) compared to £1455 (£1002) of those who underwent standard thoracic repair (n = 14).This higher cost was driven by re-interventions with the hybrid graft cohort recording four re-interventions with a mean (s.d.) cost of £22 468 (£31 639) per re-intervention, resulting in an mean (s.d.) cost of £11 234 (£19 442) across the hybrid graft group.There were no re-interventions recorded in the standard thoracic repair group.One aneurysm-related hospital admission was recorded in the standard thoracic repair group with a cost of £364, resulting in a mean (s.d.) cost to the group of £23 (£91).Twelve-month follow-up costs were low, £507 (£239) for the thoracoabdominal aneurysm repair (n = 2) subgroup, with only secondary care recorded.Due to small numbers, it is only appropriate to compare subgroups between descending thoracic aortic repair (TEVAR n = 78) and standard thoracic repair (OSR n = 14).For these subgroups, TEVAR descending thoracic repair had a much higher mean 12-month cost compared to standard thoracic repair.Higher mean costs for descending thoracic repair were caused by higher recorded hospital admissions and reinterventions in this subgroup compared to standard thoracic repair.
Discussion
Although costing is undertaken from a UK NHS perspective, the micro-costing methodology provides a detailed insight into the true resource inputs of providing OSR and TEVAR in routine clinical care.The micro-costing methodology defined as the 'direct enumeration and costing of every input consumed in the treatment of a particular patient' 17 involves a number of stages, the first being a detailed insight into the identification of all the resources involved in the provision of care.This study has relevance in international settings as the micro-costing approach used may help inform future accurate renumeration studies as resource inputs are unlikely to differ across different healthcare systems, particularly in terms of the surgical procedure itself.This is important because accurate information regarding the costs of surgical interventions is vital to inform policy and guidance 23 , suggesting these findings are transferable where national prices can be applied.
Estimated mean costs from index procedure up until 12-month follow-up are higher for OSR relative to TEVAR, which is largely driven by differences in costs up until discharge.The procedure cost for TEVAR accounts for over three-quarters of mean total cost until discharge, with these driven by the costs of the endovascular stent graft device used.While the costs of OSR procedures are lower, there are higher costs until discharge, driven by length of stay in critical care.Mean follow-up costs up at 12 months were higher for TEVAR, driven by re-interventions, although these were not enough to outweigh the higher in-hospital costs of OSR.
The NHS England tariff (reimbursement) for TEVAR and OSR procedures does not include the cost of critical care, which is determined on a trust-by-trust basis, or the cost of the endovascular stent graft, which is funded centrally 21 .For a meaningful comparison with NHS tariff, critical care and stent costs were removed from the ETTAA patient cost estimates.The NHS England tariff for TEVAR in 2018-19, the same price year that our cost analysis was conducted, was £7880 for an elective complex procedure and £7272 for an elective simple procedure.TEVAR patients in the ETTAA study had a mean (s.d.) cost of £9370 (£5584), which was higher than the tariff.Furthermore, estimated mean (s.d.) costs for patients undergoing OSR was £24 023 (£25 621), which was also significantly higher than the £15 722 tariff.These costing estimates therefore have significant implications for hospitals, suggesting provision of treatment may not be adequately compensated.Centralization of services means this burden will be on larger specialist units.There are non-financial benefits of providing these treatments and a societal need to provide a regional service, but these results suggest there will be little incentive to continue this work from a financial point of view and calls for reorganization of payments may be welcomed.At the time of the ETTAA study, stent costs were negotiated on a hospital-by-hospital basis with manufacturers, with stent costs included in this study collected directly from manufacturers.However, the NHS moving towards central purchasing 24 of endovascular stent grafts may provide the opportunity to greatly reduce TEVAR procedure costs and make substantial cost savings.The development and introduction of more expensive and complex technology such as more sophisticated arch stent grafts should be carefully considered by health systems, particularly with high costs involved in these groups.
Previous studies conducted in the USA 25,26 estimated costs of TEVAR and OSR for open elective repair of descending thoracic aortas in a single centre using the hospital's accounting system found similar results regarding in-hospital cost drivers for each procedure.Endograft costs were a predictor of TEVAR costs with postoperative complications and length of stay being a predictor of OSR hospitalization costs.There is one other UK-based economic analysis that estimated costs of OSR and TEVAR from a consecutive series of 84 patients undergoing intervention on the descending aorta over a 13-year period using pre-, peri-and postoperative data from a single centre 27 .However, the procedure resources and associated costs were not micro-costed but based on a consensus regarding resource inputs with costs estimated at a 'broad level'.Hospital costs were estimated from NHS reference costs and included staff time, consumables and length of stay.The findings of this study are like ours where OSR Gray et al. | 5 incurred higher costs relating to staff, consumables, transfusion and length of stay.However, they report that stent costs of TEVAR completely outweighed these higher costs with no difference in total costs reported overall (median (i.q.r.) OSR £15 045 (£9299-£27 571) and TEVAR £16 694 (£13 352-£21 729), P = 0.41).These median costs are also much lower than those identified in our study, even accounting for inflation, possibly due to a less-precise costing approach undertaken.Most cost analyses of TAA repairs have only analysed hospital costs during the index hospitalization.Some of these studies have shown TEVAR to be less expensive due to shorter hospital stays with lower complication rates 25,28,29 , while others have shown no difference between open and endovascular repairs 27 .Other studies have reported overall lower hospitalization costs for TEVAR relative to OSR 30 .One study has evaluated costs beyond the initial hospital stay.Karimi et al. 25 evaluated their TAA cost data in a 57-patient, single-centre cohort for 2 years postintervention.They found that in-hospital and at 2 years postintervention, TEVAR was the more cost-effective option.Gillen et al. 30 conducted an in-hospital costing of both procedures and also assessed costs at 3 years utilizing a Monte Carlo simulation model and found that costs were also lower.However, except for Glade et al. 28 , who used data from three vascular centres in Amsterdam, most of these studies estimated in-hospital costs from a single-centre institution.These details of resource utilization and associated costs were estimated based on either a retrospective medical record review 28 or individual organization's financial accounting review 25,29 .No studies estimated costs from a health system perspective, rather than costs incurred by hospitals only.In addition, none of the studies to date adopted a detailed micro-costing exercise and presented details of costing methodologies that should be adopted when undertaking costing studies 31 , particularly in relation to annuitization of costs and discounting.
There are several limitations of this study.Micro-costing provides a more accurate method of resource-use assessment in economic analyses of surgical interventions 23 .Despite the in-depth cost analysis undertaken using a whole National Health Service costing perspective, the sample size of the population for OSR (n = 35) was small relative to TEVAR (n = 115).This was due to the subsample of the ETTAA population being eligible with no recorded contraindication to either procedure in order to ensure a less-biased comparison.It was identified that the majority of patients in the ETTAA study who had an OSR procedure were not eligible for TEVAR, with the reasons reported elsewhere 11 .However, the main focus of micro-costing as a methodology is precision regarding the assessment of the economic costs of a healthcare intervention 15 and identifying key cost drivers.It has been highlighted that micro-costing studies vary widely in methodological and reporting quality, with a need to standardize methods and reporting of these studies and develop tools for their evaluation 32 .However, despite these debates in the health economics literature, sample sizes are not a research priority with a focus on accuracy and transparency.Given the detail and precision of our costing methodology, the cost estimates we present are likely to be representative of costs in routine clinical practice.
As reported previously 11 , it was clear that there were also differences in characteristics between the two surgical groups despite patients being eligible for both procedures.For example, TEVAR patients were older with smaller aneurysm size and more OSR patients had aneurysm extending into the aortic arch.Although the group included in this analysis are a subset of the larger ETTAA cohort study who were eligible for both OSR and TEVAR, there are still differences in anatomy between patients which may impact costs.Direct comparison between cohorts may result in possible bias as the sample sizes did not allow for control for potential confounding in costs.However, many of the resource inputs will be fixed in nature (for example, surgical equipment costs) and may not be strongly influenced by population characteristics or sample size.
The micro-costing methods and results presented in this study are of great value to help guide future research for the cost-effectiveness comparison of TEVAR versus OSR for treatment of distal arch/descending CTAA and are important to providers and decision makers for the purposes of developing guidelines.This study has identified and quantified the extent to which TEVAR procedure costs are driven by the high cost of endovascular stent graft and OSR procedure costs are driven by stay in critical care.Furthermore, this study has identified that NHS tariffs for TEVAR and OSR may be lower than the true cost of TEVAR and OSR procedures for the elective treatment of arch/descending thoracic aortic aneurysms, suggesting providers may not be adequately compensated.
Table 1 Baseline characteristics of patients who underwent endovascular stent grafting (TEVAR) or open surgical repair (OSR) and were eligible for both procedures
Data are presented as mean (s.d.) unless otherwise stated.*P of difference between TEVAR and OSR at baseline.
Table 2 Costs of resource use from index procedure until discharge for patients who underwent endovascular stent grafting (TEVAR) or open surgical repair (OSR)
All costs are reported in pounds Sterling (£) as mean (s.d.) unless otherwise noted.*EndovasculardeviceforTEVAR patients, surgical graft for OSR patients.†Criticalcarebeddays include intensive care unit and high-dependency unit.Gray et al. | 3with thoracoabdominal aneurysm repair having a very large overall cost £142 816 (£158 652).
Table 3 Subgroup analysis costs of resource use from index procedure until discharge
All costs are reported in pounds Sterling (£) as mean ± standard deviation unless otherwise noted.*Endovasculardevice for TEVAR patients surgical graft for OSR patients.†Criticalcare bed days include intensive care unit and high-dependency unit.
Table 4 Costs of NHS resource use from discharge to follow-up at 12 months for patients who underwent endovascular stent grafting (TEVAR) or open surgical repair (OSR)
All costs are reported in pounds Sterling (£) as mean (s.d.) unless otherwise noted.
Table 5 Subgroup analysis of costs of NHS resource use from discharge to follow-up at 12 months
All costs are reported in pounds Sterling (£) as mean (s.d.) unless otherwise noted.TEVAR = endovascular stent graft; OSR = open surgical repair. | 2023-12-16T12:39:21.131Z | 2023-12-13T00:00:00.000 | {
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245816517 | pes2o/s2orc | v3-fos-license | Fish Composition and Diversity of Four Coral Reefs in the South China Sea Based on Hand-Line Catch
: To improve the overall understanding of the fish diversity and spatial patterns of major coral reefs in the South China Sea, fish assemblage composition, dominant species, biodiversity indices, and multivariate analysis of community structure were reported for four major coral reefs based on hand-line survey data in May and September 2018. A total of five orders, 21 families, 45 genera and 121 species of fish were recorded with Perciformes (78.5%) being the most diverse. The highest number (5) of dominant species was found near Chenhang Island while the lowest (2) number was detected at Zhubi Reef. The highest abundance index (7.21) occurred at Zhubi Reef, while the Shannon–Wiener diversity (4.80), Pielou’s evenness (0.81), and Simpson’s dominance (0.95) indexes were all highest at Qiliangyu Island. Based on cluster analysis and non-metric multi-dimensional scaling (NMDS), fish communities varied more spatially than seasonally. Our results led us to hypothesize that the habitat complexity and level of anthropogenic disturbance were the main factors affecting the composition of reef-dwelling fish on each coral reef. Topography was likely responsible for most variation in the spatial pattern of fish diversity.
Introduction
Coral reefs are highly biodiverse and productive environments, and among the most obvious of the ecosystems to be affected by global climate change and anthropogenic disturbance. The conservation of coral reef ecosystems and reef fish diversity is a topical and internationally important issue in the field of marine environmental science [1], with the decreased biodiversity and functional degradation of coral reef habitats attracting considerable attention [2][3][4].
Coral reefs are the most important and distinctive ecosystems in the South China Sea (SCS), and are of great significance in the maintenance of biodiversity in this region and as fishing grounds for coral reef fisheries [5,6]. Various surveys of reef fish in this region have been performed [7][8][9][10], and extensive information regarding coral reef fish biodiversity is widely available [11][12][13][14]. New records of species and the feeding and biology of coral reef fish in the SCS have improved knowledge of species diversity, structure and function [10,12,[15][16][17][18][19].
Climate change and ocean acidification can affect coral reef fish [20][21][22][23]. For example, coral bleaching can lead to diversity loss and changes in the structure of fish assemblages [24][25][26], and loss of coral reef fish biodiversity can affect ecosystem functioning and services [27]. Therefore, research on coral reef fish diversity improves our understanding of reef fish succession and their vulnerability to environmental changes and anthropogenic disturbance [28,29]. In particular, after repeat coral bleaching and long-term anthropogenic disturbance, the composition and diversity of coral reef fish in the SCS needs to be determined in order to conserve remaining coral reefs and their fish resources. Most previous studies on coral reef fish in the South China Sea have focused on the archipelagic scale, with few studies reporting the characteristics of fish diversity on the individual reef level and the differences among reefs [6,9,11,12]. To improve the overall understanding of coral reef fish diversity in the South China Sea, in this study we analyzed the composition, dominant species, biodiversity indices, and multivariate analysis of fish assemblages from four major coral reefs of the Nansha Islands and Xisha Islands in the SCS (Qilianyu and Chenhang Islands and Zhubi and Meiji Reefs), and investigated their spatial distribution and the differences among the coral reefs. In the four coral reefs of our study, there were two previous surveys on the coral reef fish of Meiji Reef, but the investigation efforts and sample sizes were very small, and the data were very poor and there was no diversity information available. For the other three study sites, there were no survey data available on fish biodiversity. Therefore, our research is very meaningful for understanding the diversity of fish in these coral reefs.
Study Site
Reef fish assemblages were surveyed at four coral reefs in the SCS (Qilianyu and Chenhang Islands and Zhubi and Meiji Reefs; Figure 1) in the boreal spring (May) and autumn (September) of 2018. The northeast monsoon prevails from October to March of the following year, and the southwest monsoon prevails from May to September. In Chenhang Island, Zhubi Reef and Meiji Reef, the water ecosystems are being measured, and commercial fishing is strictly prohibited except for scientific fishing, but in Qilianyu Island commercial coral reef fishing is allowed by gillnet, hand-line and diving fishing. The four study sites are not currently included in the MPA.
Fish Surveys
The R/V NanFeng (66.66 m length, 12.40 m width, 1537 t GT), equipped with a motorboat, was used to perform surveys. Fish were collected by hand-line from an outboard Zhubi (10 • 54 N, 114 • 03 E) and Meiji (9 • 55 N, 115 • 32 E) Reefs are both part of the Nansha Islands. Zhubi Reef is a closed and approximately pear-shaped atoll, with a central lagoon approximately 9.5 km 2 in area, mostly 20 m deep, and 24 m at its deepest [30]. Meiji Reef is a semi-closed, oval-shaped atoll with a lagoon of maximum depth 30 m, surrounded by a ring of reef flat of approximately 30.62 km 2 [31]. The straight-line distance between Zhubi and Meiji reefs is nearly 194 km, but both are located in tropical regions with no obvious seasonal fluctuation in weather conditions.
Qilianyu ( Water temperature and salinity were determined at each reef using an YSI ProPlus meter (YSI Inc., Yellow Springs, OH, USA). In spring and autumn, the mean temperatures of Zhubi Reef in the upper 5 m of the water column were 28.69 ± 0.09 • C and 29.08 ± 0.12 • C, respectively, and for Meiji Reef, 27.95 ± 0.10 • C, and 28.50 ± 0.05 • C, respectively. Mean temperatures in the upper 5 m at Qilianyu Island in spring and autumn were 28.88 ± 0.11 • C and 29.28 ± 0.08 • C, respectively, and at Chenhang Island, 28.82 ± 0.08 • C and 29.10 ± 0.07 • C, respectively.
Fish Surveys
The R/V NanFeng (66.66 m length, 12.40 m width, 1537 t GT), equipped with a motorboat, was used to perform surveys. Fish were collected by hand-line from an outboard gasoline-powered motorboat (7.85 m length, 1.50 m width).
Each fish specimen caught was identified to the lowest taxonomic category using morphological characteristics, then frozen (−20 • C) for further shore-based analysis. The wet body mass was measured using electronic scales to the nearest 0.01 g (Sunny Hengping Instrument, Shanghai, China). The total number of catches collected was 2282 individuals.
Diversity Indices
The data were standardized according to the weight and number of the specimens per unit hook by time, and kg/(100 hooks·h) and individuals/(100 hooks·h) were used to represent the standardized catch per unit effort (CPUE) based on the weight and number of specimens. An index of relative importance [32], Margalef's richness index (D) [33], Shannon-Wiener diversity index (H ) [34,35], Simpson's diversity index (C) [36], Pielou's evenness index (J ) [37], and Jaccard similarity coefficient (J s ) [38] were calculated to evaluate the structure of coral reef fish assemblages. Among them, in order not to be influenced by inter-and intra-species individual differences in fish species, and to calculate the results more closely relative to the inter-species energy distribution, a method proposed by Wilhm [34] was used to calculate fish species diversity by replacing the number of individuals with biomass.
The index of relative importance (IRI) was calculated as: where N i is a percentage, the number of the ith species divided by the total number of all individuals of all species; W i is a percentage, the weight of ith species divided by the total weight of all specimens of all species; and F i is a percentage, the number of stations with the ith species divided by the total number of surveyed stations. Species with IRI ≥ 500, 100 ≤ IRI < 500, 10 ≤ IRI < 100, and IRI < 10 are classified as dominant, common, general, and rare, respectively [39]. D, H , C, J , and J s are calculated as follows [34][35][36][37][38]: where S is the total number of species; N is the total number of individuals in the sample; W i is the wet weight of the ith species; W is total wet weight of the species divided by the total wet weight of all specimens of all species; H max is equal to log 2 S; a is the number of species on one reef (nominally "reef A"); b is the number of species on another reef (nominally "reef B"); and c is the number of species common to reefs A and B. When 0 ≤ J s < 0.25, 0.25 ≤ J s < 0.50, 0.50 ≤ J s < 0.75, and 0.75 ≤ J s < 1.00, the composition of species on reefs A and B are considered extremely dissimilar, moderately dissimilar, moderately similar, and very similar, respectively [40].
Multivariable Statistical Analysis of Community Structure
The data were processed and analyzed using ArcGIS 10.3 and R (version 3.6.3). The dominance and diversity indices of fish assemblages were calculated depending on regions and seasons to compare spatio-temporal differences. One-way analysis of similarities (ANOSIM) was used to test the differences in species composition among assemblage structures in different regions, and the significance among the communities was described by cluster analysis. ANOSIM was followed with a SIMPER routine to identify which species is/are the most responsible for the observed spatial or temporal differences. The Bray-Curtis similarity coefficients were calculated using the fourth root-transformed data to construct a similarity matrix among the islands in different seasons. Group average clustering and non-metric multidimensional scaling (NMDS) were used to analyze the heterogeneity of fish composition between seasons in the four regions [41]. Because of the complementary nature of these two methods, they could be used together to validate each other's analytical results and to elucidate community patterns more effectively [42]. The above multivariate analysis was conducted in PRIMER 5.0 software. The strength of the NMDS analysis was measured by the stress coefficient (stress), where coefficients < 0.2, 0.1 and 0.05 indicated acceptable representation, good ranking and good representation of ranking, respectively [43].
Species Composition
On the four coral reefs, 121 fish species were caught and identified, attributed to five orders, 21 families, and 45 genera (Appendix A). The most species occurred at Qiliangyu Island, where 60 species belonging to four orders and 14 families were recorded; Meiji Reef included 53 species belonging to 14 families and four orders, and Zhubi Reef encompassed 49 species from 15 families and five orders; 38 species from 21 families and five orders were registered near Chenhang Island. There were seasonal differences in fish composition between regions. In Meiji Reef, the number of species, genera and families was lower in May than in September. In Zhubi Reef, the number in May was equal to that in September. In Qilianyu and Chenhang Islands, the numbers in May were both higher than in September (Table 1). Perciformes dominated, with 95 species in 16 families accounting for 78.5% of all species, followed by Beryciformes (13 species), Tetraodontiformes (7 species), Aulopiformes (3 species), and Clupeiformes (2 species
Dominance Degree
For all four coral reefs, the seasonal differences in the composition of dominant species of fish assemblages were significant ( Table 2). Reef fish with IRI ≥100 are presented in Table 3.
At Meiji Reef, four species were dominant, 11 were common, 19 were general and 19 were rare. Catches of dominant, common, general, and rare species accounted for 46%,
Dominance Degree
For all four coral reefs, the seasonal differences in the composition of dominant species of fish assemblages were significant (Table 2). Reef fish with IRI ≥ 100 are presented in Table 3. At Meiji Reef, four species were dominant, 11 were common, 19 were general and 19 were rare. Catches of dominant, common, general, and rare species accounted for 46%, 36%, 14%, and 4% of the total catch, respectively. There were four dominant species in both May and September, and two species among them were recurrent.
At Zhubi Reef, two species were dominant, seven were common, 23 were general and 18 were rare. Catches of dominant, common, general, and rare species accounted for 58%, 22%, 16%, and 4% of the total catch, respectively. There were seven dominant species in both May and September, and four species among them were recurrent.
At Qilianyu Island, four species were dominant, 18 were common, 34 were general and four were rare. Catch of the dominant, common, general, and rare species accounted for 35.1%, 48.5%, 16.1% and 0.3% of the total catch, respectively. There were eight dominant species in both May and September, and two species among them were recurrent.
At Chenhang Island, five species were dominant, seven were common, 12 were general and 14 were rare. Catches of dominant, common, general, and rare species accounted for 77%, 15%, 6% and 2% of the total catch, respectively. There were four dominant species in May, six dominant species in September, and four species among both were recurrent.
Diversity Indices
In terms of season (Table 4), the mean D and H of the four coral reefs were higher in May than September for the four reefs, but the mean J of the four coral reefs was lower in May than September. In terms of spatial distribution (Table 5), D values among reefs ranged 3.915-7.064, with that for Zhubi Reef being highest, and that for Chenhang Island lowest; H values ranged 3.158-4.801, with that for Qiliangyu Island being highest and that for Chenhang Island lowest; J values ranged 0.580-0.813, with that for Qiliangyu Island being highest and that for Zhubi Reef being lowest; and C values ranged 0.729-0.946, with that for Qiliangyu Island being highest and that for Zhubi Reef being lowest. J s values ranged 0.172-0.273, with that between Chenhang and Qiliangyu islands being the greatest, and that between Qiliangyu Island and Zhubi Reef being the least ( Table 6).
Community Patterns
The results of the cluster analysis among the different seasons and coral reefs showed that the fish could be divided into three communities: Community I, Community II and Community III (Figure 3). Moreover, differences among seasons and coral reefs were also visualized through non-metric multi-dimensional scaling (NMDS) ordination (Figure 4). The overall stress coefficient was 0.14, and the stress coefficient of sorting result was less than 0.2, which indicated that the graphs had interpretative meaning. The ANOSIM test showed that the difference between communities was significant (R 2 = 0.729, p < 0.05), indicating that community division was feasible.
Species Composition
Species richness is the most direct and fundamental expression of the degree of species diversity [44]. We reported the total number of fish species on four coral reefs in the
Species Composition
Species richness is the most direct and fundamental expression of the degree of species diversity [44]. We reported the total number of fish species on four coral reefs in the SCS to be much lower than that previously recorded for these areas [15,16,45,46], but higher than the number of species in the Weizhou Island Coral Reef Sea [47], possibly because of differences in the timing, duration, frequency, and season of sampling. Qilianyu Island had the most species, and Chenhang Island the least, with the number of species at Meiji and Zhubi reefs being similar. The species contribution was calculated by combining the ANOSIM and SIMPER programs, and species contributing more than 10% were identified as being most responsible for the observed spatial or temporal differences. Epinephelus merra (11.41%) was the main contributor at Meiji Reef; four species of Parupeneus trifasciatus (18.94%), Cheilinus fasciatus (18.19%), Parapercis hexophtalma (14.83%) and Cephalopholis urodeta (13.54%) were the main contributors at Zhubi Reef; two species of Lethrinus rubrioperculatus (11.70%) and Gnathodentex aureolineatus (11.55%) were the main contributors at Qilianyu Island; and four species of Pentapodus caninus (11.57%), Cephalopholis spiloparaea (10.67%), Gnathodentex aureolineatus (10.51%) and Parupeneus trifasciatuswere (10.39%) the main contributors at Chenhang Island.
Species Composition
Species richness is the most direct and fundamental expression of the degree of species diversity [44]. We reported the total number of fish species on four coral reefs in the SCS to be much lower than that previously recorded for these areas [15,16,45,46], but higher than the number of species in the Weizhou Island Coral Reef Sea [47], possibly because of differences in the timing, duration, frequency, and season of sampling. Qilianyu Island had the most species, and Chenhang Island the least, with the number of species at Meiji and Zhubi reefs being similar.
Differences in the numbers of fish species at different coral reefs may be related to reef size, habitat complexity, reef status, and different levels of anthropogenic disturbance [48][49][50]. Sandin et al. [51] found classic positive relationships between reef fish abundance and habitat area, and others have shown that increased isolation from terrestrial disturbance leads to increased biomass and abundance [52][53][54][55][56]. Meiji Reef is a semi-enclosed lagoon, with water exchange within and outside the lagoon. Zhubi Reef is a closed atoll, which, together with the reef flat barrier, prevents seawater exchange between the lagoon and the sea. Qilianyu Island comprises a series of smaller open islands. Chenhang Island is an atoll, on the same reef as Guangjin Island, with a lagoon within the atoll, with the northern part of the atoll connected to the outer sea. Differences in habitat structure among coral reefs and human activities (including pollution and overfishing) strongly influence the abundance and distribution of fish [57][58][59][60][61]. Our results led us to hypothesize that the habitat complexity and level of anthropogenic disturbance were the main factors affecting the composition of reef-dwelling fish on each coral reef. At the same time, compared with Zhubi Reef and Meiji Reef, there was obvious seasonality in the fish compositions at Qiliangyu Island and Chenhang Island, which also belong to the Xisha Islands. Qiliangyu Island and Chenhang Island are located in the northwestern part of the South China Sea, and are influenced by southwesterly and northeasterly winds and have obvious seasonal climate changes, while the sea temperature changes are jointly influenced by the ENSO and East Asian monsoon (EAM), so the fish composition showed obvious seasonal differences [62,63].
Theory suggests that the community in an undisturbed habitat will often include morphologically distinct species belonging to different phyla, while a heavily disturbed habitat has communities often comprising a few closely related species [64]. The influence of animal bait (i.e., shrimp) may bias sampling toward carnivorous fish and exclude herbivorous or omnivorous species, which can also affect fish composition in biodiversity surveys [65]. The fish species of southeastern Brazil and the Brazilian coast are dominated by the order Perciformes [66,67]. Fish in our study were also dominated by Perciformes, with the proportion on each reef being relatively high. This may be an important feature of coral reef fish community composition in the SCS. However, to determine whether the high proportion of Perciformes on the reef is a natural feature or because the habitat or sampling deviation has been disturbed would need further exploration.
Dominant Species
There were obvious differences in the dominant species of each island reef in different seasons, especially in Qilianyu Island. Dominant species occupy an important position in the ecosystem, and any changes in them can affect community structure, status and energy flow, and stability [68,69]. We reported that the dominant species on Meiji and Zhubi reefs belonged to the families Serranidae, Lutjanidae, Pentapodidae, Lethrinidae, Mullidae, and Scaridae, which is generally consistent with the types of dominant taxa found in coral reef habitats around the Nansha Islands [70]. However, the number of dominant species on Meiji Reef was lower than reported by [71], possibly because of differences in methods, duration and survey effort; Li et al. [15] did not report representatives of either the Labridae (which play an important role in maintaining reef stability) or Pomacentridae (which indicate living coral cover) on Zhubi Reef. Being a closed atoll, Zhubi Reef is more susceptible to deterioration, decline of coral cover, and habitat destruction.
Dominant species at Qiliangyu and Chenhang islands belonged to the Pentapodidae, Lethrinidae, Mullidae, and Serranidae. Based on a gillnet survey, Sun et al. [72] reported more coral reef fish at the Xisha Islands in families such as the Chaetodontidae, Labridae, Scaridae, and Acanthuridae than we did. Compared with Sun et al. [72], we reported an obvious difference in dominant taxa, especially in the Chaetodontidae, probably because of differences in sampling methods. The smaller number of advantageous categories and higher IRI at Chenhang Island affects the survival of less competitive fish, and reduces the ability of the fish community to cope with external threats, to the detriment of community stability [73,74]. Recent studies have shown that coral reefs in the Xisha Islands have deteriorated, with reduced habitat and food resources for reef fish, and with reef fish densities generally declining because of anthropogenic disturbance [15,75]. The loss of coral cover was very serious in the South China Sea. According to studies, live coral cover declined from an average of approximately 65% to approximately 20% during 1998-2007 in the major offshore atolls of the Nansha Islands and Xisha Islands [76]. In the Xisha Islands, the coral cover in 2016 was approximately 5.44% [77]. The carrying capacity of the Xisha Islands ecosystem may have been reduced because of environmental change and anthropogenic disturbance, and fish assemblages may have responded to habitat change by altering community structure, especially of dominant species.
Coral Reef Fish Diversity
Species diversity is influenced by a number of factors, and is necessary for maintaining ecosystem stability [78][79][80][81]. Fish diversity varies among regions and years [29]. We reported significant differences in fish diversity among coral reefs in the SCS, which may be a function of spatial differences among reef habitats and their fish assemblages in this region. Values of H exceeding 3 usually occur only in healthy ecosystems with high biodiversity [82]. We reported H values ranging 3.16-4.80, which was indicative of extremely high coral reef biodiversity in the SCS.
The highest values of species richness, H , J and C, occurred at Qilianyu Island, and D was second only to that at Zhubi Reef, on which basis we inferred that this environment was highly heterogeneous with many ecological niches, resulting in higher species diversity. Species richness and evenness in distribution influence biodiversity indices: the higher the evenness and richness, the higher the biodiversity [37,83]. Additionally, according to "island effect" theory, habitat area is extremely important for determining the diversity of coral reef fish [84]. As an open coral reef comprising a series of islets, Qilianyu Island has more habitat than the other enclosed or semi-enclosed atolls, and can accommodate more species. Fish assemblages at Zhubi Reef had the highest D, but H , J and C were lower. As a closed atoll, Zhubi Reef is vulnerable to environmental change and anthropogenic disturbance [16], its ecosystem is fragile, and it warrants protection. Zhang [71] and Chen et al. [39] reported fish assemblage H values at Meiji Reef of 3.58 and 0.92, while we reported a higher value of 4.37. It has been suggested that a proportion of both non-seasonal and seasonal resident fish occurs at Meiji Reef, and H values are also associated with a large number of seasonal and incidental species [29,71]. Further investigation is necessary to determine whether the higher H values of fish assemblages at Meiji Reef were because of seasonal effects. Fish assemblages at Chenhang Island had low D, H , J and C values, and the lowest species richness among reefs. A recent tendency for some Xisha Island coral reefs to eutrophicate because of anthropogenic disturbance may affect fish diversity [18,85,86]. Differences in H values across islands suggested that topographic structure might be one of the main causes of differences in spatial distribution.
The recent concept of "ecological memory" maintains that coral adaptation may be difficult because of the cumulative thermal stresses of long-term warming [61]. Longterm seawater warming has not promoted thermal adaptation of corals in the SCS, and future coral growth will be less suited to warmer conditions [87]. The cumulative thermal stress of long-term warming and regional environmental stresses caused by anthropogenic disturbance (increased terrestrial sedimentation, deteriorating water quality, etc.) may weaken coral thermal adaptation, reduce resistance and resilience, exacerbate declines in growth, and contribute to an overall decline in coral growth in the SCS. This inevitably weakens the functions of coral reef ecosystems that provide food, habitat, and breeding grounds for fish, and reduces the diversity of coral reef fish. Concurrently, changes in evenness appear to drive differences in species richness on different reefs-that is, the effects of anthropogenic disturbance may be reversible over time but may also simultaneously accumulate and significantly alter diversity at various spatial scales [88].
Community Structure
Fish species composition showed some spatial and temporal heterogeneity, which is very closely related to complex physicochemical factors and seafloor geomorphology [89]. The community structure is directly related to the ecological function of the habitat [90]. Community I was in the Zhubi Reef, which is a typical closed atoll in the northern part of the Nansha Islands in the South China Sea, with no connection to the outer sea except limited water exchange between the lagoon and the outer ocean at high tide [91]. The special geographical, topographical and hydrological environment brought a unique community pattern. Community II was in the Meiji Reef and Chenhang Island. Although they were relatively far apart and differed in fish composition, there were many common dominant species. Although fish aggregation patterns can define the composition of aggregations in different areas, changes are gradual and there are no clear aggregation boundaries, with most fish occurring in two or more communities simultaneously [92]. Community III was at Qilianyu Island, which consists mainly of a series of smaller open reefs and is distinctly different from Zhubi Reef. Among the four coral reefs, the habitat of Qilianyu is relatively more favorable. Therefore, the species richness of coral reef fish in the Qilianyu was also relatively high. In the study, for the four coral reefs, the variation among fish communities by space was greater than that by season, based on cluster analysis. The four coral reefs are all located in tropical waters, and their natural environment, such as water temperature, monsoon, salinity, has relatively small seasonal differences, but the differences among coral reefs in the complexity, depth and area are relatively greater. Therefore, the influence of geomorphology on fish communities was greater than that of season.
Conclusions
We reported fish composition, dominant species, biodiversity indices, and assemblage structures at four coral reefs in the Nansha Islands (Meji Reef and Zhubi Reef) and Xisha Islands (Chenhang Island and Qilianyu Island) in the South China Sea based on hand-line survey data in May and September 2018. Of the four reefs, a total of five orders, 21 families, 45 genera and 121 species of fish were recorded with Perciformes (78.5%) being the most diverse. The highest number (5) of dominant species was found near Chenhang Island and the lowest (2) number was detected on Zhubi Reef. The highest abundance index (7.21) occurred at Zhubi Reef, while the Shannon-Wiener diversity (4.80), Pielou's evenness (0.81), and Simpson's dominance (0.95) indexes were all highest at Qiliangyu Island. Based on cluster analysis and NMDS, the spatial distribution of fish assemblages among the reefs in different seasons could be divided into three communities. The ANOSIM test showed that the differences in fish composition among different assemblages were significant (R 2 = 0.729, p < 0.05). The variation in assemblage structures by space was greater than that by season. Our results led us to hypothesize that the habitat complexity and level of anthropogenic disturbance were the main factors affecting the composition of reef-dwelling fish on each coral reef. Topography was likely responsible for most variation in the spatial pattern of fish diversity.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author.
Acknowledgments:
We are grateful to Captain Wenming Yu and the entire crew of the Nanfeng for their participation in the sampling. We thank Yan'e Jiang, Yuyan Gong and Yutao Yang for their collaboration on the experiments.
Conflicts of Interest:
The authors declare no conflict of interest. | 2022-01-09T16:17:25.619Z | 2021-12-31T00:00:00.000 | {
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10968808 | pes2o/s2orc | v3-fos-license | Entrainment of noise-induced and limit cycle oscillators under weak noise
Theoretical models that describe oscillations in biological systems are often either a limit cycle oscillator, where the deterministic nonlinear dynamics gives sustained periodic oscillations, or a noise-induced oscillator, where a fixed point is linearly stable with complex eigenvalues and addition of noise gives oscillations around the fixed point with fluctuating amplitude. We investigate how each class of model behaves under the external periodic forcing, taking the well-studied van der Pol equation as an example. We find that, when the forcing is additive, the noise-induced oscillator can show only one-to-one entrainment to the external frequency, in contrast to the limit cycle oscillator which is known to entrain to any ratio. When the external forcing is multiplicative, on the other hand, the noise-induced oscillator can show entrainment to a few ratios other than one-to-one, while the limit cycle oscillator shows entrain to any ratio. The noise blurs the entrainment in general, but clear entrainment regions for limit cycles can be identified as long as the noise is not too strong.
Biological systems present us with a wide range of oscillators, which include cell cycles, circadian rhythms, calcium oscillations, pace maker cells, and protein responses, but it is often a challenging task to identify the minimal models behind these oscillations. The proposed models are typically categorized into two classes: (i) Limit cycle oscillator, where fixed points are linearly unstable and the oscillations are described by stable limit cycles sustained by nonlinearities of the system which are deterministic. Noise can be added on the top of the deterministic oscillations. (ii) Noise-induced oscillator, where the fixed point is linearly stable for the system without noise and the system relaxes to the fixed point with damped oscillations when temporally perturbed. Addition of noise to this type of system is known to show sustained oscillations with fluctuating amplitudes. We propose a way to distinguish the two, by using the phenomenon of entrainment to a periodic perturbation. Taking the van der Pol equation with noise as an example, we show that entrainments to all the rational ratios are seen only in the limit cycle oscillator. In the case of the noise-induced oscillator with additive external forcing, the oscillator can entrain only at one-to-one ratio, meaning that the entrainment to other than the one-to-one ratio is the sign of the dominance of the limit cycle mechanism. When the external forcing is multiplicative, we find that the noise-induced oscillator with weak nonlinearity can show some entrainment ratios other than one-to-one, but not all the ratios.
I. INTRODUCTION
Biological systems present us with a bewildering fauna of oscillators: cell cycles 1 , circadian rhythms 2-5 , calcium oscillations 6 , pace maker cells 7 , protein responses [8][9][10][11][12][13][14] , and so on. Sometimes, however, it is hard to see what are the minimal models behind these oscillations. Typically, the models are categorized into two classes: (i) Limit cycle oscillator: The fixed point is linearly unstable and the oscillations are described by stable limit cycles sustained by nonlinearity of the system in the deterministic case 11,12 . Noise (e.g. molecular noise due to limited number of copy numbers) can be added on the top of the deterministic oscillations. (ii) Noise-induced oscillator: The fixed point is linearly stable for the system without noise and the system relaxes to the fixed point with damped oscillations when temporally perturbed. Addition of noise to such a system is known to show sustained oscillations with fluctuating amplitude 13,14 . For some systems, both limit cycle oscillators (i) and noise-induced oscillators (ii) are proposed as a mechanism for the oscillation [11][12][13] .
Here, we propose a way to distinguish the two, by using the phenomenon of entrainment to a periodic perturbation.
It is well known that, when an periodic perturbation is added to a deterministic limit cycle, the system's oscillation frequency ω will be entrained to the external frequency Ω with various rational numbers of frequencies ω/Ω = P/Q for all positive integers P and Q in a finite window of the external frequency Ω, where the width of the window depends on the amplitude of the external forcing 15,16 . Entrainment, also called mode-locking, has been observed in variety of physical systems during the last decades, from onset of turbulence 17 , Josephson junctions 18,19 , one-dimensional conductors 20 , semiconductors 21,22 and crystals 23 .
It has been predicted, and verified experimentally, that the mode-locking structure possesses certain universal properties 15,16 . In biological systems, entrainment has been investigated theoretically for circadian rhythms 4,5 as well as in model systems for protein responses 25 .
Experimental observation of entrainment in biological systems is often rather difficult due to noisy signals, but it has been observed for circadian rhythms 2,3 and synthetic genetic oscillators 24 .
In this paper, we study the difference in the entrainment behavior for the limit cycle oscillators and noise-induced oscillators.
Our main question is the following: Can we distinguish the two cases by means of the entrainment behavior? We employ the famous van der Pol equation with noise as an example, because there we can easily study both cases by changing parameters. We show that entrainments to all the rational ratios are seen only in the limit cycle oscillator. In the case of the noise-induced oscillator with additive external forcing, the oscillator can entrain only at one-to-one ratio, meaning that the entrainment to other than the one-to-one ratio is the sign of the dominance of the limit cycle mechanism.
When the external forcing is multiplicative, we find that the noise-induced oscillator with weak nonlinearity can show some entrainment ratios other than one-to-one, but not all the ratios.
To confirm the generality of the entrainment behavior for the limit cycle system under weak noise, we also study a biological example, the TNF-driven oscillating NF-κB system, and confirm that P/Q entrainments can be seen.
A. van der Pol equation
Consider the following two-dimensional equation with noise: Here, d, σ, and B are parameters, and Γ i (t) are uncorrelated, statistically independent Gaussian white noise, satisfying First let us consider the deterministic case, σ = 0. The model has a fixed point at (x 1 , x 2 ) = (0, 0), and the eigenvalues around this fixed point are indicating that the system experiences a Hopf bifurcation at d = 0. When d < 0, the fixed point relaxes to the fixed point with damped oscillation with the angular frequency ω (d) = |d 2 − 4|/2, while when d > 0 and B > 0 the model shows a stable limit cycle (van der Pol oscillator).
In the stochastic case with σ > 0, however, the system shows a sustained oscillation even in the linearly stable case, d < 0, because the noise keeps activating the oscillation with frequency ω . This is the case of the linear p53 model introduced in Ref. 13 . When d > 0, σ > 0 adds fluctuations on top of the stable oscillation around the limit cycle.
B. Setup
We investigate the entrainment behavior of the model, focusing on the following three classes of parameter sets.
2. For the noise-induced oscillator, we consider the two subcategories.
(a) The linear system with a stable fixed point, with d < 0 and B = 0, i.e., the equations are linear in x and the fixed point is stable.
(b) The nonlinear system with a stable fixed point, with d < 0 and B > 0, i.e., the fixed point is linearly stable but the equations possess a nonlinear term.
When noise-induced oscillators are studied, normally only linear terms are considered. However, in reality, there are often nonlinear terms, which can play a role when distance from the stable fixed point |x| is sufficiently large. This is the reason why we consider both linear and nonlinear noise-induced oscillators.
When needed, numerical integration of stochastic differential equations are performed by using Euler method. Figure 1 shows the typical behavior of the model in each categories. The parameters are chosen so that the period and amplitude are in similar range. Without noise, the limit cycle is the only case with stable oscillation (Fig. 1a), while linear and nonlinear systems with a stable fixed point exhibit damped oscillations relaxing to the fixed point (Fig. 1bc).
When noise is added, the oscillation is perturbed for limit cycle oscillator (Fig. 1d); here the noise level is chosen so that the base oscillation is still recognizable. For linear and nonlinear noise-induced oscillators (Fig. 1ef), we observe oscillations with the expected angular frequency (ω (−0.1) ≈ 1). In order to demonstrate the difference between the two, we apply the exact same sequence of noises in both cases. We observe a bigger difference when linear noise-induced oscillator have large (|x| ≈ 1) amplitude, because the nonlinear term becomes more important. Naturally this effect depends on the value of B (data not shown).
We study these oscillators under the following two kinds of external periodic perturbation.
a. Additive forcing. The first case is an additive forcing, in the form oḟ with b. Multiplicative forcing. The second case is an multiplicative forcing (also called parametric forcing), in the form ofẋ where In the next section, we first present the behavior of the model under the additive forcing, and then show the parallel results for the multiplicative forcing.
III. RESULTS
A. Additive forcing
Linear case
In the case of the additive periodic forcing to a linear deterministic system, we have in where L is a coefficient matrix of the linearized equation, and A(t) is periodic function in time with a period T , satisfying A(t + T ) = A(t).
By expressing x(t) = d j=1 C j (t)u j , with using eigenvectors u j of the matrix L given by Lu j = λ j u j , we can show that in the long-time limit we have Note that (λ j ) < 0 because the fixed point x = 0 is stable. F n is defined by the Fourier where v t j is the left eigenvector. Therefore the solution will always be a periodic function of t with the period T in the long time limit, and contains only the frequencies that the external forcing has. In other words, the system will be always in a 1/1 entrained state if the perturbation is pure sine or cosine wave.
When Gaussian white noise is added to eq. (12), we havė In this case, we can evaluate the auto-correlation of C j (t) for large enough t 0 Namely, the response contains oscillations with the frequencies from forcing 2π/T and from the complex part of the eigenvalue λ j , and the amplitude of the latter is proportional to σ.
Numerical results
We now investigate numerically the entrainment behaviors for all three categories. First we demonstrate the behavior without noise, and then show how the noise modify this behavior.
a. Without noise. Figure 2 illustrates typical entrainment behaviors for additive forcing when noise is absent. With a limit cycle oscillator ( Fig. 2 a, d), the system's angular frequency can entrain to the external angular Ω with various ratios, while in the linear system, one-to-one entrainment occurs ( Fig. 2 b, e). The nonlinear system shows very similar behavior to the linear system, where we see only one-to-one entrainment ( Fig. 2 c, f).
In order to define the system's angular frequency in a simple way, we adopt the polar coordinate (r, θ) using as proposed in Ref. 26 . We define θ(t) so that (θ(t) − θ(0))/2π gives the winding number, i.e., how many times the orbit went around the fixed point by time t. The system's angular frequency is numerically calculated from for long enough T (typically 1000 times external forcing period). With this definition, Fig. 2(a) shows the entrainment of the ratio ω/Ω = 2/1, while Fig. 2(d) gives ω/Ω = 1/2.
b. With noise. The addition of noise blurs the entrainment behavior, as depicted in Fig. 3. For the limit cycle oscillator ( Fig. 3a and d), we can see that the noise makes the orbit irregular, which can make the phase to slip. In the linear noise-induced oscillator for small external angular frequency, we can clearly see that the noise induces the oscillation with angular frequency close to ω on top of one-to-one entrainment behavior (Fig. 3b), as expected from the auto-correlation eq. (13). When Ω is larger than ω , the external angular frequency is more visible, because the noise σ is small compared to the amplitude A for this case, though both frequencies should be present. The nonlinear noise-induced oscillator behaves again very similar to the linear case in entrainment behavior ( Fig. 3c and f). The visible difference is a suppression of large amplitude by the nonlinear term.
c. "Devil's staircase" and "Arnold's tongues". For deterministic limit cycles, the plot of ω/Ω vs Ω for a fixed amplitude of external forcing shows a infinitely complex structure with fractal nature, known as Devil's staircase 15,16,26 . For the present system of limit cycle oscillator without noise, this is also observed as shown in Fig. 4(a) (solid line). As noise increases, the phase slips occasionally, therefore narrow entrainment regions become harder to recognize (Fig. 4a, dashed and dotted line). For the systems with a stable fixed point, there is only one-to-one entrainment for the no noise case (Fig. 4b, solid line), while noise induced oscillation around the entrained solution will add some phase slips giving a change in the angular frequency when the entrainment is not so strong, resulting in an escape from the one-to-one ratio as shown in Fig. 4(b).
When entrainment regions for various values of ω/Ω are plotted in the A-Ω plain, it gives an "Arnold's tongue" structure for the deterministic limit cycles: The entrainment regions widen as the external forcing amplitude A grows, resulting in tongue-like shapes of the entrainment region -when A is large enough the tongues start to overlap 15,16 . This can be seen in the limit cycle oscillator without noise in Fig. 5 (a). When noise is added, the phase of the oscillator sometimes slips, resulting in narrower tongues (Fig. 5 b). For the noiseinduced oscillators (i.e. with a stable fixed point), there exists only 1/1 entrainment without noise, and with noise 1/1 entrainment is the only case that gives the tongue-like structure, both for the linear and nonlinear cases (Fig. 5 cd). We see other ratios of entrainment "regions", because for a given A with changing Ω, ω/Ω changes continuously outside of the entrainment region (e.g. Fig.4 b).
B. Multiplicative forcing
Linear case without noise
We next consider the multiplicative forcinġ where the matrix M(t) satisfies with T = 2π/Ω. It is known from Floquet theory 27 that the solution matrix of this equation is expressed as where and a general solution is the linear combinations of column vectors consisting of Q(t). The eigenvalues of the matrix Λ, called Floquet exponents, determine the stability of the solution: The solution will converge to the fixed point when the real parts of the Floquet exponents are all negative, and diverges if some Floquet exponent have positive real parts. Therefore, no entrainment behavior will be observed for a linear noise-induced oscillator without noise under multiplicative forcing.
In Fig.6, we show numerically calculated the maximum real part of the Floquet exponents λ R for (17) with (9) with d = −0.1 and B = 0, as a function of amplitude of forcing M and external frequency Ω. When λ R < 0 (dark blue region), |x| will exponentially decays to zero, otherwise |x| will diverge except for the marginal case λ R = 0.
Numerical results
a. Without noise. Figure 7 shows the entrainment behaviors for multiplicative forcing.
For the limit cycle oscillator (Fig. 7 ac), there is no qualitative difference from the additive noise case, i.e., the system shows entrainment with various frequency ratio ω/Ω = P/Q. For the linear system with a stable fixed point without noise, on the other hand, the system can either decay to the fixed point (Fig. 7 b) or diverge (Fig. 7 d), which can be predicted from the Flouquet exponents (Fig.6). When nonlinear term is added, it does not prevent the decay (Fig. 7 c), but the divergent behavior is suppressed and system shows the entrainment behavior (Fig. 7 f). The frequency ratio ω/Ω is not necessarily 1/1; the example in Fig. 7(f) gives ω/Ω = 3/2.
b. With noise. When noise is added, the behavior changes drastically in the noiseinduced oscillators, as shown in. Fig. 8. The noise can induce the oscillation with the angular frequency close to ω for the case where the no-noise system would decay to the fixed point (Fig. 8 b, c). On the other hand, in the linear noise-induced oscillator, adding noise does not prevent divergence (Fig. 8 e). For the parameters where no-noise system would entrain, the noise blurs the entrainment due to occasional phase slip for both limit cycle oscillator (Fig. 8 ac) and nonlinear noise-induced oscillator (Fig. 8 f).
c. "Devil's staircase" and "Arnold's tongue". We also study the "Devil's staircase" for the multiplicative forcing. For the limit cycle oscillator without noise, we again see proper devil's staircase, where noise will blur the entrainment behaviors (Fig.9 a). For the noise-induced oscillators, only the nonlinear case is studied because the linear case may diverge depending on the parameter values. Without noise, we see discrete finite regions of entrainment ( Fig.9 b squares), while noise induces the oscillations in the decaying region resulting in a continuous line ( Fig.9 b dashed line).
The Arnold's tongue structure for the limit cycle is similar to those in the additive forcing case, as seen in Fig.10(a) and (b). The Arnold's tongues for all the entrainment ratios are observed without noise, and noise makes the regions smaller. For the nonlinear system with a stable fixed point without noise, there are entrainment regions for a few rational ratios, but the ones that appear are problem specific -for instance, in the present case, the ω/Ω = 1/3 is not observed at all in Fig.10(c). With noise (Fig.10d), the entrainment regions shrinks, but at the same time the system can occasionally pass the given ratio of ω/Ω, resulting in narrow line of "fake" entrainment.
IV. BIOLOGICAL EXAMPLE: ENTRAINMENT OF TNF-DRIVEN NF-κB SYSTEM
In this section, we study a biological example, TNF-driven NF-κB system, to confirm the generality of the entrainment behavior for the limit cycle system under weak noise.
The system has been studied for the deterministic case in ref. 25 . NF-κB is a transcription factor, and it has been verified experimentally that NF-κB level in the nucleus shows sharp oscillations after treatment with tumor-necrosis factor (TNF) 33,34 . The interaction network involves a negative feedback loop between the NF-κB and an inhibitor, IkBα, which is the main mechanism for the oscillations. TNF modulates the state of the IkBα and hence affects the oscillation. TNF can be added externally to the cell, therefore it can serve as a possible probe to study the entrainment, i.e., we can use TNF level as the external forcing term. In ref. 25 , the system was modeled by 5 dimensional coupled nonlinear ODEs, The variable x 1 denotes the nuclear NF-κB level, and [T N F ] denotes the TNF level, which we shall change to a periodically external forcing onto the system. The biological meaning of the variables and parameter values are summarized in Table I. Note that [T N F ] appears twice in eq. (25) in the terms multiplied with x, therefore this is an example of multiplicative forcing. We study this system with adding a Gaussian white noise in each term, i.e., This has been studied in the no-noise case by Jensen and Krishna 25 , and it was found that the entrainments of various ratios can occur, when the frequency of the NF-κB level is determined based on the frequency of the peaks. Figure 11(c) shows an example of 1/2 entrainment, for M T N F = 0.05 and Ω = 0.0297, in the deterministic case. With weak enough noise, the entrainment is maintained (Fig. 11e), but larger noise induces phase slips ( Fig. 11f), as has been seen in the Van der Pol system.
In Fig. 12, several Devil's staircases are shown with and without noise. In Ref. 25 the Arnold tongues have been calculated, and it has been demonstrated that general P/Q entrainments occur. A characteristic observation to this system is that the tongues overlap easier for larger external frequency; e.g., M T N F ≈ 0.04 for 1/3 and 1/2 tongues to overlap, while the 2/1 and 5/2 tongues do not overlap even at M T N F = 0.1. When the Devil's staircase are calculated for overlapping region, non-smooth or irregular jumps between the steps can be seen, and is thus dependent on initial conditions in general. This is visible in our data in Fig.12, for large M T N F and larger Ω. When weak noise is added, it enables the system to jump to other overlapping tongues, which results in irregular behavior around the entrainment regions. As the noise becomes larger, the entrainment is again smoothed away by phase slips.
V. SUMMARY AND DISCUSSION
Our motivation behind this work was to ask: Can one by applying an external periodic forcing and studying entrainment determine whether an oscillating system is driven by a linear mechanism (noise induced oscillator) or a non-linear mechanism (limit cycle oscillator)? Our answer to this question is generally yes. Our obtained results on entrainment behavior of oscillators are summarized in Table II.
When the forcing is additive, there is clear difference between the limit cycle oscillators and the noise-induced oscillators. The former can entrain to any frequency ratio, while the latter shows only one-to-one entrainment. Therefore, if one see entrainment to P/Q = 1 ratio, under additive forcing, it is a sign of limit cycle oscillator.
When the forcing is multiplicative, the non-linear noise-induced oscillators can also show P/Q = 1 ratio entrainment, but not necessarily all of the rational ratios. If the system is noise-induced oscillator and the non-linear term is small, one might be able to capture the diverging tendency of the amplitude, because saturation happens when the amplitude is large enough to make the non-linear term relevant. In such a case, one might see big difference in amplitude for a fixed M with varying Ω.
We thus urge experiment to be performed on oscillating biological systems. It is well known that some proteins (p53, NF-kB, Wnt) can oscillate in cells under stress responses.
In the case of p53, both non-linear 11,12 and linear models have been proposed 13 Finally, we would like to briefly comment on "noise-induced" oscillations by mechanisms other than the linear model studied here. It has been long known that, when noise is added to excitable system with a stable fixed point, regular oscillatory behaviour can be observed at a certain level of noise (coherence resonance) 28,29 . Since the nonlinearity plays an important role in an oscillation, such a system shows mode-locking behaviour similar to the deterministic nonlinear oscillators 30 . More recently, in gene network models with negative feedback, it has been shown that the noise due to finiteness of the number of molecules can modify the condition for oscillatory behaviour 31 or enhance the oscillation 32 .
It would also be interesting to see the entrainment behaviour in such systems. The horizontal axis is the external frequency Ω, and the vertical axis is the forcing amplitude M .
Entrainment is defined as ω/Ω is within 1% of the given value. For (c), the exponentially decaying case were excluded numerically by the following way: The equations are integrated with initial condition x(1) = 1 and x(2) = 0, and if the average amplitude for 390π/Ω < t < 400π/Ω is less than 90% of the average amplitude for 200π/Ω < t < 210π/Ω, then the solution is excluded. Limit cycle entrainment to any P/Q entrainment to any P/Q with phase slips A entrainment to any P/Q entrainment to any P/Q with phase slips M Linear one-to-one entrainment * one-to-one entrainment * with phase slips A noise-induced decay or diverge noise-induced oscillation with ∼ ω M or diverge Nonlinear one-to-one entrainment * one-to-one entrainment * with phase slips A noise-induced small decay or noise-induced oscillation with ∼ ω or M some P/Q entrainment some P/Q entrainment with phase slips * All the frequencies contained in the forcing can be observed. regions are calculated from the frequency of the peaks in the the deterministic case. In the finite noise case, we define the nuclear NF-κB peak as follows: We first determined the maximum value N max and the minimum value N min of x 1 of the steady state in the deterministic simulation for the given parameters. We then calculate two thresholds, N H = (N max + N min )/2 and N L = (N max + 3N min )/4. Next we perform the corresponding simulation with finite σ. We define a switching event from the "low" state to "high" state when x 1 exceeds N H , while the reverse switching happens when x 1 becomes smaller than N L . The number of peaks are calculated from how often the "high" states are reached. This way we can filter out the wiggly motion due to the noise and thus define the overall peak. | 2013-05-17T01:52:31.000Z | 2013-01-11T00:00:00.000 | {
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44084109 | pes2o/s2orc | v3-fos-license | Short‐term outcomes of open liver resection and laparoscopic liver resection: Secondary analysis of data from a multicenter prospective study (CSGO‐HBP‐004)
Abstract The aim of the present study was to compare short‐term outcomes of laparoscopic and open liver resection (LLR and OLR, respectively), and we first analyzed a preoperatively enrolled and prospectively collected database. We carried out a secondary analysis using a preoperative enrolled database that included the details of 786 patients who had been enrolled in a previously carried out randomized controlled trial to assess short‐term outcomes, including morbidities. Statistical analyses included logistic regression, propensity score matching (PSM) with replacement, and inverse probability of treatment weighting (IPTW) analyses. Among 780 liver resections, OLR was carried out in 543 patients and LLR was carried out in 237 patients. LLR was selected in patients with a worse liver function and was related to a smaller resected liver weight and/or partial resection. Logistic regression, PSM, and IPTW analyses revealed that LLR was associated with less blood loss and a lower incidence of morbidities, but a longer operating time. LLR was found to be a preferred factor in biliary leakage by IPTW only. LLR was a preferred factor for blood loss, morbidities and hospital stay, but was associated with a longer operating time. UMIN‐CTR, UMIN000003324.
| INTRODUCTION
Laparoscopic surgical techniques have recently been applied to liver resection, [1][2][3][4][5] despite the fact that their feasibility remains controversial. Although several randomized controlled trials (RCT) have been carried out to investigate the usefulness of laparoscopic techniques in gastric and colorectal surgery, [6][7][8] laparoscopic liver resection (LLR) has a relatively short history and the surgical techniques are still under development. As a consequence, it remains difficult to control quality in RCT. Thus, most studies on LLR and open liver resection (OLR) have been retrospective in nature and have analyzed a relatively small number of patients at a single hospital, [9][10][11] whereas other studies have used a nationwide database of postoperatively enrolled patients. [12][13][14] Thus, the potential for selection bias, enrolment bias, and missing values could not be avoided.
We previously carried out a multicenter RCT in which the endpoint was short-term surgical outcome. 15 Patients in the database were classified according to the method that was used to seal the liver cut surface. Briefly, over 700 patients from 11 institutes were enrolled in the RCT. Results showed that incidence of postoperative bile leakage and bleeding among the methods of sealing did not differ to a statistically significant extent. LLR accounted for approximately one-third of the procedures that were included in the database. All of the patients were enrolled preoperatively. Perioperative factors that were recorded in the database included: liver function, hepatitis, type of resection, operating time, blood loss, resected liver weight, detailed morbidities, and hospital stay.
We are of the opinion that the database would be useful for analysis of short-term outcomes of patients undergoing LLR and OLR for several reasons: patients were enrolled preoperatively, short-term surgical outcome and all morbidities were collected prospectively, and data were obtained from a multi-institutional study and therefore showed universality. Although an RCT is necessary to make a precise comparison, the results of the analyses in the present study would provide a useful rationale for the carrying out an RCT.
The present study shows how patients were selected for LLR and compares short-term outcomes between OLR and LLR. Propensity score matching (PSM) and inverse probability of treatment weighting (IPTW) analyses were used to reduce selection bias.
Results showed that LLR was associated with less blood loss and a lower incidence of morbidities, but a prolonged operating time.
These results provide information that can be used until completion of an RCT. The results are useful for determining the indications for LLR and for obtaining informed consent from patients in whom LLR is indicated.
| Study design
In the present study, we aimed to carry out a secondary analysis of the data obtained in a previous open, multicenter RCT that was carried out to explore the efficacy of fibrin sealant (FS) with polyglycolic acid (PGA) versus fibrinogen-based collagen fleece (CF) in preventing postoperative biliary leakage and/or hemorrhage at the liver cut surface. 15 As a result of the lack of an international definition of biliary leakage 16 when planning this RCT, we defined biliary leakage as a drain bilirubin to serum bilirubin ratio of ≥5. When the ratio was 3 to <5, we re-measured the drain and serum bilirubin levels after 2 or 3 days. Postoperative hemorrhage was defined by the need for relaparotomy or transfusion to achieve hemostasis.
Patient selection was carried out as follows. Patients in whom hepatectomy was planned and who were ≥20 years of age were enrolled in the RCT and preoperatively assigned. Type of resection
| Statistical analysis
Statistical analyses were carried out according to the flow diagram in Figure 1. Methods of the analyses are described below.
The data were expressed as the mean. Differences between groups were tested using Student's t test or the chi-squared test, as appropriate. P values of <.05 were considered to indicate statistical significance. A multivariate logistic regression analysis was performed. PSM analysis was performed with replacement to increase the average quality of matching and to decrease bias. 18,19 IPTW was used to adjust for differences and reduce the impact of any treatment selection bias. 20 With this method, the weights for patients who were treated with LLR were the inverse of the propensity score (determined by logistic regression); the weights for patients who underwent OLR were the inverse of the 1-propensity score. Logistic regression was used to estimate the propensity scores. The following variables were included in the model: type of resection, type of resection, age, gender, platelet, bilirubin, albumin, prothrombin time, HBs-antigen positivity and HCV-antibody positivity. To visualize hazard ratio of surgical outcomes and similarity between analyses, we used a forest plot. All of the statistical analyses were conducted using the R software program (version 2.15.2, Foundation for Statistical Computing, Vienna, Austria, http://www.rproject.org).
| Patient flow
A total of 786 patients were enrolled in the present study. Among these patients, 780 patients underwent hepatectomy and were analyzed in the present study ( Figure 1). OLR was carried out in 543 patients and LLR was carried out in 237 patients. We first carried out a logistic regression analysis using all of the patient data. Next, we carried out a PSM analysis after confirming that the C-index was >.8 (C-index: .8093). PSM analysis was done with replacement to increase the average quality of matching and to decrease bias. 18,19 Two hundred and eighty-six patients from each group were included in the PSM analysis by replacement. Finally, we carried out an IPTW analysis to reduce selection bias and to analyze all of the data in the database.
| Demographic characteristics of the patients who underwent OLR and LLR
We summarized the demographic characteristics of the patients who underwent OLR and LLR (Table 1). In the trial database, platelet count and prothrombin time of the LLR group was lower, whereas the rate of HCV positivity was higher in comparison to the OLR group. Regarding type of liver resection, the rate of partial resection was higher in the LLR group. Liver resection weight, estimated blood loss, and duration of postoperative hospital stay in the LLR group were lower in comparison to the OLR group. Rates of biliary leakage and other postoperative adverse events were lower in the LLR group.
In summary, laparoscopic resection was selected in patients with a worse liver function (lower platelet count, lower prothrombin time, and HCV positivity). Laparoscopic operative procedures were related to a smaller liver resection weight and/or partial resection.
| Surgical outcomes of OLR and LLR (analyzed by logistic regression, PSM, and IPTW)
We analyzed estimated blood loss, operating time, biliary leakage, other adverse events and duration of postoperative hospital stay as the surgical outcomes of liver resection. Biliary leakage was defined according to the definition of the International Study Group of Liver Surgery (ISGLS). 16 With regard to blood loss, logistic regression analysis showed that LLR, albumin, and partial resection were associated with less estimated blood loss (Figure 2). After PSM, LLR remained a preferred factor ( Table 2). In the IPTW analysis, LLR, albumin, HCV positivity, and partial resection were preferred factors (Figure 3).
With regard to operative time, logistic regression analysis showed that age, albumin, HCV positivity, and partial resection were associated with a shorter operative time, whereas LLR was associated with a longer operative time (Figure 2). After PSM, LLR was still associated with a longer operative time (Table 2). In IPTW analysis, age, albumin, HBs-positivity, and partial resection were preferred factors, whereas LLR remained associated with a longer operative time (Figure 3). (Table 2). In IPTW analysis, LLR, albumin, and partial resection were preferred factors (Figure 3). Regarding biliary leakage, in logistic regression analysis, male sex and partial resection were preferred factors (Figure 2). After PSM, LLR was not associated with biliary leakage ( Table 2). In IPTW analysis, LLR, male sex, and partial resection were preferred factors (Figure 3).
Regarding length of hospital stay, LLR and partial resection were preferred factors. After PSM, LLR remained a preferred factor (Table 2). In IPTW analysis, LLR and partial resection were preferred factors ( Figure 3).
In summary, LLR was a preferred factor for estimated blood loss, adverse events and hospital stay. However, the relationship between LLR and biliary leakage depended on the type of analysis. LLR was associated with a longer operative time.
| DISCUSSION
Numerous retrospective studies have investigated the short-term outcomes of LLR. 21,22 Some were case-control studies that analyzed more than 200 patients 23,24 ; others used PSM to analyze nationwide large-scale databases. [12][13][14] However, retrospective studies are associated with certain limitations: they might lack cases and/or information, and they involve selection and enrolment biases.
Thus, retrospective studies risk underestimating the incidence of morbidities.
In the present study, we used a preoperatively enrolled and prospectively collected database to investigate perioperative morbidities; which there was no biliary leakage according to our definition, the drainage tube could be removed without any complications. However, the incidence of biliary leakage according to this definition was too low to analyze in the multivariate analysis. We therefore used the definition of the ISGLS in the present study.
Next, we compared the short-term outcomes observed in the present study with those of previous reports (Table 3). 9,13,14,[26][27][28][29][30][31][32] Almost all of the reports showed less blood loss and a shorter hospital stay; however, there were discrepancies among the reports with regard to operating time and incidence of morbidities. As Table 3 shows, in most of the previous reports, less than 100 patients underwent LLR. One report analyzed over 300 LLR patients 13 | 2018-06-05T06:27:00.401Z | 2017-10-23T00:00:00.000 | {
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27576084 | pes2o/s2orc | v3-fos-license | The effect of Bulgarian propolis against Trypanosoma cruzi and during its interaction with host cells
Propolis has shown activity against pathogenic microorganisms that cause diseases in humans and animals. The ethanol (Et-Blg) and acetone (Ket-Blg) extracts from a Bulgarian propolis, with known chemical compositions, presented similar activity against tissue culture-derived amastigotes. The treatment of Trypanosoma cruzi-infected skeletal muscle cells with Et-Blg led to a decrease of infection and of the intracellular proliferation of amastigotes, while damage to the host cell was observed only at concentration 12.5 times higher than those affecting the parasite. Ultrastructural analysis of the effect of both extracts in epimastigotes revealed that the main targets were the mitochondrion and reservosomes. Et-Blg also affected the mitochondrion-kinetoplast complex in trypomastigotes, offering a potential target for chemotherapeutic agents.
The effect of Bulgarian propolis against Trypanosoma cruzi and during its interaction with host cells
Andréia Pires Dantas, Kelly Salomão, Helene Santos Barbosa*, Solange Lisboa De Castro/ + Laboratório de Biologia Celular *Laboratório de Biologia Estrutural, Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz-Fiocruz, Av.Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brasil Propolis has shown activity against pathogenic microorganisms that cause diseases in humans and animals.The ethanol (Et-Blg) and acetone (Ket-Blg) extracts from a Bulgarian propolis, with known chemical compositions, presented similar activity against tissue culture-derived amastigotes.The treatment of Trypanosoma cruzi-infected skeletal muscle cells with Et-Blg led to a decrease of infection and of the intracellular proliferation of amastigotes, while damage to the host cell was observed only at concentration 12.5 times higher than those affecting the parasite.Ultrastructural analysis of the effect of both extracts in epimastigotes revealed that the main targets were the mitochondrion and reservosomes.Et-Blg also affected the mitochondrion-kinetoplast complex in trypomastigotes, offering a potential target for chemotherapeutic agents.
Key words: propolis -natural products -Trypanosoma cruzi -Chagas disease Chagas disease, caused by the trypanosomatid protozoan Trypanosoma cruzi, is endemic in Latin America.Initially restricted to natural ecotopes, with transmission among small autochthonous mammals by hematophagous Triatominae bugs, the occupation by humans of such ecotopes led to the establishment of the vectors in human dwellings, changing the characteristic of the disease from zoonotic to antropozoonotic.In humans, the acute phase results in 2-8% mortality, specially among children, while in the chronic phase most patients remain asymptomatic and about 30 to 40% of the cases develop symptoms of cardiac or digestive lesions.The World Health Organization estimates that about 18 million people are infected with T. cruzi (WHO 2002).The available drugs for the clinical treatment, the nitroderivatives benznidazole and nifurtimox, are unsatisfactory.They present variable healing effects during the acute phase according to the geographical region and require long term treatment, besides presenting frequent toxic side effects and limited efficacy in the chronic stage (reviewed in Coura & De Castro 2001).
In this context, there is an intense search for new synthetic compounds and natural products for treatment of Chagas disease.Thus, our group has been engaged in experimental chemotherapy studies using propolis (Higashi & De Castro 1994, De Castro & Higashi 1995, Marcucci et al. 2001, Prytzyk et al. 2003, Paulino et al. 2003, Salomão et al. 2004).Propolis, a bee product collected from different plant exudates, is a complex mixture containing known bioactive constituents, such as flavonoids, phenolic acids and esters, terpenes, sesquiterpenes, aromatic aldehydes, and alcohols (Marcucci 1995).In temperate zones, bud exudates from poplar trees (Populus spp.) are the main source of flavonoid-rich propolis, which are rich in flavonoids (reviewed in Bankova et al. 2000).
During the last decades, an increasing number of studies on the chemical composition, biological activity, and therapeutic uses of propolis has been published (reviewed in De Castro 2001).In recent studies with a Bulgarian propolis sample, we have characterized by high temperature high resolution gas chromatography coupled to mass spectrometry (HT-HRGC-MS) the composition of ethanol and acetone extracts and assayed their activity against epimastigote and trypomastigote forms of T. cruzi, as well as on some species of fungi and bacteria of medical importance (Prytzyk et al. 2003, Salomão et al. 2004).In the present work we have analyzed the activity of the two extracts from this Bulgarian propolis sample against tissue culture-derived and intracellular amastigotes of T. cruzi, as well as ultrastructural alterations induced in epimastigote and trypomastigote forms of this parasite.
Propolis sample and preparation of the extracts -A
propolis sample was collected in Burgas (Southeast Bulgaria), being the exudates collected by bees mainly from buds of Populus nigra.The resin was cut in small pieces, and after cooling bellow -10°C, extracted with 70% ethanol (1:10 w/v), under agitation at room temperature for 24 h (Bankova et al. 1999).From 4 g propolis extracted with hexane (1:25, w/v) to remove apolar components, the residue was further extracted with acetone at room temperature (Prytzyk et al. 2003).Both extracts were evaporated to dryness under vacuum at 40°C and stored in a desicator at 4°C.The extracts were named Et-Blg and Ket-Blg, and obtained in a yield of 62 and 10%, respectively.Stock solutions were prepared in dimethysulfoxide and diluted in phosphate buffered saline (PBS) before use.The final concentration of solvent did not exceed 1%, which had no effect per se on the parasites or mammalian cells.
Parasites and cell cultures -Assays were performed with the Y strain of T. cruzi (Silva & Nussenszweig 1953).Epimastigote forms were maintained in LIT medium supplemented with 10% fetal calf serum (FCS) (Camargo 1964) and harvested during the exponential phase of growth.Bloodstream trypomastigotes were obtained from infected Swiss mice.Amastigotes were collected from the supernatant of trypomastigote-infected J774G-8 macrophage cultures (De Castro et al. 1987).Primary cultures of skeletal muscle cells (SKMC) were prepared as previously described (Araújo-Jorge et al. 1986).Briefly, tissues from thigh muscles of 18-day-old mouse embryos were fragmented and dissociated with 0.05% trypsin and 0.01% versene in PBS.Thereafter, the cells were ressuspended in Dulbecco's modified Eagle medium, supplemented with 10% horse serum, 5% FCS, 2% embryo chicken extract, 2.5 mM CaCl 2 , 1 mM L-glutamine, and antibiotics (DMEM).SKMC were plated (10 5 cells/well) on 0.02% gelatin-coated glass coverslips in 24-well plates and maintained at 37°C in 5% CO 2 atmosphere (Barbosa et al. 2000).
Antitrypanosomal activity -Tissue culture-derived amastigotes were resuspended in Dulbecco's modified Eagle medium supplemented with 10% FCS (DMES) to a concentration of 10 × 10 6 parasites/ml.In 96-well plates, 100 µl of the parasite suspension was added to the same volume of Et-Blg or Ket-Blg, previously prepared at twice the desired final concentrations.The extracts were assayed in the range between 4 and 250 µg/ml.After incubation at 28°C for 24 h the number of parasites was quantified using a Neubauer chamber, and the activity of the extracts expressed as IC 50 values, corresponding to the concentration that induces 50% of parasite lysis.
T. cruzi suspensions (10 ml) were treated with Et-Blg (40 to 200 µg/ml), being the assays with epimastigotes performed in LIT at 28°C and those with trypomastigotes in DMES at 4°C.After 24 h the parasites were washed in PBS and processed for electron microscopy, as described below.
Ultrastructural analysis -For transmission electron microscopy (TEM) the parasites were fixed with 2.5% glutaraldehyde (GA), 2.5 mM CaCl 2 and 0.1 M Na-cacodylate buffer (pH 7.2) for 1 h at 4°C.After post-fixation for 1 h in a solution containing 1% OsO 4 , 0.8% potassium ferricyanide, and 2.5 mM CaCl 2 in cacodylate buffer, the material was processed for routine transmission electron microscopy and observed with an Zeiss EM10C microscope (Oberkochen, Germany).For monitoring the treatment, immediately after fixation in GA, control and treated parasites were fixed in methanol, stained with Giemsa and observed by light microscopy.
For scanning electron microscopy (SEM), the parasites were adhered to 0.1% poly-L-lysine coated coverslips and fixed in 2.5% GA in 0.1 M Na-cacodylate buffer for 30 min at room temperature, followed by rinsing in the same buffer.After post-fixation in 1% OsO 4 for 30 min at room temperature, the material was dehydrated in an ascending acetone series, dried by the critical point method with CO 2 , mounted with silver cellotape on aluminium stubs and coated with a 20 nm-thick layer of gold.The samples were examined with a Zeiss DSM 940 microscope.
Effect of Et-Blg on the interaction of T. cruzi with SKMC -After plating for 96 h, SKMC were infected with bloodstream trypomastigotes (at a 1:10 parasite:host cell ratio) in DMEM for 18 h and washed to remove non-internalized parasites.Et-Blg was added in final concentrations ranging from 3 to 12 µg/ml and this medium was changed every two days.After 24, 48, and 72 h of treatment, the cultures were fixed and processed for light microscopy and the infection levels were quantified using an Zeiss Axioplan microscope.
Effect of the extracts on amastigote forms of T. cruzi -
Both propolis extracts, Et-Blg and Ket-Blg, were active against tissue culture-derived amastigotes presenting IC 50 /24 h of 36.4 ± 4.9 µg/ml and 39.5 ± 8.2 µg/ml, respectively (Fig. 1a).In relation to control cells, treatment of of the mitochondrion, including rarefaction of the matrix, absence of crystae in the inner membrane and increase of organelle volume (Fig. 2c).Alterations were also observed in the reservosomes, which presented a heterogeneous shape and large electron-lucent inclusions (Fig. 2c).Treatment with 80 µg/ml Et-Blg led to alterations in the organization of the kinetoplast and the mitochondrion, to the formation of concentric membranous structures and large vacuoles in the cytoplasm, and to a decrease in electron density of reservosomes (Figs 2e-g).SEM revealed that T. cruzi-infected SKMC with Et-Blg led to an increase in the percent of infection inhibition (Fig. 1b), and inhibited the proliferation of intracellular parasites with an IC 50 /24 h of 2.2 ± 0.3 µg/ml (Fig. 1c).Damage to the host cells was observed only in concentrations above 25 mg/ml (data not shown).
Ultrastructural alterations induced on T. cruzi -Analysis by TEM of epimastigotes treated with 100 µg/ml Ket-Blg for 24 h showed interference on the morphology Ket-Blg induced alterations in the body shape (Fig. 2d), while Et-Blg always led to rounding and shortening of the parasites (Figs 2h,i).
Trypomastigotes treated with 100 µg/ml Et-Blg for 24 h presented a dilated mitochondrion with matrix rarefaction and scarcity of crystae, especially at the kinetoplast region (Fig. 3b).Increasing the concentration of the extract to 200 µg/ml, the ultrastructural alterations were more intense, affecting a higher number of parasites (data not shown).SEM analysis of trypomastigotes treated with 100 µg/ml Et-Blg showed rounding of the posterior region with maintenance of the normal morphology of the flagellum (Figs 3c,d).
DISCUSSION
In the present work, we demonstrated that ethanol (Et-Blg) and ketone (Ket-Blg) propolis extracts presented similar activity against tissue culture-derived amastigotes of T. cruzi, with IC 50 /24 h between 36 and 40 µg/ml.In a previous work we have observed that both extracts showed similar IC 50 values against trypomastigotes (Prytzyk et al. 2003).Evaluation of the activity of Et-Blg against T. cruzi-infected SKMC showed that intracellular parasites were about 20 times more susceptible than tissue culture-derived forms, and their proliferation was inhibited in concentrations 12.5 times lower than those which led to damage to the host cells.
Comparison between the direct effect of Et-Blg against the three forms of the parasite showed that tissue-culture derived amastigotes were more susceptible than epimastigotes (Prytzyk et al. 2003) and trypomastigotes (Salomão et al. 2004).Such difference in susceptibility to Et-Blg among the three forms of T. cruzi has been already described in treatments with other drugs (Schlemper et al. 1977, De Castro et al. 1992, 1993).
Ultrastructural alterations induced by Ket-Blg and Et-Blg in epimastigote forms were similar in several aspects, showing that the main targets were the mitochondrion and reservosomes, together with alterations in the body shape as observed by SEM.Rarefaction of the mitochondrial matrix, absence of crystae and mitochondrial swelling have been already described after treatment of the parasites with different drugs, such as inhibitors of sterol biosynthesis (Lazardi et al. 1990, Santa Rita et al. 2000, Magaraci et al. 2003) and other synthetic compounds (Bernacchi et al. 2002).The presence of heterogeneous forms and large electron-lucent inclusions in the reservosomes after treatment with Et-Blg has been already reported in the DM28 clone of T. cruzi, suggesting a rupture of the organelle (De Souza et al. 2002).Since reservosomes are acidic storage compartments in epimastigotes (Soares 1999), decrease in the electron-density of the matrix of these organelles in Et-Blg-treated parasites suggests a possible interference in the content of lipids and proteins.
Trypomastigotes treated with Et-Blg showing disruption of the kinetoplast structure besides mitochondrial alterations suggest that this complex, i.e. the mitochondrion-kinetoplast, is a potential target of the propolis extract.
It has been already shown propolis activity against pathogenic microorganisms that cause diseases in humans and animals (reviewed in De Castro 2001, Prytzyk et al. 2003, Salomão et al. 2004).The present work encourages further investigations on the effect of propolis in experimentally T. cruzi-infected mice, aiming an alternative natural product for the treatment of Chagas disease. | 2017-08-30T11:44:04.076Z | 2006-03-01T00:00:00.000 | {
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184483044 | pes2o/s2orc | v3-fos-license | DS at SemEval-2019 Task 9: From Suggestion Mining with neural networks to adversarial cross-domain classification
Suggestion Mining is the task of classifying sentences into suggestions or non-suggestions. SemEval-2019 Task 9 sets the task to mine suggestions from online texts. For each of the two subtasks, the classification has to be applied on a different domain. Subtask A addresses the domain of posts in suggestion online forums and comes with a set of training examples, that is used for supervised training. A combination of LSTM and CNN networks is constructed to create a model which uses BERT word embeddings as input features. For subtask B, the domain of hotel reviews is regarded. In contrast to subtask A, no labeled data for supervised training is provided, so that additional unlabeled data is taken to apply a cross-domain classification. This is done by using adversarial training of the three model parts label classifier, domain classifier and the shared feature representation. For subtask A, the developed model archives a F1-score of 0.7273, which is in the top ten of the leader board. The F1-score for subtask B is 0.8187 and is ranked in the top five of the submissions for that task.
Introduction
For getting feedback from costumers or users, an organization often uses forums and social media channels. Also ratings of products on rating platforms can be an useful feedback to make a product better. The feedback from a customer can be in the form of a suggestion which appears in a rating text or is directly asked from the customer. The task of suggestion mining can be defined as the extraction of sentences that contain suggestions from unstructured text (Negi et al., 2018). SemEval-2019 Task 9 Subtask A provides the challenge to do suggestion mining on data from an online suggestion forum. For that subtask, a train and validation set is provided so that it is possible to apply super-vised training. For subtask B, suggestions in hotel reviews should be identified. An additional difficulty for that subtask is that no labeled data is given except a small validation set, which is not allowed to be used for supervised training. For both tasks, silver standard datasets are allowed to use, which means that data that is likely to belong to a certain class can be taken as long as it is not manually labeled. A more detailed task description can be found in (Negi et al., 2019).
Data
The dataset for subtask A provides an overall count of 8500 examples, where 6415 examples are labeled as non-suggestion and 2085 as suggestion. Also a trial dataset is provided that contains 592 examples, divided in 296 examples for each class. Every example contains only one sentence, which could be part of a whole post in the forum where it was extracted.
The domain of software suggestion forum posts in general provides more balanced data than other domains, for example hotel reviews. Also the domain contains very specific vocabulary which is frequently used in software development, so that it can be difficult to use a trained model of this domain for other domains (Negi et al., 2018).
For subtask B, only a validation dataset is provided. The set contains an overall count of 808 examples with 404 examples for each class. As mentioned in the introduction, it is not allowed to use the validation data for supervised learning for this subtask. The data is only allowed to be used for model evaluation and error analysis and also for automatic hyperparameter tuning. The presented solution in this paper uses the validation data for early stopping at a fixed count of train steps after the best score is reached. The model state at the best score is then returned and used for the predic-tion of the test data.
For both subtasks, additional data is allowed that is not manually labeled. In this work, the hotel review dataset, which is presented in (Wachsmuth et al., 2014), is used to apply cross-domain classification for subtask B. The dataset comes with nearly 200k examples of hotel reviews without labels.
Related Work
The task of text classification improved a lot during the last years. In the past, machine learning techniques like support vector machines were used to assign a class to a text. In (Joachims, 1998), a text classification with support vector machines is presented. For that, a document vector is extracted for the whole text and used as the feature vector for the classification.
Since such methods can provide a good base line today, the increasing popularity of neural network approaches provides new methods that can classify texts more exactly. Especially the introduction of word embeddings in (Mikolov et al., 2013) was a big step forward in the field of text processing and opened new opportunities for many natural language processing tasks. Also for the task of text classification, word embeddings are useful features and can lead to good results. Since the release of these word embeddings, many other word embedding approaches have been introduced. A very recent one is shown in (Devlin et al., 2018) and is called BERT: Bidirectional Encoder Representations from Transformers. These embeddings are the result of the training process of transformer, which is described in (Vaswani et al., 2017) and delivers a state-of-the-art method for different natural language generation tasks, especially for translation.
To use the word embeddings as features for text classification, a commonly used approach is Longshort term memory (LSTM), which is described in (Hochreiter and Schmidhuber, 1997). The advantage of using LSTM cells over support vector machines as classifier is the processing of features in time steps. By passing a single word embedding into a single time step of the LSTM, every feature is processed separately. Since the features are processed one after the other, also the order of the features has influence on the classification process. In addition to that, LSTM cells have a state that enables them to save information for many previous time steps. For text classification, this can be useful when there are connections between words in a text that are far apart.
Another method to process word embeddings are convolutional neural networks (CNN), which are introduced in (LeCun and Bengio, 1998). With the ability to extract features of two-dimensional input data by defining a sliding window of variables, the method is often used for image processing. But also good results for text classification are reported, for example in (Kim, 2014). The results show that even a simple CNN with one layer performs very well and a tuning of hyperparameters brings an improvement of the performance.
For subtask B, the focus gets in the direction of methods for cross-domain classification. By the introduction of Generative Adversarial Nets (GAN) in (Goodfellow et al., 2014), a new way for training neural networks was provided that leads to new opportunities for different task, especially image generation. In (Chen and Cardie, 2018) is shown that this training technique could also be used for cross-domain classification of texts of different domains. As the main four components, a shared feature extractor, a domain feature extractor, a text classifier and a domain discriminator are introduced. The main goal of that system is to learn a domain invariant feature representation by training the shared feature extractor with the discriminator and the text classifier. The discriminator learns to separate the domains and the training goal for the shared feature extractor is to increase the loss of the discriminator. The extracted features become invariant for the domains, so that the text classifier results improve for the domain where no labels are given.
Models
In this section, the models for subtask A and B are presented. The overall idea of the model for subtask A is using an ensemble of LSTM and CNN networks. As input features, pre-trained BERT embeddings for the texts are used.
For subtask B, the idea is to extend the model from subtask A with a domain discriminator and shared features. Since that adds a lot of parameters to the model, the text classifier has a simpler structure than in subtask A and uses only CNNs for classification. The full TensorFlow implementation of the models can be found at GitHub. 1
BERT embeddings
For both subtasks, BERT embeddings are used to create a representation of the text. The Tensor-Flow implementation, which is openly available and comes with pre-trained multi-language embeddings, is taken for that. 2 The model for creating the embeddings is the small uncased model, which has been trained on lower cased wikipedia texts. It has a total count of twelve layers and a layer size of 768 in each hidden layer. The whole model has a 110 million parameters in total.
To extract the embeddings out of the model, the text gets tokenized and mapped into a sequence of integers by using the vocabulary of the pre-trained model. This representation is then given into the network, where it passes the different transformer layers. The embeddings are delivered by the hidden layers of the model. In this project, the output from the last four layers before the output layer is taken as the representation of a word, so that every word is represented by a vector of the shape (4, 768).
Subtask A
For subtask A, a text classification ensemble of LSTM and CNN is built. To bring all sentences to the same length, a maximum sequence length of 40 is defined. With that sequence length, for around 95% of all train data sentences all words are taken as input. Only for the remaining 5% which have more than 40 words, the texts are cut to the maximum sequence length. If a text is shorter than 40 words, it is filled with zeros. Using the batch size of 64, the input shape for a batch for the training process is (64, 40, 768) for every of the four extracted input features from BERT.
One problem of the data is the imbalance of the classes. When taking random batches out of the whole dataset, it is likely that the count of one class is always higher than of the other. The algorithm learns to predict the class with the higher example count with a higher probability. To avoid that, the technique of oversampling is applied to the training process. The data is separated into two sets, each for every of the two classes and then fed into the network alternately. When all examples of the class with the lower count were used as training input, the set gets repeated so that these examples occur more often as training input. 2 https://github.com/google-research/bert The model structure for subtask A can be divided into the three following main parts: • Processing of single words with dense layers.
• Processing of the whole text with LSTM cells.
• Processing of sliding windows through the text with CNN.
For the processing of the dense layer and the LSTM, separated graphs are created for each of the input features from BERT. As mentioned before, four embeddings for a word are gathered, each of a different hidden layer of the transformer. Thus four graphs of dense layers and LSTM layers are created, each for processing a different embedding type of the input text. The first step is a transformation of every single input word with a dense layer. This approach is applied to focus on single signal words that can occur in the text. For example, the occurrence of the word recommend could be a hint for a suggestion, without regarding other words. The outputs of the single word processings are concatenated and forwarded to a dense layer to reduce the dimension.
The LSTM is represented by two cells to realize a bidirectional approach. The output of the two cells is concatenated and followed by a GlobalMaxPool-Layer, which takes the maximum of the output's timestep axis to bring it into a one-dimensional representation. To do a further feature transformation, a dense layer is applied to the output. The result is concatenated to the output of the previous described single word approach.
Unlike the single word processing dense layer and the LSTM, the CNN approach processes all four BERT features for the words in the same network. When using CNNs for image processing, the colors of an image are arranged as additional channels that the CNN can process. For feeding the CNN with all BERT features for the words, they are shaped similarly to an image and can be seen as the channels of the word. By using that approach, the words are given with four channels into the CNN. The output of the CNN is then processed by a dense layer. The CNN approach can be seen as an bag of words approach, which takes the words within a sliding window until the end of text is reached. The amount of words is fixed, the approach is build for each of 2-5 words.
At the end of the processing, the dense-and LSTM-features and the CNN-features are concatenated and given to the classification layer, which is composed of two dense layers. For more robust predictions, three graphs are build to get three predictions, the final result is formed by the mean. For training the model, the cross-entropyloss is used. The model gets optimized with AdamOptimizer. On every training step, the model is validated with the provided validation dataset, and the model weights on the best F1score are taken to predict the test examples.
Subtask B
For subtask B, a similar model as in (Chen and Cardie, 2018) is built to do cross-domain classification. The model in this work is composed of three major parts: • Label classifier: Model that predicts if an example is a suggestion.
• Domain classifier: Model for the prediction of the domain of an example.
• Shared features: Model that applies a transformation on the input features.
The training of the model can be split into two phases: The pre-training phase of the supervised label classifier and the adversarial training of the domain classifier and the shared features.
In the pre-training phase, the model uses supervised training like for subtask A. In this phase, the label classifier and the shared features are trained to get the best score on the suggestion data of the domain of online suggestion forums. The shared features get the word embeddings as input and apply a CNN on each BERT embedding. The size of the CNNs is the same as the length of the embedding, so that the projected word features are of the same shape as the input features.
In the next step, the projected features are classified with the label classifier. Unlike to subtask A, only the CNN part is used for the classification because of the amount of additional parameters of the shared features and the domain classifier. The CNN works like in subtask A and processes the four BERT features as single channels. In this task, a sliding window of the word counts from 2-6 words is taken. The optimization is applied with AdamOptimizer and stops when the best score is reached on the validation data of subtask A. In phase two, the domain classifier starts with the training and learns to choose the right domain for the examples. To do that, also examples of the unlabeled hotel review dataset are given into the net. The domain classifier has the same structure as the text classifier and uses CNNs to find the right label for an example.
After one train step of the domain classifier, the shared features are retrained in an adversarial way to maximize the loss of the domain classifier. To realize this, the parameters are trained with the switched labels for the hotel review examples which are marked as suggestions for this training step.
After the training step of the domain classifier and the shared features, the validation examples of subtask B are used to predict a score. To do that, the examples are predicted with the text classifier, which uses the updated shared features to make a prediction if the example is a suggestion. Early stopping is used to find the best model with the validation data, so the model stops at the maximum F1-score for the validation set for subtask B.
Results
In this section, the results for subtask A and B are discussed. Also the test data, which has been provided to the participants after the evaluation phase, is used for the analysis.
Subtask A
For subtask A, the best model reached a F1-score of 0.7273 for the test data. This model archived a validation F1-score of 0.875 which is a noticeable difference to the test data score. To find the difference in the datasets, the example counts are compared in Table 1. It can be seen that there are differences in the ratio of the data. While the train data contains about 25% of examples for suggestions, for the test data there are only about 15%. Since oversampling is used to tackle the problem of class imbalance, this difference of train and test data should not have much influence on the result. Also the counts of predicted classes for the test set can be seen in the table, which show a similar amount like the test classes, but has some more predictions for the class of suggestions.
Another factor for the gap between validation and test score could be explained by very different examples in the two sets. In addition to that, the validation set may be better represented by the train set. That could lead to bad results for the test data when stopping at a good F1-score for the validation data. For analyzing this, another train run is started and the curve for the F1-score for the validation set and the test set plotted. The outcome can be seen in Figure 1.
It can be seen that the test F1-score is constantly lower than the validation score. Also there are much variations in the test score, even in a late train phase. Overall it can be determined that the train data describes the validation data better than the test data with the given model for subtask A.
Subtask B
The model for subtask B reached a final F1-score on the test data of 0.8187. In comparison to subtask A, it can be seen that the final score on the test data is higher, although no labeled data is given for that task except the validation data. The reason for this could be the use of the external hotel review dataset that inputs many new examples into the model. The overall count of examples for hotel reviews is much higher than the labeled data in subtask A, so that the higher score can be explained with the presence of more data. Also the validation score of 0.884 doesn't differ to the test score as much as in subtask A. This also shows that the use of external data improves the overall result of the model. The validation data for subtask B was used to apply early stopping and saving the weights at the best validation F1score. Like for subtask A, the class counts for the different datasets are shown in Table 2. It can be seen that the distribution of the classes in the test and validation set is more equal than in subtask A, what could be a reason why the model archived better results. To verify this, another training run is started to compare the test and validation F1-score over the training epochs. The results can be seen in Figure 2. The validation data score for subtask A and B and the test score for subtask B is plotted for every training step. Since the training is separated into two phases, the left graph shows the scores for the pre-training phase and the right graph for the adversarial training.
In the pre-training phase of the model, the score of the validation data of subtask A improves as expected since supervised training is performed. Also it can be seen that the subtask B data score shows a high variance over the epochs, but decreases slightly. Over all epochs, the test and validation score of subtask B is nearly the same what could be a hint that the difference between the datasets is very small. This is confirmed in phase two of the training, where the validation and test score increase in the first epochs. Although the validation score reaches a higher peak for the F1score, the peak of the test score is found in around the same region of train steps. The validation score for subtask A decreases in the adversarial train phase, what could be caused by a overfitting to the hotel review domain. Also the validation score of subtask B decreases after about 50 epochs. The reason for that could be that the amount of nonsuggestions in very high in the hotel reviews data, so that the model unlearns to distinguish between the two classes.
Conclusion and Future Work
In this work, for each subtask of SemEval-2019 Task 9 a solution is presented. For subtask A, a supervised model is built on the neural network techniques CNN and LSTM. As input features, BERT word embeddings are taken, which are pre-trained on huge datasets. One problem in subtask A is the class imbalance of the data, which is tackled with oversampling. Another problem that occurred during the evaluation of the training phase is the difference of examples in the validation and the test set, what could be one of the reasons why the validation score is much higher than the test score. In future works, it can be tried to extend the labeled data with additional unlabeled data to tackle the problem of class imbalance and too few examples. Since extending the data works for subtask B, it could also work for subtask A and the domain of suggestion forum posts. Subtask B sets the task to classify sentence as suggestion or non-suggestion for the domain of hotel reviews. Unlike to subtask A, only a small validation set is given as labeled data which is not allowed to be used for supervised training. To solve the problem of not having labeled data, the technique of cross-domain classification has been used. This is done by building a neural network model, which is trained in an adversarial way. Like in subtask A, a classification model for the suggestions is given. In addition to that, a shared feature representation and a domain classifier are added. The domain classifier is trained to assign the right domain label to a sentence. The shared feature representation is trained adversarial to the domain classifier, so that it learns to generate a global representation for both domains. For that, an unlabeled external dataset is taken which contains examples for the domain of hotel reviews.
The results for subtask B show that the adversarial training can improve the F1-score for the domain with no labeled data. This happens with the cost of lowering the score for the labeled data, on which the model was pre-trained. Also the score for the hotel review data falls down after the peak is reached. This makes it necessary to have at least a small dataset which contains labeled data for subtask B to measure the score during the training and stop when best score has been reached. For future work, it could be tried to develop a better shared features method where a good feature representation for both domains is formed. That would give a classifier that could be used to predict sentences of both domains. Another improvement could be archived by developing a model where the curve for the unlabeled data doesn't fall down that sharply in the late train phase. This could lead to a method were no labeled data is needed to stop the training at a good point. | 2019-06-12T14:58:12.845Z | 2019-01-01T00:00:00.000 | {
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18184072 | pes2o/s2orc | v3-fos-license | Roles of Gibberellin Catabolism and Signaling in Growth and Physiological Response to Drought and Short-Day Photoperiods in Populus Trees
Survival and productivity of perennial plants in temperate zones are dependent on robust responses to prolonged and seasonal cycles of unfavorable conditions. Here we report whole-genome microarray, expression, physiological, and transgenic evidence in hybrid poplar (Populus tremula × Populus alba) showing that gibberellin (GA) catabolism and repressive signaling mediates shoot growth inhibition and physiological adaptation in response to drought and short-day (SD) induced bud dormancy. Both water deprivation and SDs elicited activation of a suite of poplar GA2ox and DELLA encoding genes. Poplar transgenics with up-regulated GA 2-oxidase (GA2ox) and DELLA domain proteins showed hypersensitive growth inhibition in response to both drought and SDs. In addition, the transgenic plants displayed greater drought resistance as evidenced by increased pigment concentrations (chlorophyll and carotenoid) and reductions in electrolyte leakage (EL). Comparative transcriptome analysis using whole-genome microarray showed that the GA-deficiency and GA-insensitivity, SD-induced dormancy, and drought response in poplar share a common regulon of 684 differentially-expressed genes, which suggest GA metabolism and signaling plays a role in plant physiological adaptations in response to alterations in environmental factors. Our results demonstrate that GA catabolism and repressive signaling represents a major route for control of growth and physiological adaptation in response to immediate or imminent adverse conditions.
Introduction
Phenotypic plasticity in response to adverse conditions determines plant productivity and survival. Abiotic stress results in the largest loss in crop yields worldwide [1] and is a major threat to crop sustainability [2]. Thus, improving abiotic stress resistance is considered to be a main route for sustainable yield growth and will likely become progressively more important as arable land is becoming increasingly limited [3] due to (1) the deterioration of previously productive lands [4], (2) the predicted expansion of areas affected by droughts [5] and high salinity [6], and (3) the predicted increase in the occurrences of climatic extremes [7].
Plants reduce growth under adverse conditions as a mechanism to avoid potentially lethal stresses [8]. In addition, plants can utilize environmental cues to detect and anticipate imminent, adverse conditions and correspondingly adjust their growth [9]. For example, woody perennials (trees and shrubs) from temperate latitudes stop shoot growth in response to short-day (SD) photoperiods that signal the approaching winter and impending months of dehydration and freezing conditions. The cessation of shoot growth precedes a more permanent growth inhibition known as winter dormancy that can last months, requires development of a specialized organ (e.g., bud), and entails physiological resetting to allow resumption of growth [10].
Gibberellins (GAs) are involved in regulating several aspects of plant growth and development [11][12][13]. The GA metabolic and signaling pathways have been extensively studied. The GA 2oxidases (GA2ox) and DELLA domain proteins, like GAI (GAinsensitive) and RGL1 (repressor of ga1-3 like), are important regulators of GA levels and signaling. GA2oxs are enzymes that catalyze the 2-oxidation inactivation of both bioactive GAs and some of their precursors [14][15][16]. Overexpression of GA2oxs in transgenic plants leads to bioactive GA-deficiency and various levels of dwarfism [15,16]. GA2oxs are encoded by small gene families which regulate specific processes in plants, in part by specific expression patterns [15][16][17][18]. DELLA domain proteins are strong repressors of several GA responses and characterized by the conserved DELLA domain which mediates the susceptibility of the protein to proteolytic degradation [19]. Mutant forms of these proteins (gai and rgl1) with truncation of the DELLA domain are resistant to degradation and impart repressive blocks to several GA-mediated responses [20][21][22].
An accumulating body of evidence suggests that DELLA domain proteins and GA2oxs are involved in plant abiotic stress response. For example, activation of DELLA domain proteins appears to be crucial for restraining growth in adverse conditions [23][24][25]. DELLA proteins are believed to affect both cell expansion [11] and proliferation [26,27]. For instance, DELLA proteins have been shown to inhibit cell proliferation through elevation of cell cycle inhibitors [27], and to promote cell differentiation by reducing inhibitors of the developmental transition from mitosis to endoreduplication that modulate anaphase-promoting complex/cyclosme activity [26]. DELLA proteins not only inhibit growth but also promote plant survival under stressful conditions by limiting the accumulation of reactive oxygen species (ROS), thus delaying cell death [25]. In Arabidopsis (Arabidopsis thaliana), salt stress leads to DELLA protein stabilization and as a consequence, growth inhibition and increased plant survival [23]. In addition, a DELLA protein in Arabidopsis has been shown to bind to the promoter and increase expression of the XERICO gene, which is involved in drought response [28]. DELLA proteins have also been implicated in mediating hormonal cross-talk between GA andabscisic acid (ABA) signaling pathways [23,28]. ABA is a growth inhibiting hormone that regulates one of the two major stress signal transduction pathways in plants [29].
In addition to modulation of GA sensitivity, stressful conditions can directly influence levels of bioactive GAs. For example, cold treated Arabidopsis plants have been shown to have increased expression of three GA 2-oxidase (GA2ox) genes [24], whereas under salinity stress six GA2ox genes were shown to be up-regulated [30]. Furthermore, the cold-inducible CBF1/DREB1b protein in Arabidopsis imparts freezing tolerance, at least in part by activating the expression of GA2ox genes, which in turn leads to reductions in bioactive GAs and suppression of growth [24]. Similarly, in Arabidopsis the DWARF AND DELAYED FLOW-ERING 1 (DDF1) protein, involved in salt stress response, binds to the promoter and activates the GA2ox7 gene [30]. Though GA2oxs' role in control of seed dormancy has been well substantiated [31,32], their involvement in regulation of winter bud dormancy is based solely on correlative evidence. For instance, in several tree species, SD-induced transition to dormancy is associated with reduction in bioactive GAs [33][34][35].
Changes in GA catabolism and signaling can have profound effects on tree growth, phenology, morphology, physiological, metabolism, and gene expression [36][37][38][39]. In Populus, GA-deficient and GA-insensitive transgenics are semidwarfs of varying degrees of severity [39]. Semidwarfism has also been associated with other characteristics that could be advantageous under adverse conditions such as increased biomass allocation to roots, reduced stem elongation, and increased water use efficiency [40]. The wide diversity of effects associated with GA modulation suggests that GA metabolism and signaling could be involved in mediating plant adaptive responses to adverse environmental conditions. Here, using a diverse array of evidence, we show that both DELLA and GA2ox encoding genes in hybrid poplar (Populus tremula x Populus alba) constitute a major regulatory circuit mediating growth restraint and physiological adaptation to drought stress and SD photoperiods.
Poplar DELLA Domain and GA2ox Encoding Genes are Induced by Drought and SDs
We studied expression of four poplar DELLA protein (PtaGAI1, PtaGAI2, PtaRGL1-1, and PtaRGL1-2) and seven PtaGA2ox (PtaGA2ox1 to 7) encoding genes (Table 1) in leaves in response to drought and SD photoperiods (Figures 1 and 2). Drought treatment, imposed through water deprivation under greenhouse conditions, increased expression in two of the four DELLA protein encoding genes and four of the seven PtaGA2ox genes (Figure 1). Expression increased weekly, reaching peak levels for most genes at the end of the studied period. The largest increase in expression occurred for PtaGA2ox2 and PtaGA2ox7 which showed seven-fold induction ( Figure 1). To study the role of the same genes in growth cessation during SD-induced bud dormancy, we imposed a SD photoperiod (8 h light/16 h dark) under controlled growth chamber conditions (see Materials and methods) and followed changes in expression in the leaves on a weekly basis. Expression of three of the four DELLA protein encoding genes and three of the seven PtaGA2ox genes increased significantly ( Figure 2). There was substantial overlap in the expression of genes up-regulated by both drought and SDs (PtaRGL1-1, PtaRGL1-2, PtaGA2ox1, PtaGA2ox3, and PtaGA2ox7). Expression of only two (PtaGAI1 and PtaGA2ox2) Figure 1. Poplar DELLA domain and GA2ox encoding genes were significantly up-regulated in response to drought stress. Shown are mean6SE of RT-PCR results for three biological reps each consisting of leaf tissue pooled from 2-3 plants for well-watered control (C) and water-withholding (1-3 weeks) treatments. Expression was normalized to Ubq and Cyc. Significant differences between watered and water-withholding treatments were determined by one-way ANOVA followed by Dunnett's post-hoc test (*, P,0.05). doi:10.1371/journal.pone.0086217.g001 of the seven genes was specifically influenced by one but not the other treatment (Figures 1 and 2).
GA-deficiency and GA-insensitivity Accelerates Growth Inhibition in Response to Drought and SDs
The induction of DELLA and GA2ox genes in response to drought and/or SDs suggests that these genes may mediate growth inhibition during both responses. We took advantage of previously, well-characterized, GA-deficient (35S::PcGA2ox) and GA-insensitive (35S::rgl1 and pGAI::gai) transgenic poplar with representative, stable, and intermediate/semidwarf phenotypes (see Materials and methods) to study their response to drought stress and SD photoperiods. To ensure that any inherent differences in size between genotypes did not obscure analysis of treatment effects, relative growth rates were used to determine significant differences between WT and transgenic plants. Experiments (drought and SD photoperiod) with transgenic plants were performed in a similar fashion to that of expression analysis and as described in the Materials and methods. Prior to implementing drought and SD photoperiod experiments, weekly relative growth rates were not significantly different between transgenic and WT plants under well-watered conditions and long-day photoperiods ( Figure 3). For the drought experiment, to further facilitate valid comparisons between different genotypes, methods recommended by Verslues et al. [41] were employed; whereby, transgenic and WT plants were grown in the same pots so that roots of all genotypes would grow into the same soil and be exposed to similar conditions (see Materials and methods) ( Figure S1). Because of the leaves' importance in controlling water loss and previous results of transgenic genotypes having specific effects on leaf size [39], we measured leaf area and expansion ( Figure S1). In support of our previous findings [39], the leaf area of GA-deficient transgenics was significantly different than WT, whereas GA-insensitive transgenics was not. However, expansion rates of newly formed leaves were similar between all transgenics and WT throughout the experiment ( Figure S1). After only one week of withholding water, the gai and rgl1 expressing transgenic plants had significantly reduced weekly relative growth rates in height, diameter, and number of nodes compared to WT ( Figure 3A). Three weeks post-water deprivation, growth was virtually absent in gai/rgl1 expressing plants whereas WT, and to a lesser extent GA2ox expressing plants, did not completely cease growth until weeks five and six. Interestingly, water deprivation also affected secondary woody growth (stem diameter at the base) in the gai/rgl1 transgenics, as indicated by their significantly decreased growth rates relative to WT in weeks three through five ( Figure 3A).
We also studied transgenics' growth response to SD photoperiods that induce winter dormancy. The first response to SDs, which precedes and is a prerequisite for dormancy, is cessation of shoot growth. Poplars are highly photoperiod sensitive, and the genotype under investigation, P. tremula x alba (717 1B4), cease growth after three to five weeks under SD photoperiod [42]. All transgenics had significantly greater, early reductions in weekly relative growth rate compared to WT plants (as early as one week under SD) ( Figure 3B). WT plants had a more gradual reduction in weekly growth and, as in the drought experiment, did not completely cease growth until the fifth week under SD conditions. In contrast to drought, we did not observe any differences between transgenics and WT with respect to reduction of diameter growth under SD conditions. Despite differences in growth cessation, the timing of bud set was not significantly (P.0.05) affected and occurred around week five in both transgenics and WT (data not shown).
Physiological Changes in Response to Drought Stress
Drought stress has a profound effect on a number of physiological parameters in plants, and measurement of these parameters is useful in determining their stress response and resistance [43,44]. To test if the GA2ox and DELLA expressing transgenic plants differ with respect to their physiological responses to drought, we measured several important parameters before and during drought stress response. We quantified pigment concentrations, photosynthetic rate, transpiration, and stomatal conductance, which are frequently used to measure the degree of stress imposed on plants ( Table 2). In general, GA-insensitive and GA-deficiency had similar effects on measured responses ( Table 2). Under well-watered conditions, all transgenics had significantly higher photosynthetic rates compared to WT (Table 2). Increases in photosynthetic rate corresponded to significantly higher total chlorophyll and carotenoids for all transgenic types (Table 2). Typically, drought stress reduces photosynthesis and causes were significantly up-regulated by SD photoperiod. Shown are mean6SE of three biological reps each consisting of leaf tissue pooled from three plants subjected to long-day (LD) and SD (1, 3, and 5 weeks) treatments. Expression was normalized to Ubq and Cyc. Significant differences between LD and SD treatments were determined by oneway ANOVA followed by Dunnett's post-hoc test (*, P,0.05). doi:10.1371/journal.pone.0086217.g002 degradation of chlorophyll. In contrast to WT, photosynthetic rates, chlorophyll and carotenoids remained significantly higher in all transgenics under drought conditions ( Table 2). Under wellwatered conditions transpiration and stomatal conductance were similar in transgenic and WT plants (Table 2). However, under drought stress, transgenic plants displayed significantly higher transpiration and stomatal conductance ( Table 2). Transgenics had significantly higher water use efficiency under well-watered conditions as compared to WT. Under drought stress all transgenics showed a slight decrease in water use efficiency as compared to WT but only those expressing gai were significantly different ( Table 2).
To assess the overall stress resistance of the different genotypes, we measured wilting and electrolyte leakage (EL). The DELLA expressing transgenics showed significantly lower levels of wilting compared to WT ( Figure 4A). The GA2ox transgenics also showed a lower level of wilting but the differences were not statistically significant (P.0.05) ( Figure 4A). EL can be used to quantify the extent of cellular damage caused by stress [41]. We found significantly lower EL in all transgenics ( Figure 4B).
Delayed Senescence in GA-deficient and GA-insensitive Poplars
In support of our quantitative pigment measurements, signs of senescence after water deprivation were more visible in WT leaves compared to transgenics ( Figure 5). Percent of senescing leaves was not significantly different, but an apparent trend of more advanced senescence in WT as compared to transgenics was evident ( Figure 5).
Leaf senescence preceding winter dormancy is typically initiated by SD photoperiods but is enhanced by low temperatures (,10uC). To investigate senescence in GA-deficient and GAinsensitive plants, we subjected transgenic and WT plants to six weeks of SDs at 21uC followed by three weeks at 4uC and measured the extent of leaf senescence. In a manner similar to their response to drought, all transgenic plants showed delayed senescence when compared to WT ( Figure 6).
Increased Axillary Shoot Outgrowth in GA-deficient and GA-insensitive Poplars
As with bud set, there were no significant differences in the timing of the first bud to flush after winter dormancy between transgenics and WT ( Figure 7). However, in WT plants apical buds were always the first to initiate growth, whereas in transgenics axillary lateral buds typically flushed first ( Figure 7).
In GA2ox overexpressing transgenics, this was followed by a significant increase in lateral branch outgrowth as compared to WT plants ( Figure 8A and B). In contrast to GA2ox transgenics, the flushed axillary buds in the DELLA expressing plants never elongated and remained in a leaf rosette stage. Because of the significant increase in lateral branches in GA2ox transgenics, we measured total height and branch growth three months after bud flush. Although GA2ox transgenics were significantly shorter, their branch length was significantly greater compared to WT ( Figure 8C and D).
Differential Expression of a Large Number of Genes in GA-modified Transgenics
Because the GA-modified transgenics showed accelerated response to drought and SDs, we hypothesized that the mechanisms associated with these responses are constitutively elevated even under control conditions (well-watered and long-day photoperiods). We therefore used whole-genome poplar microarray to compare transcriptomes of transgenic and WT leaves from plants grown under a control environment. We found 2,890 differentially expressed genes (ANOVA, P,0.01) (Table S1). Gene ontology (GO) analysis was used to gain insight into the global patterns of gene expression. Consistent with our results showing increased resistance to drought stress, we found 'response to stress' (GO:0006950) in the top 10 most significantly-enriched biological categories in the transgenic plants (Table S2). Among the genes associated with stress response were orthologs of CBF1 and CBF3 that encode AP2/ERF type transcription factors, and previously, CBF1-mediated cold stress response was shown to involve reductions in bioactive GA through increased GA2ox expression, which promoted DELLA protein accumulation [24]. Table 1. Names, models, and primer sequences for genes used in expression analysis.
PtaGAI2
Potri.010G110700 TTATACCCTCAAAATTCAACCGA TACTGAGTTCGAGTCTGTGGCT Likely because GA is highly integrated into the regulatory network of other hormones [45], we found that at least one aspect of each of the five major hormones (e.g., auxin, cytokinin, brassinosteroid, ABA, ethylene) was significantly affected in the GA-modified transgenics (Table S2). GO categories associated with ethylene were prevalent in a number of categories and enrichment significance (Table S2). The trends in expression of these ethylene-associated genes are indicative of increased production and signaling through the ethylene signal transduction pathway. For example, genes encoding biosynthetic enzymes, such as acetyl-CoA carboxylase and acetyl-CoA synthetase, were upregulated while the catabolizing ETHYLENE OVERPRODU-CER 1 was down-regulated. Furthermore, downstream AP2/ERF type transcription factor genes were also up-regulated.
GA metabolism effects both cell expansion and proliferation [11]. The growth restraining effects of DELLA proteins has been shown to be, at least in part, due to regulation of cell proliferation [26,27]. Previously, Achard et al. [27] showed that DELLAs restrain cell production by causing up-regulation of cell cycle inhibitors. We found the Kip-related protein (KRP3), a negative regulator of cell division, to be up-regulated in both GA-modified transgenics (Table S1), which is suggestive of a common mechanism for growth restraint through inhibition of cell proliferation.
GA-deficiency and GA-insensitivity Share Common Transcriptome Responses with Dormancy and Drought
Because our studies suggest that GA-insensitive and GAdeficient plants have faster and more robust responses to dormancy-inducing conditions and drought, we speculated that alterations in GA metabolism and signaling in transgenics may result in transcriptome level changes that are shared with plants that are responding to stress (i.e., SD photoperiod and drought). Therefore, we compared the transcriptome of GA-deficient and GA-insensitive poplars to previously published transcriptomes of WT poplars during dormancy induction and drought response [46,47]. Unfortunately, the data for dormancy induction was based on apices; microarray data for expression changes in poplar leaves was not available. Nevertheless, transcriptome comparisons could be useful in identifying differentially expressed genes that are not tissue-confounded. Indeed, nearly a quarter of all differentially expressed genes in the GA-modified transgenics were common with the genes found to be regulated during both dormancy induction and drought (Figure 9). In addition, the transgenics' transcriptomes shared additional overlaps with either dormancy induction or drought. Among the most significantly enriched GO terms in the common transcriptome were processes associated with chloroplast biogenesis and function (Table S3).
Discussion
Survival and productivity of long-lived perennial plants in temperate zones are dependent on robust responses to prolonged and seasonal cycles of unfavorable conditions. Here we report whole-genome microarray, expression, physiological, and transgenic evidence suggesting that GA catabolism and repressive signaling mediate shoot growth inhibition in response to both drought and SD-induced bud dormancy. Both water deprivation and SDs elicited up-regulation of a suite of poplar GA2ox and DELLA protein encoding genes (Figures 1 and 2). We also found that the two environmental factors elicited similar responses in the same set of genes with the only exception being PtaGAI1 and PtaGA2ox2, which were up-regulated by one but not the other factor. Particularly instructive are the trends in expression of the GA2ox genes, for which we have performed a significant amount of previous work [17,18]. For example, we found that GA2ox3 was up-regulated by both drought and SD photoperiods (Figures 1 and 2). PtaGA2ox3 is predominantly expressed in the apex, which contains the shoot apical meristems (SAM); furthermore, it was the only gene for which we failed to regenerate RNAi-suppressed Table 2. Physiological changes in GA-deficient and GA-insensitive poplars under well-watered and drought conditions. transgenic plants, strongly suggesting that this gene is involved in organization of SAM [18]. Because growth, and particularly primary growth which originates in SAM, is most affected by drought and SDs, we believe that the up-regulation of PtaGA2ox3 is likely an important mechanism for controlling growth through modulation of SAM activity. Another gene that was up-regulated by both drought and SDs was PtaGA2ox7. We have previously found that PtaGA2ox7 and its paralog PtaGA2ox2 are predominantly expressed in roots under optimal conditions and positively regulate lateral root proliferation [18,37]. The fact that they are up-regulated by drought in leaves suggests they are involved in a mechanism for coordination of shoot-root ratio in relation to resource availability. Decreased aerial growth with increased lateral root growth would produce a plant that demands less of limited resources while still actively exploring the soil environment.
Condition
The commonalities in the transcription responses to drought and SDs are interesting because the signaling mechanisms induced by these two types of environmental cues are quite different. One involves a response to osmotic stress [48], while the other encompasses the light signaling pathway [49]. This suggests that GA2ox and DELLA protein genes are downstream of different signal transduction pathways that converge on the same types of genes. One possible convergence point could be through cross-talk with ABA biosynthesis or response. For example, DELLA proteins have been shown to regulate XERICO, which encodes a ubiquitinligase that positively regulates ABA biosynthetic encoding genes [28]. Indeed, our microarray work showed that a poplar ortholog of XERICO was highly up-regulated in the GA-modified transgenic plants. Alternatively the DELLA proteins themselves could be a convergence hub, as an increasing body of evidence suggests that they are focal points of multiple signaling pathways in plants [50]. Finally, ethylene can also play a role in the cross-talk, as our microarray results indicate a strong up-regulation of genes involved in ethylene biosynthesis and response.
Using previously characterized GA-deficient and GA-insensitive transgenics, with increased expression of GA2ox and DELLA protein encoding genes and representative semidwarf phenotypes [36,39,40], we show that GA-modified transgenics have significant changes in growth and physiological responses under both drought and SDs. Overall, overexpression of GA2ox and DELLA proteins in poplar caused hypersensitive growth inhibition under both Plants were grown for nine weeks under SD photoperiod (8 h). The temperature was maintained at 21uC for the first six weeks and reduced to 4uC for the remaining three weeks. Significant differences between transgenic and WT treatments were determined by one-way ANOVA followed by Dunnett's post-hoc test (*, P,0.05). doi:10.1371/journal.pone.0086217.g006 drought and SDs. Relative growth rates were not significantly different between transgenic and WT plants under well-watered conditions and long-day photoperiods. In comparison, transgenics generally responded to water withholding and SDs by reducing relative growth on average one to three weeks earlier than WT ( Figure 3). Nevertheless, traits were affected differently under drought and SD conditions. Decrease in GA concentration and sensitivity in transgenic plants caused inhibition of primary growth (e.g., height growth and number of internodes) under drought and/or SDs. In contrast, the transgenic GA modulation had an impact on secondary growth (e.g., stem diameter growth) only under drought but not SDs. In addition, some genotype-specific response differences were found among the transgenics under the two conditions. Most notably, under drought conditions only GAinsensitive (and not GA-deficient) transgenics had significantly reduced relative growth as compared to WT plants ( Figure 3A) (discussed below); whereas, under SD conditions all transgenic types had significantly reduced relative growth ( Figure 3B).
In addition to growth cessation, reduction of bioactive GA levels and GA signaling in poplar transgenics significantly modified and/ or delayed the onset of several other physiological changes that are typically associated with stress response. For instance, stressful conditions typically cause a reduction in photosynthesis and promote leaf senescence, which in turn negatively affects productivity [43]. Thus, delayed leaf senescence and the ability to sustain high photosynthetic capacity in adverse conditions are highly desirable traits [51]. Under drought stress, GA-deficient and GA-insensitive transgenics sustained higher rates of photosynthesis (Table 2). Although we did not measure the photosynthetic rate during SD-induced dormancy, we did observe a delay in leaf senescence in transgenic plants (Figure 6), which is suggestive of a similar response. In support of our findings, GAs have been implicated in the repression of light-regulated genes [52]. Decreases in GA levels via up-regulation of GA2ox genes is associated with increases in the expression of light-regulated genes [52], photosynthesis [53], chlorophyll content, and light harvesting chlorophyll proteins [54]. It has also been shown that DELLA proteins are directly involved in photomorphogenesis [55]. In GAdeficient and GA-insensitive transgenics,sustained increases in photosynthesis coupled to reduced shoot growth demands could facilitate shunting of resources from primary to secondary/storage metabolism and/or investment in root growth. Previously, we have shown experimental evidence supporting these hypotheses. For example, the same transgenics also accumulate increased levels of secondary metabolites [36] and show increased levels of root production [36,37,40].
In addition, carotenoids are antioxidants that can increase stress resistance via detoxification of ROS [56], and are precursors to the stress-responsive hormone ABA [57]. In Arabidopsis, stress induced DELLA protein accumulation and increased activity of ROS-detoxification enzymes led to reductions in ROS levels [25]. Our microarray data also showed enrichment of GO categories associated with stress response (Table S2, GO:0006950) in the GA-deficient and GA-insensitive transgenics. A number of genes encoding enzymes associated with detoxification of ROS were found in this category, including peroxidases, glutathione-Stransferase, superoxide dismutase, glyoxylate reductase, monodehydroascorbate reductase (Table S1 and 2). Furthermore, genes associated with ABA biosynthesis, such as nine-cis-epoxycarotenoid dioxygense 3, were also highly up-regulated. In summary, DELLA proteins and GA2oxs likely not only repress growth but enhance plant resistance to stress via activation of ROSdetoxification enzymes, increased carotenoid production and ABA biosynthesis.
To better understand the effects of overexpressing GA2ox and DELLA protein encoding genes on drought response, we must also consider the implications of GA-deficient and GA-insensitive transgenics' semidwarf phenotype. Soil drying experiments can be difficult to interpret because the plants dictate the severity of stress by controlling the balance of water uptake by roots and loss through leaves [41]. Though we implemented methods to minimize genotypic differences in stress severity, semidwarfism could result in plants experiencing a reduced severity of stress. A smaller plant may require less water and thus deplete soil moisture slower. However, it is unlikely that our results are solely reflective of disparities in soil drying causing different stress severities intransgenic and WT plants. If transgenics had experienced a reduced stress severity then we would have expected relative growth rates, similar to those experienced under well-watered conditions, to be sustained longer and delays in growth cessation as compared to WT. However, our results are quite contrary as under drought conditions GA-insensitive transgenics decreased growth much earlier while GA-deficient plants did not differ compared to WT ( Figure 3A). Hence, transgenic responses may be due to increased perception of water deficits, morphological differences, and/or altered drought resistant mechanisms.
Because GA-insensitive transgenics responded significantly earlier to water deprivation as compared to WT ( Figure 3A), it is unlikely that the hypersensitive drought response induced by GA-insensitivity is a result of increased water availability due to disparities in soil drying. Nevertheless, unlike GA-deficient transgenics, GA-insensitive transgenics had a prompt response to water withdrawal associated with aboveground growth inhibition and suggestive of enhanced drought avoidance mechanisms ( Figure 3A). However, the leaves of GA-insensitive transgenics were similar in size but had increased gas exchange when compared to WT (Table 2). Thus, the prompt drought response was likely not a result of decreased leaf water loss. This is further supported by the fact that despite their smaller leaf size, GAdeficient transgenics did not differ from WT in respect to their growth inhibition ( Figure 3A). Thus, it is more likely that GAinsensitivity caused transgenic plants to have an enhanced ability to perceive and respond to water deficits. This is strongly supported by a large body of evidence suggesting that the DELLA proteins that cause GA-insensitivity are central hubs for responding to various stresses [23][24][25]. Furthermore under drought stress, leaves of GA-insensitive transgenics were still actively assimilating carbon with increases in photosynthesis and transpiration as compared to WT (Table 2 and Figure S3). The assimilated carbon could be used towards enhancing tolerance mechanisms to protect against cellular damage (osmotic solutes, and ROS detoxification) or to increase root growth, which would be advantageous to water uptake and further promote drought avoidance. Indeed, GAinsensitivity has been previously found to be associated with increased ratios of root mass to leaf area which have been suggested could help sustain higher transpiration rates and decreases in water use efficiency [40]. In summary, the GAmodifications caused complex responses that likelycontribute to both drought avoidance and tolerance mechanisms. Similar to recent findings by Mauriat et al. [58], we show that the GA2ox expressing transgenics have increased branch production which is indicative of decreased apical dominance ( Figure 8). However, in contrast to this previous finding, in the present study, the increased branching phenotype developed only after winter dormancy. This difference could be attributed to the different promoters, genetic backgrounds or growing conditions used in this study (note sylleptic branching is very sensitive to environmental quality). It appears that the observed reduction of apical dominance caused by GA2ox up-regulation is a result of decreased auxin concentration and transport [58]. We also show that upregulation of DELLA proteins leads to a similar phenotype, suggesting that the effect of GA on apical dominance, through modulation of auxin biosynthesis and transport, is downstream of and mediated through a DELLA signaling hub. However, in the DELLA transgenics, branch outgrowth after dormancy was very short-lived and resulted in a very short, stubby branch. In contrast, GA2ox transgenic plants produced very well developed branches from almost all axillary buds. This suggests that DELLA proteins have differential effects on apical dominance before and after dormancy release. Furthermore, this would imply that apical dominance before and after dormancy may be regulated via different branches of the GA signaling pathway or by a completely different mechanism involving different hormones and regulators. These two phenotypic differences in the GA2ox and DELLA transgenics had a significant and dramatic impact on crown shape and size in the field (e.g., wide, ball-shape in GA2ox; narrow compact in the DELLA transgenics) [39].
Conclusions
In summary, we found that GA catabolism and repressive signaling are part of a highly interactive mechanism for sensing and responding to immediate (drought) and imminent (SD signaling approaching winter) conditions involving ceasing or reducing growth as well as increasing physiologically acclimative responses to stress. These findings suggest that regulation of GA metabolism and response may be a focal point for evolution of various adaptive strategies in response to short-term and prolonged unfavorable conditions. It also suggests novel venues to engineer and breed for improved stress resistance in crop plants.
Plant Material
Genetic background for all transgenic plants was the INRA 717-1B clone (Populus tremula x P. alba). For consistency, all experiments involving WT plants were performed using this same genotype. Generation of 35S::PcGA2ox, pGAI::gai, and 35S::rgl1 transgenics was previously described [17,36]. The Arabidopsis (Arabidopsis thaliana) DELLA genes gai and rgl1 have complete truncations of the DELLA domains which confers a gain-of-function dominant mutation with constitutive repression of GA signaling. The gai gene was under the control of the native Arabidopsis promoter (pGAI) while rgl1 was under the control of the cauliflower mosaic virus 35S promoter (35S). The GA2ox1 gene was from Phaseolus coccineus (PcGA2ox) and was also under the control of the 35S promoter. Because constructs were previously well-characterized with respect to transgene presence, expression, GA content and stability of phenotype over many years [37,39], for each transgenic type one representative line with multiple ramets was selected for use in subsequent studies. The differences in the levels of expression of these transgenes in independent transformation events elicits a gradient of phenotypic responses ranging from severe dwarfism to nearly wild-type like [39]. To avoid confounding effects in severely affected plants, we selected lines with intermediate (semi-dwarf) phenotypes.
Experimental Design
The drought experiment consisted of four genotypes in a completely randomized block design with eight replications. Each block represented a rectangular pot (height = 32 cm, length = 50 cm, and width = 35 cm) assigned four plants: one from each of three transgenic genotypes (35S::PcGA2ox, pGAI::gai, and 35S::rgl1) and a WT (untransformed control). Plants were randomized and spaced in a rectangular pattern (25620 cm apart from each other) within pots. The confiding nature of the pots, allowed plants to be grown in a proximity where their roots were exposed to similar conditions to minimize effects of any differences in water usage between genotypes [41]. The use of relatively large, deep pots facilitated a slow, gradual soil drying process ( Figure S2) that provided plants sufficient time for lengthy responses. Plants used in the experiment were propagated and grown in vitro for one month prior to their transfer to a greenhouse. In the greenhouse, plants were placed in a mixture of peat, top soil, perlite, and vermiculite (4:1:1:1, v/v) and grown for approximately two months prior to experiment implementation. The drought treatment consisted of three weeks of well-watered conditions (daily watering regime), followed by a drought stress imposed by completely withholding water for five weeks. A similar experimental regime was used on control plants used in expression analysis (Figure 1) with the exception that plants were grown in a completely randomized design (without blocks), in separate pots, and in a growth chamber (Conviron, Pembina, ND, USA).
SD photoperiod experiments were performed in a completely randomized design with eight replications. Propagation and acclimation of plants to the greenhouse were performed as described above for the drought experiment. After two months of greenhouse growth, plants were transferred to a growth chamber (Conviron, Pembina, ND, USA), with three weeks under LDs (16 h light/8 h dark, at 21uC) followed by six weeks of SDs (8 h light/16 h dark, at 21uC). Plants were then transferred to a cold room (4uC) for 11 weeks to fulfill the chilling requirement needed for resumption of growth. Finally, plants were transferred to LD conditions and bud flush was monitored on a daily basis.
Biometric and Physiological Measurements
Phenotypic measurements were made on a weekly basis. To avoid problems with soil level changes, all height measurements were made from permanent markers at the base of the plant stem. Net photosynthesis rate, transpiration rate, and stomatal conductance were measured with the portable photosynthesis system LI-6400 (LI-COR, Lincoln, NE, USA), using an air temperature of 25uC and photosynthetically active radiation of 1500 mmol m 22 s 21 . Instantaneous water use efficiency was calculated as net photosynthesis rate dividing by transpiration rate. Gas exchange measurements were made between 8:00 and 10:00 am weekly and at leaf plastochron index (LPI) = 1061. After five weeks of withholding water, additional measurements were made including percent leaf senescence, percent leaf wilt, and electrolyte leakage (EL). EL was measured with a conductivity meter (Model 32, Yellow Springs Instrument Inc., Yellow Springs, OH, USA) using procedures modified from Ren et al. [59] and Verslues et al. [41]. Leaves (LPI = 1061) were thoroughly washed in distilled water and three 1 cm 2 discs obtained from leaves were placed in tubes containing 15 ml of distilled water. Tubes were gently agitated for 4 h and initial conductivity was measured. Samples were then autoclaved for 15 min and conductivity was again measured. EL measurements were repeated three times per plant, and values were averaged and expressed as percentage of initial conductivity. Chlorophyll and carotenoid content was measured on non-stressed (well-watered) plants subjected to drought stress (after withholding water for five weeks). Pigments were extracted in N,N-dimethylformamide according to Porra et al. [60]. Pigments were quantified with a spectrophotometer using equations from Porra et al. [60] for chlorophyll a and b, and from Wellburn et al. [61] for carotenoid. Two 1 cm 2 discs were measured for each plant from LPI = 861. Methods similar to those in [62] were used to quantify the relative intensity of green in senescing leaves. Digital images of leaf surfaces were processed using ImageJ version 1.43 (http://rsbweb.nih.gov/ij/) and the Threshold Colour plugin (http://www.dentistry.bham.ac.uk/ landinig/software/software.html).
Expression Analysis
All samples used in expression analysis were collected at the same time weekly, immediately frozen in liquid nitrogen, and stored at 280uC for further use. Samples were analyzed in three biological replications, each consisting of tissues pooled from two to three plants. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen,Valencia, CA, USA) with an on-column DNA digestion using RNase-Free DNase (Qiagen). cDNA was synthesized from 2 mg of DNaseI-treated total RNA using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with an oligo-dT primer. One ml of RT reaction was used for RT-PCR. Primers used in the expression analyses are shown in Table 1.
Statistical Analysis
Statistical analysis was performed using SAS 9.1 (SAS Institute Inc., Cary, NC, USA). In transgenic experiments, treatments consisted of the four genotypes under investigation. For both experiments, analysis of variance (ANOVA) was used to test for overall treatment effects (a = 0.05), and separate ANOVAs were done for each week of the experiment. When significant differences were present, Tukey's Honestly Significant Difference (HSD) was used to make comparisons among WT and transgenics, or Dunnett's test (P,0.05) was used when we were interested in making comparison to WT only. For analyses of growth-related parameters, the response variable is the weekly relative growth rate, calculated as: Y = (X n+1 2X n )/X n , where Y is weekly relative growth rate, X is the measured growth parameter, and n is the week.
Microarray Hybridization and Data Analyses
We used a total of three individual genotypes as follows: WT, 35S:PcGA2ox and 35S:rgl1. Leaves from two independent biological replicates per genotype were used, each pooled from twenty clonally propagated plants. RNA was isolated as previously described using the Qiagen RNeasy Plant Kit [17]. Prior to labeling, RNA quality was assessed by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, USA) and three mg of total RNA was used to prepare biotinylated complementary RNA (cRNA). The labeling, hybridization, and imaging procedures were performed according to Affymetrix protocols at the Center for Genomics Research and Biocomputing, Oregon State University), using the Affymetrix Poplar GeneChip (Affymetrix Santa Clara, CA, USA). Collection and analysis of data were compliant with MIAME standards (Brazma et al., 2001). Data were analyzed using TM4:MeV [63,64]. Raw data was first normalized using RMA algorithm [65]. One-way ANOVA (P,0.01) was used to isolate differentially expressed genes. Gene ontology (GO) analyses for significant enrichments of various categories were performed using GOEAST [66] and the corresponding AGI loci using default parameters. Data for trancriptome changes during drought and dormancy were derived from previously published studies [46,47]. Because of the differences in the microarray platforms and genome versions used in these studies, we used the AGI of the closest Arabidopsis ortholog to compare the three data sets. We have previously validated the results from the array analysis using RT-PCR for 12 genes (six up-regulated and six down-regulated) for the roots from the same plants [37]. The microarray data have been deposited in the Gene Expression Omnibus (GEO) database as accession number GSE38390. Figure S1 Representative phenotypes and leaf measurements of GA-modified transgenic poplar. (A) Transgenic (35S::PcGA2ox; top left, pGAI::gai; bottom left, and 35S::rgl1; bottom right) and wild-type (WT; top right) plants grown in pots (25620 cm apart from each other) for three weeks under wellwatered conditions, prior to being subjected to five weeks under water-withholding conditions. (B) Bars indicate mean6SE of total area (cm 3 ) of at least eight mature leaves per a genotype. (C) Leaf expansion (cm 3 ) was measured weekly under well-watered (week 0) and water-withholding conditions (weeks 1 to 5) on eight ramets/ line and eight WT plants. Measurements in C represent the total area of expansion of the first unfurled leaf (leaf plastrochon index 1) after one week. Leaf measurements were made from digital images in ImageJ version 1.43 (http://rsbweb.nih.gov/ij/). Significant differences between transgenic and WT plants were determined by one-way ANOVA followed by Dunnett's post-hoc test (*, P,0.05). Scale bar = 25 cm (A). (TIF) Figure S2 Estimation of volumetric soil moisture content (g cm 23 ) in pots under well-water (week 0) and water-withholding conditions (weeks 1 to 5). A soil auger was used to sample whole vertical profiles of pots. Samples were oven dried at 100uC for at least 24 hrs. Values are means6SE of at least three samples taken from at least two pots. (TIF) Figure S3 Weekly responses of transgenic and WT Populus under well-watered and water-withholding conditions. The dotted line denotes the initiation of water withholding. Red lines show significant differences between weekly responses of transgenics and WT (see Material and Methods), as determined by one-way ANOVA followed by Dunnett (post-hoc test (P,0.05). (TIF) | 2016-05-31T19:58:12.500Z | 2014-01-20T00:00:00.000 | {
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